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STEM CELLS IN THE CLINIC

1. INTRODUCTION
Stem cells are basically biological cells found in all multicellular organisms, which can divide (through mitosis) and differentiate into diverse specialized cell types and can self-renew to produce more stem cells. They have the ability to divide in culture and give rise to specialized cells. They have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell. Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions. Research in the stem cell field grew out of findings by Canadian scientists Ernest A. McCulloch and James E. Till in the 1960s.

2. TYPES OF STEM CELLS

1. 2. 3. 4. 5. 6. 7. Human Embryonic Stem Cells Differentiated Human Embryonic Stem cells Core blood Stem Cells Adult hematopoietic Stem Cells Adult mesenchymal Stem Cells Adipose Tissue Stem Cells Induced Pluripotent Stem Cells

2|Page I. HUMAN EMBRYONIC STEM CELLS (HESC) Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indenitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Development of ES cells evolved out of work on mouse teratocarcinomas, tumors that arise in the gonads of a few inbred strains, and consist of a remarkable array of somatic tissues juxtaposed together in a disorganized fashion. Classical work on teratocarcinomas established their origins from germ cells in mice and provided the concept of a Stem cell (the embryonic carcinoma or EC cell) that can give rise to the multiple types of tissue found in the tumors (Kleinsmith and Pierce, 1964; review, Stevens, 1983). As mentioned earlier, Embryonic stem cells are said to be pluripotent, meaning that they have the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system). Human embryonic stem cells (hESCs) are generated by transferring cells from a pre-implantation stage embryo into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The cells divide and spread over the surface of the dish. The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide. This coating layer of cells is called a feeder layer. The mouse cells in the bottom of the culture dish provide the cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Researchers have devised ways to grow embryonic stem cells without mouse feeder cells. This is a significant scientific advance because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells .The process of generating an embryonic stem cell line is somewhat inefficient, so lines are not produced each time cells from the pre-implantation stage embryo are placed into a culture dish. However, if the plated cells survive, divide and multiply enough to crowd the dish, they are removed gently and plated into several fresh culture dishes. The process of re-plating or sub-culturing the cells is repeated many times and for many months. Each cycle of sub-culturing the cells is referred to as a passage. Once the cell line is established, the original cells yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for a prolonged period of time without differentiating, are pluripotent, and have not developed genetic abnormalities are referred to as an embryonic stem cell line. A human embryonic stem cell is also defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency. The cell surface antigens most commonly used to identify hES cells are the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. Ethical issues arise when the ES cells are used for research. The main ethical issue which comes to mind, when considering the use of embryos for clinical research, is the question of the life or the humanity of the embryo - when is the embryo a human life? The questions to be answered are: from where have the embryos come and for what purpose do they exist? Opponents of the research argue that embryonic stem cell technologies are a slippery slope to reproductive cloning and can fundamentally devalue human life. Some in the pro-life movement argue that a human embryo is already a human life that is entitled to protection. Contrarily, supporters of embryonic stem cell research argue that such research should be pursued because the resultant treatments could have significant medical potential. It is also noted that excess embryos created for in vitro fertilization could be donated with consent and used for the research. IVF embryos are the result of the fertilization of a sperm and egg in a laboratory (rather than naturally through sexual intercourse

3|Page between a man and woman) due to the inability of a couple to conceive naturally. Aside from the Ethical issues around the use of ES cells, some other issues also hinder its widespread use. One of such issues is the rejection by Natural Killer cells.

II. ADULT (HEMATOPOIETIC) STEM CELLS Adult stem cells are stem cells that can be derived from different parts of the body and, depending on where they are from, have different properties. They exist in several different tissues including bone marrow, blood and the brain. Some studies have suggested that adult stem cells are very versatile and can develop into many different cell types. However, other studies have concluded that adult stem cells are only able to develop into a limited number of cell types related to the tissue that the stem cells originally came from. Thus, they are said to be Multipotent. Hematopoietic stem cells are adult stem cells found mainly in the bone marrow and they provide the blood cells required for daily blood turnover and for fighting infections. A hematopoietic stem cell is a cell isolated from the blood or bone marrow that can renew itself, can differentiate to a variety of specialized cells, can mobilize out of the bone marrow into circulating blood, and can undergo programmed cell death, called apoptosisa process by which cells that are detrimental or unneeded self-destruct. The classic source of hematopoietic stem cells (HSCs) is bone marrow. For more than 40 years, doctors performed bone marrow transplants by anesthetizing the stem cell donor, puncturing a bonetypically a hipboneand drawing out the bone marrow cells with a syringe. About 1 in every 100,000 cells in the marrow is a long-term, blood-forming stem cell; other cells present include stromal cells, stromal stem cells, blood progenitor cells, and mature and maturing white and red blood cells. For clinical transplantation of human HSCs, doctors now prefer to harvest donor cells from peripheral, circulating blood. It has been known for decades that a small number of stem and progenitor cells circulate in the bloodstream, but in the past 10 years, researchers have found that they can coax the cells to migrate from marrow to blood in greater numbers by injecting the donor with a cytokine, such as granulocyte-colony stimulating factor (GCSF). The donor is injected with GCSF a few days before the cell harvest. To collect the cells, doctors insert an intravenous tube into the donor's vein and pass his blood through a filtering system that pulls out CD34+ white blood cells and returns the red blood cells to the donor. Of the cells collected, just 5 to 20 percent will be true HSCs. Thus, when medical researchers commonly refer to peripherally harvested "stem cells," this is something of a misnomer. As is true for bone marrow, the CD34+ cells are a mixture of stem cells, progenitors, and white blood cells of various degrees of maturity.

