CNS, central nervous system; ESC, embryonic stem cell; FDA, Food & drug administration; HLA, human leukocyte antigen; HSC, hematopoietic stem cell; ICM, inner cell mass; iPSC, induced pluripotent stem cell; IVF, in vitro fertilization; MS, multiple sclerosis; MSC, mesenchymal stem cells; SC, stem cell
Adipose tissue, Amniotic sac, Antigen, Autoimmune disease, Axon, Basophil, Benign, Blood cell, Bone marrow, Cancer, Cell, Cell division, Chemotherapy, Clinical trial, Cloning, Culture, Differentiation, Egg cell, Embryo, Eosinophil, Epidermis, Erythrocyte, Fertilization, Gene, Homeostasis, Homogenous, Human development, Immune response, Immune system, Immunosuppressant, in vitro, in vivo, Inner Cell Mass, Lesion, Leukocyte, Lymphocyte, Monocyte, Morphology, Mortality, Multipotent, Muscle cell, Myelin, Neuron, Neutrophil, Nucleus, Oncogene, Organ, Placenta, Platelet, Pluripotent, Potency, Red blood cell, Somatic cell, Tissue, Totipotent, Unipotent, Virus, Zygote
Regenerative medicine is an interdisciplinary field of medicine that focuses on replacing, regrowing, or repairing cells, tissues, and organs that have been lost or damaged during disease, to retrieve normal function [1 – 3]. The use of cell-based therapies such as the therapeutic use of stem cells is a modern and promising approach to regenerative medicine that offers the possibility to treat and potentially cure a variety of diseases by engineering a patient’s cells [4 – 6]. However, while the number of clinical trials assessing the applicability of stem cell-based therapies has increased exponentially in recent years, to date, very few have been approved for routine use . Currently, the most prevalent stem cell therapy available that is approved by the US Food and Drug Administration (FDA), is a hematopoietic (blood) stem cell transfer. This type of stem cell therapy is often used for the treatment of cancers and immune system disorders. It is likely that in the future, medicine will be increasingly focused on cell-based therapies relative to drug-based therapies, with potential applications in the treatment of numerous neurodegenerative diseases, diabetes, and genetic conditions [6, 7]. However, translating upcoming science into viable treatment options is time-consuming and there are still several obstacles to overcome.
What is a stem cell?
The human body is made up of a diverse array of cell types such as muscle cells, red blood cells, and neurons, each of which has its specific function. These specialized cells are known as ‘differentiated’ cells. However, stem cells (SC) are a group of unspecialized cells that are unique as they can develop into several different cell types [8, 9]. Specialized cells are involved in the growth and development of the body as well as the replacement of old or damaged cells through the process of cell division, in which a single cell divides to produce two “daughter cells”.
SCs exhibit two defining properties or fates [1, 8]:
- Self-renewal: the SC divides, producing at least one daughter cell that remains as a SC . This is a crucial characteristic to ensure that the SC pool is not depleted.
- Differentiation: the SC divides and does not self-renew. Rather, the daughter cells differentiate, thereby gaining a specialized function.
Following the fertilization of an egg cell, a blastocyst is formed. The blastocyst contains two types of cells, an outer layer (known as the ‘trophoblast‘), and the inner cell mass (ICM) (Fig. 1) . The blastocyst is a rapidly dividing ball of cells that is formed approximately 5 – 6 days post-fertilization . The trophoblast subsequently grows into extra-embryonic tissues (such as the placenta or amniotic sac) that support the embryo’s nutrition and development . The ICM develops three germ layers, which are groups of cells in an embryo that differentiate into the cells that form all of the organs and tissues of the human body (Fig. 1A) . These three cell layers are called the endoderm (inner layer), the mesoderm (middle layer), and the ectoderm (outer layer). Each of these layers differentiates to allow the development of different types of specialized cells . The endoderm develops into the inner cell lining of numerous organs, such as the lungs, liver, and gastrointestinal tract . The mesoderm develops into the majority of organs (i.e., blood, muscle, and bone) . The ectoderm forms the outer surfaces of the body such as the skin (‘epidermis’) and hair follicles, as well as the central nervous system (CNS)  (Fig. 2).
