Monday, August 4, 2008
Sunday, August 3, 2008
Compare the two techniques for 'stem cell' creation
But the research has proved controversial. Korean scientist Hwang Woo-suk claimed in 2005 that he had created such cell lines, but the study was later discredited. Meanwhile, critics have objected on ethical grounds, saying it is wrong to use embryos for research.
Some scientists argue that clones might not be required to harvest stem cells. Last year, researchers in Japan and the US were able to "rewind" adult cells back to their embryonic state using a new technique.
Professor Jack Price of King's College, London, is an expert on neural stem cells. He too said the Californian experiment was a small step forward but not a breakthrough.
"This constitutes technical progress," he said. "It shows that the approach using human embryos does still have promise and it does provide justification for continuing that avenue of research."
TECHNIQUES FOR MAKING 'STEM CELLS'
Therapeutic cloning produces stem cells which can develop into different types of body cell, making them ideal for research into treatment of disease.
But this technology involves the creation and destruction of embryos, which is ethically controversial. The stem cells created also run the risk of being rejected by the body.
The new technology, nuclear reprogramming, creates stem-like cells from the patient's own cells, avoiding both these problems
Some scientists argue that clones might not be required to harvest stem cells. Last year, researchers in Japan and the US were able to "rewind" adult cells back to their embryonic state using a new technique.
Professor Jack Price of King's College, London, is an expert on neural stem cells. He too said the Californian experiment was a small step forward but not a breakthrough.
"This constitutes technical progress," he said. "It shows that the approach using human embryos does still have promise and it does provide justification for continuing that avenue of research."
TECHNIQUES FOR MAKING 'STEM CELLS'
Therapeutic cloning produces stem cells which can develop into different types of body cell, making them ideal for research into treatment of disease.
But this technology involves the creation and destruction of embryos, which is ethically controversial. The stem cells created also run the risk of being rejected by the body.
The new technology, nuclear reprogramming, creates stem-like cells from the patient's own cells, avoiding both these problems
What is the history of stem cell research?
The history of stem cell research had a benign, embryonic beginning in the mid 1800's with the discovery that some cells could generate other cells. Now stem cell research is embroiled in a controversy over the use of human embryonic stem cells for research. In the early 1900's the first real stem cells were discovered when it was found that some cells generate blood cells. The history of stem cell research includes work with both animal and human stem cells. Stem cells can be classified into three broad categories, based on their ability to differentiate. Totipotent stem cells are found only in early embryos. Each cell can form a complete organism (e.g., identical twins). Pluripotent stem cells exist in the undifferentiated inner cell mass of the blastocyst and can form any of the over 200 different cell types found in the body. Multipotent stem cells are derived from fetal tissue, cord blood, and adult stem cells. Although their ability to differentiate is more limited than pluripotent stem cells, they already have a track record of success in cell-based therapies. A prominent application of stem cell research has been bone marrow transplants using adult stem cells. In the early 1900's physicians administered bone marrow by mouth to patients with anemia and leukemia. Although such therapy was unsuccessful, laboratory experiments eventually demonstrated that mice with defective marrow could be restored to health with infusions into the blood stream of marrow taken from other mice. This caused physicians to speculate whether it was feasible to transplant bone marrow from one human to another (allogeneic transplant). Among early attempts to do this were several transplants carried out in France following a radiation accident in the late 1950's. Performing marrow transplants in humans was not attempted on a larger scale until a French medical researcher made a critical medical discovery about the human immune system. In 1958 Jean Dausset identified the first of many human histocompatibility antigens. These proteins, found on the surface of most cells in the body, are called human leukocyte antigens, or HLA antigens. These HLA antigens give the body's immune system the ability to determine what belongs in the body and what does not belong. Whenever the body does not recognize the series of