III. MESENCHYMAL STEM CELLS Mesenchymal stem cells (MSCs) are Multipotent stem cells that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes, adipocytes, beta-pancreatic islets cells, and potentially, neuronal cells. MSCs are of intense therapeutic interest because they represent a population of cells with the potential to treat a wide range of acute and degenerative diseases. MSCs are advantageous over other stem cells types for several reasons. First, they avoid the ethical issues that surround embryonic stem cell research. Second, repeated studies have found that human MSCs are immuno-privileged, and therefore, represent an advantageous cell type for allogeneic

4|Page transplantation, reducing the risks of rejection and complications of transplantation. Recently, there have also been significant advances in the use of autologous MSCs to regenerate human tissues, including cartilage and meniscus, tendons, and bone fractures. Mesenchymal stem cells (MSCs) occur at low frequency in tissues and must be expanded in vitro to obtain sufficient numbers for research and therapeutic applications.

IV. ADIPOSE TISSUE STEM CELL In addition to bone marrow, adipose tissue has been identied as a source of Multipotent cells that have the capacity to differentiate to cells of adipogenic, chondrogenic, myogenic and osteogenic lineages when cultured with the appropriate lineage specic stimuli. Adipose-derived stem cells (ADSCs) may be obtained from tissue harvested through liposuction (termed processed lipoaspirate cells (PLAs)), or through abdominoplasty procedures. These cells have also been identied as mesenchymal cells because they are derived from adipose tissue which is, in turn, derived from mesenchyme, much like bone marrow. Adipose tissue stem cells (ADSCs) have been shown to be very similar to marrow-derived stem cells in morphology and phenotype. In addition to their common multipotency, several CD marker antigens found on the surface of marrow stem cells have been found on the surface of ADSCs. Adipose tissue is often available in an abundant, expendable quantity. It is also easy to harvest, in contrast to marrow stromal cell extraction which results in signicant pain. ADSCs are limited, however, by several factors. First, ADSCs have not been classied as immortal. ADSCs display obvious signs of old age, thus limiting their capacity for sub-culturing. Additionally, adipose tissue is known to vary in metabolic activity and in its capacity for proliferation and differentiation, depending on the location of the tissue depot and the age and gender of the patient.

V. INDUCED PLURIPOTENT STEM CELLS Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem celllike state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Although these cells meet the defining criteria for pluripotent stem cells, it is not known if iPSCs and embryonic stem cells differ in clinically significant ways. Mouse iPSCs were first reported in 2006, and human iPSCs were first reported in late 2007. Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expressing stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers. induced pluripotent stem cells (iPSCs) can be generated from mouse embryonic broblasts (MEF) and adult mouse tail-tip broblasts by the retrovirus-mediated transfection of four transcription factors, namely Oct3/4, Sox2, c-Myc, and Klf4 (Takahashi and Yamanaka, 2006). Mouse iPS cells are indistinguishable from ES cells in morphology, proliferation, gene expression, and teratoma formation. Furthermore, when transplanted into blastocysts, mouse iPS cells can give rise to adult chimeras, which are competent for germ line transmission (Maherali et al., 2007; Okita et al., 2007;

5|Page Wernig et al.,2007). These results are proof of principle that pluripotent stem cells can be generated from somatic cells by the combination of a small number of factors. Retroviruses are used to introduce the factors into the cells. These retro viruses contain Oct3/4, Sox2, c-Myc, and Klf4.

