Stem Cell Research and Testing Dissertation

Stem Cell Research and Testing

Stem Cell Research

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The field of Stem cell research has come out of its first phase of research to the current phase where researchers are trying to harness its efficacy in the areas of regenerative medicine that could alter our entire approach to the management of degenerative diseases. Stem cells from umbilical cord blood are found to be very effective in the treatment of hematological, neurological and immunological disorders. There is no question of doubt that research in stem cells holds great potential for the effective treatment and management of a range of degenerative disorders.

Much progress has been achieved in the field of medicine with advanced diagnostic methods, preventive and curative interventions, and an overall improvement in human disease management, yet with all the expertise, degenerative diseases such as immune disorders, neural complications, cardiovascular conditions, diabetes, etc., continue to be a major cause of concern, affecting the quality of life in a big way. Stem cell research is currently one of the most fascinating areas of medicine with great promise for curing a variety of diseases. This growing field of regenerative medicine has totally altered our perception of diseases and their treatment. Few years ago, the U.S. congress contributed a $10 million funding for establishing a national cord blood stem cell bank. [Emily Ann Meyer, 2005, 4] the capability of stem cells to differentiate into specific cell types based on their environment has created a whole new approach to disease management. Medical research is currently focused on utilizing this reparative property of stem cells as a potent treatment solution for a variety of diseases such as leukemia, cancers, spinal cord injuries, blood and immune disorders, etc. However, actualizing this new technology is not an easy task and there still remain considerable hurdles to be overcome. Asides the difficulties pertaining to the perfection of the technology, there are also strong concerns about the moral and ethical aspects, particularly of those research involving embryonic stem cells. Let us have a brief overview of the field of stem cell research and its medical utility.

Stem Cells Different Types

Stem cells are the most basic cells of living beings and they have the special property of replication as well as differentiation. Stem cells are divided into four main categories based on their potential to differentiate. They are the Totipotent, Pluripotent, Multipotent and Unipotent stem cells. Totipotent stem cells refer to the initial few cells that are formed after the fusion of the egg and the sperm. They can differentiate into embryonic and extra embryonic cell types. (all cell types of the organism) Pluripotent stem cells, as the name suggests, are capable of developing into most types of tissues and are derived from the totipotent cells. Multipotent stem cells are more committed in their biological function and capable of producing cells that are closely related. The hematopoietic stem cells are a good example of the multipotent type and they produce red blood cells, platelets and white blood corpuscles. Finally, the unipotent cells are those cells that are capable of differentiating into only one specific cell type. (ex skin cells) [Holland, Suzanne, 2001] Now let us discuss the different sources of stem cells and their applications.

Sources of Stem cells

Stem cells, the precursor cells of all other cell types, can be derived from a variety of sources. Embryonic stem cells, embryonic germ cells, adult stem cells and stem cells derived from the umbilical cord matrix, are the major sources of stem cells. As discussed above, the differentiating ability of the stem cells is related to its stage of development. Embryonic stem cells are pluripotent and retain the greatest potential to develop into any tissue type as they are derived from the embryo. They are obtained from the blastocyst (initial stage of the embryo) and the first culture of embryonic stem cells was done only in 1998. Thus, embryonic stem cell research is still in its early stages. Embryonic germ cells on the other hand are obtained from fetuses that are aborted within the first 8 weeks. [Holland, 2001] Since they are derived from early stage human embryos, there are strong ethical concerns pertaining to the field of embryonic stem cells research.

Adult stem cells are derived from mature and differentiated tissues. Stem cells from the bone marrow, skin, etc., are some examples of adult stem cells. Unlike embryonic stem cells, adult stem cell research has been in prominence over the last four decades and as early as 1968 the first bone marrow transplant was performed successfully. The main drawback with adult stem cells is that they are limited in their potential and that in vitro culturing is a very difficult task. The proliferation rate of adult stem cells is very low compared to embryonic stem cells. [LE Magazine]

Stem cells from the umbilical cord, however, were first successfully used in 1988 in the treatment of anemia. Since then stem cells from cord blood have been transplanted successfully in more than 6000 cases. [Emily Ann Meyer, 2005, 35] Because umbilical cords are discarded anyway, stem cells derived from them do not contribute to any ethical or moral issues. “Umbilical cords are a rich, non-controversial source of stem cells, but currently hospitals throw millions of them away each year because we do not have the infrastructure needed to properly collect and store them,” “The best kept medical secret has been that thousands have been successfully treated with cord blood stem cells for more than 67 diseases including Leukemia and Sickle Cell Anemia.” [Chris Smith] Today there are cord blood banks all over the world which store the stem cells obtained from the umbilical cord during childbirth as a biological insurance.

