case studies on stem cell research

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• Published: 26 February 2019

Stem cells: past, present, and future

• Wojciech Zakrzewski 1 ,
• Maciej Dobrzyński 2 ,
• Maria Szymonowicz 1 &
• Zbigniew Rybak 1  

Stem Cell Research & Therapy volume  10 , Article number:  68 ( 2019 ) Cite this article

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In recent years, stem cell therapy has become a very promising and advanced scientific research topic. The development of treatment methods has evoked great expectations. This paper is a review focused on the discovery of different stem cells and the potential therapies based on these cells. The genesis of stem cells is followed by laboratory steps of controlled stem cell culturing and derivation. Quality control and teratoma formation assays are important procedures in assessing the properties of the stem cells tested. Derivation methods and the utilization of culturing media are crucial to set proper environmental conditions for controlled differentiation. Among many types of stem tissue applications, the use of graphene scaffolds and the potential of extracellular vesicle-based therapies require attention due to their versatility. The review is summarized by challenges that stem cell therapy must overcome to be accepted worldwide. A wide variety of possibilities makes this cutting edge therapy a turning point in modern medicine, providing hope for untreatable diseases.

Stem cell classification

Stem cells are unspecialized cells of the human body. They are able to differentiate into any cell of an organism and have the ability of self-renewal. Stem cells exist both in embryos and adult cells. There are several steps of specialization. Developmental potency is reduced with each step, which means that a unipotent stem cell is not able to differentiate into as many types of cells as a pluripotent one. This chapter will focus on stem cell classification to make it easier for the reader to comprehend the following chapters.

Totipotent stem cells are able to divide and differentiate into cells of the whole organism. Totipotency has the highest differentiation potential and allows cells to form both embryo and extra-embryonic structures. One example of a totipotent cell is a zygote, which is formed after a sperm fertilizes an egg. These cells can later develop either into any of the three germ layers or form a placenta. After approximately 4 days, the blastocyst’s inner cell mass becomes pluripotent. This structure is the source of pluripotent cells.

Pluripotent stem cells (PSCs) form cells of all germ layers but not extraembryonic structures, such as the placenta. Embryonic stem cells (ESCs) are an example. ESCs are derived from the inner cell mass of preimplantation embryos. Another example is induced pluripotent stem cells (iPSCs) derived from the epiblast layer of implanted embryos. Their pluripotency is a continuum, starting from completely pluripotent cells such as ESCs and iPSCs and ending on representatives with less potency—multi-, oligo- or unipotent cells. One of the methods to assess their activity and spectrum is the teratoma formation assay. iPSCs are artificially generated from somatic cells, and they function similarly to PSCs. Their culturing and utilization are very promising for present and future regenerative medicine.

Multipotent stem cells have a narrower spectrum of differentiation than PSCs, but they can specialize in discrete cells of specific cell lineages. One example is a haematopoietic stem cell, which can develop into several types of blood cells. After differentiation, a haematopoietic stem cell becomes an oligopotent cell. Its differentiation abilities are then restricted to cells of its lineage. However, some multipotent cells are capable of conversion into unrelated cell types, which suggests naming them pluripotent cells.

Oligopotent stem cells can differentiate into several cell types. A myeloid stem cell is an example that can divide into white blood cells but not red blood cells.

Unipotent stem cells are characterized by the narrowest differentiation capabilities and a special property of dividing repeatedly. Their latter feature makes them a promising candidate for therapeutic use in regenerative medicine. These cells are only able to form one cell type, e.g. dermatocytes.

Stem cell biology

A blastocyst is formed after the fusion of sperm and ovum fertilization. Its inner wall is lined with short-lived stem cells, namely, embryonic stem cells. Blastocysts are composed of two distinct cell types: the inner cell mass (ICM), which develops into epiblasts and induces the development of a foetus, and the trophectoderm (TE). Blastocysts are responsible for the regulation of the ICM microenvironment. The TE continues to develop and forms the extraembryonic support structures needed for the successful origin of the embryo, such as the placenta. As the TE begins to form a specialized support structure, the ICM cells remain undifferentiated, fully pluripotent and proliferative [ 1 ]. The pluripotency of stem cells allows them to form any cell of the organism. Human embryonic stem cells (hESCs) are derived from the ICM. During the process of embryogenesis, cells form aggregations called germ layers: endoderm, mesoderm and ectoderm (Fig.  1 ), each eventually giving rise to differentiated cells and tissues of the foetus and, later on, the adult organism [ 2 ]. After hESCs differentiate into one of the germ layers, they become multipotent stem cells, whose potency is limited to only the cells of the germ layer. This process is short in human development. After that, pluripotent stem cells occur all over the organism as undifferentiated cells, and their key abilities are proliferation by the formation of the next generation of stem cells and differentiation into specialized cells under certain physiological conditions.

Oocyte development and formation of stem cells: the blastocoel, which is formed from oocytes, consists of embryonic stem cells that later differentiate into mesodermal, ectodermal, or endodermal cells. Blastocoel develops into the gastrula

Signals that influence the stem cell specialization process can be divided into external, such as physical contact between cells or chemical secretion by surrounding tissue, and internal, which are signals controlled by genes in DNA.

Stem cells also act as internal repair systems of the body. The replenishment and formation of new cells are unlimited as long as an organism is alive. Stem cell activity depends on the organ in which they are in; for example, in bone marrow, their division is constant, although in organs such as the pancreas, division only occurs under special physiological conditions.

Stem cell functional division

Whole-body development.

During division, the presence of different stem cells depends on organism development. Somatic stem cell ESCs can be distinguished. Although the derivation of ESCs without separation from the TE is possible, such a combination has growth limits. Because proliferating actions are limited, co-culture of these is usually avoided.

ESCs are derived from the inner cell mass of the blastocyst, which is a stage of pre-implantation embryo ca. 4 days after fertilization. After that, these cells are placed in a culture dish filled with culture medium. Passage is an inefficient but popular process of sub-culturing cells to other dishes. These cells can be described as pluripotent because they are able to eventually differentiate into every cell type in the organism. Since the beginning of their studies, there have been ethical restrictions connected to the medical use of ESCs in therapies. Most embryonic stem cells are developed from eggs that have been fertilized in an in vitro clinic, not from eggs fertilized in vivo.

Somatic or adult stem cells are undifferentiated and found among differentiated cells in the whole body after development. The function of these cells is to enable the healing, growth, and replacement of cells that are lost each day. These cells have a restricted range of differentiation options. Among many types, there are the following:

Mesenchymal stem cells are present in many tissues. In bone marrow, these cells differentiate mainly into the bone, cartilage, and fat cells. As stem cells, they are an exception because they act pluripotently and can specialize in the cells of any germ layer.

Neural cells give rise to nerve cells and their supporting cells—oligodendrocytes and astrocytes.

Haematopoietic stem cells form all kinds of blood cells: red, white, and platelets.

Skin stem cells form, for example, keratinocytes, which form a protective layer of skin.

The proliferation time of somatic stem cells is longer than that of ESCs. It is possible to reprogram adult stem cells back to their pluripotent state. This can be performed by transferring the adult nucleus into the cytoplasm of an oocyte or by fusion with the pluripotent cell. The same technique was used during cloning of the famous Dolly sheep.

hESCs are involved in whole-body development. They can differentiate into pluripotent, totipotent, multipotent, and unipotent cells (Fig.  2 ) [ 2 ].

Changes in the potency of stem cells in human body development. Potency ranges from pluripotent cells of the blastocyst to unipotent cells of a specific tissue in a human body such as the skin, CNS, or bone marrow. Reversed pluripotency can be achieved by the formation of induced pluripotent stem cells using either octamer-binding transcription factor (Oct4), sex-determining region Y (Sox2), Kruppel-like factor 4 (Klf4), or the Myc gene

Pluripotent cells can be named totipotent if they can additionally form extraembryonic tissues of the embryo. Multipotent cells are restricted in differentiating to each cell type of given tissue. When tissue contains only one lineage of cells, stem cells that form them are called either called oligo- or unipotent.

iPSC quality control and recognition by morphological differences

The comparability of stem cell lines from different individuals is needed for iPSC lines to be used in therapeutics [ 3 ]. Among critical quality procedures, the following can be distinguished:

Short tandem repeat analysis—This is the comparison of specific loci on the DNA of the samples. It is used in measuring an exact number of repeating units. One unit consists of 2 to 13 nucleotides repeating many times on the DNA strand. A polymerase chain reaction is used to check the lengths of short tandem repeats. The genotyping procedure of source tissue, cells, and iPSC seed and master cell banks is recommended.

Identity analysis—The unintentional switching of lines, resulting in other stem cell line contamination, requires rigorous assay for cell line identification.

Residual vector testing—An appearance of reprogramming vectors integrated into the host genome is hazardous, and testing their presence is a mandatory procedure. It is a commonly used procedure for generating high-quality iPSC lines. An acceptable threshold in high-quality research-grade iPSC line collections is ≤ 1 plasmid copies per 100 cells. During the procedure, 2 different regions, common to all plasmids, should be used as specific targets, such as EBNA and CAG sequences [ 3 ]. To accurately represent the test reactions, a standard curve needs to be prepared in a carrier of gDNA from a well-characterized hPSC line. For calculations of plasmid copies per cell, it is crucial to incorporate internal reference gDNA sequences to allow the quantification of, for example, ribonuclease P (RNaseP) or human telomerase reverse transcriptase (hTERT).

Karyotype—A long-term culture of hESCs can accumulate culture-driven mutations [ 4 ]. Because of that, it is crucial to pay additional attention to genomic integrity. Karyotype tests can be performed by resuscitating representative aliquots and culturing them for 48–72 h before harvesting cells for karyotypic analysis. If abnormalities are found within the first 20 karyotypes, the analysis must be repeated on a fresh sample. When this situation is repeated, the line is evaluated as abnormal. Repeated abnormalities must be recorded. Although karyology is a crucial procedure in stem cell quality control, the single nucleotide polymorphism (SNP) array, discussed later, has approximately 50 times higher resolution.

Viral testing—When assessing the quality of stem cells, all tests for harmful human adventitious agents must be performed (e.g. hepatitis C or human immunodeficiency virus). This procedure must be performed in the case of non-xeno-free culture agents.

Bacteriology—Bacterial or fungal sterility tests can be divided into culture- or broth-based tests. All the procedures must be recommended by pharmacopoeia for the jurisdiction in which the work is performed.

Single nucleotide polymorphism arrays—This procedure is a type of DNA microarray that detects population polymorphisms by enabling the detection of subchromosomal changes and the copy-neutral loss of heterozygosity, as well as an indication of cellular transformation. The SNP assay consists of three components. The first is labelling fragmented nucleic acid sequences with fluorescent dyes. The second is an array that contains immobilized allele-specific oligonucleotide (ASO) probes. The last component detects, records, and eventually interprets the signal.

Flow cytometry—This is a technique that utilizes light to count and profile cells in a heterogeneous fluid mixture. It allows researchers to accurately and rapidly collect data from heterogeneous fluid mixtures with live cells. Cells are passed through a narrow channel one by one. During light illumination, sensors detect light emitted or refracted from the cells. The last step is data analysis, compilation and integration into a comprehensive picture of the sample.

Phenotypic pluripotency assays—Recognizing undifferentiated cells is crucial in successful stem cell therapy. Among other characteristics, stem cells appear to have a distinct morphology with a high nucleus to cytoplasm ratio and a prominent nucleolus. Cells appear to be flat with defined borders, in contrast to differentiating colonies, which appear as loosely located cells with rough borders [ 5 ]. It is important that images of ideal and poor quality colonies for each cell line are kept in laboratories, so whenever there is doubt about the quality of culture, it can always be checked according to the representative image. Embryoid body formation or directed differentiation of monolayer cultures to produce cell types representative of all three embryonic germ layers must be performed. It is important to note that colonies cultured under different conditions may have different morphologies [ 6 ].

Histone modification and DNA methylation—Quality control can be achieved by using epigenetic analysis tools such as histone modification or DNA methylation. When stem cells differentiate, the methylation process silences pluripotency genes, which reduces differentiation potential, although other genes may undergo demethylation to become expressed [ 7 ]. It is important to emphasize that stem cell identity, together with its morphological characteristics, is also related to its epigenetic profile [ 8 , 9 ]. According to Brindley [ 10 ], there is a relationship between epigenetic changes, pluripotency, and cell expansion conditions, which emphasizes that unmethylated regions appear to be serum-dependent.

hESC derivation and media

hESCs can be derived using a variety of methods, from classic culturing to laser-assisted methodologies or microsurgery [ 11 ]. hESC differentiation must be specified to avoid teratoma formation (see Fig.  3 ).

Spontaneous differentiation of hESCs causes the formation of a heterogeneous cell population. There is a different result, however, when commitment signals (in forms of soluble factors and culture conditions) are applied and enable the selection of progenitor cells

hESCs spontaneously differentiate into embryonic bodies (EBs) [ 12 ]. EBs can be studied instead of embryos or animals to predict their effects on early human development. There are many different methods for acquiring EBs, such as bioreactor culture [ 13 ], hanging drop culture [ 12 ], or microwell technology [ 14 , 15 ]. These methods allow specific precursors to form in vitro [ 16 ].

The essential part of these culturing procedures is a separation of inner cell mass to culture future hESCs (Fig.  4 ) [ 17 ]. Rosowski et al. [ 18 ] emphasizes that particular attention must be taken in controlling spontaneous differentiation. When the colony reaches the appropriate size, cells must be separated. The occurrence of pluripotent cells lasts for 1–2 days. Because the classical utilization of hESCs caused ethical concerns about gastrulas used during procedures, Chung et al. [ 19 ] found out that it is also possible to obtain hESCs from four cell embryos, leaving a higher probability of embryo survival. Additionally, Zhang et al. [ 20 ] used only in vitro fertilization growth-arrested cells.

Culturing of pluripotent stem cells in vitro. Three days after fertilization, totipotent cells are formed. Blastocysts with ICM are formed on the sixth day after fertilization. Pluripotent stem cells from ICM can then be successfully transmitted on a dish

Cell passaging is used to form smaller clusters of cells on a new culture surface [ 21 ]. There are four important passaging procedures.

Enzymatic dissociation is a cutting action of enzymes on proteins and adhesion domains that bind the colony. It is a gentler method than the manual passage. It is crucial to not leave hESCs alone after passaging. Solitary cells are more sensitive and can easily undergo cell death; collagenase type IV is an example [ 22 , 23 ].

Manual passage , on the other hand, focuses on using cell scratchers. The selection of certain cells is not necessary. This should be done in the early stages of cell line derivation [ 24 ].

Trypsin utilization allows a healthy, automated hESC passage. Good Manufacturing Practice (GMP)-grade recombinant trypsin is widely available in this procedure [ 24 ]. However, there is a risk of decreasing the pluripotency and viability of stem cells [ 25 ]. Trypsin utilization can be halted with an inhibitor of the protein rho-associated protein kinase (ROCK) [ 26 ].

Ethylenediaminetetraacetic acid ( EDTA ) indirectly suppresses cell-to-cell connections by chelating divalent cations. Their suppression promotes cell dissociation [ 27 ].

Stem cells require a mixture of growth factors and nutrients to differentiate and develop. The medium should be changed each day.

Traditional culture methods used for hESCs are mouse embryonic fibroblasts (MEFs) as a feeder layer and bovine serum [ 28 ] as a medium. Martin et al. [ 29 ] demonstrated that hESCs cultured in the presence of animal products express the non-human sialic acid, N -glycolylneuraminic acid (NeuGc). Feeder layers prevent uncontrolled proliferation with factors such as leukaemia inhibitory factor (LIF) [ 30 ].

First feeder layer-free culture can be supplemented with serum replacement, combined with laminin [ 31 ]. This causes stable karyotypes of stem cells and pluripotency lasting for over a year.

Initial culturing media can be serum (e.g. foetal calf serum FCS), artificial replacement such as synthetic serum substitute (SSS), knockout serum replacement (KOSR), or StemPro [ 32 ]. The simplest culture medium contains only eight essential elements: DMEM/F12 medium, selenium, NaHCO 3, l -ascorbic acid, transferrin, insulin, TGFβ1, and FGF2 [ 33 ]. It is not yet fully known whether culture systems developed for hESCs can be allowed without adaptation in iPSC cultures.

Turning point in stem cell therapy

The turning point in stem cell therapy appeared in 2006, when scientists Shinya Yamanaka, together with Kazutoshi Takahashi, discovered that it is possible to reprogram multipotent adult stem cells to the pluripotent state. This process avoided endangering the foetus’ life in the process. Retrovirus-mediated transduction of mouse fibroblasts with four transcription factors (Oct-3/4, Sox2, KLF4, and c-Myc) [ 34 ] that are mainly expressed in embryonic stem cells could induce the fibroblasts to become pluripotent (Fig.  5 ) [ 35 ]. This new form of stem cells was named iPSCs. One year later, the experiment also succeeded with human cells [ 36 ]. After this success, the method opened a new field in stem cell research with a generation of iPSC lines that can be customized and biocompatible with the patient. Recently, studies have focused on reducing carcinogenesis and improving the conduction system.

Retroviral-mediated transduction induces pluripotency in isolated patient somatic cells. Target cells lose their role as somatic cells and, once again, become pluripotent and can differentiate into any cell type of human body

The turning point was influenced by former discoveries that happened in 1962 and 1987.

The former discovery was about scientist John Gurdon successfully cloning frogs by transferring a nucleus from a frog’s somatic cells into an oocyte. This caused a complete reversion of somatic cell development [ 37 ]. The results of his experiment became an immense discovery since it was previously believed that cell differentiation is a one-way street only, but his experiment suggested the opposite and demonstrated that it is even possible for a somatic cell to again acquire pluripotency [ 38 ].

The latter was a discovery made by Davis R.L. that focused on fibroblast DNA subtraction. Three genes were found that originally appeared in myoblasts. The enforced expression of only one of the genes, named myogenic differentiation 1 (Myod1), caused the conversion of fibroblasts into myoblasts, showing that reprogramming cells is possible, and it can even be used to transform cells from one lineage to another [ 39 ].

Although pluripotency can occur naturally only in embryonic stem cells, it is possible to induce terminally differentiated cells to become pluripotent again. The process of direct reprogramming converts differentiated somatic cells into iPSC lines that can form all cell types of an organism. Reprogramming focuses on the expression of oncogenes such as Myc and Klf4 (Kruppel-like factor 4). This process is enhanced by a downregulation of genes promoting genome stability, such as p53. Additionally, cell reprogramming involves histone alteration. All these processes can cause potential mutagenic risk and later lead to an increased number of mutations. Quinlan et al. [ 40 ] checked fully pluripotent mouse iPSCs using whole genome DNA sequencing and structural variation (SV) detection algorithms. Based on those studies, it was confirmed that although there were single mutations in the non-genetic region, there were non-retrotransposon insertions. This led to the conclusion that current reprogramming methods can produce fully pluripotent iPSCs without severe genomic alterations.

During the course of development from pluripotent hESCs to differentiated somatic cells, crucial changes appear in the epigenetic structure of these cells. There is a restriction or permission of the transcription of genes relevant to each cell type. When somatic cells are being reprogrammed using transcription factors, all the epigenetic architecture has to be reconditioned to achieve iPSCs with pluripotency [ 41 ]. However, cells of each tissue undergo specific somatic genomic methylation. This influences transcription, which can further cause alterations in induced pluripotency [ 42 ].

Source of iPSCs

Because pluripotent cells can propagate indefinitely and differentiate into any kind of cell, they can be an unlimited source, either for replacing lost or diseased tissues. iPSCs bypass the need for embryos in stem cell therapy. Because they are made from the patient’s own cells, they are autologous and no longer generate any risk of immune rejection.

At first, fibroblasts were used as a source of iPSCs. Because a biopsy was needed to achieve these types of cells, the technique underwent further research. Researchers investigated whether more accessible cells could be used in the method. Further, other cells were used in the process: peripheral blood cells, keratinocytes, and renal epithelial cells found in urine. An alternative strategy to stem cell transplantation can be stimulating a patient’s endogenous stem cells to divide or differentiate, occurring naturally when skin wounds are healing. In 2008, pancreatic exocrine cells were shown to be reprogrammed to functional, insulin-producing beta cells [ 43 ].

The best stem cell source appears to be the fibroblasts, which is more tempting in the case of logistics since its stimulation can be fast and better controlled [ 44 ].

• Teratoma formation assay

The self-renewal and differentiation capabilities of iPSCs have gained significant interest and attention in regenerative medicine sciences. To study their abilities, a quality-control assay is needed, of which one of the most important is the teratoma formation assay. Teratomas are benign tumours. Teratomas are capable of rapid growth in vivo and are characteristic because of their ability to develop into tissues of all three germ layers simultaneously. Because of the high pluripotency of teratomas, this formation assay is considered an assessment of iPSC’s abilities [ 45 ].

Teratoma formation rate, for instance, was observed to be elevated in human iPSCs compared to that in hESCs [ 46 ]. This difference may be connected to different differentiation methods and cell origins. Most commonly, the teratoma assay involves an injection of examined iPSCs subcutaneously or under the testis or kidney capsule in mice, which are immune-deficient [ 47 ]. After injection, an immature but recognizable tissue can be observed, such as the kidney tubules, bone, cartilage, or neuroepithelium [ 30 ]. The injection site may have an impact on the efficiency of teratoma formation [ 48 ].

There are three groups of markers used in this assay to differentiate the cells of germ layers. For endodermal tissue, there is insulin/C-peptide and alpha-1 antitrypsin [ 49 ]. For the mesoderm, derivatives can be used, e.g. cartilage matrix protein for the bone and alcian blue for the cartilage. As ectodermal markers, class III B botulin or keratin can be used for keratinocytes.

Teratoma formation assays are considered the gold standard for demonstrating the pluripotency of human iPSCs, demonstrating their possibilities under physiological conditions. Due to their actual tissue formation, they could be used for the characterization of many cell lineages [ 50 ].