3. CLINICAL PRACTICE WITH STEM CELLS

a) BONE MARROW TRANSPLANT A bone marrow transplant, more commonly known as a stem cell transplant, replaces damaged bone marrow with healthy bone marrow stem cells. In patients with leukemia, aplastic anemia, and some immune deficiency diseases, the stem cells in the bone marrow malfunction, producing an excessive number of defective or immature blood cells (in the case of leukemia) or low blood cell counts (in the case of aplastic anemia). The immature or defective blood cells interfere with the production of normal blood cells, accumulate in the bloodstream and may invade other tissues. Large doses of chemotherapy and/or radiation are required to destroy the abnormal stem cells and abnormal blood cells. These therapies, however, not only kill the abnormal cells but can destroy normal cells found in the bone marrow as well. Similarly, aggressive chemotherapy used to treat some lymphomas and other cancers can destroy healthy bone marrow. A bone marrow transplant enables physicians to treat these diseases with aggressive chemotherapy and/or radiation by allowing replacement of the diseased or damaged bone marrow after the chemotherapy/radiation treatment. While bone marrow transplants do not provide 100 percent assurance that the disease will not recur, a transplant can increase the likelihood of a cure or at least prolong the period of disease-free survival for many patients. There are three kinds of bone marrow transplants: Autologous bone marrow transplant: "Auto" means "self." Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment and stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments are done, your stems cells are put back in your body to add to your normal blood cells. This is called a "rescue" transplant. Allogeneic bone marrow transplant: "Allo" means "other." Stem cells are removed from another person, called a donor. Most times, the donor must at least partly match you genetically. Special blood tests are done to determine if a donor is a good match for you. A brother or sister is most likely to be a good match. However, sometimes parents, children, and other relatives may be good matches. Donors who are not related to you may be found through national bone marrow registries. Umbilical cord blood transplant: Stem cells are removed from a newborn baby's umbilical cord immediately after birth. The stem cells are stored until they are needed for a transplant. Umbilical cord blood cells are so immature; there is less of a need for matching. Bone marrow transplants are complicated procedures with significant risks. In some cases the transplanted cells (graft cells) recognize the recipient's cells as 'foreign' and try to attack them. This

6|Page is known as graft versus host disease (GvHD). The risk of infection is also increased because the immune system is weakened as you are conditioned (prepared) for the transplant.

b) STEM CELLS AND MYOCARDIAL INFARCTION Myocardial infarction (MI), commonly known as heart attack, is a leading cause of death and disability in Western societies. In an acute myocardial infarction, the flow of blood from a blood vessel in the heart is blocked, whereby the cardiac muscle receives insufficient oxygen and heart tissue dies. In many cases, the supply of blood in the deadened portion of the heart can be restored via the so-called balloon technique. But the heart suffers permanent damage, primarily to the left ventricle. Standard treatment following MI has traditionally involved thrombolytic therapy - the use of drugs to break up blood clots. More recently, coronary angioplasty (also known as percutaneous coronary intervention) has become an intervention of choice. This treatment involves the inflation of a balloon in the coronary artery to compress obstructions and restore functionality, and has been credited with significant reductions in death and disability rates. Both of these treatments, however, are limited in that they can only interrupt an ongoing process: neither can improve nor restore cardiac function that has been lost as a result of damage caused by MI. The potential capability of both embryonic and adult stem cells to develop into these cells types in the damaged heart is now being explored as part of a strategy to restore heart function to people who have had heart attacks or have congestive heart failure. It is important that work with stem cells is not confused with recent reports that human cardiac myocytes may undergo cell division after myocardial infarction. This work suggests that injured heart cells can shift from a quiescent state into active cell division. This is not different from the ability of a host of other cells in the body that begin to divide after injury. There is still no evidence that there are true stem cells in the heart which can proliferate and differentiate. What is the evidence that such an approach to restoring cardiac function might work? In the research laboratory, investigators often use a mouse or rat model of a heart attack to study new therapies. To create a heart attack in a mouse or rat, a ligature is placed around a major blood vessel serving the heart muscle, thereby depriving the cardio-myocytes of their oxygen and nutrient supplies. During the past year, researchers using such models have made several key discoveries that kindled interest in the application of adult stem cells to heart muscle repair in animal models of heart disease. Orlic and colleagues reported on an experimental application of hematopoietic stem cells for the regeneration of the tissues in the heart. In this study, a heart attack was induced in mice by tying off a major blood vessel, the left main coronary artery. Through the identification of unique cellular surface markers, the investigators then isolated a select group of adult primitive bone marrow cells with a high capacity to develop into cells of multiple types. When injected into the damaged wall of the ventricle, these cells led to the formation of new cardio-myocytes, vascular endothelium, and smooth muscle cells, thus generating de novo myocardium, including coronary arteries, arterioles, and capillaries. The newly formed myocardium occupied 68 percent of the damaged portion of the ventricle nine days after the bone marrow cells were transplanted, in effect replacing the dead myocardium with living, functioning tissue. The researchers found that mice that received the transplanted cells survived in greater numbers than mice with heart attacks that did not receive the mouse stem cells. Follow-up experiments are now being conducted to extend the posttransplantation analysis time to determine the longer-range effects of such therapy. The partial repair of the damaged heart muscle suggests that the transplanted mouse hematopoietic stem cells responded to signals in the environment near the injured myocardium. The cells migrated to the