antigens on the cell walls, it creates antibodies and other substances to destroy the cell. A bone marrow transplant between identical twins guarantees complete HLA compatibility between donor and recipient. These were the first kinds of transplants in humans. It was not until the 1960's that physicians knew enough about HLA compatibility to perform transplants between siblings who were not identical twins. In 1973 a team of physicians performed the first unrelated bone marrow transplant. It required 7 transplants to be successful. In 1984 Congress passed the National Organ Transplant Act, which among other things, included language to evaluate unrelated marrow transplantation and the feasibility of establishing a national donor registry. This led ultimately to National Marrow Donor Program (NDWP) a separate non-profit organization that took over the administration of the database needed for donors in 1990. The 1990's saw rapid expansion and success of the bone marrow program with more than 16,000 transplants to date for the treatment of immunodeficiencies and leukemia. Adult stem cells also have shown great promise in other areas. These cells have shown the potential to form many different kinds of cell types and tissues, including functional hepatocyte-like (liver) cells. Such cells might be useful in repairing organs ravaged by diseases. In 1998, James Thompson (University of Wisconsin - Madison) isolated cells from the inner cell mass of early embryos, and developed the first embryonic stem cell lines. In the same year, John Gearhart (Johns Hopkins University) derived germ cells from cells in fetal gonadal tissue (primordial germ cells). Pluripotent stem cell "lines" were developed from both sources. The blastocysts used for human stem cell research typically come from in vitro fertilization (IVF) procedures. The ethical concerns over this type of embryonic stem cell research has been expressed in the following US legal regulations: In 1973 a moratorium was placed on government funding for human embryo research. In 1988 a NIH panel voted 19 to 2 in favor of government funding. In 1990, Congress voted to override the moratorium on government funding of embryonic stem cell research, which was vetoed by President George Bush. President Clinton lifted the ban, but changed his mind the following year after public outcry. Congress banned federal funding in 1995. In 1998 DHHS Secretary Sullivan extended the moratorium. In 2000, President Bill Clinton allowed funding of research on cells derived from aborted human fetuses, but not from embryonic cells. On August 9, 2001, President George W. Bush announced his decision to allow Federal funding of research only on existing human embryonic stem cell lines created prior to his announcement. His concern was to not foster the continued destruction of living human embryos. In 2004, both houses of Congress have asked President George W. Bush to review his policy on embryonic stem cell research. President George W. Bush released a statement reiterating his moral qualms about creating human embryos to destroy them, and refused to reverse the federal policy banning government funding of ESC research (other than for ESC lines established before the funding ban). In the November 2004 election, California had a Stem Cell Research Funding authorization initiative on the ballot that won by a 60% to 40% margin. It established the "California Institute for Regenerative Medicine" to regulate stem cell research and research facilities. It authorizes issuance of general obligation bonds to finance institute activities up to $3 billion dollars subject to an annual limit of $350 million.
V. Human Stem Cells and the Treatment of Disease
A major goal of stem cell research is to provide healthy differentiated cells that, once transplanted, could repair or replace a patient’s diseased or destroyed tissues. In pursuit of this goal, one likely approach would start by isolating stem cells that could be expanded substantially in vitro. A large number of the cultivated stem cells could then be stored in the frozen state, extensively tested for safety and efficacy as outlined above, and used as reproducible starting material from which to prepare differentiated cell preparations that will express the needed beneficial properties when they are transplanted into patients with specific diseases or deficiencies.To make more concrete both the potential of this approach and the obstacles it faces, we will summarize, as a case study example, some current information on the properties of cells derived from human stem cell populations that have been used in an animal model of Type-1 diabetes. But before doing so, we discuss an obstacle to any successful program of stem cell-based transplantation therapy: the problem of immune rejection of the transplanted cells.