Directed differentiation

To be useful in therapy, stem cells must be converted into desired cell types as necessary or else the whole regenerative medicine process will be pointless. Differentiation of ESCs is crucial because undifferentiated ESCs can cause teratoma formation in vivo. Understanding and using signalling pathways for differentiation is an important method in successful regenerative medicine. In directed differentiation, it is likely to mimic signals that are received by cells when they undergo successive stages of development [ 51 ]. The extracellular microenvironment plays a significant role in controlling cell behaviour. By manipulating the culture conditions, it is possible to restrict specific differentiation pathways and generate cultures that are enriched in certain precursors in vitro. However, achieving a similar effect in vivo is challenging. It is crucial to develop culture conditions that will allow the promotion of homogenous and enhanced differentiation of ESCs into functional and desired tissues.

Regarding the self-renewal of embryonic stem cells, Hwang et al. [ 52 ] noted that the ideal culture method for hESC-based cell and tissue therapy would be a defined culture free of either the feeder layer or animal components. This is because cell and tissue therapy requires the maintenance of large quantities of undifferentiated hESCs, which does not make feeder cells suitable for such tasks.

Most directed differentiation protocols are formed to mimic the development of an inner cell mass during gastrulation. During this process, pluripotent stem cells differentiate into ectodermal, mesodermal, or endodermal progenitors. Mall molecules or growth factors induce the conversion of stem cells into appropriate progenitor cells, which will later give rise to the desired cell type. There is a variety of signal intensities and molecular families that may affect the establishment of germ layers in vivo, such as fibroblast growth factors (FGFs) [ 53 ]; the Wnt family [ 54 ] or superfamily of transforming growth factors—β(TGFβ); and bone morphogenic proteins (BMP) [ 55 ]. Each candidate factor must be tested on various concentrations and additionally applied to various durations because the precise concentrations and times during which developing cells in embryos are influenced during differentiation are unknown. For instance, molecular antagonists of endogenous BMP and Wnt signalling can be used for ESC formation of ectoderm [ 56 ]. However, transient Wnt and lower concentrations of the TGFβ family trigger mesodermal differentiation [ 57 ]. Regarding endoderm formation, a higher activin A concentration may be required [ 58 , 59 ].

There are numerous protocols about the methods of forming progenitors of cells of each of germ layers, such as cardiomyocytes [ 60 ], hepatocytes [ 61 ], renal cells [ 62 ], lung cells [ 63 , 64 ], motor neurons [ 65 ], intestinal cells [ 66 ], or chondrocytes [ 67 ].

Directed differentiation of either iPSCs or ESCs into, e.g. hepatocytes, could influence and develop the study of the molecular mechanisms in human liver development. In addition, it could also provide the possibility to form exogenous hepatocytes for drug toxicity testing [ 68 ].

Levels of concentration and duration of action with a specific signalling molecule can cause a variety of factors. Unfortunately, for now, a high cost of recombinant factors is likely to limit their use on a larger scale in medicine. The more promising technique focuses on the use of small molecules. These can be used for either activating or deactivating specific signalling pathways. They enhance reprogramming efficiency by creating cells that are compatible with the desired type of tissue. It is a cheaper and non-immunogenic method.

One of the successful examples of small-molecule cell therapies is antagonists and agonists of the Hedgehog pathway. They show to be very useful in motor neuron regeneration [ 69 ]. Endogenous small molecules with their function in embryonic development can also be used in in vitro methods to induce the differentiation of cells; for example, retinoic acid, which is responsible for patterning the nervous system in vivo [ 70 ], surprisingly induced retinal cell formation when the laboratory procedure involved hESCs [ 71 ].

The efficacy of differentiation factors depends on functional maturity, efficiency, and, finally, introducing produced cells to their in vivo equivalent. Topography, shear stress, and substrate rigidity are factors influencing the phenotype of future cells [ 72 ].

The control of biophysical and biochemical signals, the biophysical environment, and a proper guide of hESC differentiation are important factors in appropriately cultured stem cells.

Stem cell utilization and their manufacturing standards and culture systems

The European Medicines Agency and the Food and Drug Administration have set Good Manufacturing Practice (GMP) guidelines for safe and appropriate stem cell transplantation. In the past, protocols used for stem cell transplantation required animal-derived products [ 73 ].

The risk of introducing animal antigens or pathogens caused a restriction in their use. Due to such limitations, the technique required an obvious update [ 74 ]. Now, it is essential to use xeno-free equivalents when establishing cell lines that are derived from fresh embryos and cultured from human feeder cell lines [ 75 ]. In this method, it is crucial to replace any non-human materials with xeno-free equivalents [ 76 ].

NutriStem with LN-511, TeSR2 with human recombinant laminin (LN-511), and RegES with human foreskin fibroblasts (HFFs) are commonly used xeno-free culture systems [ 33 ]. There are many organizations and international initiatives, such as the National Stem Cell Bank, that provide stem cell lines for treatment or medical research [ 77 ].

Stem cell use in medicine

Stem cells have great potential to become one of the most important aspects of medicine. In addition to the fact that they play a large role in developing restorative medicine, their study reveals much information about the complex events that happen during human development.

The difference between a stem cell and a differentiated cell is reflected in the cells’ DNA. In the former cell, DNA is arranged loosely with working genes. When signals enter the cell and the differentiation process begins, genes that are no longer needed are shut down, but genes required for the specialized function will remain active. This process can be reversed, and it is known that such pluripotency can be achieved by interaction in gene sequences. Takahashi and Yamanaka [ 78 ] and Loh et al. [ 79 ] discovered that octamer-binding transcription factor 3 and 4 (Oct3/4), sex determining region Y (SRY)-box 2 and Nanog genes function as core transcription factors in maintaining pluripotency. Among them, Oct3/4 and Sox2 are essential for the generation of iPSCs.

Many serious medical conditions, such as birth defects or cancer, are caused by improper differentiation or cell division. Currently, several stem cell therapies are possible, among which are treatments for spinal cord injury, heart failure [ 80 ], retinal and macular degeneration [ 81 ], tendon ruptures, and diabetes type 1 [ 82 ]. Stem cell research can further help in better understanding stem cell physiology. This may result in finding new ways of treating currently incurable diseases.

Haematopoietic stem cell transplantation

Haematopoietic stem cells are important because they are by far the most thoroughly characterized tissue-specific stem cell; after all, they have been experimentally studied for more than 50 years. These stem cells appear to provide an accurate paradigm model system to study tissue-specific stem cells, and they have potential in regenerative medicine.

Multipotent haematopoietic stem cell (HSC) transplantation is currently the most popular stem cell therapy. Target cells are usually derived from the bone marrow, peripheral blood, or umbilical cord blood [ 83 ]. The procedure can be autologous (when the patient’s own cells are used), allogenic (when the stem cell comes from a donor), or syngeneic (from an identical twin). HSCs are responsible for the generation of all functional haematopoietic lineages in blood, including erythrocytes, leukocytes, and platelets. HSC transplantation solves problems that are caused by inappropriate functioning of the haematopoietic system, which includes diseases such as leukaemia and anaemia. However, when conventional sources of HSC are taken into consideration, there are some important limitations. First, there is a limited number of transplantable cells, and an efficient way of gathering them has not yet been found. There is also a problem with finding a fitting antigen-matched donor for transplantation, and viral contamination or any immunoreactions also cause a reduction in efficiency in conventional HSC transplantations. Haematopoietic transplantation should be reserved for patients with life-threatening diseases because it has a multifactorial character and can be a dangerous procedure. iPSC use is crucial in this procedure. The use of a patient’s own unspecialized somatic cells as stem cells provides the greatest immunological compatibility and significantly increases the success of the procedure.

Stem cells as a target for pharmacological testing

Stem cells can be used in new drug tests. Each experiment on living tissue can be performed safely on specific differentiated cells from pluripotent cells. If any undesirable effect appears, drug formulas can be changed until they reach a sufficient level of effectiveness. The drug can enter the pharmacological market without harming any live testers. However, to test the drugs properly, the conditions must be equal when comparing the effects of two drugs. To achieve this goal, researchers need to gain full control of the differentiation process to generate pure populations of differentiated cells.

Stem cells as an alternative for arthroplasty

One of the biggest fears of professional sportsmen is getting an injury, which most often signifies the end of their professional career. This applies especially to tendon injuries, which, due to current treatment options focusing either on conservative or surgical treatment, often do not provide acceptable outcomes. Problems with the tendons start with their regeneration capabilities. Instead of functionally regenerating after an injury, tendons merely heal by forming scar tissues that lack the functionality of healthy tissues. Factors that may cause this failed healing response include hypervascularization, deposition of calcific materials, pain, or swelling [ 84 ].

Additionally, in addition to problems with tendons, there is a high probability of acquiring a pathological condition of joints called osteoarthritis (OA) [ 85 ]. OA is common due to the avascular nature of articular cartilage and its low regenerative capabilities [ 86 ]. Although arthroplasty is currently a common procedure in treating OA, it is not ideal for younger patients because they can outlive the implant and will require several surgical procedures in the future. These are situations where stem cell therapy can help by stopping the onset of OA [ 87 ]. However, these procedures are not well developed, and the long-term maintenance of hyaline cartilage requires further research.

Osteonecrosis of the femoral hip (ONFH) is a refractory disease associated with the collapse of the femoral head and risk of hip arthroplasty in younger populations [ 88 ]. Although total hip arthroplasty (THA) is clinically successful, it is not ideal for young patients, mostly due to the limited lifetime of the prosthesis. An increasing number of clinical studies have evaluated the therapeutic effect of stem cells on ONFH. Most of the authors demonstrated positive outcomes, with reduced pain, improved function, or avoidance of THA [ 89 , 90 , 91 ].

Rejuvenation by cell programming

Ageing is a reversible epigenetic process. The first cell rejuvenation study was published in 2011 [ 92 ]. Cells from aged individuals have different transcriptional signatures, high levels of oxidative stress, dysfunctional mitochondria, and shorter telomeres than in young cells [ 93 ]. There is a hypothesis that when human or mouse adult somatic cells are reprogrammed to iPSCs, their epigenetic age is virtually reset to zero [ 94 ]. This was based on an epigenetic model, which explains that at the time of fertilization, all marks of parenteral ageing are erased from the zygote’s genome and its ageing clock is reset to zero [ 95 ].

In their study, Ocampo et al. [ 96 ] used Oct4, Sox2, Klf4, and C-myc genes (OSKM genes) and affected pancreas and skeletal muscle cells, which have poor regenerative capacity. Their procedure revealed that these genes can also be used for effective regenerative treatment [ 97 ]. The main challenge of their method was the need to employ an approach that does not use transgenic animals and does not require an indefinitely long application. The first clinical approach would be preventive, focused on stopping or slowing the ageing rate. Later, progressive rejuvenation of old individuals can be attempted. In the future, this method may raise some ethical issues, such as overpopulation, leading to lower availability of food and energy.

For now, it is important to learn how to implement cell reprogramming technology in non-transgenic elder animals and humans to erase marks of ageing without removing the epigenetic marks of cell identity.

Cell-based therapies

Stem cells can be induced to become a specific cell type that is required to repair damaged or destroyed tissues (Fig.  6 ). Currently, when the need for transplantable tissues and organs outweighs the possible supply, stem cells appear to be a perfect solution for the problem. The most common conditions that benefit from such therapy are macular degenerations [ 98 ], strokes [ 99 ], osteoarthritis [ 89 , 90 ], neurodegenerative diseases, and diabetes [ 100 ]. Due to this technique, it can become possible to generate healthy heart muscle cells and later transplant them to patients with heart disease.

Stem cell experiments on animals. These experiments are one of the many procedures that proved stem cells to be a crucial factor in future regenerative medicine

In the case of type 1 diabetes, insulin-producing cells in the pancreas are destroyed due to an autoimmunological reaction. As an alternative to transplantation therapy, it can be possible to induce stem cells to differentiate into insulin-producing cells [ 101 ].

Stem cells and tissue banks

iPS cells with their theoretically unlimited propagation and differentiation abilities are attractive for the present and future sciences. They can be stored in a tissue bank to be an essential source of human tissue used for medical examination. The problem with conventional differentiated tissue cells held in the laboratory is that their propagation features diminish after time. This does not occur in iPSCs.

The umbilical cord is known to be rich in mesenchymal stem cells. Due to its cryopreservation immediately after birth, its stem cells can be successfully stored and used in therapies to prevent the future life-threatening diseases of a given patient.

Stem cells of human exfoliated deciduous teeth (SHED) found in exfoliated deciduous teeth has the ability to develop into more types of body tissues than other stem cells [ 102 ] (Table  1 ). Techniques of their collection, isolation, and storage are simple and non-invasive. Among the advantages of banking, SHED cells are:

Guaranteed donor-match autologous transplant that causes no immune reaction and rejection of cells [ 103 ]

Simple and painless for both child and parent

Less than one third of the cost of cord blood storage

Not subject to the same ethical concerns as embryonic stem cells [ 104 ]

In contrast to cord blood stem cells, SHED cells are able to regenerate into solid tissues such as connective, neural, dental, or bone tissue [ 105 , 106 ]

SHED can be useful for close relatives of the donor

Fertility diseases

In 2011, two researchers, Katsuhiko Hayashi et al. [ 107 ], showed in an experiment on mice that it is possible to form sperm from iPSCs. They succeeded in delivering healthy and fertile pups in infertile mice. The experiment was also successful for female mice, where iPSCs formed fully functional eggs .

Young adults at risk of losing their spermatogonial stem cells (SSC), mostly cancer patients, are the main target group that can benefit from testicular tissue cryopreservation and autotransplantation. Effective freezing methods for adult and pre-pubertal testicular tissue are available [ 108 ].

Qiuwan et al. [ 109 ] provided important evidence that human amniotic epithelial cell (hAEC) transplantation could effectively improve ovarian function by inhibiting cell apoptosis and reducing inflammation in injured ovarian tissue of mice, and it could be a promising strategy for the management of premature ovarian failure or insufficiency in female cancer survivors.

For now, reaching successful infertility treatments in humans appears to be only a matter of time, but there are several challenges to overcome. First, the process needs to have high efficiency; second, the chances of forming tumours instead of eggs or sperm must be maximally reduced. The last barrier is how to mature human sperm and eggs in the lab without transplanting them to in vivo conditions, which could cause either a tumour risk or an invasive procedure.

Therapy for incurable neurodegenerative diseases

Thanks to stem cell therapy, it is possible not only to delay the progression of incurable neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease (AD), and Huntington disease, but also, most importantly, to remove the source of the problem. In neuroscience, the discovery of neural stem cells (NSCs) has nullified the previous idea that adult CNS were not capable of neurogenesis [ 110 , 111 ]. Neural stem cells are capable of improving cognitive function in preclinical rodent models of AD [ 112 , 113 , 114 ]. Awe et al. [ 115 ] clinically derived relevant human iPSCs from skin punch biopsies to develop a neural stem cell-based approach for treating AD. Neuronal degeneration in Parkinson’s disease (PD) is focal, and dopaminergic neurons can be efficiently generated from hESCs. PD is an ideal disease for iPSC-based cell therapy [ 116 ]. However, this therapy is still in an experimental phase ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539501 /). Brain tissue from aborted foetuses was used on patients with Parkinson’s disease [ 117 ]. Although the results were not uniform, they showed that therapies with pure stem cells are an important and achievable therapy.

Stem cell use in dentistry

Teeth represent a very challenging material for regenerative medicine. They are difficult to recreate because of their function in aspects such as articulation, mastication, or aesthetics due to their complicated structure. Currently, there is a chance for stem cells to become more widely used than synthetic materials. Teeth have a large advantage of being the most natural and non-invasive source of stem cells.

For now, without the use of stem cells, the most common periodontological treatments are either growth factors, grafts, or surgery. For example, there are stem cells in periodontal ligament [ 118 , 119 ], which are capable of differentiating into osteoblasts or cementoblasts, and their functions were also assessed in neural cells [ 120 ]. Tissue engineering is a successful method for treating periodontal diseases. Stem cells of the root apical areas are able to recreate periodontal ligament. One of the possible methods of tissue engineering in periodontology is gene therapy performed using adenoviruses-containing growth factors [ 121 ].

As a result of animal studies, dentin regeneration is an effective process that results in the formation of dentin bridges [ 122 ].

Enamel is more difficult to regenerate than dentin. After the differentiation of ameloblastoma cells into the enamel, the former is destroyed, and reparation is impossible. Medical studies have succeeded in differentiating bone marrow stem cells into ameloblastoma [ 123 ].

Healthy dental tissue has a high amount of regular stem cells, although this number is reduced when tissue is either traumatized or inflamed [ 124 ]. There are several dental stem cell groups that can be isolated (Fig.  7 ).

Localization of stem cells in dental tissues. Dental pulp stem cells (DPSCs) and human deciduous teeth stem cells (SHED) are located in the dental pulp. Periodontal ligaments stem cells are located in the periodontal ligament. Apical papilla consists of stem cells from the apical papilla (SCAP)

Dental pulp stem cell (DPSC)

These were the first dental stem cells isolated from the human dental pulp, which were [ 125 ] located inside dental pulp (Table  2 ). They have osteogenic and chondrogenic potential. Mesenchymal stem cells (MSCs) of the dental pulp, when isolated, appear highly clonogenic; they can be isolated from adult tissue (e.g. bone marrow, adipose tissue) and foetal (e.g. umbilical cord) [ 126 ] tissue, and they are able to differentiate densely [ 127 ]. MSCs differentiate into odontoblast-like cells and osteoblasts to form dentin and bone. Their best source locations are the third molars [ 125 ]. DPSCs are the most useful dental source of tissue engineering due to their easy surgical accessibility, cryopreservation possibility, increased production of dentin tissues compared to non-dental stem cells, and their anti-inflammatory abilities. These cells have the potential to be a source for maxillofacial and orthopaedic reconstructions or reconstructions even beyond the oral cavity. DPSCs are able to generate all structures of the developed tooth [ 128 ]. In particular, beneficial results in the use of DPSCs may be achieved when combined with other new therapies, such as periodontal tissue photobiomodulation (laser stimulation), which is an efficient technique in the stimulation of proliferation and differentiation into distinct cell types [ 129 ]. DPSCs can be induced to form neural cells to help treat neurological deficits.

Stem cells of human exfoliated deciduous teeth (SHED) have a faster rate of proliferation than DPSCs and differentiate into an even greater number of cells, e.g. other mesenchymal and non-mesenchymal stem cell derivatives, such as neural cells [ 130 ]. These cells possess one major disadvantage: they form a non-complete dentin/pulp-like complex in vivo. SHED do not undergo the same ethical concerns as embryonic stem cells. Both DPSCs and SHED are able to form bone-like tissues in vivo [ 131 ] and can be used for periodontal, dentin, or pulp regeneration. DPSCs and SHED can be used in treating, for example, neural deficits [ 132 ]. DPSCs alone were tested and successfully applied for alveolar bone and mandible reconstruction [ 133 ].

Periodontal ligament stem cells (PDLSCs)

These cells are used in periodontal ligament or cementum tissue regeneration. They can differentiate into mesenchymal cell lineages to produce collagen-forming cells, adipocytes, cementum tissue, Sharpey’s fibres, and osteoblast-like cells in vitro. PDLSCs exist both on the root and alveolar bone surfaces; however, on the latter, these cells have better differentiation abilities than on the former [ 134 ]. PDLSCs have become the first treatment for periodontal regeneration therapy because of their safety and efficiency [ 135 , 136 ].

Stem cells from apical papilla (SCAP)

These cells are mesenchymal structures located within immature roots. They are isolated from human immature permanent apical papilla. SCAP are the source of odontoblasts and cause apexogenesis. These stem cells can be induced in vitro to form odontoblast-like cells, neuron-like cells, or adipocytes. SCAP have a higher capacity of proliferation than DPSCs, which makes them a better choice for tissue regeneration [ 137 , 138 ].

Dental follicle stem cells (DFCs)

These cells are loose connective tissues surrounding the developing tooth germ. DFCs contain cells that can differentiate into cementoblasts, osteoblasts, and periodontal ligament cells [ 139 , 140 ]. Additionally, these cells proliferate after even more than 30 passages [ 141 ]. DFCs are most commonly extracted from the sac of a third molar. When DFCs are combined with a treated dentin matrix, they can form a root-like tissue with a pulp-dentin complex and eventually form tooth roots [ 141 ]. When DFC sheets are induced by Hertwig’s epithelial root sheath cells, they can produce periodontal tissue; thus, DFCs represent a very promising material for tooth regeneration [ 142 ].

Pulp regeneration in endodontics

Dental pulp stem cells can differentiate into odontoblasts. There are few methods that enable the regeneration of the pulp.

The first is an ex vivo method. Proper stem cells are grown on a scaffold before they are implanted into the root channel [ 143 ].

The second is an in vivo method. This method focuses on injecting stem cells into disinfected root channels after the opening of the in vivo apex. Additionally, the use of a scaffold is necessary to prevent the movement of cells towards other tissues. For now, only pulp-like structures have been created successfully.

Methods of placing stem cells into the root channel constitute are either soft scaffolding [ 144 ] or the application of stem cells in apexogenesis or apexification. Immature teeth are the best source [ 145 ]. Nerve and blood vessel network regeneration are extremely vital to keep pulp tissue healthy.

The potential of dental stem cells is mainly regarding the regeneration of damaged dentin and pulp or the repair of any perforations; in the future, it appears to be even possible to generate the whole tooth. Such an immense success would lead to the gradual replacement of implant treatments. Mandibulary and maxillary defects can be one of the most complicated dental problems for stem cells to address.

Acquiring non-dental tissue cells by dental stem cell differentiation

In 2013, it was reported that it is possible to grow teeth from stem cells obtained extra-orally, e.g. from urine [ 146 ]. Pluripotent stem cells derived from human urine were induced and generated tooth-like structures. The physical properties of the structures were similar to natural ones except for hardness [ 127 ]. Nonetheless, it appears to be a very promising technique because it is non-invasive and relatively low-cost, and somatic cells can be used instead of embryonic cells. More importantly, stem cells derived from urine did not form any tumours, and the use of autologous cells reduces the chances of rejection [ 147 ].

