Stem Cells

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INTRODUCTION :Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). The definition of hematopoietic stem cells has undergone considerable revision in the last two decades. The hematopoietic tissue contains cells with long-term and short-term regeneration capacities and committed multipotent, oligopotent, and unipotent progenitors. Recently, long-term transplantation experiments point toward a clonal diversity model of hematopoietic stem cells. Here, the HSC compartment consists of a fixed number of different types of HSC, each with epigenetically preprogrammed behavior. This contradicts older models of HSC behavior, which postulated a single type of HSC that can be continuously molded into different subtypes of HSCs. HSCs constitute 1:10.000 of cells in myeloid tissue The search for stem cells began in the aftermath of the bombings in Hiroshima and Nagasaki in 1945. Those who died over a prolonged period from lower doses of radiation had compromised hematopoietic systems that could not regenerate either sufficient white blood cells to protect against otherwise nonpathogenic infections or enough platelets to clot their blood. Higher doses of radiation also killed the stem cells of the intestinal tract, resulting in more rapid death. Later, it was demonstrated that mice that were given doses of whole body X-irradiation developed the same radiation syndromes; at the minimal lethal dose, the mice died from hematopoietic failure approximately two weeks after radiation exposure. Significantly, however, shielding a single bone or the spleen from radiation prevented this irradiation syndrome. Soon thereafter, using inbred strains of mice, scientists showed that whole-body-irradiated mice could be rescued from otherwise fatal hematopoietic failure by injection of suspensions of cells from blood-forming organs such as the bone marrow. In 1956, three laboratories demonstrated that the injected bone marrow cells directly regenerated the blood-forming system, rather than releasing factors that caused the recipients’ cells to repair irradiation damage. To date, the only known treatment for hematopoietic failure following whole body irradiation is transplantation of bone marrow cells or HSCs to regenerate the blood-forming system in the host organisms. All these needs facilitated an efficient means for preservation of hematopoietic cells such that they could be replenished as and when a need arises. Hence a variety of techniques were devised to achieve this purpose. Cell therapies based on hematopoietic stem cells (HSCs) have become the standard of care for a variety of diseases, and the number of patients and disorders being treated using HSCs continues to grow. Effective methods of preservation are critical for the clinical implementation of HSCbased therapies. Preservation permits the completion of safety and quality control testing, transportation to the site of use and coordination of the therapy with patient care regimes. Recent advances in preservation technology can also be used to reduce cell losses during cell processing for preservation and improve the safety of HSCs used therapeutically. Finally, the growth of HSC therapy worldwide has created interest in alternative methods of preservation. Improved methods for preserving HSCs will improve not only therapies based on these cells but can improve and speed development of preservation protocols for other cell types used therapeutically. The role of hematopoietic stem cell transplantation in the treatment of hematologic and

nonhematologic malignancies are rapidly expanding. In certain situations fresh stem cells can be employed in the setting of allogeneic transplantation. If the transfer from donor to recipient can be established within 72 hr, protocols for preliminary storage at suprafreezing temperatures are in place. However, the current therapeutic strategies demand that the progenitor cells are cryopreserved for virtually all autologous and many allogeneic transplants. This strategy has been proven to be safe and not associated with significant adverse outcomes regarding failure to engraft, graft versus host disease (GVHD), or engraftment failure. The cryopreservation process is of importance for all types of stem cell collection, but is perhaps particularly critical for umbilical cord blood (UCB). The UCB is usually stored by either public or private cord blood banks. Public cord blood banks are usually nonprofit organizations, which offer the donor unit to matching recipients via national or international registries to potential recipients in need. Cord blood banks store a donor specimen for the donor or in the case of public banks for an unknown recipient for an indeterminate time span. There are now about 170,000 frozen units in 37 cord-blood registries in 21 countries. Two thousand nine hundred units have been transplanted to date, with adults having received about one third of those units.

HEMATOPOIETIC STEM CELLS DIFFERENTIATION

The cryopreservation process entails the following general components: 1. Harvesting of the donor cells, which entails the actual collection of the specimen and the reduction of bulk. 2. Addition of cryopreservatives 3. The actual freezing procedure 4. Assessment of the viability of the frozen unit after about 72 hr 5. The thawing procedure 6. The washing and conditioning of the donor unit prior to transplant No single cryopreservation method has been universally used. Variations in technique occur between different transplant centers. Hematopoietic Progenitor Stem Cells are collected in a minimally manipulated fashion as defined by the Foundation for the Accreditation of Hematopoietic Cell Therapy (FAHCT) with a minimal cell dose of at least 2.5 × 106–5.0 × 106 CD34(+) cells/kg body weight, as currently considered standard. The specimen is then centrifuged to develop the cell rich pellet. In autologous transplants donor plasma is used. The supernatant out of this cryoprecipitation process is used for the reliquidification of the pellet cells, after the addition of a solution of heparinized Plasmalyte solution and 10% DMSO (Dimethylsulfoxide). This usually eventuates into a cellular concentration of 500 × 10−6 cells in the cryopreservate. We store the bone marrow or peripheral blood stem cell product at initially −4°C. Then the sample is frozen down to the target temperature of −156°C (when stored in the vapor phase) to −196°C (when stored in the liquid phase), depending on where in the container the specimen is stored. To guarantee the integrity of the donor unit prior to myeloablative therapy, a viability reassessment of the unit is performed using a Trypan Blue assay, and if this is equivocal a propidium iodide assay. Prior to the actual stem cell infusion the sample is rapidly thawed in a 37°C water bath. Several elements of the cryopreservative procedure are still matter of debate. Factors such as freezing temperature, freezing rate, cryopreservatives, durability, thawing temperature, and thawing rate affect the rate of cryopreservation and hence should be maintained. TEMPERATURE The temperatures used for the cryopresevation of hemopoeitic stem cells over the past fifteen years has been −196, −156, or −80°C, reflecting the storage temperatures in liquid and vapor phase nitrogen and in cryopreservation mechanical freezers, respectively. The development of the use of cryopreservative temperatures was from lower temperatures of around −196°C in