A. Will Stem Cell-Based Therapies Be Limited by Immune Rejection?
Much of the impetus for human stem cell research comes from the hope that stem cells (or, more likely, differentiated cells derived from them) will one day prove useful in cell transplantation therapies for a variety of human diseases. Such cell transplantation would augment the current practice of whole organ transplantation. To the extent that the healing process works with in vitro derived cells, the need for organ donors and long waiting lists for organ donation might be reduced or even eliminated.Will the recipient (patient) accept or reject the transplanted human cells? In principle, the problem might seem avoidable altogether: adult stem cells could be obtained from each individual patient needing treatment. They could then be grown or modified to produce the desired (autologous and hence rejection-proof) transplantable cells. But the logistical difficulties in processing separate and unique materials for each patient suggest that this approach may not be practical. The cost and time required to produce sufficient numbers of well-characterized cells suitable for therapy suggest that it will be cells derived from one or another unique stem cell line that will be used to treat many (genetically different) individual patients (allogeneic cell transplantation).When allogeneic organ or tissue transplantation is currently done using, for example, bone marrow, kidney, or heart, powerful immunosuppressive drugs—carrying undesirable side effects—must be used to prevent immunological rejection of the transplanted tissue.5 Without such immunosuppression, the patient’s T-lymphocytes and natural killer (NK) cells recognize surface molecules on the transplanted cells as “foreign” and attack and destroy the cells. Also, in whole organ transplantation, donor T-lymphocytes and NK cells, entering the recipient with the transplanted organ, can also destroy the tissues of the transplant recipient (called “graft versus host” disease).Are the differentiated derivatives of human stem cells as likely to incite immune rejection, when transplanted, as are solid organs? Do their surfaces carry those protein antigens that will be recognized as “foreign”? Experiments have been done to examine human ESC and MSC preparations growing in vitro for the expression of surface molecules known to play important roles in the immune rejection process. Drukker and coworkers39 showed that embryonic stem cells in vitro express very low levels of the immunologically crucial major histocompatibility complex class I (MHC-I) proteins on their cell surface. The presence of MHC-I proteins increased moderately when the ESCs became differentiated, whether in vitro or in vivo. A more pronounced increase in MHC-I antigen expression was observed when the ESCs were exposed to gamma-interferon, a protein produced in the body during immune reactions. Thus, under some circumstances, human ESC-derived cells can express cell surface molecules that could lead to immune rejection upon allogeneic transplantation.Similarly, Majumdar and colleagues showed that human mesenchymal stem cells in vitro express multiple proteins on their cell surfaces that would enable them to bind to, and interact with, T-lymphocytes. They also observed that gamma-interferon increased expression of both human leukocyte antigen (HLA) class I and class II molecules on the surface of these MSCs.40 These results indicate that it will probably not be possible to predict, solely on the basis of in vitro experiments, the likelihood that transplanted allogeneic MSCs would trigger immune rejection processes in vivo.Many further studies in this area are badly needed. At this time there is insufficient information to determine which, if any, of the approaches to get around the rejection problem will eventually prove successful.
B. Case Study: Stem Cells in the Future Treatment of Type-1 Diabetes?
1. The Disease and Its Causes.
The human body converts the sugar glucose into cell energy for heart and brain functioning, and indeed, for all bodily and mental activities. Glucose is derived from dietary carbohydrates, is stored as glycogen in the liver, and is released again when needed into the bloodstream. A protein hormone called insulin, produced by the beta cells in the islets of the pancreas, facilitates the entrance of glucose from the bloodstream into the cells, where it is then metabolized. Insulin is critical for regulating the body’s use of glucose and the glucose concentration in the circulating blood.The body’s failure to produce sufficient amounts of insulin results in diabetes, an extremely common metabolic disease affecting over 10 million Americans, often with widespread and devastating consequences. In some five to ten percent of cases, known as Type-1 diabetes (or “juvenile diabetes”), the disease is caused by “autoimmunity,” a process in which the body’s immune system attacks “self.”xiv T-lymphocytes attack the patient’s own insulin-producing beta cells in the pancreas. Eventually, this results in destruction of ninety percent or so of the beta cells, resulting in the diabetic state.With a deficiency or absence of insulin, the blood glucose becomes elevated and may lead to diabetic coma, a fatal condition if untreated. Chronic diabetes, both Type-1 and the much more common Type-2 diabetes (which is not autoimmune, but largely genetic), causes late complications in the retina, kidneys, nerves, and blood vessels. It is the leading cause of blindness, kidney failure, and amputations in the U.S. and a major cause of strokes and heart attacks.Type-1 diabetes is a devastating, lifelong condition that currently affects an estimated 550,000-1,100,000 Americans,41 including many children. It imposes a significant burden on the U.S. healthcare system and the economy as a whole, over and above the disabilities and impairments borne by individual sufferers. Recent estimates suggest that treatment of all forms of diabetes costs Americans a total of $132 billion per year.42 At 5-10 percent of all diabetes cases, the costs of Type-1 diabetes can be estimated as $6.5-$13 billion per year.