Use of graphene in stem cell therapy

Over recent years, graphene and its derivatives have been increasingly used as scaffold materials to mediate stem cell growth and differentiation [ 148 ]. Both graphene and graphene oxide (GO) represent high in-plane stiffness [ 149 ]. Because graphene has carbon and aromatic network, it works either covalently or non-covalently with biomolecules; in addition to its superior mechanical properties, graphene offers versatile chemistry. Graphene exhibits biocompatibility with cells and their proper adhesion. It also tested positively for enhancing the proliferation or differentiation of stem cells [ 148 ]. After positive experiments, graphene revealed great potential as a scaffold and guide for specific lineages of stem cell differentiation [ 150 ]. Graphene has been successfully used in the transplantation of hMSCs and their guided differentiation to specific cells. The acceleration skills of graphene differentiation and division were also investigated. It was discovered that graphene can serve as a platform with increased adhesion for both growth factors and differentiation chemicals. It was also discovered that π-π binding was responsible for increased adhesion and played a crucial role in inducing hMSC differentiation [ 150 ].

Therapeutic potential of extracellular vesicle-based therapies

Extracellular vesicles (EVs) can be released by virtually every cell of an organism, including stem cells [ 151 ], and are involved in intercellular communication through the delivery of their mRNAs, lipids, and proteins. As Oh et al. [ 152 ] prove, stem cells, together with their paracrine factors—exosomes—can become potential therapeutics in the treatment of, e.g. skin ageing. Exosomes are small membrane vesicles secreted by most cells (30–120 nm in diameter) [ 153 ]. When endosomes fuse with the plasma membrane, they become exosomes that have messenger RNAs (mRNAs) and microRNAs (miRNAs), some classes of non-coding RNAs (IncRNAs) and several proteins that originate from the host cell [ 154 ]. IncRNAs can bind to specific loci and create epigenetic regulators, which leads to the formation of epigenetic modifications in recipient cells. Because of this feature, exosomes are believed to be implicated in cell-to-cell communication and the progression of diseases such as cancer [ 155 ]. Recently, many studies have also shown the therapeutic use of exosomes derived from stem cells, e.g. skin damage and renal or lung injuries [ 156 ].

In skin ageing, the most important factor is exposure to UV light, called “photoageing” [ 157 ], which causes extrinsic skin damage, characterized by dryness, roughness, irregular pigmentation, lesions, and skin cancers. In intrinsic skin ageing, on the other hand, the loss of elasticity is a characteristic feature. The skin dermis consists of fibroblasts, which are responsible for the synthesis of crucial skin elements, such as procollagen or elastic fibres. These elements form either basic framework extracellular matrix constituents of the skin dermis or play a major role in tissue elasticity. Fibroblast efficiency and abundance decrease with ageing [ 158 ]. Stem cells can promote the proliferation of dermal fibroblasts by secreting cytokines such as platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and basic fibroblast growth factor. Huh et al. [ 159 ] mentioned that a medium of human amniotic fluid-derived stem cells (hAFSC) positively affected skin regeneration after longwave UV-induced (UVA, 315–400 nm) photoageing by increasing the proliferation and migration of dermal fibroblasts. It was discovered that, in addition to the induction of fibroblast physiology, hAFSC transplantation also improved diseases in cases of renal pathology, various cancers, or stroke [ 160 , 161 ].

Oh [ 162 ] also presented another option for the treatment of skin wounds, either caused by physical damage or due to diabetic ulcers. Induced pluripotent stem cell-conditioned medium (iPSC-CM) without any animal-derived components induced dermal fibroblast proliferation and migration.

Natural cutaneous wound healing is divided into three steps: haemostasis/inflammation, proliferation, and remodelling. During the crucial step of proliferation, fibroblasts migrate and increase in number, indicating that it is a critical step in skin repair, and factors such as iPSC-CM that impact it can improve the whole cutaneous wound healing process. Paracrine actions performed by iPSCs are also important for this therapeutic effect [ 163 ]. These actions result in the secretion of cytokines such as TGF-β, interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor-AA (PDGF-AA), and basic fibroblast growth factor (bFGF). Bae et al. [ 164 ] mentioned that TGF-β induced the migration of keratinocytes. It was also demonstrated that iPSC factors can enhance skin wound healing in vivo and in vitro when Zhou et al. [ 165 ] enhanced wound healing, even after carbon dioxide laser resurfacing in an in vivo study.

Peng et al. [ 166 ] investigated the effects of EVs derived from hESCs on in vitro cultured retinal glial, progenitor Müller cells, which are known to differentiate into retinal neurons. EVs appear heterogeneous in size and can be internalized by cultured Müller cells, and their proteins are involved in the induction and maintenance of stem cell pluripotency. These stem cell-derived vesicles were responsible for the neuronal trans-differentiation of cultured Müller cells exposed to them. However, the research article points out that the procedure was accomplished only on in vitro acquired retina.

Challenges concerning stem cell therapy

Although stem cells appear to be an ideal solution for medicine, there are still many obstacles that need to be overcome in the future. One of the first problems is ethical concern.

The most common pluripotent stem cells are ESCs. Therapies concerning their use at the beginning were, and still are, the source of ethical conflicts. The reason behind it started when, in 1998, scientists discovered the possibility of removing ESCs from human embryos. Stem cell therapy appeared to be very effective in treating many, even previously incurable, diseases. The problem was that when scientists isolated ESCs in the lab, the embryo, which had potential for becoming a human, was destroyed (Fig.  8 ). Because of this, scientists, seeing a large potential in this treatment method, focused their efforts on making it possible to isolate stem cells without endangering their source—the embryo.

Use of inner cell mass pluripotent stem cells and their stimulation to differentiate into desired cell types

For now, while hESCs still remain an ethically debatable source of cells, they are potentially powerful tools to be used for therapeutic applications of tissue regeneration. Because of the complexity of stem cell control systems, there is still much to be learned through observations in vitro. For stem cells to become a popular and widely accessible procedure, tumour risk must be assessed. The second problem is to achieve successful immunological tolerance between stem cells and the patient’s body. For now, one of the best ideas is to use the patient’s own cells and devolve them into their pluripotent stage of development.

New cells need to have the ability to fully replace lost or malfunctioning natural cells. Additionally, there is a concern about the possibility of obtaining stem cells without the risk of morbidity or pain for either the patient or the donor. Uncontrolled proliferation and differentiation of cells after implementation must also be assessed before its use in a wide variety of regenerative procedures on living patients [ 167 ].

One of the arguments that limit the use of iPSCs is their infamous role in tumourigenicity. There is a risk that the expression of oncogenes may increase when cells are being reprogrammed. In 2008, a technique was discovered that allowed scientists to remove oncogenes after a cell achieved pluripotency, although it is not efficient yet and takes a longer amount of time. The process of reprogramming may be enhanced by deletion of the tumour suppressor gene p53, but this gene also acts as a key regulator of cancer, which makes it impossible to remove in order to avoid more mutations in the reprogrammed cell. The low efficiency of the process is another problem, which is progressively becoming reduced with each year. At first, the rate of somatic cell reprogramming in Yamanaka’s study was up to 0.1%. The use of transcription factors creates a risk of genomic insertion and further mutation of the target cell genome. For now, the only ethically acceptable operation is an injection of hESCs into mouse embryos in the case of pluripotency evaluation [ 168 ].

Stem cell obstacles in the future

Pioneering scientific and medical advances always have to be carefully policed in order to make sure they are both ethical and safe. Because stem cell therapy already has a large impact on many aspects of life, it should not be treated differently.

Currently, there are several challenges concerning stem cells. First, the most important one is about fully understanding the mechanism by which stem cells function first in animal models. This step cannot be avoided. For the widespread, global acceptance of the procedure, fear of the unknown is the greatest challenge to overcome.

The efficiency of stem cell-directed differentiation must be improved to make stem cells more reliable and trustworthy for a regular patient. The scale of the procedure is another challenge. Future stem cell therapies may be a significant obstacle. Transplanting new, fully functional organs made by stem cell therapy would require the creation of millions of working and biologically accurate cooperating cells. Bringing such complicated procedures into general, widespread regenerative medicine will require interdisciplinary and international collaboration.

The identification and proper isolation of stem cells from a patient’s tissues is another challenge. Immunological rejection is a major barrier to successful stem cell transplantation. With certain types of stem cells and procedures, the immune system may recognize transplanted cells as foreign bodies, triggering an immune reaction resulting in transplant or cell rejection.

One of the ideas that can make stem cells a “failsafe” is about implementing a self-destruct option if they become dangerous. Further development and versatility of stem cells may cause reduction of treatment costs for people suffering from currently incurable diseases. When facing certain organ failure, instead of undergoing extraordinarily expensive drug treatment, the patient would be able to utilize stem cell therapy. The effect of a successful operation would be immediate, and the patient would avoid chronic pharmacological treatment and its inevitable side effects.

Although these challenges facing stem cell science can be overwhelming, the field is making great advances each day. Stem cell therapy is already available for treating several diseases and conditions. Their impact on future medicine appears to be significant.

After several decades of experiments, stem cell therapy is becoming a magnificent game changer for medicine. With each experiment, the capabilities of stem cells are growing, although there are still many obstacles to overcome. Regardless, the influence of stem cells in regenerative medicine and transplantology is immense. Currently, untreatable neurodegenerative diseases have the possibility of becoming treatable with stem cell therapy. Induced pluripotency enables the use of a patient’s own cells. Tissue banks are becoming increasingly popular, as they gather cells that are the source of regenerative medicine in a struggle against present and future diseases. With stem cell therapy and all its regenerative benefits, we are better able to prolong human life than at any time in history.

Abbreviations

Basic fibroblast growth factor

Bone morphogenic proteins

Dental follicle stem cells

Dental pulp stem cells

Embryonic bodies

Embryonic stem cells

Fibroblast growth factors

Good Manufacturing Practice

Graphene oxide

Human amniotic fluid-derived stem cells

Human embryonic stem cells

Human foreskin fibroblasts

Inner cell mass

Non-coding RNA

Induced pluripotent stem cells

In vitro fertilization

Knockout serum replacement

Leukaemia inhibitory factor

Monocyte chemotactic protein-1

Fibroblasts

Messenger RNA

Mesenchymal stem cells of dental pulp

Myogenic differentiation

Osteoarthritis

Octamer-binding transcription factor 3 and 4

Platelet-derived growth factor

Platelet-derived growth factor-AA

Periodontal ligament stem cells

Rho-associated protein kinase

Stem cells from apical papilla

Stem cells of human exfoliated deciduous teeth

Synthetic Serum Substitute

Trophectoderm

Vascular endothelial growth factor

Transforming growth factors

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Wojciech Zakrzewski, Maria Szymonowicz & Zbigniew Rybak

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Zakrzewski, W., Dobrzyński, M., Szymonowicz, M. et al. Stem cells: past, present, and future. Stem Cell Res Ther 10 , 68 (2019). https://doi.org/10.1186/s13287-019-1165-5

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Viterbi Conversations in Ethics

Stem Cells: A Case for the Use of Human Embryos in Scientific Research

Embryonic stem cells have immense medical potential. While both their acquisition for and use in research are fraught with controversy, arguments against their usage are rebutted by showing that embryonic stem cells are not equivalent to human lives. It is then argued that not using human embryos is unethical. Finally, an alternative to embryonic stem cells is presented.

INTRODUCTION

Embryonic stem cells have the potential to cure nearly every disease and condition known to humanity. Stem cells are nature’s Transformers. They are small cells that can regenerate indefinitely, waiting to transform into a specialized cell type such as a brain cell, heart cell or blood cell [1]. Most stem cells form during the earliest stages of human development, immediately when an embryo is formed. These cells, known as embryonic stem cells (ESCs), eventually develop into every single type of cell in the body. As the embryo develops, adult stem cells (ASCs) replace these all-powerful embryonic stem cells. ASCs can only become a number of different cells within their potency. This limited application means an adult mesenchymal stem cell cannot become a neural cell.

By harnessing the unique ability of embryonic stem cells to transform into functional cells, scientists can develop treatments for a number of diseases and injuries, according to the California Institute for Regenerative Medicine, a private organization which awards grants for stem cell research [1]. For example, scientists at the Cleveland Clinic converted ESCs into heart muscle cells and injected them into patients who suffered from heart attacks. The cells continued to grow and helped the patients’ hearts recover [2].

With this enormous potential to cure devastating diseases, including heart failure, spinal cord injuries and Alzheimer’s disease, governments and research organizations have the moral imperative to support and encourage embryonic stem cell research. President Barack Obama signed an executive order in 2009 loosening federal funding restrictions on stem cell research, saying, “We will aim for America to lead the world in the discoveries it one day may yield.” [3]. The National Institute of Health and seven state governments, including California, Maryland and New York, followed Obama’s lead by creating programs that offered over $5 billion in funding and other incentives to scientists and research institutions for stem cell research [4].

A MIRACLE CURE

Scientists believe that harnessing the capability of embryonic stem cells will unlock the cure for countless diseases. “I am very excited about embryonic stem cells,” said Dr. Dieter Egli, professor of developmental cell biology at Columbia University. “They will lead to unprecedented discoveries that will transform life. I have no doubt about it.” [5]. The results thus far are inspiring. In 2016, Kris Boesen, a 21-year-old college student from Bakersfield, California, suffered a severe spinal cord injury in a car accident that left him paralyzed from the neck down. In a clinical trial conducted by Dr. Charles Liu at the University of Southern California Keck School of Medicine, Boesen was injected with 10 million embryonic stem cells that transformed into nerve cells [6]. Three months after the treatment, Boesen regained the use of his arms and hands. He could brush his teeth, operate a motorized wheelchair, and live more independently. “All I’ve wanted from the beginning was a fighting chance,” he said. The power of stem cells made his wish possible [6].

Embryonic stem cell treatments may also cure type 1 diabetes. Type 1 diabetes, which affects 42 million worldwide, is an autoimmune disorder that results in the destruction of insulin-producing beta cells found in the pancreas [7]. ViaCyte, a company in San Diego, California, is developing an implant that contains replacement beta cells originating from embryonic stem cells [7]. The implant will preserve or replace the original beta cells to protect them from the patient’s immune system [7]. The company believes that if successful, this strategy will effectively cure type 1 diabetes. Patients with the disease will no longer have to closely monitor their blood sugar levels and inject insulin [7]. ViaCyte projects that an experimental version of this implant will become available by 2020 [7].

Ultimately, scientists believe they will grow complex organs using stem cells within the next decade [8]. Over 115,000 people in the United States need a life-saving organ donation, and an average of 20 people die every day due to the lack of available organs for transplant, according to the American Transplant Foundation [9]. Three-dimensional printing of entire organs derived from stem cells holds the most promise for solving the organ shortage crisis [8]. Researchers at the University of California, San Diego have successfully printed part of a functional liver [8]. While the printed liver is not ready for transplant, it still performs the functions of a normal liver. This has helped scientists reduce the need for often cruel and unethical animal testing. The scientists expose drugs to the printed liver and observe how it reacts. The liver’s response closely mimics that of a human being’s and no living animals are harmed in the process [8].

HUMAN CELLS OR HUMAN LIFE?

Research using embryonic stems cells provides an unprecedented understanding of human development and the potential to cure devastating diseases. However, stem cell research has generated controversy among religious organizations such as the Catholic Church as well as the “pro-life” movement [3]. That is because scientists harvest stem cells from embryos donated by fertility clinics. Opponents of embryonicstem cell research equate the destruction of an embryo to the murder of an innocent human being [10]. Pope Benedict XVI said that harvesting stem cells is “not only devoid of the light of God but is also devoid of humanity” [3]. However, this view does not reflect a reasonable understanding and interpretation of basic biology. Researchers typically harvest embryonic stem cells from an embryo five days after fertilization [1]. At this stage, the entire embryo consists of less than 250 cells, smaller than the tip of a pin. Of these cells, only 30 are embryonic stem cells, which cannot perform any human function [11]. For comparison, an adult has more than 72 trillion cells, each with a specialized function [3]. Therefore, this microscopic blob of cells in no way represents human life.

With no functional cells, there exist no characteristics of a human being. Fundamentalist Christians believe that the presence or absence of a heartbeat signifies the beginning and end of a human life [10]. However, at this stage there is no heart, not even a single heart cell [10]. Some contend that brain activity, or the ability to feel, defines a human being. Michael Gazzaniga, president of the Cognitive Neuroscience Institute at the University of California, Santa Barbara, explains in his book,  The Ethical Brain,  that the “fertilized egg is a clump of cells with no brain.” [12]. There is no brain nor nerve cells that could allow this cellular object to interact with its environment [12]. The only uniquely human feature of embryonic cells at this stage is that they contain human DNA. This means that a 5-day-old human embryo is effectively no different than the Petri dishes of human cells that have grown in laboratories for decades with no controversy or opposition. Therefore, if the cluster of cells in the earliest stage of a human embryo is considered a “human life,” a growing plate of skin cells must also be considered “human life.” Few would claim that a Petri dish of human cells is morally equivalent to a living human or any other animal. Why, then, would a microscopic collection of embryonic cells have the same moral status as an adult human?

The status of the human embryo comes from its  potential  to turn into a fully grown human being.  However, the potential of this entity to become an individual does not logically mean that it has the same status as an individual who can think and feel. If this were true, virtually every cell grown in a laboratory would be subject to the same controversy. This is because scientists have developed technology to convert an ordinary cell such as a skin cell into an embryo [10]. Although this requires a laboratory with special conditions, the normal development of a human being also requires special conditions in the womb of the mother. Therefore, almost any cell could be considered a potential individual, so it is illogical to conclude that a cluster of embryonic cells deserves a higher moral status.

THE FATE OF UNUSED EMBRYOS

Hundreds of thousands of embryos are destroyed each year in a process known as in vitro fertilization (IVF), a popular procedure that helps couples have children [13]. Society has an ethical obligation to use these discarded embryos to make medical advancements rather than simply throw them in the trash for misguided ideological and religious reasons as opponents of embryonic stem cell research desire.

With IVF, a fertility clinician harvests sperm and egg cells from the parents and creates an embryo in a laboratory before implanting it in the woman’s womb. However, creating and implanting a single embryo is expensive and often leads to unsuccessful implantation. Instead, the clinician typically creates an average of seven embryos and selects the healthiest few to implant [13].

This leaves several unused embryos for every one implanted. The couple can pay a fee to preserve the unused embryos by freezing them or can donate them to another family. Otherwise, they are slated for destruction [14]. A 2011 study in the “Journal of the American Society for Reproductive Medicine” found that 19 percent of the unused embryos are discarded and only 3 percent are donated for scientific research [14]. Many of these embryos could never grow into a living person given the chance because they are not healthy enough to survive past early stages of development [14]. If a human embryo is already destined for destruction or has no chance of survival, scientists have the ethical imperative to use these embryos to research and develop medical treatments that could save lives. The modern version of the Hippocratic oath states, “I will apply, for the benefit of the sick, all measures which are required [to heal]” [10]. Republican Senator Orrin Hatch of Utah supports the pro-life movement, which recognizes early embryos as human individuals. However, even he favors using the leftover embryos for the greater good. “The morality of the situation dictates that these embryos, which are routinely discarded, be used to improve and save lives. The tragedy would be in not using these embryos to save lives when the alternative is that they would be discarded.” [3]

ALTERNATIVES TO EMBRYONIC STEM CELLS

Although scientists have used embryonic stem cells (ESCs) for promising treatments, they are not ideal, and scientists hope to eliminate the need for them. Primarily, ESCs come from an embryo with different DNA than the patient who will receive the treatment, meaning they are not autologous. ESCs are not necessarily compatible with everyone and could cause the immune system to reject the treatment [11]. The most promising alternative to ESCs are known as induced pluripotent stem cells. In 2008, scientists discovered a way to reprogram human skin cells to embryonic stem cells [15]. Scientists easily obtained these cells from a patient’s skin, converted them into the desired cell type, then transplanted them into the diseased organ without risk of immune rejection [15]. This eliminates any ethical concerns because no embryos are harvested or destroyed in the process. However, induced stem cells have their own risks. Recent studies have shown that they can begin growing out of control and turn into cancer [3]. Several of the first clinical trials with induced stem cells, including one aimed at curing blindness by regenerating a patient’s retinal cells, were halted because potentially cancerous mutations were detected [3].

Scientists believe that induced stem cells created in a laboratory will one day completely replace embryonic stem cells harvested from human embryos. However, the only way to create perfect replicas of ESCs is to thoroughly understand their structure and function. Scientists still do not completely understand how ESCs work. Why does a stem cell sometimes become a nerve cell, sometimes become a heart cell and other times regenerate to produce another stem cell? How can we tell a stem cell what type of cell to become? To develop a viable alternative to ESCs, scientists must first answer these questions with experiments on ESCs from human embryos. Therefore, extensive embryonic stem cell research today will eliminate the need for embryonic stem cells in the future.

The Biomedical Engineering Society Code of Ethics calls upon engineers to “use their knowledge, skills, and abilities to enhance the safety, health and welfare of the public.” [16] Stem cell research epitomizes this. Stem cells hold the cure for numerous diseases ranging from spinal cord injuries to organ failure and have the potential to transform modern medicine. Therefore, the donation of human embryos to scientific research falls within most conventional ethical frameworks and should be allowed with minimal restriction.

Because of widespread ignorance about the science behind stem cells, ill-informed opposition has prevented scientists from receiving the funding and support they need to save millions of lives. For example, George W. Bush’s religious opposition to stem cell research resulted in a 2001 law severely limiting government funding for such research [3]. Although most opponents of stem cell research compare the destruction of a human embryo to the death of a living human, the biology of these early embryos is no more human than a plate of skin cells in a laboratory. Additionally, all embryos sacrificed for scientific research would otherwise be discarded and provide no benefit to society. If society better understood the process and potential of embryonic stem cell research, more people would surely support it.

Within the next decade, stem cells will likely provide simple cures for diseases that are currently untreatable, such as Alzheimer’s disease and organ failure [1]. As long as scientists receive support for embryonic stem cell research, stem cell therapies will become commonplace in clinics and hospitals around the world. Ultimately, the fate of this new medical technology lies in the hands of the public, who must support propositions that will continue to allow and expand the impact of embryonic stem cell research.

By Jonathan Sussman, Viterbi School of Engineering, University of Southern California

ABOUT THE AUTHOR

At the time of writing this paper, Jonathan Sussman was a senior at the University of Southern California studying biomedical engineering with an emphasis in biochemistry. He was an undergraduate research assistant in the Graham Lab investigating proteomics of cancer cells and was planning to attend an MD/PhD program.

[1] “Stem Cell Information”,  Stem Cell Basics , 2016.  [Online]. Available at:  https://stemcells.nih.gov/info/basics/3.htm  [Accessed 11 Oct. 2018].