the 1980s to around −80°C in the 1990s. For umbilical cord isolated stem cells similar trends are observed. The standard temperatures currently in use are −196 to −80°C. Secondary to the recent reports about the spread of infectious agents (i.e., aspergillus as well as viral spread), through the liquid phase of the nitrogen tanks, the currently recommended optimal storage conditions are in the vapor nitrogen phase, at −156°C. Mechanical freezers might represent a viable alternative. Also, several studies examined the possibility to store HSCs at supra freezing temperature, at 4°C. A preclinical study that examined PBSCs mobilized in autologous plasma with post storage clonogenic and viability assays suggested that a storage up to five days is safe. A small case study by Ruiz-Arguelles et al. successfully used PBPCs after 96 hr storage at 4°C for rescue after high dose chemotherapy. FREEZING RATE The rate of freezing was widely debated in the literature. The controlled rate freezing technique is still considered standard, mainly due to the fact that the heat liberation at the transition or eutactic point (about 4°C) is deemed detrimental to the stem cell population. At this point the water molecules within the frozen unit are in a precise molecular order, what eventuates in the thermodynamic liberation of fusion heat. In controlled rate freezing, the concentrated stem cells are frozen down at a rate of 1–2°C/min up to a temperature point of about −40°C. Then, the freezing process down to a target of −120° C is performed at a faster pace, about 3–5°C/min. For umbilical cord stem cells, bone marrow, and PBSCs the controlled rate freezing process is considered standard, and was in different reports found to be superior to uncontrolled freezing approaches. This procedure is time consuming and requires staff with a specific expertise. Hence, the use of uncontrolled rate freezing in which the specimen is first cooled down to −4°C and then directly deposited into a freezer at −80°C or put into liquid phase nitrogen has been evaluated. Several reports established that the uncontrolled method is safe and reveals comparable results to the controlled rate process for BM and PBSCs. A controlled study performed by Perez-Oteyza et al. showed that the controlled and uncontrolled rate freezing approach are comparable in terms of viability testing and that only a statistically significant decrease in the CFU-GM clonality assay could be detected in the uncontrolled freezing situation. Recent studies suggested that uncontrolled freezing is also a viable approach for UCB stem cells. No consensus exists about the relevance of the compensation for fusion heat during the freezing procedure. However, a study of Balint et al. outlined the importance of this intervention, comparing five different freezing protocols. The protocols using a five step controlled freezing approach compensating for the fusion heat achieved better post thawing viability. DURABILITY The actual durability, defined as the time that stem cells can be preserved, is still unclear. The viability of the stem cells in cryopreservation has been questioned in different studies after a time course of cryopreservation of 6 months. Further studies have demonstrated a prolonged freezing time with complete preservation of stem cell function. Several substitute assays are used to estimate the functional hematopoietic reconstitutive capacity of the first frozen and then

thawed specimens. While BFU-E and CFU-GM appear to be compromised earlier in the course of cryopreservation, the recovery of nucleated cells (NC) and CD34+ cells and the actual engraftment in NOD/SCID mice seems to be preserved for a longer period of time. Those observations have been initially made in bone marrow and PBSC, and similar observations have been made with UBC stem cells. The NOD/SCID mouse assay is currently considered the most valuable assay to assess the functionality for hematologic recovery of HSC preparations, but is not routinely practical. After Kobylka et al. and Mugishima et al. proved the durability after 12 and 15 years with flow cytometric and clonogenic assays, respectively, and Broxmeyer et al. performed a reassessment of his long-term preserved CB units, a durability of up to 15 years was established by using hematopoietic reconstitution in NOD/SCID mice. Clinical validity of preclinical studies was documented in anecdotal reports when successful trilineage engraftment was achieved with BM, stored for 7 years. A systematic review evaluating the combined experience of the Brigham and Women’s Hospital and the EBMT Group noticed that HSC can be effectively cryopreserved for up to 11 years. A retrospective study from Seattle revealed full trilineage recovery in patients receiving HSC, stored for up stored for up to 7.8 years without consistent detrimental effects. CELL CONCENTRATION Reinfusion of cryopreserved cells has been associated with varying toxicities, which were partially attributed to the total volume and the cryopreservatives in the solution. Concern was raised in the past that a high cell concentration in the cryopreservate can eventuate in toxicity to the cells. Hence, the initially proposed concentration of cryopreserved cells was suggested to be not over 2 × 10−7/ml NC. This would eventuate in a cryopreservation volume of about 7 l per patient. The storage space needed and the labor to wash the graft prior to reinfusion would be immense. After initial murine models were found safe, Rowley et al. established that high cell concentrations (up to 5.6 × 10−8 cells/ml) in the cryopreservate are well tolerated, not associated with significant adverse effect to the cells, and resulted in good clinical outcomes. Similar conclusions were drawn from subsequent studies by Kawano et al. [42] and Cabezudo et al. For practical purposes a cell concentration of 200 × 10−6/ml appears achievable. CRYOPRESERVATIVES Cryopreservatives are necessary additives to stem cell concentrates, since they inhibit the formation of intra and extracellular crystals and hence cell death. The standard cryoprotectant is DMSO, which prevents freezing damage to living cells. It was initially introduced into medical use as an anti-inflammatory reagent and is still occasionally used in auto-immune disorders. Usually it is used at concentrations of 10% combined with normal saline and serum albumin. This was established to be a safe and non-stem cell toxic agent. However, DMSO is associated with a clinically significant side effect profile. Nausea, vomiting, and abdominal cramps occur in about half of all the cases. Other side effects encompass cardiovascular, respiratory, CNS, renal, hemolytic, and hepatotoxic presentations. Case fatalities attributed to DMSO toxicities have also been reported. A recent multinational questionnaire based survey, including data from 97 EBMT transplant centers, revealed that DMSO related toxicities other than nausea and vomiting are observed in