There are several different types of SCs. These can be categorized according to their differentiation potential. Differentiation potential, or potency, refers to how many different cell types an SC can differentiate into. Totipotent SCs have the highest differentiation potential and are capable of generating all of the different types of cells that are required for the growth and development of a whole new organism [1, 8, 9]. This includes both cells that differentiate into the three germ layers and cells that differentiate into the cells that make up the extra-embryonic tissues [1, 8, 9]. Pluripotent SCs can give rise to cells of all three germ layers of the blastocyst, but not to cells that make up the extra-embryonic tissues [1, 8, 9]. Multipotent SCs can only develop into cells of a specific germ layer or lineage [1, 8, 9]. Finally, unipotent SCs have the narrowest potency and can only differentiate into one particular cell type.
[1, 8, 9].
Figure 1: Human Development & Changes in Stem Cell Potency
Stem cells are not homogenous throughout human development . Totipotent stem cells are the most undeveloped and are present during the period between fertilization and the morula stage . Cells that make up the morula continue to divide, forming the blastocyst. In the blastocyst, potency changes, and the stem cells become pluripotent . Stem cells extracted from the inner cell mass of the blastocyst are called embryonic stem cells and these cells are responsible for fetus development. Stem cells in the fetus and extra-embryonic tissues are multipotent. Adult tissue stem cells can either be multipotent or unipotent, to support growth and homeostasis. Figure created on BioRender.com.
SCs can also be categorized according to their origin. Human embryonic stem cells (ESCs) are isolated from the ICM of the blastocyst, approximately five to seven days after fertilization [1, 6, 9, 10]. ESCs are pluripotent, meaning that they can differentiate into cells of all three germ layers and are involved in whole-body development (Fig. 2) . ESCs can divide indefinitely to provide a constant SC pool for transplantation. Therefore, ESCs represent an ideal source of SCs for regenerative medicine . However, the application of ESCs has been limited due to ethical and religious concerns [8, 9]. Currently, ESCs arise from unused, donated embryos following in vitro fertilization (IVF) treatment . In other words, the isolation of ESCs requires the destruction of an embryo [6, 10, 11]. Furthermore, ESCs have the potential to form teratomas . Teratomas are a rare type of tumor formed from germ cells, that may contain many different tissue types . For example, teratomas can consist of hair, muscle, teeth, and bone . Germ cells are sexual reproductive cells including both sperm and egg cells. Teratomas can either be benign (non-cancerous) or malignant (cancerous). Another limitation of ESCs is the immune incompatibility between donor cells and the patient recipients [10, 11]. The human immune system has evolved numerous mechanisms to prevent pathogens and foreign organisms from invading. One of these immune mechanisms is the ability to recognize specific proteins on the surface of our cells (antigens), which are known as the human leukocyte antigens (HLAs). HLAs are unique to a person, and therefore cells of the body will express the same HLA type on their surface. The immune system recognizes any cells that are expressing a different HLA type as being “non-self”, and attacks them. This can lead to the rejection of cells transplanted from another person. As a result, it is essential to try to identify donors that have a similar HLA type to the patient, to optimize immune compatibility.
The risk of immune rejection often means that patients are required to take immunosuppressant drugs for the rest of their lives. Immunosuppressants act by reducing the activity of the immune system to lower the risk of transplant rejection. However, the suppressive effect on the immune system causes individuals to be at an increased risk of repeated infections.
Figure 2: Differentiation of Human Embryonic Stem Cells (ESCs)
Embryonic stem cells are isolated from the ICM of a blastocyst. These cells are capable of self-renewal and differentiation into cells of all three germ layers. Figure created on BioRender.com.
Tissue-derived (‘somatic’) SCs, on the other hand, are extracted either from fetal tissues such as the placenta, and umbilical cord, or from amniotic fluid, or adult tissues . Fetal-derived SCs are multipotent but do not present ethical challenges as no embryos are harmed or destroyed in the process of deriving these cells . Adult SCs can be found in many tissues of the developed human body . Adult SCs normally exist in an inactive state (‘quiescence’) and only begin to divide to replace the cells that are lost following an injury [8, 9]. The first group of adult SCs to be identified was hematopoietic stem cells (HSCs), which are found in the bone marrow. These cells were first discovered in 1909, by Alexander Friedenstein . HSCs are responsible for generating all blood cell types. Adult SCs are multipotent but are normally present only in low numbers in adult tissues . This means that there is a limited quantity of these cells available for therapeutic use. A further limitation is that isolating these cells can be difficult .