7|Page damaged region of the ventricle, where they multiplied and became "specialized" cells that appeared to be cardio-myocytes. Cells with phenotypic and functional characteristics similar to the fetal angioblast also are present in adult human bone marrow. Endothelial progenitor cells (EPCs) represent a subset of HSCs that are able to acquire an endothelial phenotype. In vitro EPCs express the HSC markers CD34 and the endothelial marker Flk-1 (vascular endothelial growth factor receptor-2 (VEGFR-2)). EPC can be isolated directly from the bone marrow or from the peripheral circulation and expanded in vitro. Preclinical trials indicated that EPCs contribute to 125% of vessel formation after ischemic injury for several diseases. They promote neovascularization by secreting pro-angiogenic growth factors and stimulate re-endothelialization thereby contribute to vascular homeostasis and perhaps myogenesis. In the animal experiments injection of EPCs into infarcted myocardium improved LVF and inhibited fibrosis. Although there was no change in the non-infarcted regions of the heart, there was a significant reduction in collagen deposition and apoptosis of cardio-myocytes and an improvement in cardiac function on echocardiography. It appeared that neovascularization induced by these cells led to the prevention of apoptosis and LV remodeling and led to some degree of cardio-myocyte regeneration.

c) STEM CELLS AND CNS REPAIR Demonstration of neural differentiation potential of MSCs in several in vitro and in vivo studies suggests the potential usefulness of MSCs in the treatment of various CNS disorders. Recent multiple studies demonstrated that nave or neurally modified MSCs derived from different tissue sources exerted therapeutic effect in several animal models of spinal cord injury (SCI). However, the precise mechanisms by which transplantation of MSCs promote functional recovery after SCI is still unclear. A number of mechanisms have been suggested, including the promotion of axon regeneration, neuroprotection, modulation of the immune responses, and trans-differentiation into neural cell types (Chamberlain, Fox, Ashton, & Middleton, 2007; Dezawa, 2002; Enzmann, Benton, Talbott, Cao, & Whittemore, 2006; Keilhoff, Goihl, Stang, Wolf, & Fansa, 2006). The immunosuppressive properties of MSCs (Bartholomew et al., 2002a; Corcione et al., 2006; Di Nicola et al., 2002; X. X. Jiang et al., 2005) may combine to reduce the acute inflammatory response to SCI and hence reduce cavity formation as well as decrease astrocyte and microglia/macrophage reactivity (Abrams et al.,2009; Himes et al., 2006; Neuhuber, Timothy Himes, Shumsky, Gallo, & Fischer, 2005) in injured spinal cords. The therapeutic effect of MSCs on axonal growth could be exerted by creation of a favorable environments such as cellular bridges, guiding strands and scaffolds, secretion of trophic factors, cytokines and production of extracellular matrix (Fuhrmann et al., 2010; Gu et al., 2010; Hofstetter et al., 2002; Neuhuber et al., 2004). There is currently a great deal of interest in the use of MSCs to treat several neurological diseases such as Parkinsons disease, Alzheimers disease, amyotrophic lateral sclerosis, and multiple sclerosis. a number of studies have examined the ability of MSCs to differentiate into dopamine-producing cells, re-innervate the striatum, and ameliorate behavioral deficits in Parkinsonian models. Varying degrees of success have been achieved in vitro, including dopaminergic marker expression, and dopamine secretion in response to depolarization (Dezawa et al., 2004; Fu et al., 2006; Guo et al., 2005; Suon, Yang, & Iacovitti, 2006; Trzaska, Kuzhikandathil, & Rameshwar, 2007; Trzaska & Rameshwar, 2011). In addition, engraftment and functional improvement were demonstrated following transplantation of undifferentiated (Hellmann, Panet, Barhum, Melamed, & Offen, 2006; Y.

8|Page Li et al., 2001) and neurally differentiated MSCs (Dezawa et al., 2004; Fu et al., 2006) in hemiparkinsonian rodents. However, only relatively low efficiencies of dopaminergic differentiation were achieved, and comparisons between the varying methods have not been performed, resulting in difficulties with identifying the optimal methodology. These studies suggest that complex mechanisms might underline the therapeutic effect of MSCs in these animal parkinsonian models and neuro-protection could be the most important ones. Unlike Parkinson disease, which is a slower degenerative disease and affects a specific area of the brain, amyotrophic lateral sclerosis (ALS) presents quite a challenge for cellular therapy because of the distributed cell loss throughout the body and the requirement to properly re-innervate muscle tissue.