about one out of 50 transplants with a mean incidence of 2.2% of all administered units. Cardiovascular side effects were the most frequently observed group of adverse events witnessed in 27% of the participating centers. Respiratory events were observed in 17%, CNS toxicities, including seizures, in 5%, and renal adverse effects in 5%. Based on these toxicity considerations, newer approaches have been tried. Lower dosages of DMSO, varying from 2.2 to 6% have been established to be efficacious in bone marrow and PBSCs as well as for UCB. On the contrary, a study compared the 10% DMSO concentration to lower concentrations with different freezing rates. The 10% DMSO cryopreservation proved to be superior to lower concentrations in this in vitro study. To enhance the effect of the cryopreservative, the combination of DMSO and the extracellular cryoprotectant hydroxyethyl starch (HES) has been used with success in PBSC’s and bone marrow grafts and UCB cells. Alternative preservation methods for cryopreservation are propylene glycol, a combination of alpha tocopherol, catalase, and ascorbic acid and the glucose dimer trehalose as intra and extracellular cryoprotectant. Interesting preclinical data suggests that activation of caspases, particularly during the thawing process, can induce apoptosis and hence contribute to the cryoinjury to transplant grafts. Addition of the caspase inhibitor zVAD-fmk as cryopreservative presents a future perspective. THAWING Several techniques for the thawing procedure have been proposed. The standard method is warming in a water bath at 37°C until all ice crystals disappear. A study compared the thawing of cryopreserved units in a warm water bath with dry heat applied by gel pads at 37°C. The viability and clonogenic potential were comparable, with a trend towards less infectious contamination in the dry method. Different studies examined the preservation of function when thawed units were incubated at 0–37°C. No significant differences were detected in a study by Yang et al. who compared an incubation of the thawed unit at 0, 20, and 37°C for 20 min. The used cryopreservative proved to be nontoxic to the stem cells during the cryopreservation process, as already established by previous studies. Reducing the DMSO content at thawing temperature is an intriguing concept, because of the clinical toxicity profile of this cryopreservative. Hence, the effect of reducing the DMSO content in the thawing solution by virtue of washing or dilution has been explored. Minor or no effect on the stem cell viability has been observed. An automated method to wash out the cryopreservative has proven feasible in pre-clinical models. WASHING PROCEDURE For stem cells of cord, peripheral blood, and bone marrow origin, the process of washing out the cryopreservative after the thawing can still be considered standard, since the DMSO is assumed to have toxic effect on the stem cells. This has been questioned by several more recent reports, which suggested stem cell resistance against DMSO exposure. The wash out of the most popular cryopreservative has conceivable benefits for the recipient, i.e. reduction of toxicity, since the degree of DMSO toxicity is proportional to the amount of DMSO contained in the infused stem cell solution. It was also suggested that wash out of DMSO can enhance engraftment. This has been disputed. The current standard washing protocol follows the New York Blood Center protocol, in which the two step dilution of the thawed stem cell unit with

2.5% human serum albumin and 5% dextran 40 is followed by centrifugation at 10°C for 10 min. The supernatant is then removed and HSA and dextran solution is again added twice to a final DMSO concentration of less then 1.7%. The washed solution is infused as soon as possible. Although this procedure has been established to be safe and associated with a reasonable recovery of NC and progenitor assays, it is also very labor intensive and not free of cell loss. Recently new automated cell washing devices have been introduced with promising results.

Emerging Technology for cryopreservation of HSC’s
Advances in preservation science need to be supported by advances in preservation technology to improve the overall efficacy of the preservation process. New technologies to improve cell processing for preservation has the potential to benefit HSC-based therapies and other types of cell therapies under development. When transferred from an isotonic solution to a CPA solution containing DMSO, cells exhibit a rapid efflux of water as the cell attempts to reduce the difference in chemical potential between intracellular and extracellular solutions. Slowly, the DMSO from the surrounding solution permeates the cell membrane. Both the rate of volume change and the absolute volume changes experienced by the cell can produce cell lysis. Cells also experience volumetric excursions upon dilution or removal from a cryopreservation solution. Transfer of a cell equilibrated with a CPA solution into an isotonic solution will produce a rapid influx of water to decrease the chemical potential of the intracellular solution followed by a slow efflux of DMSO. Cells are much more sensitive to lysis upon expansion (versus dehydration) so post-thaw removal protocols are critical for preventing cell losses. Current processing protocols for cord blood include a dilution step with a dextran/albumin solution in order to reduce osmotic stresses during washing of the cells. Cell losses can result not only from introduction or removal of CPA solutions but also from exposure to the solution. Early studies demonstrated the sensitivity of HSCs to DMSO both pre freeze and post thaw. Thus, processing protocols require that after DMSO is added to the cell suspension it be placed immediately into a controlled rate freezer or mechanical freezer to begin the freezing process. Although the sensitivity to DMSO was found to be less than previously thought, conventional wisdom still minimizes exposure to DMSO both pre freeze and post thaw. Thus, cryopreserved HSC suspensions are routinely thawed at the patient bedside and infused directly into the patient. The direct infusion of cryopreserved HSC products has however been associated with adverse reactions in patients. Less severe adverse reactions, including nausea, hypotension, dyspnea, chills and cardiac arrhythmia, are experienced by the majority of patients. Smaller numbers of patients experience more severe reactions, including cardiac arrest, transient heart blockage, neurological toxicity, renal failure and respiratory arrest. Microfluidics is one technology under investigation that could be used to improve preservation processing of cells. In this microfluidic device, diffusion-based extraction can be used to remove DMSO without centrifugation. This type of device has several advantages, including low power requirements, portability, and controllability of cell motion.