2. Current Therapy Choices and Outcomes.
The current treatment of Type-1 diabetes consists of insulin injections, given several times a day in response to repeatedly measured blood glucose levels. Although this treatment is life-prolonging, the procedures are painful and burdensome, and in many cases they do not adequately control blood glucose concentrations. Whole pancreas transplants can essentially cure Type-1 diabetes, but fewer than 2,000 donor pancreases become available for transplantation in the U.S. each year, and they are primarily used to treat patients who also need a kidney transplant. Like all recipients of donated organs, pancreas transplant recipients must continuously take powerful drugs to suppress the immunological rejection of the transplanted pancreas.In addition to treatment with whole pancreas transplantation, small numbers of Type-1 diabetes patients have been treated by transplantation of donor pancreatic islets into the liver of the patient coupled with a less intensive immunosuppressive treatment (the Edmonton protocol).43 Expanded clinical trials of this procedure are currently underway. Scientists are also evaluating methods of slowing the original autoimmune destruction of pancreatic beta cells that produces the disease in the first place.Whole pancreas and islet cell transplants ameliorate Type-1 diabetes, but there is nowhere near enough of these materials to treat all in need. To overcome this shortage, people hope that human stem cells can be induced—at will and in bulk—to differentiate in vitro into functional pancreatic beta cells, available for transplantation. Of course, it would still be crucial to prevent immunological destruction of the newly transplanted stem cell-derived beta cells.
indicate, cells derived from some human stem cells transplanted into specific strains of mice mimicking major aspects of Type-1 human diabetes51 were able to reverse high blood glucose concentrations. Although these results are encouraging, the transplant rejection question remains unanswered because the likely immune rejection of the transplanted human cells was prevented in these experiments by using special strains of immunodeficient mice that lack the capacity to recognize and attack foreign cells.No tumors were observed in the transplanted mice, but the experiments were terminated after about three months, an insufficient time for much tumor development to occur. Because many Type-1 diabetes patients are children and because a largely effective therapy (insulin injection) is currently available, the introduction of islet cell transplant therapy will need a high degree of certainty that the introduced cells or their derivatives will not become malignant over the course of the patient’s life. Stringent tests of the cancer-causing potential of candidate cell preparations will be required, including multi-year studies in animals that live longer than mice or rats. Long-term follow-up of children and adult patients who had received bone marrow transplants many years ago has revealed an increased risk of severe neurologic complications52 and a variety of types of cancer.53
C. Therapeutic Applications of Mesenchymal Stem Cells (MSCs)
Before stem cell based therapies are used to treat human diseases, they will have to gain approval through the Food and Drug Administration (FDA) regulatory process. The first step in this process is filing an Investigational New Drug (IND) application. As of July 2003, four IND applications have been filed for clinical applications of mesenchymal stem cells. The disease indications include: (1) providing MSC support for peripheral blood stem cell transplantation in cancer treatment, (2) providing MSC support for cord blood transplantation in cancer treatment, (3) using MSCs to stimulate regeneration of cardiac tissue after acute myocardial infarction (heart attack), and (4) using MSCs to stimulate regeneration of cardiac tissue in cases of congestive heart failure. The first two applications are currently in Phase II of the regulatory process, with pivotal Phase III trials scheduled to begin in 2004.54
D. Evaluating the Different Types of Stem Cells
A major unresolved issue at present involves the therapeutic potential of human adult stem cells compared with embryonic stem cells. The answer may well be different for different diseases and for patients of different ages. For example, in treating an elderly patient with Parkinson’s Disease, the use of adult stem cells may be appropriate even if these cells may have a more limited number of cell divisions remaining. On the other hand, treating a child with Type I Diabetes, one may want to use embryonic stem cells because of their potentially greater longevity, or other factors. The only valid way to resolve these questions is by instituting rigorous therapeutic trials which test the efficacy of the different types of stem cells in treating a variety of different diseases to determine their comparative efficacy. Clearly, such trials would be a long-term endeavor, since it would take years to obtain answers to these very critical questions.