Ethical Controversies

Among the different sources of stem cells discussed above, research involving adult stem cells is free of any controversy. With the consent of a willing adult, stem cells thus derived and used for research and treatment procedures is without any ethical disagreement. However, much heated debate exists in the use of embryos as a source of stem cells for research. As we discussed above, working with embryonic stem cells, as well as embryonic germ cells, involves the use of human embryos and aborted fetuses. [Holland, 2001] in the case of embryonic germ cells, the ethical controversy arises naturally from the existing controversy concerning abortion itself. For those people who are against abortion, it then becomes a difficult proposition to convince the medical utility value of working with fetal tissues.[ Chapman] Thus, in the case of EG cell research, the first ethically debatable measure is already undertaken with the completion of the abortion process. There are documented cases where non-profit tissue banks have sold cadavers donated to them for research purposes for huge sums of money. As Holland puts it “Skins, tendons, heart valves, veins, and corneas are listed at about $110,000. Add bone from the same body, and one cadaver can be worth about $220,000.” [Holland, 2001, 266] So there is always the possibility that abortion clinics might try to profit from the market for embryonic germ cell research supplying fetuses to the research centers with or without the knowledge of the concerned pregnant woman.

During his tenure, president Mr. Bush vetoed the embryonic stem cell research bill owing to the ethical and moral consequences involved. Quoting Mr. Bush, “If this bill were to become law, American taxpayers would, for the first time in our history, be compelled to fund the deliberate destruction of human embryos, and I’m not going to allow it.” [CNN] However, the medical community was very upset with the veto, which blocks federal funds for embryonic stem cell research. For those suffering from degenerative diseases like diabetes and other immune disorders, embryonic stem cell research could prove to be potentially life saving. As the chairperson of the American diabetes association says, “a devastating setback for the 20.8 million American children and adults with diabetes — and those who love and care for them.” [CNN] in Canada also, federal funds for EG cell research is controlled by regulations that require that the research does not affect the decision of the pregnant woman and that such research could only be carried out upon personal consent of the pregnant woman. [CIHR]

Addressing the ethical issues involved in embryonic stem cell research, the bioethics committee of the U.S. government had proposed 4 alternative methods of obtaining stem cells for research that are free of such controversies. These include 1) extracting cells from dead embryos 2) Extracting cells from embryos without affecting them (‘non harmful biopsy’) 3) by obtaining cells from artificially created embryo like cell system which however lacks the potential for embryogenesis but still has limited cellular division. 4) ‘dedifferentiation of Somatic cells’ to restore their pluripotency. [Council on Bioethics] While these alternative approaches were being pursued vigorously over the last few years, recent policy changes on embryonic research has opened new vistas for stem cell research. With the recent change in policy by president Obama lifting the ban on federal funding for embryonic stem cell research it is hoped that the progress in embryonic stem cell research would be hastened. In the words of Obama, “Today, with the executive order I am about to sign, we will bring the change that so many scientists and researchers, doctors and innovators, patients and loved ones have hoped for, and fought for, these past eight years: We will lift the ban on federal funding for promising embryonic stem cell research,” President Obama further said. “We will vigorously support scientists who pursue this research. And we will aim for America to lead the world in the discoveries it one day may yield.” [Dan Childs] With this change of stance more and more stem cell lines that were previously banned by the Bush government policy are now open for researchers increasing the scope and hastening the development of life saving stem cell therapy for a variety of chronic conditions.