Conclusion
Much of the preservation practice at the present time could be described as ‘empirical cryopreservation’ in which protocols are developed by varying solution composition and cooling rate to determine the ‘optimal’ protocol for cell preservation. This approach does little to determine specific mechanisms of damage and to facilitate the development of rational approaches to cell preservation. Recent advances in molecular biology and adaptation of organisms to stress are permitting us to gain insight into the molecular level mechanisms of damage and to develop methods of stabilizing cells. Further work is needed to identify subcellular sites of damage and methods of stabilizing those structures against the stresses of freezing and thawing. The development of the next generation of cryoprotective agents is needed and will require rational methods of designing or screening of molecules with protective properties. This work will permit us to advance the science of preservation and provide the foundation for transforming cell preservation for HSCs and other cell types. Effective methods of preservation also required development of the supporting relevant technology. Preservation technology has changed little since the 1950s. Current technologies such as microfluidics can be adapted to the cell preservation process to improve the introduction and removal of the specialized solutions used in cell preservation and thereby improves the overall safety and efficacy of the process. Further technology development is needed. Specifically, knowledge of the temperature history of each vial/bag during freezing and during storage is important for process monitoring and quality control. The basic technology for temperature monitoring of each unit exists but has not been adapted to cell preservation. The preservation process is only as good as the container that contains the product. Containers that do not fail at liquid nitrogen temperatures and permit sterile introduction and removal of cells have been developed but need to be expanded to provide a full range volumes used in cell preservation and integrate easily into inventory control systems to facilitate product retrieval from a repository. The spreading of HSCbased therapies around the globe has led to interest in different methods of preserving cells for clinical use. Further work is needed to develop a ‘menu’ of methods that can be used to effectively preserve HSCs by methods appropriate for the clinical cell processing laboratory. The development and validation of these methods is, however, hampered by limitations in the assay of post-thaw viability. Cells that have been subjected to the stresses of freezing and thawing do not respond in the same manner to assays as fresh, non frozen cells. Much has been done to standardize assays performed on fresh products, and those efforts need to be extended to frozen and thawed products. The development of more representative post-thaw viability assays will benefit existing cell preservation processes as well as the development and validation of new processes.

Applications in Regenerative Medicine:Promising cures for blood-related diseases, such as leukemia and lymphoma, hematopoietic stem cells (HSCs) have been heavily researched for decades. However, like many significant findings in science, their discovery was not made in search for such a cure, but stumbled upon while dealing with another serious medical issue of the time: radiation. While trying to treat people exposed to lethal doses of radiation during World War II, transplants from the spleen and bone marrow were found to rescue these victims. It was not until later that scientists determined that the HSCs present in these tissues were what was restoring the damaged tissues, observed by performing transplants using lethally irradiated mouse and rat models. HSCs in humans were further characterized and cultured in the 1980s. The formation of the National Marrow Donor Program during this time also greatly improved the availability of these cells for research. Not only have HSCs been successfully used clinically in humans since the 1950s, but to this day they are still one of the few adult stem cells to be tested for clinical uses and as regenerative medicine. It is now not only better understood how HSCs from a donor animal can save a lethally irradiated recipient animal, but how HSCs can be used in many other medical applications as well. HSCs are able to give rise to all cells in the hematopoietic system, which includes myeloid elements (i.e. red blood cells, white blood cells, platelets) and the lymphatic system (i.e. T-Cells). Because radiation generally targets rapidly dividing cells, including bone marrow cells and cells in the lymphatic system, HSCs have the ability to replenish the supply of cells most damaged by radiation. While HSCs can be collected from adult bone marrow, some fetal tissues (liver, spleen, thymus), umbilical chords, and peripheral blood, in recent years there has been a great shift towards obtaining HSCs mainly from peripheral blood, using a much simpler and less controversial procedure, though achieving a large enough number of HSCs for transplants is still an obstacle to overcome. HSCs are now being used to treat cancers of the hematopoietic system (leukemia and lymphomas), replenish cells lost to high-doses of chemotherapy, and fight against autoimmune diseases, in addition to other medical applications. Regenerative Medicine is likely to involve the implantation of new tissue in patients with damaged or diseased organs. A substantial obstacle to the success of transplantation of any cells, including stem cells and their derivatives, is the immune- mediated rejection of foreign tissue by the recipient’s body. In current stem cell transplantation procedures with bone marrow and blood, success can hinge on obtaining a close match between donor and recipient tissues and on the use of immunosuppressive drugs, which often have severe and life-threatening side effects. To ensure that stem cell-based therapies can be broadly applicable for many conditions and individuals, new means to overcome the problem of tissue rejection must be found. Although ethically controversial, somatic cell nuclear transfer, a technique that produces a lineage of stem cells that are genetically identical to the donor, promises such an advantage. Other options for this purpose include genetic manipulation of the stem cells and the development of a very large bank of embryonic stem cell lines. In conjunction with research on stem cell biology and the development of stem cell therapies, research on approaches that prevent immune rejection of stem cells and stem cell-derived tissues should be actively pursued.