VI. Private Sector Activity
In the United States, much of the basic research on animal stem cells and human adult stem cells has been publicly funded. Yet before 2001, research in the U.S., using human ESCs could only be done in the private sector (the locus also of much research on animal and human adult stem cells). The current state of knowledge about human ESCs (and also about human MSCs) reflects pioneering and on-going stem cell research funded by the private sector in the U.S.54,55 For example, the work that led to the 1998 reports of the first isolation of both ESCs and EGCs, was funded by Geron Corporation. Embryonic and adult stem cell research is today vigorously pursued by many companies and supported by several private philanthropic foundations,56 and the results of some of this research have been published in peer-reviewed journals.57 Private sector organizations have pursued and been awarded patents on the stem cells themselves and methods for producing and using them to treat disease. As noted above, at least one company (Osiris Therapeutics) has protocols under review at the FDA for clinical trials with MSCs. It seems likely that private sector companies will continue to play large roles in the future development of stem cell based therapies.
VII. Preliminary Conclusions
While it might be argued that it is too soon to attempt to draw any conclusions about the state of a field that is changing as rapidly as stem cell research, we draw the following preliminary conclusions regarding the current state of the field.Human stem cells can be reproducibly isolated from a variety of embryonic, fetal, and adult tissue sources. Some human stem cell preparations (for example, human ESCs, EGCs, MSCs, and MAPCs) can be reproducibly expanded to substantially larger cell numbers in vitro, the cells can be stored frozen and recovered, and they can be characterized and compared by a variety of techniques. These cells are receiving a large share of the attention regarding possible future (non-hematopoietic) stem cell transplantation therapies.Preparations of ESCs, EGCs, MSCs, and MAPCs can be induced to differentiate in vitro into a variety of cells with properties similar to those found in differentiated tissues.Research using these human stem cell preparations holds promise for: (a) increased understanding of the basic molecular process underlying cell differentiation, (b) increased understanding of the early stages of genetic diseases (and possibly cancer), and (c) future cell transplantation therapies for human diseases.The case study of developing stem cell-based therapies for Type-1 diabetes illustrates that, although insulin-producing cells have been derived from human stem cell preparations, we could still have a long way to go before stem cell-based therapies can be developed and made available for this disease. This appears to be true irrespective of whether one starts from human embryonic stem cells or from human adult stem cells. The transplant rejection problem remains a major obstacle, but only one among many.Human mesenchymal stem cells are currently being evaluated in pre-clinical studies and clinical trials for several specific human diseases.Much basic and applied research remains to be done if human stem cells are to achieve their promise in regenerative medicine.58 This research is expensive and technically challenging, and requires scientists willing to take a long perspective in order to discover, through painstaking research, which combinations of techniques could turn out to be successful. Strong financial support, public and private, will be indispensable to achieving success.