Umbilical Cord Stem cells

Compared to the controversies surrounding the embryonic stem cell research, umbilical cord stem cells hold no ethical or moral dilemma. Since there is no fetus or embryo involved in the process, and the very fact that the placenta is usually a biological waste, the case for cord stem cell research is very encouraging and without any controversies. Thus far it was believed that stem cells derived from umbilical cord, being very few in number could hardly be used in lieu of bone marrow transplant for adults. However, an interesting characteristic of cord stem cells is that they can proliferate rapidly unlike adult stem cells. Furthermore, immunological rejection is much less severe in the case of cord blood transplantation compared with Peripheral blood stem cell transplant or bone marrow transplant. Even in the case of allogenic transplantations, cord blood cells are less likely to trigger immunological complications compared with adult stem cells as they are found to be immunologically downregulated. (Reduced cytokine production) [C a JONES] Further, the risk of acquiring viral and other microbial infections from cord blood transplantation is very minimal compared to adult stem cells.

Cord Blood (Treatment of Cancer)

Patients suffering from Leukemia and other serious forms of cancer have to undergo heavy irradiation, which destroys their bone marrow. Invariably such patients require bone marrow transplant, which is not so easily possible given the dearth of matching donors. Dr. Mary Laughlin, from the University hospitals of Cleveland says, “Out of every 10 adults who needs a stem cell transplant because of cancer or some other disease, only two have a brother or a sister who are perfect donor matches.” [ACS] Given this bleak situation the possibility of using umbilical cord blood for grafting assumes great significance. “The other eight must search for donors. Between 3,000 and 5,000 adults die every year in the U.S. because they can’t find donors. Now we know that even if you can’t find a perfect bone marrow donor, umbilical cord blood can provide a successful graft.”[ACS] Further, the potential for acquiring GVHD (Graft vs. Host Disease) by using cord blood is considerably low when compared to bone marrow grafting. Dr. Mary Laughlin and fellow researchers studied 68 transplant patients of whom 66 received mismatched transplant. It was found that the occurrence of GVHD among the subjects was around 38%, a rate that is significantly low compared to the usual rate of 75% observed among matched bone marrow transplants. Also, at the end of 40 months, 19 of the 66 patients managed to survive of which 18 patients were completely cured of their disease. As Dr. Herman Kattlove of the American Cancer Society says, “Survival doesn’t seem to be affected by receiving a cord blood graft from an unrelated and mismatched donor. “This can be life-saving for patients who can’t find a matching graft.” [ACS]

Cord blood (Spinal Cord Injuries)

Spinal injuries are very crippling resulting in patients losing their neurological functions. It has been proved by several animal studies that human umbilical cord blood can be a therapeutically potent intervention for spinal injuries. Dr. Saporta, a researcher from the University of South Florida, who conducted a study on mice found out that “HUCB (human umbilical cord blood) cells have an amazing affinity for going where they are needed and take up residence within the nervous system. Our results indicate that cord blood stem cells may provide a useful and novel treatment option for patients with spinal cord injury, but more studies are needed.” [USF] in another study involving 3 groups of mice it was found that the group that received HUCB along with brain derived neurotrophic factor (BDNF) showed the greatest recovery compared with the group that received only the HUCB and the control group. [Kuh SU, 2005] However, the first major human case study is of a 39-year-old woman from South Korea. The patient who was confined to a wheelchair for the last 19 years due to a spinal injury showed significant improvement in neurological functions within 41 days of treatment with umbilical cord stem cells. (Injected directly into the injury site) . The patient was even able to walk with the help of a walker. MRI scans confirmed regeneration of spinal cord at the injured site. [Kang KS, 2004]

Stem Cells in Gene Therapy

Stem cell-based gene therapy involving both Mesenchymal stem cells and Haematopoietic Stem Cells are thought to be the hope for a cure for many inherited genetic disorders. Over the last decade we have achieved huge strides in terms of our understanding of the behavior of stem cells in different tissue microenvironments. In particular, the controversies surrounding embryonic stem cell research as well as the high neoplastic potential of embryonic stem cells has motivated researchers to focus on the HSC and the MSC as other important modalities in the developing field of genetic therapy. The special feature of MSC’s is their ability to suppress host immune function by modulating the dendritic T cells, which result in the production of suppressor T cells. This along with MSC’s ability to target cells selectively enable them as excellent vehicles for delivery of therapeutic proteins at the appropriate tissue environment. Using a variety of techniques such as adeno viral vectors, lentivirus transduction or other non-viral methods such as electroporation or ‘liposome-based transfection’ it is possible to achieve effective transgene delivery. [Jakob Reiser, 2005]