[2] Cleveland Clinic, “Stem Cell Therapy for Heart Disease | Cleveland Clinic”, 2017.  [Online]. Available at:  https://my.clevelandclinic.org/health/diseases/17508-stem-cell-therapy-for-heart-disease  [Accessed 14 Oct. 2018].

[3] B. Lo and L. Parham, “Ethical Issues in Stem Cell Research”,  Endocrine Reviews , 30(3), pp.204-213, 2009.

[4] G. Gugliotta, “Why Many States Now Have Stem Cell Research Programs”, 2015. [Online]. Available at:  http://www.governing.com/topics/health-human-services/last-decades-culture-wars-drove-some-states-to-fund-stem-cell-research.html  [Accessed 14 Oct. 2018].

[5] D. Cyranoski, “How human embryonic stem cells sparked a revolution”,  Nature Journal , 2018. [Online]. Available at:  https://www.nature.com/articles/d41586-018-03268-4  [Accessed 11 Oct. 2018].

[6] K. McCormack, “Young man with spinal cord injury regains use of hands and arms after stem cell therapy”, The Stem Cellar, 2016. [Online]. Available at:  https://blog.cirm.ca.gov/2016/09/07/young-man-with-spinal-cord-injury-regains-use-of-hands-and-arms-after-stem-cell-therapy/  [Accessed 11 Oct. 2018].

[7] A. Coghlan, “First implants derived from stem cells to ‘cure’ type 1 diabetes”,  New Scientist , 2017. [Online]. Available at:  https://www.newscientist.com/article/2142976-first-implants-derived-from-stem-cells-to-cure-type-1-diabetes/  [Accessed 11 Oct. 2018].

[8] C. Scott, “University of California San Diego’s 3D Printed Liver Tissue May Be the Closest We’ve Gotten to a Real Printed Liver”,  3DPrint.com | The Voice of 3D Printing / Additive Manufacturing , 2018. [Online]. Available at:  https://3dprint.com/118932/uc-san-diego-3d-printed-liver/  [Accessed 11 Oct. 2018].

[9] American Transplant Foundation, “Facts and Myths about Transplant”. [Online]. Available at:  https://www.americantransplantfoundation.org/about-transplant/facts-and-myths/  [Accessed 11 Oct. 2018].

[10] A. Siegel, “Ethics of Stem Cell Research”,  Stanford Encyclopedia of Philosophy , 2013. [Online]. Available at:  https://plato.stanford.edu/entries/stem-cells/  [Accessed 11 Oct. 2018].

[11] I. Hyun, “Stem Cells – The Hastings Center”,  The Hastings Center , 2018. [Online]. Available at:  https://www.thehastingscenter.org/briefingbook/stem-cells/  [Accessed 11 Oct. 2018].

[12] M. Gazzaniga, “The Ethical Brain”,  New York: Harper Perennial , 2006.

[13] M. Bilger, “Shocking Report Shows 2.5 Million Human Beings Created for IVF Have Been Killed | LifeNews.com”,  LifeNews , 2016. [Online]. Available at:  https://www.lifenews.com/2016/12/06/shocking-report-shows-2-5-million-human-beings-created-for-ivf-have-been-killed/  [Accessed 11 Oct. 2018].

[14] Harvard Gazette, “Stem cell lines created from discarded IVF embryos”, 2008. [Online]. Available at:  https://news.harvard.edu/gazette/story/2008/01/stem-cell-lines-created-from-discarded-ivf-embryos/  [Accessed 11 Oct. 2018].

[15] K. Murray, “Could we make babies from only skin cells?”, CNN, 2017. [Online]. Available at:  https://www.cnn.com/2017/02/09/health/embryo-skin-cell-ivg/index.html  [Accessed 11 Oct. 2018].

[16] Biomedical Engineering Society, “Biomedical Engineering Society Code of Ethics”, 2004. [Online]. Available at:  https://www.bmes.org/files/CodeEthics04.pdf  [Accessed 11 Oct. 2018].

Neurology and Neurosurgery

• Study finds stem cell therapy is safe and may benefit people with spinal cord injuries

May 23, 2024

Mayo Clinic researchers have demonstrated the safety and potential benefit of stem cell regenerative medicine therapy for patients with subacute and chronic spinal cord injury.

The results of the phase 1 Clinical Trial of Autologous Adipose-Derived Mesenchymal Stem Cells in the Treatment of Paralysis Due to Traumatic Spinal Cord Injury, known as CELLTOP, were published in Nature Communications.

Illustration shows the process of fat harvest via biopsy, adipose-derived mesenchymal stem cells (AD-MSC) preparation and administration of treatment.

All trial participants had experienced traumatic spinal injury classified as grade A or B on the American Spinal Injury Association Impairment Scale (AIS). Stem cell treatment was initiated on average 11 months after injury. Participants were evaluated over a two-year period.

Key findings:

• Stem cells were successfully manufactured, and products were delivered to all 10 enrolled participants.
• No serious adverse effects occurred among any participants. The most commonly reported side effects were headache and musculoskeletal pain, which resolved with over-the-counter treatment.
• Seven participants demonstrated improvement, with each moving up at least one AIS grade.

As reported earlier in Mayo Clinic Proceedings, the first participant in the phase 1 trial was a superresponder who, after stem cell therapy, saw significant improvements in the function of his upper and lower extremities.

"Future research may show whether stem cells in combination with other therapies could be part of a new paradigm of treatment to improve outcomes for patients," says Mohamad Bydon, M.D. , a neurosurgeon at Mayo Clinic in Rochester, Minnesota, and the first author of both studies. "Not every patient who receives stem cell treatment is going to be a superresponder. One objective in our future studies is to delineate the optimal treatment protocols and understand why patients respond differently."

Dr. Bydon notes that stem cells' mechanism of action isn't fully understood. The researchers are analyzing changes in participants' MRI and cerebrospinal fluid to identify avenues for potential regeneration. Work is also underway on a larger, controlled trial of stem cell regenerative therapy.

"For years, treatment of spinal cord injury has been limited to stabilization surgery and physical therapy," Dr. Bydon says. "Many historical textbooks state that this condition does not improve. We have seen findings in recent years that challenge prior assumptions. This research is a step forward toward the ultimate goal of improving treatments for patients."

For more information

Bydon M, et al. Intrathecal delivery of adipose-derived mesenchymal stem cells in traumatic spinal cord injury: Phase I trial . Nature Communications. 2024;15:2201.

Bydon M, et al. CELLTOP clinical trial: First report from a phase I trial of autologous adipose tissue-derived mesenchymal stem cells in the treatment of paralysis due to traumatic spinal cord injury . Mayo Clinic Proceedings. 2020;95:406.

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Embryonic Stem Cell Research An Ethical Dilemma

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Introduction

In November 1998, two teams of U.S. scientists confirmed successful isolation and growth of stems cells obtained from human fetuses and embryos. Since then, research that utilizes human embryonic cells has been a widely debated, controversial ethical issue. Human embryonic cells possess the ability to become stem cells, which are used in medical research due to two significant features. First, they are unspecialized cells, meaning they can undergo cell division and renew themselves even with long periods of inactivity. Secondly, stem cells are pluripotent, with the propensity to be induced to become specified tissue or any “organ-specific cells with special functions” depending on exposure to experimental or physiologic conditions, as well as undergo cell division and become cell tissue for different organs.

The origin of stem cells themselves encapsulates the controversy: embryonic stem cells, originate from the inner cell mass of a blastocyst, a 5-day pre-implantation embryo. The principal argument for embryonic stem cell research is the potential benefit of using human embryonic cells to examine or treat diseases as opposed to somatic (adult) stem cells. Thus, advocates believe embryonic stem cell research may aid in developing new, more efficient treatments for severe diseases and ease the pain and suffering of numerous people. However, those that are against embryonic stem cell research believe that the possibility of scientific benefits of research do not outweigh the immoral action of tampering with the natural progression of a fetal development and interfering with the human embryo’s right to live. In light of these two opposing views, should embryonic stem cells be used in research? It is not ethically permissible to destroy human embryonic life for medical progress.

Personhood and the Scientific Questionability of Embryonic Stem Cell Research

The ethics behind embryonic stem cell research are controversial because the criteria of ‘personhood’ is “notoriously unclear.” Personhood is defined as the status of being a person, entitled to “moral rights and legal protections” that are higher than living things that are not classified as persons. Thus, this issue touches on existential questions such as: When does life begin? and What is the moral status that an embryo possesses? There is a debate on when exactly life begins in embryonic development and when the individual receives moral status. For example, some may ascribe life starting from the moment of fertilization, others may do so after implantation or the beginning of organ function. However, since the “zygote is genetically identical to the embryo,” which is also genetically identical to the fetus, and, by extension, identical to the baby, inquiring the beginning of personhood can lead to an occurrence of the Sorites paradox, also acknowledged as “the paradox of the heap.”

The paradox of the heap arises from vague predicates in philosophy. If there is a heap of sand and a grain is taken away from that heap one by one, at what point will it no longer be considered a heap – what classifies it as a heap? The definition of life is similarly arbitrary. When, in the development of a human being, is an embryo considered a person with moral standing? The complexity of the ethics of embryonic stem cell research, like the Sorites paradox, demonstrates there is no single, correct way to approach a problem; thus, there may be multiple different solutions that are acceptable. Whereas the definition of personhood cannot be completely resolved on a scientific basis, it serves a central role in the religious, political, and ethical differences within the field of embryonic stem cell research. Some ethicists attempt to determine what or who is a person by “setting boundaries” (Baldwin & Capstick, 2007).

Utilizing a functionalist approach, supporters of embryonic stem cell research argue that to qualify as a person, the individual must possess several indicators of personhood, including capacity, self-awareness, a sense of time, curiosity, and neo-cortical function. Proponents argue that a human embryo lacks these criteria, thereby is not considered a person and thus, does not have life and cannot have a moral status. Supporters of stem cell research believe a fertilized egg is just a part of another person’s body until the cell mass can survive on its own as a viable human. They further support their argument by noting that stem cell research uses embryonic tissue before its implantation into the uterine wall. Researchers invent the term “pre-embryo” to distinguish a pre-implantation state in which the developing cell mass does not have the full respects of an embryo in later stages of embryogenesis to further support embryonic stem cell research. Based on this reductionist view of life and personhood, utilitarian advocates argue that the result of the destruction of human embryos to harvest stem cells does not extinguish a life. Further, scientists state that any harm done is outweighed by the potential alleviation of the suffering enduring by tremendous numbers of people with varying diseases. This type of reasoning, known as Bentham’s Hedonic (moral) calculus, suggests that the potential good of treating or researching new cures for ailments such as Alzheimer’s disease, Parkinson’s disease, certain cancers, etc. outweighs any costs and alleviate the suffering of persons with those aliments. Thus, the end goal of stem cell use justifies sacrificing human embryos to produce stem cells, even though expending life is tantamount to murder. Opponents of embryonic stem cell research would equate the actions done to destroy the embryos as killing. Killing, defined as depriving their victims of life, will therefore reduce their victims to mere means to their own ends. Therefore, this argument touches on the question: if through the actions of embryotic stem cell research is “morally indistinguishable from murder?” (Outka, 2013). The prohibition of murder extends to human fetuses and embryos considering they are potential human beings. And, because both are innocent, a fetus being aborted and an embryo being disaggregated are direct actions with the intention of killing. Violating the prohibition of murder is considered an intolerable end. We should not justify this evil even if it achieves good. Under the deontological approach, “whether a situation is good or bad depends on whether the action that brought it about was right or wrong,” hence the ends do not justify the means. Therefore, under this feeble utilitarian approach, stem cell research proceeds at the expense of human life than at the expense of personhood.

One can reject the asserted utilitarian approach to stem cell research as a reductionist view of life because the argument fails to raise ethical concerns regarding the destruction embryonic life for the possibility of developing treatments to end certain diseases. The utilitarian approach chooses potential benefits of stem cell research over the physical lives of embryos without regard to the rights an embryo possesses. Advocates of embryonic stem cell research claim this will cure diseases but there is a gap in literature that confirms how many diseases these cells can actually cure or treat, what diseases, and how many people will actually benefit. Thus, killing human embryos for the potentiality of benefiting sick people is not ethically not ethically permissible.

Where the argument of personhood is concerned, the development from a fertilized egg (embryo) to a baby is a continuous process. Any effort to determine when personhood begins is arbitrary. If a newborn baby is a human, then surely a fetus just before birth is a human; and, if we extend a few moments before that point, we would still have a human, and so on all the way back to the embryo and finally to the zygote. Although an embryo does not possess the physiognomies of a person, it will nonetheless become a person and must be granted the respect and dignity of a person. Thus, embryotic stem cell research violates the Principle of “Full Human Potential,” which states: “Every human being […] deserves to be valued according to the full level of human development, not according to the level of development currently achieved.” As technology advances, viability outside the womb inches ever closer to the point of inception, making the efforts to identify where life begins after fertilization ineffectual. To complicate matters, as each technological innovation arrives, stem-cell scientists will have to re-define the start of life as many times as there are new technological developments, an exhausting and never-ending process that would ultimately lead us back to moment of fertilization. Because an embryo possesses all the necessary genetic information to develop into a human being, we must categorically state that life begins at the moment of conception. There is a gap in literature that deters the formation of a clear, non-arbitrary indication of personhood between conception and adulthood. Considering the lack of a general consensus of when personhood begins, an embryo should be referred to as a person and as morally equivalent to a fully developed human being.

Having concluded that a human embryo has the moral equivalent of a fully-fledged human being, this field of research clearly violates the amiable rights of personhood, and in doing so discriminates against pre-born persons. Dr. Eckman asserts that “every human being has a right to be protected from discrimination.” Thus, every human, and by extension every embryo, has the right to life and should not be discriminated against their for “developmental immaturity.” Therefore, the field of embryonic stem cell research infringes upon the rights and moral status of human embryos.

Principle of Beneficence in Embryonic Stem Cell Research

The destruction of human embryos for research is not ethically permissible because the practice violates the principle of beneficence depicted in the Belmont Report, which outlines the basic ethical principles and guidelines owed to human subjects involved in research. Stem cell researchers demonstrate a lack of respect for the autonomy and welfare of the human embryos sacrificed in stem cell research.

While supporters of embryonic stem cell research under the utilitarian approach argue the potential benefits of the research, the utilitarian argument however violates the autonomy of the embryo and its human rights, as well as the autonomy of the embryo donors and those that are Pro-Life. Though utilitarian supporters argue on the basis of rights, they exclusively refer to the rights of sick individuals. However, they categorically ignore the rights of embryos that they destroy to obtain potential disease curing stem cells. Since an embryo is regarded as a human being with morally obligated rights, the Principle of Beneficence is violated, and the autonomy and welfare of the embryo is not respected due to the destruction of an embryo in stem cell research. Killing embryos to obtain stem cells for research fails to treat embryos as ends in an of themselves. Yet, every human ought to be regarded as autonomous with rights that are equal to every other human being. Thus, the welfare of the embryo is sacrificed due to lack of consent from the subject.

The Principle of Beneficence is violated when protecting the reproductive interests of women in infertility treatment, who are dependent on the donations of embryos to end their infertility. Due to embryonic stem cell research, these patients’ “prospects of reproductive success may be compromised” because there are fewer embryos accessible for reproductive purposes. The number of embryos necessary to become fully developed and undergo embryonic stem cell research will immensely surpass the number of available frozen embryos in fertility clinic, which also contributes to the lack of embryos available for women struggling with infertility. Therefore, the basis of this research violates women’s reproductive autonomy, thus violating the Principle of Beneficence.

It is also significant to consider the autonomy and welfare of the persons involved. The autonomous choice to donate embryos to research necessitates a fully informed, voluntary sanction of the patient(s), which poses difficulty due to the complexity of the human embryonic stem cell research. To use embryos in research, there must be a consensus of agreement from the mother and father whose egg and sperm produced the embryo. Thus, there has to be a clear indication between the partners who has the authority or custody of the embryos, as well as any “third party donors” of gametes that could have been used to produce the embryo because these parties’ intentions for those gametes may solely have been for reproductive measures only. Because the researchers holding “dispositional authority” over the embryos may exchange cell lines and its derivatives (i.e., genetic material and information) with other researchers, they may misalign interests with the persons whose gametes are encompassed within the embryo. This mismatch of intent raises complications in confidentiality and autonomy.

Lastly, more ethical complications arise in the research of embryonic stem cells because of the existence viable alternatives that to not destroy human embryos. Embryonic stem cells themselves pose as a higher health risk than adult stem cells. Embryonic stem cells have a higher risk of causing tumor development in the patient’s body once the cells are implanted due to their abilities to proliferate and differentiate. Embryonic stem cells also have a high risk of immunorejection, where a patient’s immune system rejects the stem cells. Since the embryonic stem cells are derived from embryos that underwent in vitro fertilization, when implanted in the body, the stem cell’s marker molecules will not be recognized by the patient’s body, resulting in the destruction of the stem cells as a defensive response to protect the body (Cahill, 2002). With knowledge of embryonic stem cells having higher complications than the viable adult stem cells continued use of embryonic stem cells violates the Principle of Beneficence not only for the embryos but for the health and safety of the patients treated with stem cells. Several adult stem cell lines (“undifferentiated cells found throughout the body”) exist and are widely used cell research. The use of adult stem cells represents research that does not treat human beings as means to themselves, thus, complying with the Principle of Beneficence. This preferable alternative considers the moral obligation to discover treatments, and cures for life threating diseases while avoiding embryo destruction.

It is not ethically permissible to destroy human embryonic life for medical progress due to the violations of personhood and human research tenets outlined in the Belmont Report. It is significant to understand the ethical implications of this research in order to respect the autonomy, welfare, beneficence, and basic humanity afforded to all parties involved. Although embryonic stem cell research can potentially provide new medical advancements to those in need, the harms outweigh the potential, yet ill-defined benefits. There are adult stem cell alternatives with equivalent viability that avoid sacrificing embryos. As society further progresses, humans must be cautious of compromising moral principles that human beings are naturally entitled to for scientific advancements. There are ethical boundaries that are crossed when natural processes of life are altered or manipulated. Though there are potential benefits to stem cell research, these actions are morally and ethically questionable. Thus, it is significant to uphold ethical standards when practicing research to protect the value of human life.

Shamblott, M. J., J. Axelman, S. Wang, E. M. Bugg, J. W. Littlefield, P. J. Donovan, P. D. Blumenthal, G. R. Huggins, and J. D. Gearhart. “Derivation of Pluripotent Stem Cells from Cultured Human Primordial Germ Cells.” Proceedings of the National Academy of Sciences 95, no. 23 (November 10, 1998): 13726–31. doi:10.1073/pnas.95.23.13726.

National Institutes of Health, U.S. Department of Health and Human Services. “Stem Cell Basics I.” Stem Cell Information , 2016. https://stemcells.nih.gov/info/basics/1.htm .

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Home » 5 stem cell success stories

5 stem cell success stories

Dec 2, 2019 | Blog

Stem cell science is rapidly advancing, and more and more stem cell success stories are reported all the time. Once the stuff of science fiction, the promise of stem cells are now a life-changing reality for many people around the world.

These powerful cells have the unique ability to self-renew and develop into various different cell types that can treat a vast array of conditions.

First reported in the Daily Mail , these five stem cell success stories tell the inspiring story of people who recently received stem cell-based therapies.

Reema Sandhu, Multiple Sclerosis

Reema Sandhu’s stem cell success story started five years ago, when she was diagnosed with multiple sclerosis . The condition affects the brain and spinal cord, resulting in a wide range of life-altering symptoms such as visual impairments, muscle spasms and memory problems.

Despite receiving high dose drugs for her condition, Reema did not see any improvement and suffered from several side-effects.

After years of frustration, she decided to opt for private treatment and following a dose of chemotherapy to destroy her immune system, began to receive stem cell treatments in January.

She then received an autologous stem cell transplant, where her own stem cells were collected from her blood and returned to her body via intravenous infusion.

Significant improvements were immediately noticeable, particularly with Reema’s brain function. Two months after the transplant, her vision was restored and she returned to work.

These positive outcomes suggest that Reema’s MS has stopped progressing as a result of her stem cell transplant.

Dave Randle, Heart Attack

After suffering a heart attack in 2016, Dave Randle was left with significant heart failure and a terrifying warning from consultants: he would be dead by Christmas.

However, after discovering that stem cells could treat damaged hearts , Dave signed up for treatment at Bart’s Hospital earlier this year.

He received injections for five consecutive days that encouraged his bone marrow to release stem cells into his blood stream. These cells were then isolated and infused back into his heart.

Just weeks after the transplant, Dave’s stem cell success story had a happy ending – he began to feel better and doctors noticed substantial improvements.

George Norton, Acute Lymphoblastic Leukaemia

In 2005, George Norton was diagnosed with a form of blood cancer called Acute Lymphoblastic Leukaemia (ALL) .

After a relapse in 2014, George received a run of chemotherapy followed by a stem cell transplant from a donor through the Anthony Nolan charity, which works with leukaemia and haematopoietic stem cell transplants.

The aim of the transplant was to create a new, healthy immune system to fight the cancer and kill any leukaemia cells in the body.

Since then, George has led a healthy life free of leukaemia.

Andrew Robinson, Arthritis

47-year-old Andrew Robinson had one of the most promising stem cell success stories.

He was told that he would need a knee replacement after suffering from years of pain and swelling due to knee arthritis . However, Andrew was then recommended an alternative to knee replacement: a chondrotissue graft procedure.

This procedure involves inserting a ‘scaffold’ into the bone, which fosters the growth of new cartilage by releasing stem cells collected from the bone marrow.

Andrew was able to walk again just 10 weeks after treatment, and has now returned to his active lifestyle.

Deepan Shah, Crohn’s disease 

Having endured aggressive Crohn’s disease throughout his childhood, Deepan Shah was referred for a clinical trial investigating the use of stem cells to reset the immune system and stop its attacks on the gut.

Treatment began with chemotherapy followed by injections to encourage stem cell growth, which were then collected and infused into his body. Soon after treatment, Deepan was able to come off his medication.

Whilst Deepan still has Crohn’s disease and occasionally experiences symptoms, he is now able to lead a normal life.