Recent developments in the biological sciences have made it possible to create SCs from differentiated somatic cells in a laboratory [1, 9]. A somatic cell is any cell of the body, excluding sperm, eggs, or undifferentiated cells. The first evidence that creating SCs might be possible, came from the work of John Gurdon . His research involved removing the nucleus from a fertilized frog egg cell and replacing it with the nucleus extracted from an intestinal cell (Fig. 3) in a process called ‘somatic cell nuclear transfer’ . The intestinal cell is fully differentiated and does not exhibit any potency. However, Gurdon found that the resulting egg cells were able to develop into a frog embryo . The results of this experiment provided ground-breaking proof that somatic cells (such as skin or blood cells) could be reprogrammed to differentiate into SCs . Somatic cell nuclear transfer offers an alternative method to the conventional method of using unwanted embryos from IVF for obtaining ESCs. Using somatic cell nuclear transfer, a blastocyst can be grown following somatic cell nuclear transfer, and pluripotent ESCs can be isolated from the ICM . This makes it possible to produce an SC line that is genetically identical to the patients. The technique is known as therapeutic cloning . This strategy reduces immune incompatibility, as a patient’s cells are used, and therefore the ESCs exhibit the patient´s unique HLA type. This avoids the risk of an immune response being raised against the transplant and the risk of rejection. However, similarly to normal ESCs, this technique raises ethical concerns. Although a clone of the parent is produced, the process does involve the destruction of potential life . Therapeutic cloning is extremely technology-intensive with low yields and to date, researchers have been unsuccessful in applying therapeutic cloning for human use in medicine .
Figure 3: Nobel Prize-Winning Experiments in Physiology or Medicine (2012) Gurdon and Yamanaka’s discovery that somatic cells can be reprogrammed into pluripotent stem cells earned them the Nobel Prize in Physiology or Medicine in 2012. Figure adapted from BioRender.com
In 2006, scientific pioneers, Shina Yamanaka and Kazutoshi Takahashi demonstrated the possibility of genetic reprogramming in a laboratory [1, 6, 9, 10]. The researchers were able to successfully reprogram differentiated mouse and human cells to generate stem-like cells (Fig. 3) [1, 9, 10]. This required the overexpression of four genes which were termed the ‘Yamanaka factors’, Oct4/3, Sox2, Kfl4, and c-Myc [1, 6, 9, 10]. Genes are the codes that provide cells with the specific instructions that they need to be able to produce specific proteins. The four genes that the researchers reintroduced are known to be involved in ensuring that SCs remain as SCs during embryonic development. The cells produced are known as ‘induced pluripotent SCs’ (iPSCs) and can be stimulated to generate different cell types . Therefore, the generation of iPSCs in laboratories provides an unlimited source of human stem cells to be used for therapeutic purposes in medicine.
The introduction of iPSCs has revolutionized the field of regenerative medicine. These cells exhibit very similar characteristics to ESCs, as both cell types are capable of self-renewing and differentiating into cells that make up each of the three different germ layers . iPSCs and ESCs also have similar morphologies (shapes) and growth patterns . Similar to ESCs, iPSCs can proliferate indefinitely, meaning that there is a constant supply of SCs that can be utilized in regenerative medicine . This is in contrast to adult SCs, where the ability of the cells to divide and replenish themselves reduces with age. Finally, iPSCs can be generated by reprogramming a patient’s somatic cells [6, 9]. This eliminates the risks associated with immune incompatibility following transplantation and the need for immunosuppressants. Therefore, iPSCs provide an accessible, pluripotent source of SCs, overcoming the requirement for human embryos in SC therapy.