Several studies over the last few years have confirmed that genetically modified MSC’s could be used as effective tools for the expression of recombinant proteins, the sustained production of which could reverse the progression in many cases of degenerative neural diseases such as Parkinson’s, Alzheimer’s, etc. Kurozumi observed the effect of genetically modified MSC’s that increased the synthesis of brain-derived neurotrophic factor (BDNF) in rodents recovering from ischaemia. As a control group, the researchers used rats that were only treated with unmodified MSC’s. Over a 14-day period of observation it was found that the experimental group that were treated with the genetically modified MSC’s (expressing BDNF) showed significant reduction in infarction in the cerebral artery compared with the control group. This clearly suggests that genetically modified MSC’s that are programmed to express BDNF may increase the prognosis of stroke-affected victims and could possibly completely restore the normal functioning. [Jakob Reiser, 2005]

Transduced MSC’s can be used for treating blood related disorders. Several animal studies have attested to the ability of transduced MSC’s to differentiate while still maintaining the ability to express the desired protein. One study involved the administration of human MSC’s programmed to express hIL-3 by ‘subcutaneous, intravenous, and intraperitoneal’ routes. Peripheral blood count measured 3 months later showed levels of hIL-3 in the range of 100-800-page/ml, clearly indicating sustained systemic expression by Mesenchymal stem cells. [Jakob Reiser, 2005]

Stem Cell Therapy for Cardiovascular Diseases

Since cardiovascular diseases represent one of the single most prevalent and life threatening conditions worldwide, stem cell-based regeneration of defective or failing cardiac tissue promises a new ray of hope for millions of patients. Already scientists have managed to successfully isolate and differentiate stem cells into cardiomyocytes. However, the problem was that stem cells being by nature pluripotent carried a high risk for contamination with other cell types. Hattan (2005) used a fluorescence-activated cell seperater to isolate mice cardiomyogenic cells from mice bone marrow cells. After testing the purified cells for their gene expression the researchers then transplanted the cells to mice heart. The sorted cells started beating 3 weeks after isolation and purification and expressed genes such as alpha-skeletal actin, beta myosin, MLC-2v, and CaV1.2 that are specific for cardiomyocytes. When these isolated and expanded cells were later transplanted into the left ventricle of the adult mice they worked well with the cardiomyocytes of the recipient and lived for up to 3 months clearly suggesting that stem cell-based treatment would be an effective treatment for cases of heart muscle failures and other cardiac anomalies. [Hattan, 2005] Similarly, MSC’S that are designed to differentiate into cells (expressing the mHCN2, pacemaker gene) that have similar functions of the Sino atrial node cells could restore the natural cardiac signaling mechanism in patients suffering from aberrations in the sinus node. [Jakob Reiser]

Stem Cells for Diabetes (Vascular Regeneration)

Diabetes mellitus is another pandemic global condition with potential life threatening complications. Already results from several animal studies have attested that pancreatic function could be vastly improved and glucose metabolism restored using embryonic as well as adult stem cells that differentiate into insulin generating cells. However researchers also report from studies that cells derived from stem cells do not have the entire functionality of natural pancreatic cells that leaves more scope for future study in this direction. “Everything we’ve learned says we will get there. If there are seven steps to turn an ES cell into a pancreatic beta cell, we’ve solved two of the steps,” says Doug Melton. “We will get there, but it may be a year or a decade.” [CAMR] However, besides having the potential for restoring pancreatic function in the near future, stem cell based therapy is already being used in the treatment of complications that arise in diabetic patients. [Soria 2008] Particularly, stem cell-based vascular regeneration is saving many diabetic patients facing ischemic limb amputation. Kawamura studied 30 patients with diabetic ischemic limbs (and scheduled for amputation) and treated them with ‘autologous peripheral blood stem cell’ (PBSC) implantation. Granulocyte Colony Stimulate Factor was administered for 4 days prior to collection of PBSC. The PBSC thus collected was then divided into units of 0.5-1.0 mL and implanted at the ischemic locations. The patients were then monitored over the next several days and limb temperature regularly measured using a thermograph. In 21 of the 30 patients limb temperature normalized indicating vascular regeneration and recovery. PBSC therapy saved 22 of the 30 patients from imminent limb amputation. [Kawamura 2005]

Stem Cell Therapy for Gliomas

Stem cell therapy holds immense potential and hope for glioma patients, as inspite of the other interventions the survival rate for such patients is only 1 year or 3 years at best for anaplastic astrocytoma. Studies have shown that a group of cells known as the Brain tumor Stem Cells (BTSC’s) are responsible for persistent tumors that do not respond effectively to chemotherapy. Glioma researchers believe that these BTSC’s may either be derived from the progenitor stem cells that have mutated and become neoplastic or they could have developed from mature neuron cells as a result of mutations. However, it is exactly this similarity between stem cells and the BTSC’s that has prompted much attention to research based on using the stem cells to better track down BTSC’s and to provide more effective mesenchymal stem cell (MSC) and Neural stem cell (NSC) therapies for glioma.