These stem cell success stories demonstrate the life-changing potential of regenerative medicine in the treatment of a wide array of conditions – from arthritis to blood cancers and more. Find out more here .

• https://www.dailymail.co.uk/health/article-7674229/Patients-reveal-reaped-remarkable-benefits-stem-cells.html

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• Research article
• Open access
• Published: 12 May 2020

Ethical challenges regarding the use of stem cells: interviews with researchers from Saudi Arabia

• Ghiath Alahmad   ORCID: orcid.org/0000-0002-3331-4378 1 ,
• Sarah Aljohani 1 &
• Muath Fahmi Najjar 1  

BMC Medical Ethics volume  21 , Article number:  35 ( 2020 ) Cite this article

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With the huge number of patients who suffer from chronic and incurable diseases, medical scientists continue to search for new curative methods for patients in dire need of treatment. Interest in stem cells is growing, generating high expectations in terms of the possible benefits that could be derived from stem cell research and therapy. However, regardless of the hope of stem cells changing and improving lives, there are many ethical, religious, and political challenges and controversies that affect the research, and mandated to establish ethical guidelines and regulations. In Saudi Arabia, key stakeholders play an active role in discussing the ethics of stem cell research and therapy. The focus of the study was to explore professionals’ perceptions related to the ethical challenges of using stem cells in research and treatment in Saudi Arabia.

A qualitative research study was conducted to explore and describe the perceptions of 25 professionals employed at different tertiary hospitals in the various regions of Saudi. A thematic analysis was performed to search for and identify the most significant perceptions shared by the participants. Four themes were generated based on the ethical challenges of four areas related to stem cell use, including (1) forbidden and permitted sources of stem cells, (2) informed consent, (3) beneficence, and (4) ethical regulations and guidelines.

The study identified that there is a growing need to advance the knowledge, education, and awareness related to stem cell research and treatment in Saudi Arabia.

Peer Review reports

Literature highlights the significance of understanding stem cell therapy and research to support the development of regenerative medicine [ 1 , 2 , 3 ]. McLaren reported that there are millions of individuals suffering from and succumbing to “incurable degenerative diseases of the nervous system, heart, liver, pancreas, and other organs” annually [ 3 ]. Similarly, Lovell-Badge discussed the fact that stem cells offer great hope for patients with enervating illnesses such as “diabetes, Parkinson’s, and Huntington’s diseases” [ 4 ]. In this context, medical practitioners consider stem cells as the hope and light for many patients who are suffering and in dire need of a cure [ 1 ]. However, as indicated by several authors, the use of stem cells, present many ethical, political, and even religious challenges, either related to resources, use, or rights of donors [ 5 , 6 ]. Bharadwaj mentioned the increasing movements of social and government concerns regarding stem cell research and clinical medication in countries such as the United States, United Kingdom, and Japan. More recently, emerging countries also incorporated stem cell therapies in their practices [ 7 ].

Lo and Parham provided a classification of the different ethical issues based on the four phases of stem cell research. The first phase is the “donation of biological material,” highlighting the problem of “informed and voluntary consent” [ 2 ]. The second phase, research with human embryonic stem cells, creates several ethical issues. These issues include the “destruction of embryos and the creation of embryos for research purposes” as well as financial compensation to oocyte donors, medical hazards related to the retrieval of the oocyte, and the need to protect the reproductive interests of women undergoing infertility treatment [ 2 ]. The third phase of the research is using stem cell lines obtained from other institutions leading to the issue of adverse legal and ethical principles [ 2 ]. The fourth and last step is the use of stem cells in clinical trials, encompassing both the advantages and disadvantages of the trial and informed consent [ 2 ].

Stem cell research in Saudi Arabia

Many Arabic countries conduct research with stem cells, as evidenced by the hundreds of scientific papers published in this field. Saudi Arabia is ahead in stem cell research as many universities, such as King Saud University and King Faisal Specialized Hospital and Research Center, started stem cell research more than 20 years ago [ 8 ]. In addition, many other research institutions, established later, play a leading role in this field such as King Abdullah International Medical Research Center with a specialized stem cell research department, including a stem cell registry containing more than 10,000 donors and the Cord Blood Bank [ 9 ].

From an ethical perspective, Saudi Arabia was the first country in the region to have ethical regulations related to the use of and research with stem cells. The Research Ethics Law promulgated in 2010 and its implementing regulation in 2012 includes all ethical guidelines to control stem cell research [ 10 ]. This was followed in 2014 by Jordan where a specific law about stem cell is announced [ 11 ]. In addition to these national laws, many research centers have institutional guidelines, for example King Faisal Specialized Hospital and Research Center and King Abdullah International Medical Research Center.

Though there is an abundance of stem cell research, the ethical component has not been researched in depth. There is no literature related to the views of physicians and researchers about the ethical challenges of using stem cells. It is important to explore this important issue to enhance the ethical component and maintaining the progress of stem cell research through finding appropriate solutions of all ethical challenges and obstacles.

Research design

A qualitative research design was used to explore and describe the perceptions and experiences of participants regarding the ethical challenges of using stem cells in a “subjective and reflexive manner” [ 12 ]. The aim was to gather, explore, analyze, and extract the most meaningful perceptions of the sample using interviews. The qualitative approach was deemed appropriate for the study to emphasize the content of the data to explore a specific phenomenon.

Data collection

We collected data from professionals employed at tertiary hospitals from various regions in Saudi Arabia where stem cell research have been conducted or are being conducted. The target population included physicians from any medical specialty or non-physician researchers doing stem cell research. We visited the potential hospitals and presented the objectives and purpose of the study. We collected contact information of potential participants with the permission from the gatekeepers of the hospitals and other pertinent representatives. We used a snowball sampling technique, defined by Clark and Creswell as the sampling of individuals based on the recommendations and suggestions of others [ 12 ]. Once a participant agreed to participate, we actively inquired from the participant to identify other possible candidates for the study. The technique allowed us to recruit 25 participants. The demographic characteristics of the sample are displayed in Table  1 .

We conducted individual semi-structured interviews with open-ended questions and audiotaped the interviews. Before conducting the interviews, written informed consent was obtained. The consent form highlighted voluntary participation, no monetary reward or promise. The privacy and confidentiality clauses were explained thoroughly. The interviews were held in a private room at the preferred time and date of the participants. Each interview lasted between 40 and 60 min.

Data analysis

After completing the 25 interview transcripts, data analysis commenced. The analysis involved identifying, analyzing, and reporting the most frequent and meaningful patterns or themes [ 12 ]. The analysis followed Braun and Clarke’s six-step process [ 13 ]. First, we familiarized ourselves with the interviews and actively read and reread the transcripts and generated initial codes as the second step. In the third step, we searched for themes across the data and the initial codes were categorized in the themes. We were mindful of the three objectives and purposes of the study to identify the most important points and concepts. The fourth step entailed the constant review of the themes, with the original data or the interviews. In the fifth step, the themes were named. Finally, the last step is the creation of the report as presented in the next section. We used the NVivo12 by QSR software to assist in the management and systematic tabulation of the themes.

A thematic analysis was performed to search for the most significant and meaningful responses from the sample. The thematic analysis resulted in four themes to address the three key objectives of the study. From the participant perspective, some sources are forbidden due to ethical issues. In addition, researchers and professionals must obtain an informed consent at all times (following the IRB) and follow the international regulations regarding stem cell research. The participants expressed the need to clarify the purpose of the research and storage procedures. Table  2 displays the themes in response to the study objectives.

Theme I: an exploration of the sample’s views regarding forbidden and permitted sources of stem cells

The participants’ position regarding the sources of stem cells can be classified in two categories: permitted and forbidden resources. The majority felt that some sources of stem cells should be forbidden because they may lead to serious religious issues, but some sources were considered safe and acceptable.

Adult stem cells as a source was considered safe providing the extraction is done within the prescribed processes and guidelines. An interviewee said, “S ources which are like skin liver heart these are allowed… Adult stem cells- allowed.” A second participant added, “Adult stem cells [are] approved for clinical use.”

Pluripotent stem cells are becoming an acceptable resource of stem cells, due to their positive and safe characteristics. An interviewee shared that the use of pluripotent stem cells is continuously advancing with the hope of curing different diseases, saying: “ Why not? It’s a new science, and the people are trying to use pluripotent stem cells in another type of... a different kind of disease, and there is a lot of clinical intervention a lot of clinical trials still under investigation there is no clear answer.” Another participant indicated that pluripotent stem cells are similar to adult stem cells and can easily be replicated, saying: “Pluripotent stem cells it’s actually an adult cell you reprogramed the genetic and you move it back so you can do anything with it.”

According to our interviewees, the umbilical cord is a safe and promising stem cell source. A participant commented, “umbilical cord is one of the best sources of stem cells.”

The participants also indicated that the placenta is a permitted source, and they experienced no issues as the placenta was used previously to extract stem cells. A participant stated that using the placenta is not harmful, saying, “We used to collect stem cells from the placenta. It is not invasive.”

Obtaining stem cells from fetuses were perceived differently. One group clearly and completely forbids any use of stem cells from any fetus, either intentionally aborted or accidently miscarried, regardless of the age of fetus. One of the interviewees responded that the use is strictly prohibited, stating, “This is forbidden” and a second indicated clearly and strongly, “Miscarried fetuses before reaching [120 days] the same, the same, forbidden.” This group of professionals stated that the aborted fetus is rejected as the institutions and stakeholders are aware that the use of such a source may lead to ethical dilemmas. As one of the participants expressed, “there may be ethical issues that go along with the use of an aborted fetus.” In addition, the use of embryonic stem cells may lead to more serious and critical religious issues. A participant stated, “That they must adhere to the religious teaching that one must not touch or alter the fetus.”

However, some of the participant accepted fetuses aborted for therapeutic reasons, but forbid stem cells obtained from fetuses aborted for non-therapeutic reasons. An interviewee said, “However, these source of stem cells that we’re using is probably less chaotic, and hence should be utilized or the regulations should be applied like any other biological materials.” Spontaneously miscarried fetuses can be accepted as a resource of stem cells, if they are less than 120 days of age. One interviewee said, “If the fetus is less than 120 days old, it is not considered a human, and we can use its stem cells.”

Theme II: an exploration of professionals’ opinions regarding the ethical challenges of securing informed consent, with IRB approval

The second thematic category explored the sample’s perceptions concerning the challenges related to obtaining informed consent for stem cell research. The participants emphasized the importance of informed consent to guarantee the voluntary participation of donors. One of the interviewees said, “We do have consent actually, we never collect stem cells without taking a consent from the patient. Sure, sure yeah, we take the permission before we start collecting the cells.” However, for umbilical cord blood, consent is obtained from parents, usually during the routine visits to clinics during the pregnancy, as expressed by one interviewee.

According to the participants, the consent should explain and clearly describe the purpose of the research. A participant said: “I think that the donor should be informed what exactly we are doing with tissue that he has that we take it from him.” Another said, “The scope of the research should be properly presented. Such practice will protect both parties, the researcher and donor, from future issues.”

All donor rights should be mentioned in the informed consent. One of the interviewees said, “Donors’ rights musts be clarified and explained to them, including, but not limited to, withdrawal right.”

The explanation should be in understandable, clear language and the terms and conditions of the forms should be simplified. The communication must be sufficient to ensure the donor understand fully. A participant narrated, “The researchers must take the time to orient and explain the content of the informed consent to the volunteers.” The informed consent documents should have been reviewed and approved by an ethics committee. A participant discussed the process of procuring the form, as follows: We submit the consent to the research office as a part of the submitted research proposal and then you will get IRB approval for all proposals including the informed consent.”

Theme III: an exploration of the professionals’ perceptions regarding the ethical challenges related to the benefit resulting from the stem cell research

The participants described the potential value of the sources of stem cells in the field of medicine and research. According to the majority, any use of stem cells should be beneficial, either to the donors or to the public. A participant said, “Even if there is no direct benefit to the donors themselves, but at least stem cells research should have some potential benefits to others.” A second opinion was “There are different applications and uses of the umbilical cord and it can save many patients today.” When using stem cells in treatment, the approved procedures should be followed meticulously, as explained by one of the interviewees, “Not following approved methods may lead to serious consequences.”

The sample emphasized the responsibility of being transparent when informing and communicating with the patient about any potential benefit. “It is very important not give the patients false hope about treatment by stem cells,” as expressed by one of the interviewees. A second participant was concerned about false hopes based on wrong assumptions, “I am very sad to see hopeless people spend all their earnings and energy in trying something that can never be a success.”

Theme IV: an exploration of stem cell research regulations

Four subthemes were developed related to the regulations related to stem cell research theme, including the importance of following international regulations, the need to use international guidelines based on Islamic laws (fatwas) and beliefs, no national law related to stem cells, and the need to increase the researchers’ knowledge about the ethical guidelines of stem cells.

The majority of the sample considered following regulations and guidelines consistently every time stem cells are used as an important issue. A participant explained, “ We have to follow the set procedures by regulators and the law because it involves safety of patients.” Our findings indicate that our researchers are using international regulations in their current practice and research with stem cells. One researcher said, “ Actually we are already use international guidelines. We used to follow that when I was getting my training in the west, and we here continue do the same.” Another researcher justified why international regulations should be followed, “Following the international regulations on stem cell research is needed for two reasons: the ethical principles are the same, and we are in many cases part of international multicenter research.” However, when applying these international regulations, Islamic law and fatwas should be taken in account. One of the participants explained, “Nothing that contradicts Sariah is acceptable, and this is true when it comes to stem cells, especially when it comes to the permitted or forbidden resources.”

The participants also indicated a lack of standardization in the local setting. A participant narrated that currently, they follow only the international regulations for their clinical trials and research. The participant described the increasing need for a targeted local policy related to stem cells, saying: “There is no national standardization as far as I know.” According to a second participant, the main issue in Saudi is the actual lack of a national law related to stem cell research and therapy, he commented, “Ethics must be a priority in Saudi to be able to create laws that would be in line with the local religious or spiritual beliefs.” Another participant also expressed the need to create local guidelines, which should match the international guidelines and not contradict the Islamic law. A last perspective was as follows: “Definitely, it’s good to have a supporting in fatwa for our patients satisfaction. This is because patients and the community rely on the fatwa more than the IRB. They don’t know about the IRB. So, I think that’s why the need the fatwa. I think we would reassure our patients about that.”

Many participants admitted that researchers lack adequate knowledge and information regarding ethical considerations and guidelines about stem cells research. One of the participants said, “The researchers themselves need to be trained or oriented in a formal setting to become aware about the ethical guidelines of using stem cells. ”

Though researchers are doing research in the stem cell field, they realize the ethical challenges they are facing in their research. Having spent a significant part of their scientific life in Western countries, they are aware of ethical issues; however at the same time, their cultural and religious background plays a role in their perceptions regarding the ethical challenges and how to deal with them.

The first point to manage appropriately is the source of stem cells, classified in permitted and forbidden sources. While the sample accepts adult stem cells in general, they have a different point of view regarding embryonic stem cells. These stem cells are affected by ethical, legal, and religious considerations, especially regarding the method of obtaining the cells and more specifically, when it results in destroying embryos who may have a degree of dignity and humanity, similar to other researchers, societies and universities in the west [ 14 ]. A particular concern is if the fetus is more than 120 days old, the time of soul installment according to Islamic law [ 15 ], which is in the middle between the two opposing opinions: the first sees embryos less than complete and conscious persons, while the second sees them equal to all human beings and should not be treated differently. The sensitivity related to using stem cells from embryonic sources resulted in a significant increase in the interest of adult stem cells in medical research, even though the lesser importance they have.

The researchers’ points of view about permitted and forbidden sources, as stated in the Saudi law of ethics of research on living creatures and its implementing regulations [ 10 ], match almost completely except for limiting the use of pluripotent stem cells to laboratories only. The researchers did not mention extra fetuses (extra fertilized eggs) which, as prescribed in the Saudi law, are not permitted as a source of stem cells. The reason may be because it is neither a common practice nor legal to use this source. Researchers where satisfied with the available sources, namely cord blood and imported cell lines.

The source of stem cells was not the only point discussed by the sample. They highlighted several factors to be considered to ensure stem cell research is ethical. The points are a component of the general rules related to conducting ethical medical research, locally and internationally [ 16 ]. It was expected that the sample would mention obtaining informed consent prior to any stem cell donation, adult or embryonic, before use. Informed consent should also be obtained from donors of adult stem cells. However, for embryonic stem cells, consent should be obtained from the parents. It is noteworthy that the researchers highlighted the importance of clarifying the purpose of stem cell donation to the potential donors to avoid any possibility of employing practices that may invalidate the consent. Mandating review and approval of an ethics committee of the informed consent form protect the donors who may miss understanding some points in the informed consent documents. Although there is no direct benefit to the donors from stem cell research, the altruism principle is an important motivation to donate stem cells, which is supported by studies in other regions [ 17 ]. The participants mentioned the importance that research should not be futile but have a direct or indirect benefit. The researchers recognize that stem cells have the potential of future success and many people, especially patients with chronic diseases and difficult to treat diseases, have placed their hope on stem cells. So, it is understandable that the sample mentioned repeatedly that patients should be warned against false hope and they emphasized the importance of transparency in stem cell treatment or research; the idea that is highlighted by other researchers [ 18 ].

The awareness of researchers about the importance of respecting and complying to international guidelines can be understood in the context of receiving tertiary education abroad where they internalized the international guidelines and conducted research according to these guidelines. Frequently, the current research in Saudi Arabia is a continuation of their previous research during their training. The second reason which explains the importance of following international guidelines is that the majority of research is multi-center international studies and following the same principles is essential for success.

The harmonization of ethical and legal rules related to stem cell research with the Islamic point of view is important due to two reasons. Firstly, the acceptance or willing participation of potential donors will significantly increase if they are informed that the research is in line with Islamic law, and secondly, the Research Ethics Law in Saudi Arabia mandates that all practices in stem cell research should be in line with Islamic rules to be allowed and legitimate. However, the sample where not sufficiently aware of the regulation related to stem cell research mentioned in the Saudi Law of Research Ethics [ 10 ]. This caveat reflects a lack of responsibility about keeping themselves updated, as the law is readily available on the website of the National Committee of Bioethics www.kacst.edu.sa . From another perspective, the offices responsible in the National Committee of Bioethics should promote the law efficiently to raise awareness in researchers and donors.

In conclusion, the participants of the study indicate various ethical challenges regarding the use of stem cells in research. For the majority of the participants, specific stem cell sources are forbidden in Saudi. Particularly, embryonic stem cells as the use may result in serious religious issues. The participants also reject aborted or (some) miscarried fetuses as a source. In response to the second objective, the ethical principles and challenges related to stem cell research were identified. The sample emphasized the importance of always securing IRB approval of the informed consent documents. Informed consent should include an explanation of the scope of the research and the participants’ rights, in simple understandable language to ensure complete understanding.

The majority of the participants reported that they already follow the international regulations related to stem cell research, which they had been exposed to during their studies and training, mostly in Western countries. However, surprisingly, they are not necessarily aware of existing national local laws, which reflects a critical need of research ethics education in general and in stem cell ethics in particular, through courses, conferences, and university programs-which are currently lacking in Saudi. Also, conducting analytic and comparative studies about stem cells in Saudi research ethics law may help to increase awareness among researchers. Additional in-depth research to include different categories with different levels will be very important at the next stage.

Availability of data and materials

The datasets generated during the study are available from the corresponding author on reasonable request.

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Acknowledgements

We would like to thank the experts involved in stem cell research who agreed to participate in our study.

This study was funded by King Abdullah International Medical Research Center. There was no role of the funding body in the study design, collection, analysis, interpretation of data and in the manuscript writing.

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Ghiath Alahmad, Sarah Aljohani & Muath Fahmi Najjar

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(GA) designed, directed, analyzed and interpreted the study interviewee data and he was the major contributor in writing the manuscript. (MN) carried out the interviews with study subjects and he helped in drafting the manuscript. (SA) helped in interviewing, drafting, and reviewing the final manuscript. All authors read and approved the final manuscript.

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Alahmad, G., Aljohani, S. & Najjar, M.F. Ethical challenges regarding the use of stem cells: interviews with researchers from Saudi Arabia. BMC Med Ethics 21 , 35 (2020). https://doi.org/10.1186/s12910-020-00482-6

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Stem cell therapies

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Bubble Boy, Berlin Patient and Butterfly Patient

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Three examples of stem cell therapies have made history: the cure of X-SCID-affected Jack Crick, the cure of the Berlin patient who, with the help of stem cells, escaped both HIV and leukemia and the rescue of the butterfly child Hassan, who was close to death before genetically modified stem cell therapy saved his life and gave him a future. Their cases, however, have remained highly regarded individual cases to this day. All patient stories are interesting against the background that they embody the future potential and the associated hope in stem cell therapy - but still represent the exceptional situation.

Patient Jack Crick

2004 Born in May 2004 X-SCID is diagnosed in September. The search for a suitable bone marrow donor unsuccessful. Gene therapy using the patient's own blood stem cells is successful, even without chemotherapy. 2011 Patient has had no symptoms since.

Attending Physician Bobby Gaspar, Great Osmond Street Hospital, London

SCID stands for severe combined immunodeficiency;

A weakening of the immune system due to the absence or lack of lymphocyte function; mutations in genetic information (DNA) cause the disruption of T cell development. The patients' immune systems cannot cope with the pathogens in our normal environment and the affected children must therefore live in a sterile environment.

Collection of hematopoietic stem cells (CD34-positive); inserting the corrected gene into the test tube using a retroviral vector (taken from the mouse leukemia virus); stem cells are returned to the patient.

Advantages: Use of own cells instead of foreign cells; low risk of rejection or incompatibility; no suitable donor necessary.

Discussion The introduction of a new gene can lead to changes in cell properties or to degeneration (cancer). Cases are known where patients developed leukemia. The trigger seems to be the retroviral vector. Research is being carried out on the use of newer HIV-derived lentiviral vectors, for example, which have a much better safety profile.