HSC PLASTICITY: IMPORTANT FEATURE FOR REGENERATIVE MEDICINE
HSCs, have the capacity to differentiate into a much wider range of tissues than previously thought possible. It has been claimed that, following reconstitution, bone marrow cells can differentiate not only into blood cells but also muscle cells (both skeletal myocytes and cardiomyocytes), brain cells, liver cells, skin cells, lung cells, kidney cells, intestinal cells,and pancreatic cells. Bone marrow is a complex mixture that contains numerous cell types. In addition to HSCs, at least one other type of stem cell, the mesenchymal stem cell (MSC), is present in bone marrow. MSCs, which have become the subject of increasingly intense investigation, seem to retain a wide range of differentiation capabilities in vitro that is not restricted to mesodermal tissues, but includes tissues normally derived from other embryonic germ layers (e.g., neurons). Many experiments have spawned the idea that HSCs may not be entirely or irreversibly committed to forming the blood, but under the proper circumstances, HSCs may also function in the regeneration or repair of non-blood tissues which is a basis of regenerative medicine. This concept has in turn given rise to the hypothesis that the fate of stem cells is “plastic,” or changeable, allowing these cells to adopt alternate fates if needed in response to tissue-derived regenerative signals (a phenomenon sometimes referred to as “transdifferentiation”). This in turn seems to bolster the argument that the full clinical potential of stem cells can be realized by studying only adult stem cells, foregoing research into defining the conditions necessary for the clinical use of the extensive differentiation potential of embryonic stem cells. Several investigators have formulated criteria that must be fulfilled to demonstrate stem cell plasticity. These include:(i)
(ii) (iii)

clonal analysis, which requires the transfer and analysis of single, highly purified cells or individually marked cells and the subsequent demonstration of both “normal” and “plastic” differentiation outcomes, robust levels of “plastic” differentiation outcome, as extremely rare events are difficult to analyze and may be induced by artefact, and demonstration of tissue-specific function of the “transdifferentiated” cell type.

There is a growing body of evidence that HSCs are plastic—that, at least under some circumstances, they are able to participate in the generation of tissues other than those of the blood system. A few studies have shown that HSCs can give rise to liver cells. These findings have scientists speculating about the biological response of HSCs to disease or tissue damage and about the early differentiation of the embryonic tissues into discrete layers. It was unexpected that a component of blood could cross over a developmental separation to form a tissue type that ordinarily has a completely different embryonic origin. The findings noted above and other reports of cardiac and muscle tissue formation after bone marrow transplantation in mice and of the development of neuron-like cells from bone marrow have raised expectations that HSCs will eventually be shown to be able to give rise to multiple cell types from all three germ layers. One study has, in fact, demonstrated that a single HSC transplanted into an irradiated mouse generated not only blood components (from the mesoderm layer of the embryo), but also epithelial cells in the lungs, gut, (endoderm layer) and skin (ectoderm layer).

If HSCs are truly multipotent, their potential for life-saving regenerative therapies may be considerably expanded in the future. The full potential of bone marrow transplantation to restore a healthy blood system in every needy patient is currently limited by the unavailability of HSCs in the quantity and purity that are crucial for successful transplantation. Because of their relative rarity (one in every 10,000 bone marrow cells) and the difficulty of separating them from other components of the blood, so-called bone marrow stem cell transplants are generally impure. The significance of such impurity is great.

Therapeutic Use of Hematopoietic Stem Cells:
Most diseases and disorders are caused by or at least involve damaged body tissues and insufficient repair. For example: 1. Cancer chemotherapy and radiation therapy destroy many other non-cancerous cells in the body, including those of the bloodstream and immune system. 2. Hematopoietic disorders involve abnormal growth and/or destruction of certain types of blood cells. 3. Heart failure, generally regarded as incurable, involves damage to heart muscle, which the body cannot repair 4. Liver failure involves progressive destruction of liver cells 5. Stroke involves damage and/or death of brain cells resulting from a lack of oxygen and nutrient-carrying blood 6. Type 2 diabetes, the most common form of the disorder, involves a progressive decrease in the body’s ability to use insulin and to produce the hormone; its complications are due to progressive destruction of tissues in the eye (diabetic retinopathy, which can lead to blindness), kidney (diabetic nephropathy, which can lead to kidney failure), and nerves (diabetic neuropathy, which can lead to decreased sensation in the limbs and limb amputation) 7. Osteoarthritis involves destruction of cartilage tissue around joints 8. Parkinson’s disease, Alzheimer’s disease and other central nervous system disorders involve destruction of certain neurons in the brain 9. Various autoimmune disorders involve immune system attack and destruction of the lining around nerves (multiple sclerosis), the cells lining the intestine (ulcerative colitis), cartilage in joints (rheumatoid arthritis), and other specific tissues for specific diseases 10. Spinal cord injuries involve trauma and destruction of nerve tissue in the spinal cord 11. Aging involves a general deterioration of the body’s tissues

Hematopoietic stem cell therapy offers the potential to help repair and renew the damaged tissues associated with these and other diseases and disorders.

HSC’s for administration of grafts (GVHD)
All cells of the body express on their surface a set of molecules called histocompatibility (i.e. tissue compatibility) antigens. If a patient receives a transplant of cells from a donor that has histocompatibility antigens different from his own, the patient’s body will recognize and react to the cells as foreign. To increase the likelihood that histocompatibility antigens will match, it is preferred that donors be a related sibling of the transplant recipient. Even if their histocompatibility antigens do match, however, transplants can be contaminated by T cells from the donor’s immune system. That contamination can cause the recipient’s body to reject the material or can produce an immune reaction in which the T cells of the transplant attack the tissues of the recipient’s body, leading to a potentially lethal condition known as graft versus host disease. Although autologous transplants, in which material from a person is implanted into the same person (for example, when a cancer patient stockpiles his own blood in advance of chemotherapy or irradiation) solve the problem of immune system rejection, the inability to purify the material leads to the risk that diseased or cancerous cells in the transplant will later be reintroduced in the patient. In contrast, transplants of highly purified and concentrated populations of HSCs in mice have been shown to greatly reduce the incidence of graft versus host disease. Purified and concentrated populations of autologous HSCs transplanted in breast cancer patients after chemotherapy have been shown to engraft more swiftly and with fewer complications. Transplants of concentrated HSCs also have been shown to repopulate the blood more readily, reducing the period during which an individual is vulnerable to infection. There is also evidence that transplants derived from umbilical cord blood are less likely to provoke graft versus host disease, possibly because the cells in cord blood are immature and less reactive immunologically. The quantity of HSCs present in cord blood and its attached placenta is small and transplants from cord blood take longer to graft, but for children, whose smaller bodies require fewer HSCs, cord blood transplants are valuable, especially when there is no related sibling to donate HSCs. Banks of frozen umbilical cord and placenta blood (drawn out of the umbilical vein of the cord) are an important source of HSCs because the histocompatibility markers on the cells in these tissues can be identified and catalogued in advance of the need for a transplant.