Tumor Tropism

Tumor tropism exhibited by stem cells makes them effective in delivering therapy as stem cells can easily pass through the blood brain barrier as well as reach the tumor site. In vitro studies indicate ligand receptor affinity as the basis for this tumor tropism of stem cells. In particular EGF (epidermal growth factor) and VGEF (vascular endothelial growth factor) receptors are found to be directly involved in increased invasion and penetration of the gliomal cells by the NSC. This property of stem cells makes them effective vehicles of drug delivery to the tumor site. Baressi et al. (2003) confirmed the regression of intracranial tumors upon treatment with neural stem cells that were engineered to convert 5-FC to 5-FU in the tumor environment. This approach of using the stem cells to carry and convert a non-toxic pro-drug into toxic drug at the tumor site offers great therapeutic scope. Miletic (2007) found that genetically modified Mesenchymal Stem Cells that were engineered to express HSV-tk in the tumor microenvironment was very effective in treatment of gliomal rats. Sonabend (2008) also reported the effectiveness of using stem cells as viral delivery vehicles. In this study the researchers delivered ‘replication-competent oncolytic adenovirus’ (CRAd) away from the tumor site in rodents with intracranial gliomas. It was observed that the mesenchymal stem cells that were used as viral delivery vehicles traveled to the tumor site and provided oncolytic viral loads that were 46 times more compared to the loads achieved by direct viral injection. This is an exiting area of stem cell application that is intensely pursued by researchers. [Thomas Kosztowski, 2009]

Recently, research by the ‘International Stem Cell Corporation’ has shown the possibility of retinal progenitor cells restoring vision in animal models. Dr. Hans Keirstead, chief researcher of the project said, “Intact layers of retinal progenitor cells have been shown to restore lost visual responses in several retinal degeneration rodent models,.” He further added that, “Thus, we are developing intact retinal layers derived from International Stem Cell’s human parthenogenetic stem cells which could become a sustainable, FDA-approved therapeutic supply for patients with retinal degenerative diseases..” [Medical News Today] This is promising news for millions of people who are visually impaired due to degerative retinal disorders. While the vast promises of stem cell research are no doubt encouraging there are still some hurdles to cross before we can perfect stem cell based therapy. Some clinical and animal studies have shown certain complicating conditions resulting from stem cell therapies.

Potential Complications (Some Recent studies)

Results from some recent clinical studies have shown that more research is necessary to ensure the safety of stem cell therapy. This particular study by Ninette (2009) has shown that stem cell therapy could have serious complications. The subject of this study was a young boy suffering from ataxia telangiectasia (at), a very rare form of immunodeficiency disease. As a stem cell based therapeutic intervention, researchers administered human fetal neural cells by way of intracerebellar and intrathecal injections. This intervention was repeated several times. However, four years after the therapy the patient was diagnosed with brain tumor and tumor in the spinal cord. Surgical removal of the spinal tumor and further diagnosis by way of chromosome probes using fluorescent hybridization techniques and analysis of polymorphic microsatellite markers revealed the tumor to be of non-host origin. Further tests for the Human leukocyte antigen (HLA) revealed that the tumor could be associated with atleast two of the stem cell donors. As a very first case reporting the possibility of the stem cell transplantation as one of the potential pathways of gliomagenesis in humans, this research assumes great significance. [Ninette, (2009)] Earlier animal research using HE’s derived dopaminergic neurons resulted in significant improvement in motor skills of parkinsonian rats but also resulted in an uncontrolled growth of neuroepithelial cells. The results from this study clearly suggests that stem cell therapy, though having immense potential in the field of regenerative medicine, is plagued with the potential for developing phenotypic instability and tumor development. [Roy, 2006]