Numerous challenges still need to be overcome before iPSCs can be widely used in medicine. For example, cells are often reprogrammed through the delivery of Yamanaka factors via a non-pathogenic (non-disease causing) virus. The virus which has been engineered to carry the genes encoding the four Yamanaka factors inserts its DNA into the DNA of a patient´s cells (host genome), and subsequently begins to overexpress the four factors. However, currently, the precise location of where the integration of viral DNA occurs cannot be controlled. This creates a risk for insertional mutagenesis. Furthermore, the two Yamanaka factors, c-Myc, and Klf4, (Kruppel-like factor 4) are both oncogenes. Oncogenes are genes with an increased potential for causing cancer . Together, these overexpressed factors can increase the risk of cancer development in patients that are receiving iPSC stem cell therapy. While these are just two of the challenges presented by iPSCs, they illustrate the critical importance of having stringent regulations in place globally to ensure that high-quality and low risk iPSCs can be consistently produced and that they are safe for therapeutic use.
Many diseases, injuries, and cancers can lead to irreversible damage to cells, tissues, or organs. To develop SC therapies, researchers extract or grow SCs in the laboratory and then induce them to differentiate into specialized cells. Differentiated cells can then be transplanted into patients to specifically target their disease (Fig. 4). For example, functional heart muscle cells (cardiomyocytes) can be transplanted into the hearts of patients following myocardial infarction (heart attack) .
This strategy of manipulating SCs to differentiate into the desired cell types is often referred to as ‘directed differentiation’ . This process of differentiation is essential, as the direct transplantation of pluripotent SCs can lead to teratoma formation in patients [6, 9]. To avoid the risk of such deleterious effects, it is crucial to ensure that transplanted cells are pure and are not contaminated with undifferentiated cells. For successful directed differentiation, scientists must mirror the molecular signals that are received throughout human development, including the molecular signals from the SCs niche . The SC niche is the in vivo microenvironment in which SCs are located. It plays a crucial role in the determination of SC’s fate. SC fates can include self-renewal, differentiation, death, and quiescence. Molecular signals can inform a cell to differentiate into a specific cell type via the activation of certain genes and the inhibition of certain other genes. Accurately reflecting the correct developmental conditions to promote homogenous differentiation to a particular desired cell type poses a significant challenge in the laboratory. The results of directed differentiation often remain time-consuming and of low purity .
Directed differentiation is common to both naturally derived ESCs and iPSCs (Fig. 3). The first clinical trials for the application of these cell types in medicine, began in 2011 and 2014 respectively [14, 15]. Since then, an exponential number of studies (with over 6000 trials worldwide) and clinical trials have begun to study the potential of SC-based therapies in regenerative medicine .
Figure 4: Workflow for iPSC-based Cell Therapy
Somatic cells, such as skin or blood cells, are isolated from the patient and reprogrammed using Yamanaka factors (Oct4/3, Sox2, Kfl4, and c-Myc) to produce iPSCs. iPSCs are subsequently differentiated in vitro into the required cells and subsequently transplanted back into the patient to eventually replace any dysfunctional cells. Figure adapted from Biorender.com.
Examples of Stem-Cell Therapies in Medicine
Bone Marrow Transplants
Currently, the most well-studied and widespread use of SCs in medicine is for HSC transplantation, which is also known as bone marrow transplantation [6, 9]. As mentioned, HSCs are multipotent SCs that are involved in producing red blood cells (‘erythrocytes’), white blood cells (‘leukocytes’), and platelets. Each of these has an important role in the body. Red blood cells are required for transporting oxygen to and removing carbon dioxide from cells and tissues throughout the body. White blood cells play an important role in disabling and eliminating pathogens. Platelets are crucial for blood clotting and to prevent excessive bleeding. HSCs are mainly found in the bone marrow, a soft tissue that is found in the center of bones. They can also be extracted from the umbilical cord or the blood .
Figure 5: Stem cell differentiation from bone marrow
The bone marrow contains hematopoietic stem cells that can differentiate into all blood cell types, including erythrocytes, and leukocytes, as well as cell fragments (‘platelets’). There are various types of leukocytes present in the immune system, including neutrophils, eosinophils, basophils, monocytes, and lymphocytes. Figure adapted from BioRender.com.
The use of HSC transplantation has been studied since the 1950s and is now mainly used as a treatment for leukemia, which is a cancer of the blood cells [9, 17]. In leukemia patients, the hematopoietic system is dysfunctional, resulting in high numbers of white blood cells . These blood cells are often not fully developed . HSC transplantation can either be used to replace the abnormal bone marrow in leukemia patients or to replenish depleted bone marrow following cancer treatment such as treatment with chemotherapy .