Search terms : Bubble Boy, David Vetter, Jameson Golliday

Patient Timothy Ray Brown

1966 Born in Seattle, USA 1995 Diagnosis: HIV positive until 2006 Treatment using highly active antiretroviral therapy (HAART): 600 mg Efavirenz, 200 mg Emtricitabine and 300 mg Tenofovir 2006 Diagnosis of acute myeloid leukemia (AML); chemotherapy treatment 2007 Treatment by allogeneic stem cell transplantation from a donor with a mutation in the CCR5 cell surface receptor. The mutation prevents the HI virus from penetrating the cells. since 2007 HI virus no longer detectable using common procedures 2008 Leukemia identified again; second stem cell transplantation (same donor) since 2008HI virus undetectable using common procedures; leukemia treatment successful; neurological disorders diagnosed

09/2020 Died of another attack of leukemia

HLA-type: B57

Attending Physician

Dr. Gero Hütter, Benjamin-Franklin-Klinikum Charité Berlin (bis 2009)

Bone marrow donor

HLA type: B57 Mutation: delta 32 on receptor CCR5

Transplantation of allogeneic stem cell transplantation of a donor with a mutation in the cell surface receptor CCR5. The mutation prevents the HI virus from entering the cells.

It is not clear whether this individual case is reproducible. The procedure is very expensive. In 2012, Steven Yukl (University of California, San Francisco) investigated nine billion patient blood cells using polymerase chain reaction (PCR). After several attempts he identifies fragments of the virus genome in the blood plasma. Douglas Richman (University of California, San Diego) also conducts blood tests and finds no residues. He considers contamination in the Yukl test possible; in addition, PCR is highly sensitive and error-prone.

Search terms: The Berlin Patient, Mississippi Baby, London Patient

Patient Hassan

Born in 2008  started treatment at the age of 7 2015:  After fleeing from Syria to Germany, he suffered infections and chronic skin lesions as a  result of the inherited disorder epidermolysis bullosa. 2015-2016:  Skin graft involving 80 percent of the patient’s skin. Since then, the patient has been largely  free of symptoms.

Attending Physician Tobias Rothoeft, Kinderklinik Bochum  Tobias Hirsch, Universitätsklinikum Bergmannsheil (plastic surgeon) Michele De Luca, Center for Regenerative Medicine at the University of Modena (stem cell researcher)

Epidermolysis bullosa

Epidermolysis bullosa is an inherited disorder. Children with this condition are sometimes called butterfly children, because their skin is as fragile as a butterfly’s wings. This is because the upper layer of the skin (epidermis) is not properly attached to the layer underneath (dermis). People with this disease have a defective LAMB3 gene. This gene encodes the laminin-332 protein.

Skin cells from Hassan were sent to Italian experts in Modena for culturing. The scientists used retroviral vec�tors to insert a healthy LAMB3 gene into the skin cells. Retroviral vectors are viruses which have been specially modified to carry genes into cells. The genetically modified stem cells in the piece of skin were then cultured in a clean room laboratory to produce large pieces of skin suitable for grafting. Over a series of three operations in Germany, scientists then grafted the cultured tissue. In total, they replaced 80 percent of Hassan’s skin.

The new skin contains roughly the same amount of the laminin-332 anchor protein as normal, healthy skin.

There are around 35,000 children with epidermolysis bullosa in Europe. The severity of the disease varies great�ly. Until now, no treatment aimed at eliminating the underlying cause of the condition has been available. All gene therapies carry a risk that the new gene could be inserted into the wrong place in the genome, however. This can disrupt cell regulatory processes and cause cancer. The treatment Hassan underwent was risky and laborious. It was justified by the extent of his suffering and the fact that there was no prospect of his suffering being relieved by any other treatment.

Search terms: Patient Hassan, butterfly child

( https://www.spiegel.de/gesundheit/diagnose/gentherapie-junge-erhaelt-neue-haut-a-1177073.html )

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Stem Cell Therapy: From Idea to Clinical Practice

Regenerative medicine is a new and promising mode of therapy for patients who have limited or no other options for the treatment of their illness. Due to their pleotropic therapeutic potential through the inhibition of inflammation or apoptosis, cell recruitment, stimulation of angiogenesis, and differentiation, stem cells present a novel and effective approach to several challenging human diseases. In recent years, encouraging findings in preclinical studies have paved the way for many clinical trials using stem cells for the treatment of various diseases. The translation of these new therapeutic products from the laboratory to the market is conducted under highly defined regulations and directives provided by competent regulatory authorities. This review seeks to familiarize the reader with the process of translation from an idea to clinical practice, in the context of stem cell products. We address some required guidelines for clinical trial approval, including regulations and directives presented by the Food and Drug Administration (FDA) of the United States, as well as those of the European Medicine Agency (EMA). Moreover, we review, summarize, and discuss regenerative medicine clinical trial studies registered on the Clinicaltrials.gov website.

1. Introduction

Despite the progress in medical science, there still exist various diseases in the world for which there is no suitable treatment. People affected by incurable disorders typically use treatment methods intended to decrease the somatic and psychological symptoms and, in these situations, the physician offers treatment methods only to manage the disease, not treat it. Therefore, researchers are attempting to develop new treatment methods to not only control the symptoms of, but also to treat those diseases for which no cure is available at present.

Regenerative medicine is considered a promising new source of treatment for untreatable diseases in modern science [ 1 ]. Regenerative medicine is a multidisciplinary field including cell biology, genetic, biomechanics, material science, and computer science [ 2 , 3 ], the ultimate target of which is returning normal function to defective cells and tissues [ 4 ]. Since the discovery of stem cells and the spread of awareness regarding their unique properties, they have been defined as therapeutic agents for organ and tissue repair, and so are widely considered good candidates for regenerative medicine, due to their many potential applications [ 5 ]. Regenerative medicine is now regarded as an alternative to traditional drug-based treatments by researchers who study its potential applications in various diseases, including degenerative diseases, among others [ 6 , 7 , 8 , 9 , 10 ]. The main concept of regenerative medicine is implied tissue/organ regeneration using cells and, to reach this target, different kinds of cells have been used. However, various studies have indicated that cell therapy is restricted by a few limitations. In recent years, different alternatives have been introduced for cell therapy in order to resolve these limitations, including the improved application of stem cells for the restoration of tissue, such as the combination of cells with scaffolds, cell cultures with suitable biochemical properties, gene editing, and the immunomodulation of stem cells, as well as the use of stem cell derivatives [ 11 , 12 , 13 , 14 , 15 ]; however, the use of these alternatives clinically may be postponed, as more preclinical studies are required due to their status as newer technologies [ 16 ].

Stem cells are a group of immature cells that have the potential to build and recover every tissue/organ in the body due to their unique proliferative, differentiation, and self-renewal abilities [ 17 ]. Stem cells provide therapeutic effects which improve physical development by regenerating damaged cells to assist in organ recovery. Relying on the natural abilities of stem cells, researchers have used their biological mechanisms for stem-cell-based therapy. The mechanisms of action through which stem cells can promote the regeneration of tissue are diverse, including (1) inhibition of inflammation cascades [ 18 , 19 ], (2) reduction of apoptosis [ 20 , 21 ], (3) cell recruitment [ 22 , 23 ], (4) stimulation of angiogenesis [ 24 , 25 ], and (5) differentiation [ 26 ]. The cause of a disease is a vital consideration in selecting the proper stem cell mechanism and in the regeneration of tissue/organs using stem cells. Many examinations must be carried out to determine the main mechanisms involved in treatment when these cells are to be used in clinical practice, and the convergence of stem cell therapeutic mechanisms and disease mechanisms is expected to increase the chance of developing cures through stem cell applications.

From 1971 to 2021, 40,183 research papers were published regarding stem-cell-based therapies. All of these studies were conducted around discoveries and for the goal of “Stem Cell Therapy” based on the therapeutic efficacy of stem cells [ 27 ]. As basic stem cell research has soared over the past few years, “translation research”, a relatively new field of research, has recently greatly developed, making use of basic research results to develop new treatments. Although many articles on stem-cell-based therapies are published annually and their number increases every year, the number of clinical trial studies has not increased rapidly. Furthermore, among these studies, only a small portion of them can receive full regulatory approval for verification as treatment methods. Although one reason for this difference is due to the need for various prerequisite preclinical studies before carrying out a clinical trial study, the main reason is due to the sharply defined guidelines which prevent the translation of many preclinical studies to clinical trials.

In this review, we provide a general overview regarding the translation of stem cell therapies from idea to clinical service. Understanding the step-by-step knowledge underlying the translation of ideas to medical services is the first step in introducing a new treatment method. In this review, we divide this pathway into four levels, including idea evaluation, preclinical studies, clinical trial studies, and clinical practice. We focus not only on understanding each level’s requirements, but also discuss how an idea is assessed during the transition from one level to the next and, finally, move on to marketing.

2. From Idea to Preclinical Study

If a researcher has an idea regarding regenerative medicine using stem cells that inspires their use in a study, it must first be evaluated. During the evaluation step, it is important to select the target disease and make sure that the mechanism causing the disease is understood. Disease-related mechanisms refer to the cellular and molecular processes by which a particular disorder is caused [ 28 , 29 ], and stem-cell-based therapies are considered a treatment method intended to compensate for the disruption caused by such mechanisms in order to finally restore the defective tissue. Multiple mechanisms cause diseases [ 30 , 31 , 32 ]; however, stem cells, with their tremendous differentiation, self-renewal, angiogenesis, anti-inflammation, anti-apoptotic, and immunomodulatory potentials, as well as their capacity for induction of growth factor secretion and cell signaling, can affect these mechanisms [ 33 , 34 , 35 , 36 , 37 ].

After subject evaluation, preclinical studies should be carried out to determine whether the idea has any potential to treat the disease, and the safety of the final product should be assessed in an animal model of the target disease [ 38 , 39 , 40 ]. Preclinical studies are composed of in vitro and in vivo studies. In vitro experiments are performed with biological molecules and cells based on various hypotheses and, during the in vitro evaluation, a new treatment method is assayed in this controlled environment [ 39 ]. In contrast, during in vivo studies, as controlling all biological entities is impossible, the new product may be affected by various factors and thus present different effects. The general purpose of a preclinical study is to present scientific evidence supporting the performance of a clinical study, and the following are required for a decision to move forward to clinical study: (i) the feasibility and establishment of the rationale (e.g., validation, separation of active ingredients in vitro, and determination of its mechanism in vivo), (ii) establishment of a pharmacologically effective capacity (e.g., secure initial dose verification), (iii) optimization of administration route and usage (e.g., safe administration method, repeated administration, and interval verification), (iv) identification and verification of the potential activity and toxicity (e.g., toxicity analysis according to single and repetitive testing), (v) identification of the potential for special toxicity (e.g., genetic, carcinogenic, immunological, and neurotoxic analyses), and (vi) determination of whether to continue or discontinue development of the treatment [ 41 , 42 ].

3. From Preclinical Study to Clinical Trial

In principle, any idea regarding stem cell therapy should be assessed using comprehensive studies (i.e., in vitro and in vivo) before a clinical trial is considered, and the results of these studies should be proved by competent authorities. It can be easy during an in vitro study to create manipulative biological environments such as through the use of genetic mutation, drug testing, and pharmaceuticals, and it is easy to observe changes through the application of manipulated variables through living cells [ 43 , 44 , 45 ]. However, given the many associated variables, such as molecular transport through circulating blood and organ interactions, it is hard to say whether such a study can completely mimic the in vivo environment [ 43 , 44 , 45 ]. Before application in patients, in vivo experiments are conducted after in vitro experiments in order to overcome these weaknesses.

Many researchers use rodents for in vivo studies, due to their anatomical, physiological, and genetic similarities to humans, as well as their other unique advantages including small size, ease of maintenance, short life cycle, and abundant genetic resources [ 46 ]. The strength of in vivo studies is that they can supplement the limitations of in vitro studies, and the outcomes of their applications can be inferred in humans through the use of human-like biological environments. To establish in vivo experiments for stem cell therapies, the most correlated animal model should be selected depending on the specific safety aspects to be evaluated. Where possible, cell-derived drugs made for humans should be used for proof-of-concept and safety studies [ 47 ]. Homogeneous animal models can also be utilized as the most correlated systems in proof-of-concept studies [ 48 ].

Furthermore, in vivo studies require ethical responsibilities and obligations to be upheld according to experimental animal ethics. In other words, unnecessary and unethical experiments must be avoided. Summing up the above, we can see that both in vitro and in vivo approaches are used in preclinical studies, which should be carried out before clinical trial applications based on various interests.

Several factors must be considered in different in vitro and in vivo studies, including cell type determination, cell dose specification, route of administration, and safety and efficiency.

3.1. Stem Cell Source Determination

As expectations rise for regenerative treatment through the application of stem cell therapies, the number of applications of various types and stem cell sources has increased, and stem cell therapies have diversified from autologous to allogenic to iPSCs. These stem cell treatments can vary in risk, depending on the cell manufacturing process [ 49 ], among other factors, and in clinical experience, such that all types of stem cell treatments must be evaluated on the same basis [ 50 ]. Therefore, the strengths and weaknesses of each type of stem cell should be identified in order to determine the maximum therapeutic effect of stem cells in various diseases. This will enable us to build disease-targeted stem cells by applying the appropriate stem cells to the appropriate diseases. Below, we briefly discuss the characteristics of various stem cells.

3.1.1. Mesenchymal Stem Cells (MSCs)

MSCs are lineage-committed cells that divide into mesenchymal systems, primarily fatty cells, chondrocytes, and osteocytes [ 51 ]. It is well known that MSCs can be differentiated into dry cells, nerve cells, glioma cells, and skeletal muscle cells under proper in vitro culture conditions [ 52 , 53 , 54 , 55 , 56 , 57 ]. MSCs are primarily derived from myeloid and adipose tissues [ 58 , 59 ]. At present, MSCs are also isolated from many other tissues, such as the retina, liver, gastric mucosa, tendon, cartilage, placenta, cord blood, and blood [ 60 , 61 , 62 , 63 ]. The biggest characteristics of MSCs are their immunosuppressive functions, which prevent the proliferation of activated T cells through immunosuppressive cytokine secretion and suppression of programmed cell death signaling [ 64 , 65 ]. Due to this role, they have been spotlighted as a potential treatment for immune-related inflammation and disease. The initial clinical application of MSCs was in a case of patients with severe graft versus host disease (GVHD), and these cells have since been well applied in clinical practice, as evidenced through various studies [ 66 , 67 , 68 ].

MSCs have a variety of characteristics according to their organ of origin [ 69 ]. BM-MSCs, which are isolated from bone marrow, are useable in both autologous and allogenic contexts, and can perform stromal functions. However, the process of cell isolation from bone marrow is not only accompanied by the risk of pain and infection, but also has a lower efficiency of collection than other MSC sources. Furthermore, these cells have a longer doubling time (DT) in comparison to MSCs derived from other sources (approximately 60 h) [ 70 ]. Compared to BM-MSCs, AD-MSCs are not only easy to collect, but are also 100 to 500 times more efficient to harvest and have a shorter DT (approximately 20 h) [ 71 ]. However, these are adipose-derived stem cells that have a strong characteristic of adipogenic differentiation, such that they can be suggested as a valid alternative to BM-MSCs, but their nature must be considered regarding proper culture and body environment. Furthermore, there are concerns that these factors may affect the efficacy of treatment, as the amount of cytokines secreted is significantly lower when compared to BM-MSCs [ 72 ]. MSCs extracted from the umbilical cord (UC-MSCs) have come into the spotlight to compensate for these issues: UC-MSCs not only have the advantage of being easily collected compared to other stem cells, but also avoid ethical or donor age issues. They have superior proliferation and differentiation capabilities compared to BM-MSCs and AD-MSCs, and their DT has been reported as 24 h [ 69 , 73 ]. UC-MSCs are currently a subject of concern, as although they are easy to store frozen for a long time (e.g., in a cord blood bank), the cell survival rate and success rate during extraction are not high, due to exposure to cryogenic protectors during cryogenic storage [ 73 ]. Furthermore, as the cells are isolated from other organs, they have limited self-renewal capacity, and their senescence is faster than in other stem cells in long-term cultivation [ 66 , 74 ].

3.1.2. Hematopoietic Stem Cells (HSCs)

HSCs can be differentiated into cells from all hematopoietic systems present in the bone marrow and chest glands, namely myeloid cells and lymphocytes. HSCs can be obtained at good levels from adult bone marrow, the placenta, and cord blood. They can cause immunological problems such as transplant rejection. Nevertheless, they have been shown to be an effective treatment method in various diseases, including leukemia, malignant lymphoma, and regenerative anemia, as well as congenital metabolism, congenital immunodeficiency, nonresponsive autoimmune disease, and solid cancer to date. Furthermore, HSCs are the only stem cell type approved for stem cell treatment by the Food and Drug Administration (FDA) [ 75 , 76 ].

3.1.3. Embryonic Stem Cells (ESCs)

ESCs have established cell lines that can be maintained through in vitro culture. They are pluripotent cells that can be differentiated into almost any type of cell present in the body, and can be differentiated in vitro by adding external factors to the culture medium or by genetic modification. However, they may form teratomas, which are composed of various forms of cells derived from the endoderm, mesoderm, and exoderm, when transplanted into an acceptable host [ 77 ].

3.1.4. Induced Pluripotent Stem Cells (iPSCs)

iPSCs are artificially created stem cells. These cells are made by reprogramming adult somatic cells such as fibroblast cells. They share many of the characteristics of ESCs, including self-renewability, pluripotent differentiation, and malformed species performance. Unfortunately, these cells have little scientific evidence regarding changes in cell-specific regulatory pathways, gene expression, and epigenetic regulation. These characteristics pose a risk of tissue chimerism or cell dysfunction [ 78 ].

In summary, although the FDA-approved stem cell type is HSCs from healthy donors, a variety of issues have been raised, including a lack of donors and immune rejection. Therefore, we need to understand the characteristics of stem cells in order to handle them accordingly and overcome their disadvantages while maximizing their advantages. As stem cells derived from various sources have different characteristics, capabilities, potential, and efficiency, selecting the right source of stem cells that is appropriate for the target can be effective in assuring treatment efficiency.

3.2. Cell Dose Specification

The effective range of administration (i.e., dosage) of stem cells or stem-cell-derived products used in treatment should be determined through in vivo and in vitro studies. The safe and effective treatment capacity must be identified and, where possible, the minimum effective capacity must also be determined. When administered to vulnerable areas such as the central nervous system and myocardium, it has been reported that conducting normal dosage determination tests is unlikely. Thus, if the results of nonclinical studies can safety demonstrate treatment validity, it may be appropriate to conduct early human clinical trials with doses that may indicate therapeutic effects [ 79 ].

Will a high cell dose have better effects, considering only the effectiveness of stem cells? We answer this question below. An increasing dose of CD34 + cells (0.5 × 10 5 per mouse) has been shown to have positive effects, stimulating multilineage hematopoiesis at early stages and increasing the magnitude of reconstitution at post-transplant stages. Furthermore, improved T-cell reconstitution was correlated with higher cell doses of stem cells, compared to lower cell doses [ 80 ]. However, a few studies related to acute myeloblastic leukemia (AML) have reported that high doses of HSCs were correlated with restored function and rapid hematological and immunological recovery, but these results were not unconditional. In this study, a higher dose of HSCs (≥7 × 10 6 /kg) resulted in poorer outcomes and a higher relapse rate than the lower dose of HSCs (<1 × 10 6 /kg) [ 81 ]. In preclinical studies on heart disease, Golpanian et al. have demonstrated, through comparison of some preclinical studies for optimized cell dose, the therapeutic effects of stem cell types (i.e., allogenic and autologous MSCs), as well as the proper cell dose of stem cells and route of administration (direct epicardial and intravenous) in heart disease. Their results showed that the total number of cells used was different, but were inconsistent with the hypothesis that a higher number of cells would have higher therapeutic efficacy [ 82 ]. Therefore, these conclusions suggest that the currently reported data do not provide a decisive answer, such that sufficient and detailed early-stage studies may be needed before proceeding with clinical trials.

3.3. Route of Administration

Stem cells have been extensively studied under various disease conditions, depending on their type and characteristics. At this time, the route of administration should not be overlooked in favor of the number of stem cells transplanted. Several reports have shown that engraftment ability typically has a lower rate of reaching target organs relative to the number of transplanted cells, and does not have a temporary longer duration [ 83 , 84 ].

The methods of stem cell administration can largely be divided into local and systemic transmission. Local transmission involves specific injections through various manipulations and direct intra-organ injections, such as intraperitoneal (IP), intramuscular, and intracardiac injections. Systemic transmission uses vascular pathways, such as intravenous (IV) and intra-arterial (IA) methods. According to the publications in the literature, IV is the most common method, followed by intrasplenic and IP [ 85 , 86 , 87 ]. In a liver disease model, IV was shown to be not only suitable for targeting the liver, but also showed better liver regeneration effects than other routes of administration [ 85 , 88 ]. Intracardial injection showed better cell retention in heart disease, while intradermal injection showed better treatment in skin diseases [ 89 , 90 ]. Hence, we can determine that, in the context of these various diseases, the routes of administration should be different depending on the target organ. Many researchers have suggested that intravascular injection is a minimally invasive procedure, but it also poses a risk of clogged blood vessels, such that direct intravascular injection increases the risk of requiring open-air operations [ 91 ]. Clinical trials have reported that the number of cells and treatment efficacy under the same conditions, as in preclinical studies, are not significant, but also differ in significance depending on the route of administration [ 92 , 93 ]. Therefore, researchers should continue to study which cells are appropriate for a given route of administration—even within the same disease—based on many precedents [ 82 ]. In addition, researchers should explore the appropriate routes of administration for safer and more effective therapeutic effects.

3.4. Manipulation of Cell Transplantation for Safety and Efficiency Improvement of Administration

All medical treatments have benefits and risks. It is not particularly safe to apply these unproven stem cell treatments to patients. As expectations for regenerative treatment through stem cell therapies increase, the application of various administration pathways, including through the spinal cord, subcutaneous, and intramuscular, as well as the stem cell therapies themselves, have been diversifying, from autologous to homogenous to iPS. These stem cell treatments can vary in risk, depending on the cell type manufacturing process among other factors, and they differ in clinical experience, such that all types of stem cell treatments must be evaluate on the same basis. Furthermore, it should only be in limited and justified contexts that stem cells which can proliferate and have all-purpose differentiation remain in a final product.