Faraci (2002) was one of the earlier studies that focused on the complications of stem cell therapy. This study focused on the adverse effects following hematopoietic stem cell transplantation in children with hematological as well as non-hematological diseases. This research studied cases of both autologus and allogenic transplants. This study was comprehensive and stretched over a period of 16 years from 1985 to 2001. A total of 272 pediatric subjects at the G. Gaslini Children’s Research Institute were analyzed for the development of neurological complications subsequent to hematopoietic stem cell transplantation. (Using medical records) Results from the observations indicated that stem cell therapy was a high risk factor for developing severe neurologic events (SNE). In all, 37 children (13.6%) developed SNE within a mean period of 90 days following the HSCT. Of these, 11 children (30%) succumbed to the complications. The results also indicated that transplantation from allogenic sources resulted in severe graft-vs.-host disease (GvHD). [Faraci (2002)]. A more recent study also reported the risk of allogenic donors in stem cell transplantation. This Japanese study by Makoto (2008) observed a 49-year-old patient who received ‘allogenic peripheral blood stem cell transplantation’ from a HLS identical brother. The subject developed donor cell leukemia (DCL) 27 months post the stem cell transplantation. [Makoto,, 2008]

Stem Cells and Drug Development

Stem cell research has important promises in the field of new drug development. As Doug Melton from the Harvard stem cell institute says, “In the next decade, most advances will come from drugs that affect progression of disease. And we’ll get there by using hE’s cells as test beds for new therapeutics,” [CAMR] Culturing stem cells and differentiating them into the required cell types would offer an excellent laboratory medium that replicates the in vivo conditions for the purposes of testing out drug pathways and interactions. This would eliminate the need for animal testing, which is itself steeped in ethical controversy. Also since animal models do not reflect human conditions effectively, they are unreliable. In vitro stem cell experiments can therefore provide a new, alternative and more accurate tool for testing and development of drugs, speeding up the entire process considerably. As an FDA report reads, “…a 10-percent improvement in predicting failures before clinical trials could save $100 million in development costs per drug.” With the knowledge gained from embryonic stem cell research, scientists have identified a new form of stem cell derived from genetically engineered adult somatic cells known as ‘induced pluripotent cells’. As says Kevin Eggan, Harvard University researcher says, “We are doing very careful comparisons of how well iPS cells and hE’s cells make motor neurons, and how functional those cells truly are,,.” “NIH should be funding both activities.” [CAMR]


The field of Stem cell research has come out of its first phase of research to the current phase where researchers are trying to harness its efficacy in the areas of regenerative medicine that could alter our entire approach to the management of a range of degenerative diseases. With the present government lifting the curbs on embryonic stem cell research and promising more funding, much progress is expected in the next few years. While the debate on embryonic stem cell continues, Umbilical cord derived stem cell has opened up new vistas in medical research. This developing medical frontier holds great promise for those suffering from intractable medical conditions. The fact that cord blood does not create any ethical concerns is an encouraging aspect for researchers who have recently been plagued by controversies relating to embryonic stem cell research. Also, HUCB cells have a high proliferating rate compared to adult stem cells. Results from studies conducted so far are proving that they are effective in inducing neurological regeneration in the treatment of hematological defects suggesting the great reparative potential of cord blood, and its effectiveness in chronic disease management. The difficulties in obtaining donors for bone marrow transplant and the high rate of immunological complications associated with such treatment, suggest umbilical cord blood grafting as a comparatively easier as well as safer therapeutic procedure for patients suffering from advanced stages of various types of cancer. Furthermore, the new and developing field of ‘Induced Pluripotent stem cells’ is holding great promise without any ethical or moral stains. Though not a cure all solution, there is no question of doubt that research in stem cells has great potential in the effective treatment and management of a variety of degenerative disorders and chronic conditions.


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16) Kawamura a, Horie T & Tsuda I (Feb, 2005),’Prevention of Limb Amputation in Patients with limb Ulcers by autologous peripheral blood mononuclear cell implantation’, Therapeutic Apheresis and Dialysis 9(1):59-63

17) Kang KS, “A 37-year-old spinal cord-injured female patient, transplanted of multipotent stem cells from human UC blood, with improved sensory perception and mobility, both functionally and morphologically: a case study.” Cytotherapy 2005; 7(4): 368-73.

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