HSC’s for treatment of cancer:Cancer is typically treated with surgery and/or chemotherapy and radiation therapy. These latter two forms of treatment destroy cancer cells, but destroy other non-cancerous cells in the body as well – including red blood cells, white blood cells and platelets. Indeed, when levels of these essential blood cells are reduced below a critical level, chemotherapy must be reduced or suspended to avoid dangerous side effects, such as life-threatening infections, severe fatigue, and depression.

These three main types of blood cells are made primarily in the bone marrow from multipotent hematopoietic stem cells; thus, HSC therapy would seem a very logical and promising supportive treatment for patients undergoing chemotherapy or radiation therapy. By helping regenerate these blood cell families, chemotherapy could continue for its full course, often at even higher doses, which can significantly improve the prognosis. Supporting this idea is the data described in which HSC therapy has been used to treat blood and immune system cancers and other disorders, helping to reconstitute normal blood and immune cell production. Data from the Institute also shows HSC therapy provides good hematopoieitic support to patients undergoing cancer therapies. Professor Baltaytis and his colleagues have used HSC therapy for years as supportive treatment before and during cancer chemotherapy, and improved outcomes for many cancer patients. In addition, the impact of HSC supportive therapy during cancer treatment may go beyond hematological support. For example, one published report from the Ukraine showed that in 20 patients with pancreatic cancer, known to be extremely difficult to treat, administration of HSC s in conjunction with standard treatment, compared to standard treatment alone, significantly improved patients’ self-reported quality of life and decreased their fatigue.

Liver Disease:Among diseases of the liver, hepatitis and liver cirrhosis are among the most difficult to treat. Hepatitis is an inflammation of the liver that is caused by various viruses and other factors. Over time, hepatitis seriously damages the liver and can eventually lead to hospitalization and even death. There are at least six different strains of the hepatitis virus, including hepatitis A, B, C, D, E and G. Hepatitis B and C are considered the most serious and common. Unfortunately, the efficacy of even the most advanced current therapies for chronic viral hepatitis is still rather low. For example, current antiviral interferon therapies result in sustained elimination of the hepatitis C virus in patients 20 to 90 percent of the time, depending on the subtype of the virus and patient factors. Hepatitis C is associated with liver cirrhosis 40 percent of the time, liver cancer 65 percent of the time, and the need for liver transplantation 30 percent of the time. Sociomedical problems related to liver transplantation – currently the only treatment for patients at the terminal phase of illness – are well known. For example, because of the shortage of donated livers, over 50 percent of patients on the waiting list die while waiting for transplantation surgery. Using laparoscopic methods, scientists implanted stem cells into the livers of nine patients suffering from liver cirrhosis and waiting for a liver transplantation. The patients showed significant improvements in liver functioning, symptoms of liver insufficiency and quality of life. All the patients lived more than 14 months, allowing enough time to undergo a liver transplantation operation.

HSC’s for treatment of radiation injury:Since hematopoietic cells are sensitive to radiation, hematopoietic stem cell (HSC) transplantation is essential for treatment of patients with radiation injury. HSC transplantation provides a prototype for the regeneration strategy. Although bone marrow and peripheral blood

stem cells have been applied for HSC transplantation, recently cord blood has also attracted our attention. However, number of the HSCs available from a single donor is not necessarily sufficient for transplantation to adults. Thus a new technology for amplifying HSCs ex vivo could help support future clinical applications of HSCs. Amplification of HSCs could provide a particular progress to the regeneration strategy. Then we started our research focusing on molecular mechanisms regulating HSCs. We isolated a mammalian homologue of Drosophila Polycomb-group genes (PcG) and developed a mutant mice lacking the gene. And we firstly proved that PcG are required for sustaining the activity of HSCs, providing us a new clue for elucidating molecular mechanisms underlying HSC regulation. Interestingly we recently uncovered that the PcG complex is involved in licensing of DNA replication through the interaction with Geminin, an inhibitor of DNA licensing factor Cdt. And further we found that Hox genes, HSC regulators as well as target genes of the PcG complex, are also involved in the regulation of Geminin. Detailed analysis of these molecules is in progress. A molecular mechanism supporting HSCs could further help understand molecular bases underlying stemness in the other adult stem cells as well as cancer stem cells including leukemic stem cells.