During bone marrow transplants, healthy HSCs are transplanted into the patient to replicate and restore the ability of the body to produce functional blood cells (Fig. 5) . Such transplantations can either be autologous (using the patient’s own SCs), allogenic (receiving SCs from a donor), or syngeneic (receiving SCs from an identical twin) .
Figure 6: Workflow for autologous hematopoietic stem cell transplants Procedure in which a patient’s healthy hematopoietic stem cells (HSCs) are collected from the bloodstream, frozen, and given back to the patient after chemotherapy treatment. The healthy HSCs are used to replace diseased or damaged blood cells. Figure adapted from Biorender.com.
Bone marrow transplants face certain limitations. Firstly, there are restricted numbers of cells available for transplant, which are simultaneously difficult to extract . Furthermore, it is necessary to identify donors who carry the same antigens (HLA type) to reduce the risks of immune rejection [9, 17]. Furthermore, HSC transplantation is a risky surgery that is associated with numerous risks and complications. The treatment-related mortality following HSC transplantation is high, given that it is estimated that approximately 15% of patients die by thirty days post-transplantation . Although some of these deaths may be due to the underlying disease, rather than the risks of the procedure, this high mortality has resulted in HSC transplantation being reserved only for patients with life-threatening diseases . The most common complications of the procedure are infection, graft-versus-host disease, and malignancies [17, 19].
Neurodegenerative diseases such as multiple sclerosis (MS), Alzheimer’s, and Parkinson’s, are characterized by a loss of or damage to neurons or connections in the nervous system. This damage leads to a decline in cognitive, mobility, and coordination. Such diseases have previously been very difficult to treat as neurons are thought to be post-mitotic, meaning that the cells no longer have the ability to divide and replicate. In other words, the loss of neurons during disease is permanent. SC-based therapies have allowed neural cells to be produced in vitro and can offer a novel treatment strategy to both delays and prevent disease [6, 9].
MS is a chronic autoimmune disorder that often causes patients to suffer from loss of mobility, muscle control, and vision . The loss of control of body functions over time eventually leads to irreversible disability and loss of functional independence. Symptoms such as pain, fatigue, and muscle weakness usually begin to appear between the ages of 20 to 40 years and result from a patient’s immune system attacking the myelin sheath of neurons . The myelin sheath is an insulating layer that surrounds the axons of nerves in the central nervous system (CNS). The myelin sheath is crucial for the quick transmission of electrical signals in the CNS to muscles. Although the underlying cause of MS remains unknown, SC-based therapies are currently being tested to halt the destruction of myelin. These therapies are showing great promise for the treatment of this debilitating disease.
SC therapies for MS are strongly focused on the application of mesenchymal stem cells (MSCs) . MSCs are found in several tissues, including the bone marrow, umbilical cord, adipose (fat) tissue, and blood [8, 11]. MSCs are pluripotent and are thought to exhibit strong anti-inflammatory properties . The procedure is autologous, involving the extraction of bone marrow from the patient . The MSC population can be isolated from the bone marrow, expanded to produce more identical cells, and then returned to the patient’s body intravenously via an infusion . Alternatively, MSCs can be derived from the placenta or umbilical cord. This therapy is based on the expectation that transplanted MSCs will travel to sites of injury in the brain and CNS to inhibit additional damage to myelin sheath . Therefore, MSC therapy is expected to exert restorative effects following neurodegeneration.
A Phase II trial was conducted to investigate the effectiveness of MCS therapy in 2015-2018 at the MS Center at the Hadassah Medical Center in Israel . This therapy was developed by the biotechnology company, NeuroGenesis . The trial found that the administration of MSC therapy (referred to as NG-01) directly into the spinal canal of MS patients, resulted in neuroprotective effects. It was found that symptoms were either significantly alleviated or eliminated in over 70% of the trial participants . The treatment halted disease progression in 60% of patients and improvements in clinical symptoms continued to be observed for up to four years following MSC therapy . The treatment also resulted in less frequent relapses and brain lesions as well as improved cognitive function [21, 22]. These results provide encouraging insights for the use of SC-based therapies to treat MS and similar neurodegenerative diseases . Tal Gilat, the CEO of NeuroGenesis, has mentioned plans for conducting further phase II trials, which are to begin later in 2022 .