Unfortunately, the only safe stem cells that have been employed in regenerative medicine so far are omnipotent stem cells, such as HSCs and MSCs, which are isolated from their self-origin [ 94 ]. Unfortunately, potential clinical applications using iPSCs and ESCs face many hurdles, as they present higher risk, including the possibility of rejection, teratoma formation, and genomic instability [ 95 ]. Hence, many researchers have attempted to overcome stem cell tracking for safety assessment. To check the engraftment and the remaining amount of stem cells, they have been labeled using BrdU, CM-Dil, and iron oxide nanoparticles, and visualized using Magnetic resonance imaging (MRI) [ 84 , 96 , 97 ].

A close analysis of the distribution patterns of administrative sites and target organs is required, as well as whether a distribution across the body is expected, and the organ that the cells are predicted to be distributed through should undergo a full-term analysis, including evaluation at administrative sites. To date, studies have reported assessments in the brain, lungs, heart, spleen, testicles, ovaries, kidneys, pancreas, bone marrow, blood, and lymph nodes, including areas of administration [ 98 ].

Some researchers have carried out the detection of transplanted UC-MSCs delivered by IV injection in the lung, heart, spleen, kidney, and liver. According to their results, the transplanted cells were not detected in other organs, except the lung and liver, for 7 days. In the lung and liver, the detected cells persisted at least 7 days after the transplant [ 99 ]. Furthermore, in a study comparing BM-MSCs and UC-MSCs in terms of cell tracking, they reported on the persistence of stem cells according to the route of administration used. In the results of the comparison of intracardiac and intravenous routes, the transplanted stem cells were detected in the lung for 10 days, but the signal disappeared after 21 days [ 100 ]. In other research, the stem cells were transplanted with using a biomaterial scaffold. The AD-MSCs were transplanted with hyaluronic acid/alginate hydrogel through intradermal injection, and could be detected by CM-Dil staining for 30 days [ 101 ]. These studies may show that the transplanted cells localized to the damaged organs through their homing ability, but the results of these previous studies seem to indicate that the residual volume and the residual date vary significantly depending on the target disease, organs, and type of stem cells. The cell residual means the survival of the cell, which represents the risk of formation of tumors. To overcome the problem of teratoma formation, the following results have been reported: According to one study, ESCs showed the following rates of teratoma formation: 100% under the kidney capsule, 60% intratesticular, 25–100% subcutaneous, and 12.5% intramuscular. To overcome this problem, the investigators performed a co-injection with Matrigel into an animal model. According to their results, subcutaneous implantation of ESCs in the presence of Matrigel appeared to be the most efficient, reproducible, and easiest approach for preventing teratoma formation, other than only using ESCs [ 102 ]. Moreover, cellular products derived from iPSCs have higher potential as potential cell sources in personalized medicine [ 103 ]. Their applicability is currently limited due to concerns regarding the potential risk of serious transplant-related side effects, such as tumor formation due to residual pluripotent cells [ 104 ]. Hence, a recent study reported the establishment of an optimized tool for therapeutic intervention that allows for controlled specific and selective ablation of iPSCs through the use of LVCAGs–transgenic iPSCs [ 104 ].

Unlike MSCs, which are generally considered immune-tolerant as an immunomodulator, transplantation of ESCs and HSCs requires close examination of the matching of histocompatibility antigen (HLA) between the donor and beneficiary [ 105 , 106 ]. Although homogeneous mesenchymal stem cells are known to have immunogenicity in immune-active rodent models and are quickly removed from the peripheral blood, studies have shown that a few MSCs remain for weeks to months. Therefore, it is recommended to conduct a study to assess the persistence of MSCs in the cell preparations administered, in order to assess the risk of stem cell removal. Therefore, for stem cell therapies that have undergone extensive in vitro manipulation such as long-term cell culture—including those derived from ESCs and iPSCs—both oncogenicity and genetic stability must be evaluated before clinical research begins. Furthermore, we must constantly review and study the latest research on safety, as well as the effects of regeneration using stem cells, and discuss and study the potential of regenerative medicine [ 107 , 108 , 109 , 110 , 111 ].

As discussed earlier, in vitro and in vivo preclinical studies are the direction of current research, and encompass the tasks that need to be completed. If we reinforce the current strengths and weaknesses based on the preceding content, we are already a step closer to developing stem cell treatments.

4. From Clinical Trial to Clinical Practice

Before a treatment is applied in humans (i.e., patients), preclinical study must involve checking whether the effect of treatment will be positive or negative and, if there are any negative effects, the researcher must check the safety possibilities at every step. Due to concerns relating to treatment using stem-cell-based products, deciding whether preclinical studies are sufficient for translating to clinical trials raises several issues that must be assessed by competent authorities. An application for a clinical trial should be submitted to the Food and Drug Administration (FDA), the European Medicine Agency (EMA), or another organization, based on the country [ 112 ].

The FDA is responsible for certifying clinical trial studies for stem-cell-based products in the United States [ 113 ]. If a new drug is introduced to a clinical investigator which has not been approved by the FDA, an Investigational New Drug (IND) application may need to be submitted [ 114 ]. The IND application includes data from animal pharmacology and toxicology studies, clinical protocols, and investigator information [ 115 ]. A lack of preclinical support (e.g., in vitro and in vivo studies) can lead to required modification or disapproval. If the FDA has announced that an IND requires modifications (meaning that the application is intended to secure approval but has not yet been approved), the results of the preclinical studies were deemed insufficient or inadequate for translation to clinical trial study, such that further study must be completed, after which an amended IND should be submitted.

The FDA has published guidelines for the submission of an IND in the Code of Federal Regulations (CFR). These regulations are presented in 21 CFR part 210, 211 (Current Good Manufacturing Practice (cGMP)), 21 CFR part 312 (Investigational New Drug Application), 21 CFR 610 (General Biological Product Standards), and 21 CFR 1271 (Human Cells, Tissues, and Cellular and Tissue-Based Products) [ 116 , 117 , 118 ]. These guidelines have been issued for the development of stem cell products with the highest standards of safety and potential effective translation to clinical trial studies.

The FDA issued 21 CFR parts 210 and 211to ensure the quality of the final products [ 119 ]. The 21 CFR part 210 contains the minimum current good manufacturing practice (cGMP) considered at the stages of manufacturing, processing, packing, or holding of a drug, while the 21 CFR part 211 contains the cGMP for producing final products. The 21 CFR 211 includes FDA guidelines for personnel, buildings and facilities, equipment, and control of components, process, packaging, labeling, holding, and so on, all of which are critical for pharmaceutical production [ 116 , 117 , 118 , 119 , 120 , 121 ]. The requirements for IND submission and conducting clinical trial studies, reviewed by the FDA in the 21 CFR part 312 (Investigational New Drug Applications), includes exemptions that are described in detail in 312.2 (general provisions). Such exemptions do not require an IND to be submitted, but other studies must present an IND based on 21 CFR part 312. The section, 21 CFR part 312, provides different information, including the requirements for an IND, its content and format, protocols, general principles of IND submission, and so on. In addition, the FDA describes the administrative actions of IND submission, the responsibilities of sponsors and investigators, and so on, in this section [ 116 , 117 , 122 ]. The 21 CFR part 610 contains general biological product standards for final product characterization. The master cell bank (MCB) or working cell bank (WCB) used as a source for stem-cell-based final products must be tested before the release or use of the product in humans. The MCB and WCB should be tested for sterility, mycoplasma, purity, identity, and potency, among other tests based on the final products (e.g., viability, stability, phenotypes), before use at the clinical level. The FDA provides all required information regarding general biological product standards in this section, including release requirements, testing requirements, labeling standards, and so on [ 116 , 117 , 123 , 124 ]. The 21 CFR part 1271 focuses on introducing the regulations for human cells, tissues, and cellular and tissue-based products (HCT/P’s), in order to ensure adequate control for preventing the transmission of communicable disease from cell/tissue products. Current Good Tissue Practice (GTP) is a part of 21 CFR part 1271, where the purpose of GTP is to present regulations for the establishment and maintenance of quality control for prevention of introduction, transmission, or spread of communicable diseases, including regulations for personnel, procedures, facilities, environmental control, equipment, and so on [ 125 , 126 , 127 , 128 ].

The EMA is an agency in the European Union (EU) which is responsible for evaluating any investigational medical products (IMPs) in order to make sure that the final product is safe and efficient for public use. When planning to introduce a new drug for a clinical trial in Europe, one may be required to submit clinical trial applications to the EMA for IMPs. Clinical trial applications for IMPs include summaries of chemical, pharmacological, and biological preclinical data (e.g., from in vivo and in vitro studies) [ 129 ]. The EMA has presented different regulations to support the development of safe and efficient products for public usage, including Regulation (EC) No. 1394/2007, Directive 2004/23/EC, Directive 2006/17/EC, Directive 2006/86/EC, Directive 2001/83/EC, Directive 2001/20/EC, and Directive 2003/94/EC.

Regulation (EC) No. 1394/2007 defines the criteria for regulation regarding ATMPs. Advanced therapy products (ATMPs) are focused on gene therapy medicinal products (GTMP), somatic cell therapy medicinal products (sCTMP), tissue-engineered products (TEP), and combined ATMPs, which refers to a combination of two different medical technologies. Regulation (EC) No. 1394/2007 includes the requirements to be used in development, manufacturing, or administration of ATMPS [ 130 , 131 , 132 ]. Directive 2004/23/EC, Directive 2006/17/EC, and Directive 2006/86/EC define standards for safety and quality, as well as technical requirements for donation, procurement, testing, preservation, storage, and distribution of tissue and cells intended for human applications [ 133 , 134 , 135 ]. Directive 2001/83/EC applies to medicinal products for human use [ 136 ]. Directive 2001/20/EC presents the regulations for the implantation of products in clinical trials in the EU [ 137 ]; however, this directive will be replaced by regulation (EU) No. 536/2014. Regulation (EU) No. 536/2014 was adapted by the European Parliament in 2014, and provides regulation for clinical trials on medical products intended for human use. The new EU regulation comes into effect on 31 January 2022 and aims to coordinate all clinical trials performed throughout the EU, using clinical trials submitted into CTIS (Clinical Trials Information System). The definition of regulation (EU) No. 536/2014 as a homogeneous regulation serves an important role in the EU, as all member states of the EU can be involved in multicenter clinical trials using international coordination, thus allowing larger patient populations [ 138 ]. Directive 2003/94/EC provides Good Manufacturing Practice (GMP) Guidelines in relation to medicinal products or IMPs intended for human use [ 139 ]. All process and application requirements for the IMP application are present in the regulations and directives of the EMA. After presenting an IND/IMP to the regulatory authority responsible for clinical trial oversight (FDA or EMA), the application will be reviewed in accordance with the FDA/EMA criteria and, if assured of the protection of humans enrolled in the clinical study, the application will be approved by the investigational review boards (IRBs) in the United States or Ethics Committees (ECs) in the European Union. Clinical trial studies are composed of different steps where, at each step, products are assessed using different quality and quantity measurements by the responsible agency. An efficient clinical trial study should address the safety and efficiency of new stem cell products in each of the different steps, and it is important to complete each step based on defined instructions and regulations, as the results of previous steps are needed to move forward.

Almost all clinical trial studies that have been approved for testing in humans have been registered online ( https://www.clinicaltrials.gov/ accessed 12 December 2021). Our search on this website revealed more than 6500 records for interventional studies registered using “Stem Cells” up to December 2021. The recorded clinical trials can be analyzed from different aspects.

Recruiting status: The recruiting status of these studies indicated that 18% of these studies were ongoing (recruitment) and 42% were completed ( Figure 1 ). Although completed, suspended, terminated, and withdrawn studies are all terms used for studies that have ended, each is used to describe a different status. Completed studies are those that have ended normally and the participants were completely enrolled in the study. Suspended, terminated, and withdrawn studies are studies that stopped early; however, the participant enrolment status differs between them. A suspended study may start again, but nobody can continue to participate in terminated or withdrawn studies [ 140 , 141 ].

Status of clinical trials using stem cells.

Type of disease: Stem-cell-based therapy is a new approach for the treatment of various diseases in different clinical trial studies. Blood and lymph diseases are the most common diseases that have benefited from this new approach ( Figure 2 ). Blood and lymph diseases refer to any type of disorder related to blood and lymph deficiency or abnormality, such as anemia, blood protein disorder, bone marrow disease, leukemia, hemophilia, thalassemia, thrombophilia, lymphatic disease, lymphoproliferative disease, thymoma, and so on. In addition, various clinical trial studies have been performed using stem cells to treat immune system disease; neoplasm, heart, and blood disease; and gland- and hormone-related disease ( Figure 2 ). However, this does not mean that all of these studies had great results, nor does it mean that all of these studies introduced a new treatment method; some of these clinical trial studies were only intended to increase treatment efficiency, compare different types of treatment methods, or analyze various parameters after the administration of stem cells into the body.

Diseases considered in clinical trials using stem cells.

Autologous vs. Allogenic: Stem-cell-based products for use in clinical trial studies can be divided into two categories: autologous and allogeneic stem cells. In autologous stem cell therapy, the stem cells are collected from the patient’s own body. Culture-expanded autologous stem cells are autologous stem cells that are expanded before transplantation, and can be divided into two groups: modified and unmodified expanded autologous stem cells. If autologous stem cells were transplanted to the donor immediately after collection, this is a nonexpanded autologous stem cell treatment. The use of these cells usually has fewer restrictions for receiving clinical trial authorization. The classification of allogenic stem cells is similar to that of autologous stem cells, except that allogeneic stem cells are collected from a healthy donor. The use of these cells requires more prerequisite tests, in order to check the donor’s health. Allogenic stem cells have been used more than autologous stem cells in the clinical trial studies (46.34% vs. 44.51%), as shown in Figure 3 .

Applied stem cell types in clinical trials using stem cells.

Phase: Clinical trial studies are conducted in different phases. In each phase, the purpose of study, the number of participants, and the follow-up duration may differ. A new phase of clinical trials should not be started unless the results of the completed phase(s) have been reviewed by competent authorities, in order to that certify the results of the completed phase(s) are valid for authorization of the start a new phase of the clinical trial. For this purpose, at the end of each phase of a clinical trial study, competent authorities evaluate whether the new drug is safe, efficient, and effective for the treatment of the target disease ( Figure 4 ).

Status of clinical phase within clinical trials using stem cells.

Early Phase I emphasizes the effects of the drug on the human body and how the drug is processed in the body.

Phase I of a clinical trial is carried out to ensure that a new treatment is safe and to determine how the new medicine works in humans. The FDA has estimated that about 70% of the studies pass this phase.

In Phase II, the accurate dose is determined and initial data on the efficiency and possible side effects are collected. The FDA has estimated that roughly 33% of the studies move to the next phase.

Phase III evaluates the safety and effectiveness of products. The result of this phase is submitted to the FDA/EMA for new product approval, which allows manufacturing and marketing of the drug. The FDA has estimated that 25%–30% of the drugs pass at this phase.

Phase IV take place after the approval of new products and is carried out to determine the public safety of the new product [ 142 , 143 , 144 ].

The number of participants and the duration: A new stem cell product is eligible for marketing after completing successful clinical trial phases. As the new product has been used on volunteers and the effects/side effects of the drug have also been followed for a long time throughout the different phases, it is now possible to make a decision regarding its introduction to the market for public use. The number of participants and the duration of long-term follow-up in each study and each phase differ ( Figure 5 and Figure 6 ). The number of volunteers that participate in each phase of a clinical trial study varies, as each phase has a different target. The FDA has recommended 20–80, 100–300, and several hundred to thousands of volunteers for Phase I, Phase II, and Phase III, respectively [ 144 , 145 ]. Although the FDA has defined a range for enrolments per phase, the number of participants can vary depending on the type of disease. The number of participants for clinical studies in rare diseases will be lower than when studying common diseases. Searching for stem cells in clinicaltrial.gov, studies can be found with only one participant (e.g., NCT02235844, NCT02383654, NCT03979898, and NCT01142856). The sponsor/investigator must provide the FDA with strong documentation regarding the selection of such a number of volunteers. The volunteers for each clinical trial study, before attending, should be informed about the enrolment criteria of each study, possible side effects, and the advantages of the study.

Enrolment of clinical trials using stem cells.

The duration of each clinical trial study using stem cells.

Age of participants: Roughly 190,000 people participated in all the completed clinical trial studies using stem cells that had been registered. Each clinical study was performed in different age groups, which differed among the various studies depending on the type of drug, type of disease, and sponsor decision, as shown in Figure 7 .

The age of patients participating in clinical trials using stem cells.

Number of clinical trial studies: The number of clinical trial studies increased gradually from 2000 to 2014, although it fluctuated after 2014 but did not change significantly ( Figure 8 ). The reason for this increase in 2014 is not clear, but it may have been related to the introduction of the first advanced medicinal therapy product containing stem cells (Holoclar) by the EMA in 2014–2015 [ 146 ].

The proportion of clinical trials using stem cells by year: ( A ) the proportion of new clinical trial studies using stem cells by year (green bar) and the proportion of registration results accordingly (orange color line); ( B ) the proportion of completed registered clinical trial studies using stem cells by year (blue bar) and the updated results of completed clinical trial studies using stem cells by year (orange line).

Place of study: According to economic website reports, the cell therapy market has grown significantly in recent years, and it is expected to grow more in the coming years; therefore, many countries have begun research in this field. Our data from clinicaltrial.gov showed that the United States has conducted the most clinical trials using stem cells ( Figure 9 ). Government agencies, industry, individuals, universities, and private organizations have all invested in stem-cell-based therapy. The number of stem-cell-based companies has rapidly increased in recent years, and a brief overview of the submitted clinical trial studies indicated that the studies were mostly aimed at introducing therapeutic products for clinical applications. Therefore, we can expect the introduction of stem-cell-based products to the market.

The registered and completed clinical trial studies using stem cells according to participating countries: ( A ) top 10 participating countries with registered clinical trials using stem cells; and ( B ) top 10 countries based on the completion of registered clinical trials using stem cells.

As indicated above, translational research from the laboratory to clinical services has many layers which must be passed through, each with its own requirements and measurements. Therefore, the only way to introduce a new stem-cell-based product onto the market is for competent authorities to make sure that the discovery is safe and effective for its intended human use, and that the product has successfully passed all of the clinical trial stages.

5. Challenges and Future Directions

One of the most important issues regarding the introduction of a new product for use in humans through a clinical trial is evaluation of its safety. Although many clinical trials have been performed using stem cells for the treatment of various diseases, as stem-cell-based therapies are one of the newest groups of therapeutic products in medicine, it is very hard to introduce new products based on stem cells onto the market, as many different parameters must be evaluated. There are several concerns regarding stem-cell-based therapies, including genetic instability after long-term expansion, stem cell migration to inappropriate regions of the body, immunological reaction, and so on. However, all challenges depend on the type of stem cell (e.g., embryonic stem cell, adult stem cell, iPS), type of disease, route of administration, and many other factors. Almost all researchers in the field of stem cell therapy believe that despite stem cells having great potential to treat disease through their intrinsic potential, unproven stem-cell-based therapies that have not been shown to be safe or effective may be accompanied by very serious health risks. In order to receive clinical trial approval from a competent regulatory authority, different tests must be performed for each study phase, and the results of one study should not be generalized to another study. The FDA and EMA have defined different regulations to ensure that stem-cell-based products are consistently controlled through the use of different preclinical studies (in vitro and in vivo). Based on these preclinical data, the FDA and EMA have the authority to approve a clinical trial study, as discussed in this review.

Another challenge that researchers and companies face is the duration of a clinical trial study before a stem-cell-based product can be introduced onto the market. At present, hematopoietic progenitor cells are the only FDA-approved product for use in patients with defects in blood production, while other stem-cell-based products used in clinical trials have not yet been introduced to the market.

In the past few years, several clinical trials have been conducted using stem cells, most of which have indicated the safety and high efficiency of stem-cell-based therapies. An attractive future option for regenerative medicine is the use of cell derivatives, including exosomes, amniotic fluid, Wharton’s jelly, and so on, for the treatment of diseases. Recently, the safety and efficiency of these products have been evaluated and optimized in preclinical studies. In addition, regenerative medicine using modified stem cells and combinations of stem cells with scaffolds and chemicals to overcome stem cell therapy challenges and increase the associated efficiency are two important future directions of research. However, establishing a safe method for stem cell modification and moving this technology toward clinical trial studies requires many preclinical studies.

The regenerative medicine market is developing and, due to encouraging findings in preclinical studies and predictable economic benefits, competition has increased between companies focused on the development of cell products. Therefore, government agencies, industries, individuals, universities, and private organizations have invested heavily into the development of the regenerative medicine market in recent years, such that we can be more hopeful about the future of stem-cell-based therapies.

6. Conclusions

In recent years, regenerative medicine has become a promising treatment option for various diseases. Due to their therapeutic potential, including the inhibition of inflammation or apoptosis, cell recruitment, stimulation of angiogenesis, and differentiation, stem cells can been seen as good candidates for regenerative medicine. In the last 50 years, more than 40,000 research papers have focused on stem-cell-based therapies. In this review study, we present a general overview of the translation of stem cell therapy from scientific ideas to clinical applications. Multiple mechanisms causing disease could be reversed by stem cells, due to their tremendous therapeutic potential. However, preclinical studies including in vitro and in vivo experiments are necessary to evaluate the potential of stem-cell-based treatments. Through preclinical research, it is possible to present scientific evidence and optimal treatment options for subsequent clinical studies. Before starting a clinical trial based on preclinical data, the application must be approved by a relevant regulatory administration, such as the FDA, EMA, or another organization. If the application is for the use of a new drug (including stem cells) which has never been tested before, the submission of an IND is required for FDA approval. Approximately 50% of clinical trials using stem cells take 2 to 5 years to complete. To minimize possible side effects, every new stem cell product should be approved for clinical marketing only after completing Phase I–IV clinical trials successfully. Interestingly, the number of stem-cell-based companies aimed at introducing clinical applications has rapidly increased in recent years. Therefore, it may be possible to find stem-cell-based products on the clinical market in the near future. As described in this paper, there are several steps that should be carried out on the path from the laboratory to the clinical setting. To develop new stem-cell-based medicine for the clinical market, researchers should follow the guidelines suggested by the relevant authorities. Through these well-controlled development processes, researchers can achieve safe and effective stem-cell-based therapies, thus brings their research ideas into the clinical field.