Inflammatory Bowel Disease:Inflammatory bowel disease (IBD) includes Crohn's disease and ulcerative colitis. Chron’s disease is a chronic inflammatory disease of the intestines. It primarily causes ulcerations (breaks in the lining) of the small and large intestines, but can affect the digestive system anywhere from the mouth to the anus. Ulcerative colitis is also a chronic inflammatory condition that involves only the colon. The exact cause of IBD is unknown. Some scientists suspect it may be triggered by infections from certain bacteria; others believe it is an autoimmune disorder in which the immune system attacks the cells lining the digestive tract. Recent research suggest IBD may involve genetic predisposing factors. Currently there is no medical cure for IBD, and current treatments are not very effective. Once the diseases begin, they tend to fluctuate between periods of inactivity (remission) and activity (relapse); the treatment goal is to promote longer periods of remission and reduce the frequency and duration of relapses. Current approaches include treatment, as necessary, with antiinflammatory medications, immune suppressing agents, anti-diarrheal medications and/or other drugs. Dietary modifications, such as reducing fiber intake and consuming a liquid diet, can be helpful. If portions of the intestine become severely diseased, surgery may be required. Because IBD involves immune system destruction of cells in the intestine, stem cells therapy has the potential to help treat the disease by regenerating some of the destroyed tissue and/or favorably modulating the immune system so it is less prone to attacking the intestinal cells. Data from the Institute for Cryobiology and Cryomedicine showed good responses in a majority of patients with ulcerative colitis, Chron’s disease, or another disorder, intestinal adhesions.

Recent Advances in Adult Stem Cell Research
A recent novel approach for new stem cell generation that merits mentioning here is pluripotent stem cell induction. This technique attempts to induce differentiated somatic cells into pluripotent stem cells by introducing certain factors that can induce reprogramming of the cells, renders them pluripotent. Pluripotent stem cell induction was first introduced by Takahashi and Yamanaka in 2006. After inserting genes of certain transcription factors known to induce pluripotency into the retroviral vector gene, the researchers then introduced the pluripotency inducting factor genes into mice fibroblasts by means of retroviral transduction. The resulting stem cells, called “induced pluripotent stem (iPS) cells”, successfully demonstrated certain pluripotent traits after gene integration and expression.

“Converting ADULT Stem Cells Shows Promise for Parkinson’s,” The New York Times, March 6, 2009, In the March 6th issue of the New York Times, it was reported that researchers at the Whitehead Institute in Cambridge, Mass., have converted skin cells from people with Parkinson’s disease into the general type of neuron that the disease destroys. The new approach, though it requires further work, would in principle allow the brain cells that are lost in Parkinson’s to be replaced with cells that carried no risk of immune rejection, since they would be the patients’ own. Scientists said that the method worked in five patients whose skin cells were transformed in the test tube into neurons that produce dopamine, a chemical that transmits messages between neurons in certain regions of the brain. It is the loss of dopamine-producing nerve cells that leads to Parkinson’s symptoms, which can include muscle rigidity, tremors and slowed movement. Dr. Anders Bjorklund, a pioneer in using stem cell therapy to treat Parkinson’s, called the Whitehead team’s work “an important new step” in the development of the cell reprogramming technology, although more work is needed. “Multiple Route Bone Marrow ADULT Stem Cell Injections Show Promise To Treat Spinal Cord Injury,” ScienceDaily, March 28, 2009, On March 28th, 2009 Science Daily reported about a study conducted by DaVinci Biosciences, in collaboration with Luis Vernaza Hospital in Ecuador. The study demonstrates that administering ADULT autologous bone marrow derived hematopoietic stem cells via multiple routes is feasible, safe, and most importantly, improves the quality of life for both acute and chronic spinal cord injury (SCI) patients. The study documents eight spinal cord injury patients (four acute and four chronic)who experienced varying degrees of improvement in their quality of life, such as increased bladder control, regained mobility and sensation. Most importantly, the study demonstrated no adverse effects such as tumor formation, increased pain, and/or deterioration of function following administration of autologous bone marrow derived stem cells. The study published in Cell Transplantation, which used stem cells derived from the patient’s own bone marrow, documents the restoration of significant movement, sensation, and bladder function in patients suffering from a spinal cord injury.
Adult Stem Cell Research Shows Further Insulin Independence for Type 1 Diabetes Patients Washington, DC (LifeNews.com) New research using adult stem cells is showing further insulin independence for Type 1 diabetes patients. The study, led by Richard Burt of Northwestern University's Feinberg School of Medicine in Chicago, is the second in the last two years to show significant progress in diabetes using the noncontroversial hematopoietic stem cells. It showed patients receiving injections with these stem cells were able to go as long as four years without having to rely on insulin shots. In a new paper published in the Journal of the American Medical Association, Burt and his colleagues showed how the majority of patients with type 1, or juvenile, diabetes who underwent a certain type of hematopoietic stem cell transplantation became insulin free. Several became insulin free for more than

three years, with good glycemic control, and also increased C-peptide levels, an indirect measure of betacell function.

A previous study found that the use of HSC in 15 patients with newly diagnosed type 1 diabetes resulted in the majority of patients becoming insulin free during the follow-up, which averaged about 19 months. The scientists used HSCT, hematopoietic stem cell transplant, which relies on a patient's own blood stem cells, and involves the removal and treatment of the stem cells and their return to the patient by intravenous injection. Patients remained continuously insulin free for an average time of 31 months -- with a range of 14-52 months.