Interestingly, an alternative SC-based treatment to MSC therapy against progressive MS is also being explored. This strategy involves autologous HSC transplantation . As mentioned previously, HSCs are involved in the production of white blood cells, which play very important roles in the immune system. This treatment option aims to limit the destruction of the myelin sheath by re-setting the patients’ immune system . Chemotherapy is used to obliterate dysfunctional immune cells, after which healthy HSCs are introduced to replace and rebuild the immune system.
A study at the MS clinic at Ottowa General Hospital, Canada examined the effectiveness and safety of this treatment in MS patients. No relapses were recorded in MS patients following transplantation . This clinical trial was conducted by Francis Brunet and his colleagues. It has been found that HSC transplantation is most effective in patients who are younger than 45 years and who are in the early stages of disease progression . The procedure is not a viable option for older patients or those with long-term progressive MS, due to the increased risks of mortality [21, 24]. In other words, as expressed by Brunet, “Bone marrow transplant is effective for patients who are adequately selected” . However, HSC transplantation still poses increased long-term risks for infection post-procedure, as well as a higher risk of developing cancer, autoimmune conditions, or fertility problems . These risks can further be accompanied by side effects from chemotherapy, including fatigue, hair loss, and loss of appetite [21, 25].
Both HSC and MSC therapeutic strategies are designed to halt the degradation of the myelin sheath that surrounds neurons to stabilize resulting MS symptoms and prevent relapses. However, HSC transplantation is a much more aggressive and invasive procedure. Therefore, the upcoming development of MSC therapy provides the opportunity for the development of a safer and more effective treatment with limited risk for immune rejection. This is particularly important for those whose health is too poor to survive chemotherapy.
The current standard protocol for pharmacological testing involves testing drugs on animals, (most commonly on rodents) before conducting clinical trials in humans . While this strategy has been powerful to date, it is well-known that the effects observed in rodents do not necessarily reflect the biology in humans [26, 27]. For example, drugs that are toxic to the human liver might not harm a rat. Adam Rosenthal, at the biopharmaceutical company, iPierian, states that “Many of the animal models out there are poor, demonstrating great efficacy in the mouse, but not repeating in man during late-stage clinical trials” .
SCs offer a unique opportunity for the advanced screening of pharmaceutical compounds and drugs [9, 26, 27]. Pluripotent SCs can be induced to produce specific tissue lines, allowing drug compounds to be tested on the target organ in vitro. Such models can be used to assess drug toxicity as well as to identify novel drug compounds . Drugs can also be screened using cancer cell cultures . This strategy minimizes the requirement for animal models and human volunteers in clinical trials before the drug can be approved for use in humans .
The role of stem cells in testing toxicity has been studied by Stephen Minger, a stem-cell biologist at GE Healthcare . Minger and colleagues analyzed numerous drugs which had been previously approved by the US Food and Drug Administration (FDA) following animal studies and clinical trials . Many of these FDA-approved drugs had toxic effects on humans after being available on the market for some time . When tested against heart cells, the results showed that the known toxic drugs negatively affected human cell lines . Using stem cells for pharmacological screening gives a more accurate reflection of human biology and can help scientists to detect some of the potentially harmful side-effects of drugs at an earlier stage . This can, in turn, help companies and governments to reduce some of the high costs that they incur during the drug development process . Such a strategy must incorporate genetic diversity in the cell lines that are tested, to appropriately mirror the variation observed in patients suffering from a particular disease .
iPSCs currently harbor the most promising source of SCs for use in SC-based therapies, overcoming previous ethical and immunologic concerns from ES cells. iPSCs can differentiate into all types of human cells and tissues, and therefore provide patient- and disease-specific cells. Nonetheless, there are still several challenges that need to be overcome to allow the widespread use of iPSCs in clinical therapy. This includes enhancing the genetic stability of iPSCs, reducing cancer risk, and developing integration-free methods for iPSC generation. Once these issues have been successfully addressed, SC-based therapies will likely become an important treatment option for a wide range of diseases including type I diabetes, Parkinson’s disease, and MS.
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