Author Contributions

All authors have read and agreed to the published version of the manuscript.

This review funded by National Institutes of Health grant: R01HD087417-01A1, R01HD094378-01, R01HD094380-01, R01HD100367-01, R01HD100563, R01HD100563.

Conflicts of Interest

The author has no conflicts of interest to declare.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Developmental Biology and Stem Cell

The Department of Genetics and Genome Sciences has a strong focus on developmental and stem cell biology.  We use a variety of cell and animal models to study the impact of genetic and epigenetic aberrations on normal development and their contribution to disease. 

The advent of embryonic and induced pluripotent stem cell technology has enabled temporal access to disease-relevant cells and tissues. Many labs throughout the department are using pluripotent stem cell technology to define the mechanisms underlying normal development and a spectrum of disorders including the labs of Tony Wynshaw-Boris (autism and microcephaly), Ann Harris (cystic fibrosis), Paul Tesar (multiple sclerosis and other myelin disorders), Ashleigh Schaffer (neurogenetic disorders), Fulai Jin (regulation of pluripotency), Helen Miranda (motor neuron disorders), Yan Li (diabetes), and Peter Scacheri (CHARGE syndrome). 

Mouse models and advancements in genome engineering such as CRISPR/Cas9 are being used to study endocrine disorders in David Buchner ’s lab, cystic fibrosis in the labs of Mitch Drumm , Craig Hodges , and Ron Conlon , social behavioral disorders in Tony Wynshaw-Boris ’ lab, and neurogenetic disorders in the labs of Ashleigh Schaffer and Paul Tesar .

The department also has a longstanding interest in germ cell biology and current efforts are focused on germline ovarian stem cell biology led by Helen Salz , spermatogenesis led by Shih-Hsing Leir and Ann Harris , and ovarian insufficiency led by David Buchner .  

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• Published: 14 February 2023

Progress and challenges in stem cell biology

• Effie Apostolou 1 ,
• Helen Blau 2 ,
• Kenneth Chien 3 ,
• Madeline A. Lancaster 4 ,
• Purushothama Rao Tata 5 ,
• Eirini Trompouki 6 ,
• Fiona M. Watt 7 ,
• Yi Arial Zeng 8 , 9 &
• Magdalena Zernicka-Goetz 10 , 11  

Nature Cell Biology volume  25 ,  pages 203–206 ( 2023 ) Cite this article

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Since stem cells were first discovered, researchers have identified distinct stem cell populations in different organs and with various functions, converging on the unique abilities of self-renewal and differentiation toward diverse cell types. These abilities make stem cells an incredibly promising tool in therapeutics and have turned stem cell biology into a fast-evolving field. Here, stem cell biologists express their view on the most striking advances and current challenges in their field.

Effie Apostolou: induced pluripotency — the continued reprogramming revolution

The seminal discovery of pluripotency induction achieved by means of transcription factors or chemical cocktails has revolutionized multiple biomedical fields and shed light on processes including development, aging, regeneration and cancer. Over the past 15 years, many burning questions around reprogramming mechanisms, trajectories and translational limitations have been addressed.

High-throughput functional screens identified critical regulators and barriers of reprogramming, while multimodal omics studies helped with constructing four-dimensional (4D) roadmaps of the complex transcriptional, epigenetic, topological, proteomic and metabolic changes that somatic cells undergo upon loss of their initial identity and acquisition of pluripotency. Parallel studies have also identified potentially detrimental, long-lasting aberrations that are introduced along the way. Moreover, single-cell technologies during cellular reprogramming captured intriguing intermediate and refractory states, reminiscent of early embryonic fates, senescence response, regeneration or tumorigenesis.

Despite this progress, important gaps remain and new questions continually arise. What are the cause-and-effect relationships during the multi-layered molecular chain reaction of reprogramming, and which factors lie at the top of the regulatory hierarchy? How can we reproducibly and deterministically reprogram cell identity, if we know the start and end points, to enable efficient and safe generation of any therapeutically relevant cell type from easily accessible tissues? How can we either avoid or rationally exploit the epigenetic variability of induced pluripotent stem cells? Can we capture, and propagate in vitro, transient intermediate cell states of biomedical relevance? Future studies using advanced engineering approaches for acute and reversible perturbations in defined time windows will be critical to address the functional interconnections of various reprogramming regulators and enable fine-tuning toward end states of interest. Moreover, ongoing single-cell efforts to map the continuum of cell states in early embryos and tissues or synthetic structures will determine more definitively the degree to which reprogramming intermediates recapitulate physiological or pathological transitions. Together with continuously improved computational approaches and modelling, these efforts will enable accurate predictions of critical conditions and cocktails for precise, reproducible and error-free cell fate engineering. These engineered fates can ultimately expand the toolbox for generating complex tissues and organoids for disease modelling and drug screening and for understanding and ameliorating hallmarks of ageing and cancer.

Helen Blau: multiple strategies to augment muscle regeneration and increase strength

Mobility is a major determinant of quality of life. Elderly patients with sarcopenia or patients with heritable muscle-wasting disorders suffer from a debilitating loss of muscle strength for which there is no approved treatment. COVID-19 highlighted the need for strategies to strengthen atrophied diaphragm muscles after ventilator support. Although our knowledge of stem cell function in regeneration has markedly increased, major knowledge gaps and challenges remain.

First, muscle stem cells (MuSCs) are a heterogeneous population that diverges over time and in response to disease or ageing. Targeting the functional subset of MuSCs is an unmet challenge. Second, understanding the role of the microenvironment and the muscle stem cell niche in muscle stem cell behaviour is key. Data are emerging showing that MuSCs respond not only to biochemical but also to biomechanical cues and that the elasticity of the niche matters. This suggests that stiffer fibrotic muscles, characteristic of muscular dystrophy or ageing, will harbour stem cells with impaired regenerative function. The development of hydrogels that can stiffen or soften on demand, while maintaining stem cells in a viable state, could provide new molecular and signalling insights into stem cell mechanosensing mechanisms, how they change with ageing and how they can be overcome. Third, from advances in single-cell and single-nuclei RNA sequencing, we are gaining knowledge of the gene expression patterns of the complex, diverse array of cell types that populate the niche. However, these technologies entail tissue destruction and therefore do not provide spatial information regarding cell–cell interactions that are crucial to maintaining stem cell quiescence and inducing stem cell activation and efficacious regeneration. There is a great need for spatial proteomics and multiplexed imaging modalities that preserve information about cell location and the dynamics of cell–cell interactions characteristic of regeneration, disease and ageing. Finally, inflammation has beneficial roles in wound healing, but is deleterious when chronic, as in aged muscles. Finding ways to rejuvenate muscle form and function remains a major challenge. The discovery of the prostaglandin degrading enzyme 15-PGDH, an immune modulator, as a pivotal molecular determinant of muscle ageing is a notable step in that direction. Remarkably, overexpression of 15-PGDH for one month in young adult mouse muscles induces atrophy and weakness, whereas inhibition of 15-PGDH in aged mouse muscles results in a 15% increase in muscle mass, strength and exercise performance. Solving these challenges will pave the way for new, effective stem cell-targeted therapeutic agents to regenerate and rejuvenate muscle.

Kenneth Chien: heart progenitors rebuild cardiac muscle

Rebuilding the failing human heart with working muscle is the holy grail of regenerative medicine. Although initial therapeutic attempts with non-cardiac cells have proven unfruitful, mouse studies have shown the potential to create de novo cardiomyocyte-like cells in situ by direct reprogramming via gene transfer. A novel class of adult claudin-6 + epicardial progenitors can convert to muscle, contributing to regeneration of the injured vertebrate heart. The studies point to a key role of tight junction proteins in the formation of a honeycomb-like regenerative structure. Although the adult human epicardium lacks these specific progenitors, uncovering their regenerative molecular pathways could identify new signals that can restore the myogenic potential of non-human epicardial cells via conversion to a progenitor state.

Thus far, the most advanced stem cell therapeutic agents are based on human embryonic stem (ES) cells for the generation of either cardiomyocytes or human ventricular progenitors (HVPs) for transplantation in large animals following cardiac injury. Issues of scalability, efficacy, clear evidence of working ventricular muscle grafts, lack of teratoma formation and tissue integration have all been largely addressed, moving both ES-cell-derived cell types toward the clinic with large pharma partners. However, additional issues remain, including safety (arrhythmias), durability (rejection) and the development of clinically tractable in vivo delivery systems. Our work on cardiogenesis over two decades recently led to the discovery of HVPs, which can migrate toward the injury site, prevent fibrosis via fibroblast repulsion, and proliferate to form large human ventricular muscle grafts to improve function in failing pig hearts. Additional work is ongoing, but early returns support the therapeutic potential of HVPs with minimal major side effects, with a two-year projected timeline for a first-time-in-human study. Prevention of rejection with optimal drug regimens, hypoimmune ES cell lines and new tolerization strategies, as well as novel catheters for in vivo delivery, are on the horizon. With these advances, HVPs might eventually provide new hope for patients with near-end-stage heart failure and no other options.

Madeline A. Lancaster: next-generation human neural stem cell models

The field of neural stem cell biology has made great strides in the past decade. What started out with neural stem cells that were cultured ex vivo to generate neurons and glia has evolved into a diverse field of ever-more-complex tools to model not just individual cells, but whole 3D neural tissues in a dish called neural organoids. Such organoids mimic not only the cellular makeup of the developing brain, but also local tissue architecture, with recent methods even demonstrating morphogenetic movements of neurulation.

Organoids and other in vitro models of the nervous system are becoming increasingly complex, for example through the use of so-called assembloids to combine different regions and examine their integration. Neural organoids also enable extensive neuronal maturation, even reaching hallmarks seen in the postnatal brain. However, as these models increase in complexity, so too do the challenges. With increasing size and maturity, the lack of vasculature becomes problematic. Although promising results have come from in vivo transplantation and integration of endothelial cells, vascularization leading to more advanced tissue development remains to be demonstrated. This challenge will likely represent one of the most difficult hurdles not just for the neural organoid community, but for the field of organoids as a whole, and creative approaches will be needed.

Brain organoids are already paving the way to fundamental discoveries in human neurobiology and are providing new understanding of disease pathogenesis. The future will hold new insight into why the human brain is unique, as well as how to prevent and treat various neurological conditions. Organoids may hold the key to these insights, but they cannot be the only tool, and it will be important to use them as complementary approaches alongside more established methods. Marrying in vitro and in vivo approaches will be the key to uncovering fundamental processes of neurobiology and answer age-old questions such as how genetics influence connectivity, how networks of neurons compute and how information is stored in the brain. The brain is still a largely uncharted territory, and powerful techniques combined with creative minds are needed to untangle its mysteries.

Purushothama Rao Tata: phenotypic and functional interrogation of lung biology at single-cell resolution

Lung tissues are relatively quiescent at homeostasis, but they respond rapidly to regenerate lost cells after injury. Early lineage tracing studies in animal models showed that this regeneration is driven predominantly by several ‘professional’ and facultative stem and progenitor cells in different regions of the lung, including basal and secretory cells in the airways and type 2 pneumocytes in the alveoli. These studies also uncovered a remarkable plasticity of some differentiated cell populations that contribute to regeneration following severe injury. More recently, multiple groups have used single-cell omics approaches to catalogue lung cells and their associated molecular signatures in great detail. Remarkably, in the case of the human lung, these efforts have identified previously unknown and uncharacterized cell types located in discrete regions. These cell populations are often quite heterogeneous, and include transitory states enriched in lungs from patients with respiratory disease. Significantly, these cell types are not found in the mouse, the animal model most commonly used for lung research. Consequently, there is an urgent need to develop new experimental tools to test their normal in vivo function and role in regeneration and disease.

To address this problem, efforts are underway by several groups, including our own, to develop genetically engineered ferrets and pigs as new animal models. Similarly, analytical tools are being optimized to infer cell lineages in human lungs based on clonally amplified genetic variants (single-nucleotide polymorphisms or mitochondrial heteroplasmy). In the case of ex vivo organotypic cultures, such as those derived from human induced pluripotent stem cells or primary foetal or adult lung progenitors, there remain many challenges. These include attaining or retaining mature cell types in the correct ratios to match those in normal in vivo lung tissue. To overcome this challenge, collaborative efforts are underway between lung stem cell biologists and bioengineers to generate new scaffolds to reassemble and mimic the cell–cell interactions found in native lung tissue niches. Taken together, these new approaches have the potential to identify the genetic circuits that regulate normal and disease-associated human lung cell states, establish scalable disease models and, ultimately, develop cell-based therapies to treat degenerative lung diseases.

Eirini Trompouki: the time journey of blood stem cells

Haematopoietic stem and progenitor cells (HSPCs) are critical for sustaining lifelong haematopoiesis via their extensive self-renewal and multilineage differentiation capacities. The secrets to how HSPCs acquire these capacities reside in the enigmatic process through which they are generated during an embryonic endothelial-to-haematopoietic transition (EHT). On the other end of the spectrum, age alters HSPCs, resulting in defective haematopoiesis. The most critical problems in HSPC biology relate to these lifetime bookends. Recently, human HSPC development was addressed in a spatial and single-cell manner, revealing that a haematopoietic stem cell (HSC) transcriptional signature is established after the emergence of HSCs along with continuously evolving cell surface markers, while haematopoietic heterogeneity already starts to be established at the haemogenic endothelium stage. Single-cell transcriptomics also led to the identification of a progenitor population that is responsive to retinoic acid and gives rise to haemogenic endothelial cells. Our group and others pinpointed the importance of DNA and RNA sensors in EHT. We and others found that transposable elements and R-loops trigger innate immune sensors to induce sterile inflammation that enhances EHT. Another layer of regulation lies in the interaction between HPSCs and other cells, such as macrophages or T cells, that are proposed to perform quality control of HSPCs during development and adulthood, respectively. Despite this progress, however, we still cannot faithfully recapitulate EHT in vitro and produce the massive quantities of HPSCs required for transplantations and gene therapy. Therefore, I think one of the most important aspects of haematology in the near future will be generating and maintaining good quality and quantity of HSPCs in vitro.

Ageing of HSPCs, on the other hand, is especially relevant because the population of the Earth is continuously ageing. An interesting feature of ageing that is lately gaining more and more attention is clonal haematopoiesis, which has been linked to haematological (and other) diseases. Inflammation, chemotherapy and irradiation have been shown by many groups to be advantageous for mutated clones. It is interesting to speculate that a collection of stressful moments experienced during life are ‘memorized’ by HSPCs and aided by clonality to instigate ageing. It was recently demonstrated that epigenetic memory is a feature not only of immune cells but also of HSCs. Further research needs to show whether every stress in life could be depicted in our genome as ‘memory’ and finally constitute the intricate mechanism of ageing.

Fiona M. Watt: understanding epidermal stem cell biology through data integration

Although mammalian skin contains many different cell types, the best-characterized stem cell population is in the epidermis, the multilayered epithelium that forms the skin surface. Autologous sheets of cultured epidermis were one of the first cell therapies involving ex vivo expansion of stem cells to be validated clinically, dating back to the early 1980s. That approach has been refined over the years, and the life-saving effects of combining cell and gene therapy to treat blistering skin disorders have been demonstrated unequivocally. In parallel with the development of techniques to culture human epidermis, the mouse became a key model for stem cell studies because of the availability of tools to target the different epidermal layers and the demonstration that genetic lesions in humans could be phenocopied in the mouse. With the advent of extensive single-cell RNA sequencing (scRNA-seq) databases for healthy and diseased human skin, it is essential that stem cell researchers use these resources both to validate their experimental models and to design new experiments. We need to look hard at the extent to which mouse models are still appropriate for modelling healthy and diseased human skin.

A very exciting challenge we face is data integration. There are many different axes along which integration can be achieved. One is spatiotemporal — the ability to correlate changes in cell types and states as a function of time and distribution within the skin. I am particularly intrigued by the possibility of correlating macroscopic skin features that are captured by optical coherence tomography with features obtained via spatial transcriptomics. Another example is integrating epidermal datasets from transcriptomics, proteomics, lipidomics and glycomics to gain a more holistic understanding of the nature of the stem cell state. In our enthusiasm for scRNA-seq, we risk ignoring the central dogma that DNA makes RNA that makes protein, and failing to remember the importance of protein modifications and turnover. I believe that by integrating epidermal stem cell responses to different extracellular cues, whether physical or biochemical, we will gain new insights into stem cell function and find switches between cell states that are conserved between tissues.

Yi Arial Zeng: the journey to islet regeneration

The islets of Langerhans are endocrine regions of the pancreas containing hormone-producing cells. β-cells produce and secrete insulin — the hormone that lowers blood glucose levels. Insufficient numbers of functional β-cells are associated with both type 1 and late-stage type 2 diabetes. With 1 in 11 people being diabetic, there is a great need to understand how the adult islet mass is maintained and how β-cells are regenerated to guide new therapies.

Stimulation of in situ islet regeneration is one approach for replenishing β-cells, through the formation of new progenitor-derived β-cells and enhanced proliferation of existing β-cells. Although the existence of islet progenitors in postnatal life has long been debated, recent work using mouse models has reported their existence in adults, leading to exciting opportunities for dissecting the activation mechanisms of these progenitors during homeostasis, regeneration and aging. It is noteworthy that neogenesis from progenitors and β-cell replication are not mutually exclusive: the proliferative β-cell subpopulation could possibly be the progeny of the progenitors, or there could be parallel proliferative pathways. Considering that relatively few insulin-secreting cells are needed to ameliorate hyperglycaemia, in vivo transdifferentiation represents another promising route. It has been reported that pancreatic exocrine cells and gut cells can be transdifferentiated into insulin-secreting cells. Collectively, these approaches aim to offer therapeutic strategies to stimulate in situ regeneration.

Pancreatic islet transplantation from donors is a recognized approach for replacing lost or damaged β-cells. Because of the shortage of donors, ongoing efforts aim to identify a renewable supply of human β-cells. A promising idea involves the differentiation of human pluripotent stem cells into β-like cells, and clinical trials using these β-like cells are underway. However, one may ask whether transplanting only mature β-cells is optimal, as proper glucose regulation requires coordination between various islet cell types. Will it be advantageous to produce whole islets in vitro rather than differentiating cells solely into β-like cells? Murine adult islet progenitors can generate organoids that contain all endocrine cell types of the intact islet and are proven to ameliorate diabetes in murine models. More work will be needed to establish the identity of these progenitors in the human pancreas and to translate the organoid culture system to human cells. As our understanding of islet regeneration matures, therapeutic transplant options will continue to emerge.

Magdalena Zernicka-Goetz: stem cells in modelling embryology

ES cells, derived from the pluripotent epiblast, can host transgenes and be reintroduced back into the embryo to generate a chimeric animal and a pure breeding line in future generations. A stunning application of ES cells in recent years has been their use to generate embryo-like structures in vitro. Several approaches have advanced our quest to recapitulate embryogenesis.

A 2D method using exclusively ES cells cultured as micropatterns offered a powerful route toward understanding how different cell types are established and signal between themselves. A second model, in which large aggregates of ES cells are treated with chemicals and growth factors, generated 3D structures developing many aspects of the segmental body plan, although still lacking body regions, particularly those required for forebrain development.

The importance of extraembryonic signalling was recognized through a series of whole-embryo models. The first such model, built from ES cells alone, pointed to the role of signals normally provided by the extraembryonic primitive endoderm, which can be replaced by the extracellular matrix to polarize ES cells to form a rosette-like structure that undertakes lumenogenesis. The second model, built from ES cells and trophectoderm stem cells, taught us that this interaction alone is sufficient to establish amniotic cavity and posterior embryo identity to induce mesoderm and germ cells. By incorporating a third stem cell type, extraembryonic endoderm cells, we achieved the formation of the anterior signalling centre and anterior–posterior patterning. Recently, additional approaches we and others undertook led to the generation of embryo models that were capable of developing much further to establish brain and heart structures and initiate organogenesis. Such whole-embryo-like models have brought insight into the biophysical and biochemical factors mediating stem cell self-organization and defining the cellular constituents, the chemical environment and the physical context required for embryo assembly.

Despite this progress, challenges remain. Cell fate specification relies on chemical cross-talk within and between lineages. Cell fate decisions must be spatiotemporally coordinated by establishing and interpreting gradients of numerous diffusible signalling proteins. We have much to learn about these combinatorial effects and about how to improve the efficiency with which different cell types combine to form embryo-like structures. A deep understanding of the components of cellular, biochemical and biophysical networks will be crucial to reaching this goal. Computational modelling will allow us to predict and guide self-organizational outcomes through exploitation of the capacity of cell communication to promote self-organization in vivo. It would also be powerful to advance our abilities to culture model embryos and replicate the maternal environment by delivering suitable nutrients to the circulatory system of the developing structure. These problems are also inherent to the assembly of synthetic organs, and I am certain that we will see a cross-talk between these different disciplines of synthetic biology for mutual benefit.

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Authors and affiliations.

Sanford I. Weill Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA

Effie Apostolou

Donald E. and Delia B. Baxter Foundation Professor for Stem Cell Biology, Stanford University School of Medicine, Stanford, CA, USA

Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden

Kenneth Chien

MRC Laboratory of Molecular Biology, Cambridge, UK

Madeline A. Lancaster

Department of Cell Biology and Duke Regeneration Center, Duke University School of Medicine, Durham, NC, USA

Purushothama Rao Tata

IRCAN Institute for Research on Cancer and Aging, INSERM Unité 1081, CNRS UMR 7284, Université Côte d’Azur, Nice, France

Eirini Trompouki

Directors’ Research Unit, European Molecular Biology Laboratory, Heidelberg, Germany

Fiona M. Watt

State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China

Yi Arial Zeng

School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

Magdalena Zernicka-Goetz

Stem Cells Self-Organization Group, Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

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Correspondence to Effie Apostolou , Helen Blau , Kenneth Chien , Madeline A. Lancaster , Purushothama Rao Tata , Eirini Trompouki , Fiona M. Watt , Yi Arial Zeng or Magdalena Zernicka-Goetz .

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Apostolou, E., Blau, H., Chien, K. et al. Progress and challenges in stem cell biology. Nat Cell Biol 25 , 203–206 (2023). https://doi.org/10.1038/s41556-023-01087-y

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DOI : https://doi.org/10.1038/s41556-023-01087-y

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