Barriers for HSC research in Regenerative Medicine:Irving Weissman, who presented research findings on HSC transplantations at a workshop, has explored many ways to improve the identification and purification of HSCs by looking for proteins on the surface of the stem cells that can be closely associated only with HSCs. Finding the specific profile of proteins that identifies HSCs, particularly those called long-term HSCs, is important, because these cells are believed to hold the key to future HSC therapies. Obtaining purified HSCs is a major challenge, and purification in a clinical setting is expensive and difficult. Another major barrier to progress in HSC research and transplantation therapy is that it has not been possible to culture HSCs in vitro (outside the body), although recent studies of mouse HSCs grown in combination with components of the bone marrow have offered some preliminary promise. This stubborn and not insignificant obstacle is faced by researchers with all types of adult stem cells. If it were possible to expand the numbers of stem cells by growing them in culture or to stimulate their expansion in vivo (in the living body), the prospects for patients in need of stem cell transplants would be significantly improved. However, finding a way to get HSCs to proliferate is not enough. In the long run, it is necessary to understand not only what activates HSCs to self-renew, but also what controls their decisions to differentiate into the various components of the blood and prevents them from developing into leukemic cells. One barrier in understanding the capabilities of adult stem cells is the relationship of the cellular environment to the concept of plasticity in adult stem cells. Markus Grompe showed that HSCs and pancreatic stem cells can give rise to liver cells called hepatocytes that will repopulate a diseased mouse liver, demonstrating the plasticity of adult stem cells. However, in his experiment, HSCs and pancreatic stem cells were very inefficient in repopulating the liver relative to the ability of transplanted hepatocytes themselves. This could mean that the plasticity of adult stem cells is a marginal capacity that can be exploited only with a much greater understanding of the environmental signals that influence adult stem cells. No one knows what steps HSCs or pancreatic cells go through in generating a hepatocyte, or what signals cause them to migrate to the liver in the first place. Moreover, some of the apparent plasticity in adult stem cells is difficult to interpret because it has been accomplished in abnormal environments, for example, in mice that are immunologically impaired or sub-lethally irradiated. A major limitation relevant to immediate development of therapies based on adult stem cells is the inability to maintain these cells in culture for very long before they differentiate into their mature progeny. One can envision two therapeutic approaches to stem cells. In the first, stem cells themselves are implanted in a diseased or injured organ in the hope that they will give rise

to the mature cells needed by that organ. In the second, the stem cells are stimulated to differentiate into the needed mature tissue outside the body, and that tissue is implanted in the organ. That adult stem cells are difficult to isolate, purify, and culture causes problems for either approach, although even the ability to culture stem cells for a limited time, including in the presence of other cells, could have therapeutic potential. An example is the use of autologous skin grafts for burn patients, in which healthy skin (which contains skin stem cells) is removed from the patient, cultured briefly outside the body, and grafted onto the patient’s injured tissue. The grafts are not able to regenerate hair follicles and sweat glands, but are otherwise able to function normally. However, with a few exceptions, the appropriate culture conditions to sustain most adult stem cells indefinitely have yet to be found. Very few stem cells, strictly defined, have even been isolated from adult human organs, in part because they constitute only a tiny fraction of the cells present and are not likely to be very distinct from the partially differentiated cells they give rise to as they mature and differentiate. A comparison of mouse and human HSCs shows that only about half of the genes expressed in the mouse HSCs correspond to genes expressed in human HSCs, so there are going to be differences as we move from experiments with mice to regenerative therapies in humans. Even the genetic programs that control the differentiation of human fetal liver stem cells and human HSCs, both of which evolve into the components of the blood, seem to be very different. We need to understand much more about the differences between mouse and human stem cells if we are to harness their potential. Rigorous experimentation will be needed to evaluate the implications of this basic research finding for regenerative medicine.

Conclusion:
Regenerative medicine when engaged with hematopoietic stem cells, which have an almost unlimited proliferation potential accompanied by an ability to differentiate. Thus, hematopoietic stem cells form an essential element in regenerative (or reparative) medicine, including guided regeneration. Instead of implantation of a prosthetic device or transplantation of donor organ, regenerative medicine relies on the versatile genetic information stored tightly packed in the cell nuclei, from where it is released (copied and then translated into proteins) to regulate the repair of cells, tissues and organs in an intelligent way. When such multipotent stem cells are harvested from autologous sources, immunological rejection and the burden of the use of immunosuppressive (cytotoxic) drugs are avoided. Endogenous stem cells can be activated with proper growth and differentiation stimuli to maintain or augment bodily functions. Subcutaneous fat tissue forms an autologous (personal) MSC source, which can be simply collected using a needle for fat aspiration. Cells isolated are allowed to expand and then driven to differentiate as such, in tissue engineering devices or bioreactors before applying back to body as cells, tissues or organs, either locally or systemically into circulation. They help the target site(s) to regenerate so that structure and function are not replaced but restored. Restriction to the use of hematopoietic stem cells avoids the ethical issues associated with the use of embryonic stem cells. Autologous stem cells have a known source of origin and donor cells cannot transmit any new infections or prions to the recipient. Such cells can be used at once

or stored in biobanks for eventual future use. Versatility of the cell-based therapies can be enhanced by genetic manipulation of the cells in the laboratory, coding of a missing protein such as hormone or enzyme for the life-time of the recipient. For experimental work markers useful in tracing of the transferred cells can be added to study the long-term effects. Regenerative medicine provides new insight in realms comprising cellular proliferation, effects of humoral and matrix signaling on cells, angiogenesis, tissue remodelling, naïve and adaptive immunity and other basics in cell biology. Still, regenerative medicine is in its infancy and to facilitate the process, 13 EU countries have joined forces in an attempt to improve networking and clarify where the frontiers and future needs are in this complex multidisciplinary high-tech, high-medicine field. In general, in regenerative medicine, apart from input in basic science, proof of principle experiments and randomized clinical trials are ahead, for which separate support will be sought for. In the most futuristic views, the human body is seen to undergo repeated and adequately timed maintenance of the failing body parts with cycles of healing cell therapies. Adult stem cell research has shown variable degree of success in treating disorder in hematopoietic system, heart damage, and cartilage defect in human. Hematopoietic stem cell research experiences fewer ethical obstacles compared to embryonic stem cell research, however, adult stem cell may be unable to achieve certain therapeutic benefit that could only be achieved by utilizing embryonic stem cell. Nevertheless, all hematopoietic stem cell research should be continued with support from all sides to ultimately achieve genuinely useful results in the field of Regenerative Medicine.

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