A. Will Stem Cell-Based Therapies Be Limited by Immune Rejection?
Much of the impetus for human stem cell research comes from the hope that stem cells (or, more likely, differentiated cells derived from them) will one day prove useful in cell transplantation therapies for a variety of human diseases. Such cell transplantation would augment the current practice of whole organ transplantation. To the extent that the healing process works with in vitro derived cells, the need for organ donors and long waiting lists for organ donation might be reduced or even eliminated.Will the recipient (patient) accept or reject the transplanted human cells? In principle, the problem might seem avoidable altogether: adult stem cells could be obtained from each individual patient needing treatment. They could then be grown or modified to produce the desired (autologous and hence rejection-proof) transplantable cells. But the logistical difficulties in processing separate and unique materials for each patient suggest that this approach may not be practical. The cost and time required to produce sufficient numbers of well-characterized cells suitable for therapy suggest that it will be cells derived from one or another unique stem cell line that will be used to treat many (genetically different) individual patients (allogeneic cell transplantation).When allogeneic organ or tissue transplantation is currently done using, for example, bone marrow, kidney, or heart, powerful immunosuppressive drugs—carrying undesirable side effects—must be used to prevent immunological rejection of the transplanted tissue.5 Without such immunosuppression, the patient’s T-lymphocytes and natural killer (NK) cells recognize surface molecules on the transplanted cells as “foreign” and attack and destroy the cells. Also, in whole organ transplantation, donor T-lymphocytes and NK cells, entering the recipient with the transplanted organ, can also destroy the tissues of the transplant recipient (called “graft versus host” disease).Are the differentiated derivatives of human stem cells as likely to incite immune rejection, when transplanted, as are solid organs? Do their surfaces carry those protein antigens that will be recognized as “foreign”? Experiments have been done to examine human ESC and MSC preparations growing in vitro for the expression of surface molecules known to play important roles in the immune rejection process. Drukker and coworkers39 showed that embryonic stem cells in vitro express very low levels of the immunologically crucial major histocompatibility complex class I (MHC-I) proteins on their cell surface. The presence of MHC-I proteins increased moderately when the ESCs became differentiated, whether in vitro or in vivo. A more pronounced increase in MHC-I antigen expression was observed when the ESCs were exposed to gamma-interferon, a protein produced in the body during immune reactions. Thus, under some circumstances, human ESC-derived cells can express cell surface molecules that could lead to immune rejection upon allogeneic transplantation.Similarly, Majumdar and colleagues showed that human mesenchymal stem cells in vitro express multiple proteins on their cell surfaces that would enable them to bind to, and interact with, T-lymphocytes. They also observed that gamma-interferon increased expression of both human leukocyte antigen (HLA) class I and class II molecules on the surface of these MSCs.40 These results indicate that it will probably not be possible to predict, solely on the basis of in vitro experiments, the likelihood that transplanted allogeneic MSCs would trigger immune rejection processes in vivo.Many further studies in this area are badly needed. At this time there is insufficient information to determine which, if any, of the approaches to get around the rejection problem will eventually prove successful.
B. Case Study: Stem Cells in the Future Treatment of Type-1 Diabetes?
1. The Disease and Its Causes.
The human body converts the sugar glucose into cell energy for heart and brain functioning, and indeed, for all bodily and mental activities. Glucose is derived from dietary carbohydrates, is stored as glycogen in the liver, and is released again when needed into the bloodstream. A protein hormone called insulin, produced by the beta cells in the islets of the pancreas, facilitates the entrance of glucose from the bloodstream into the cells, where it is then metabolized. Insulin is critical for regulating the body’s use of glucose and the glucose concentration in the circulating blood.The body’s failure to produce sufficient amounts of insulin results in diabetes, an extremely common metabolic disease affecting over 10 million Americans, often with widespread and devastating consequences. In some five to ten percent of cases, known as Type-1 diabetes (or “juvenile diabetes”), the disease is caused by “autoimmunity,” a process in which the body’s immune system attacks “self.”xiv T-lymphocytes attack the patient’s own insulin-producing beta cells in the pancreas. Eventually, this results in destruction of ninety percent or so of the beta cells, resulting in the diabetic state.With a deficiency or absence of insulin, the blood glucose becomes elevated and may lead to diabetic coma, a fatal condition if untreated. Chronic diabetes, both Type-1 and the much more common Type-2 diabetes (which is not autoimmune, but largely genetic), causes late complications in the retina, kidneys, nerves, and blood vessels. It is the leading cause of blindness, kidney failure, and amputations in the U.S. and a major cause of strokes and heart attacks.Type-1 diabetes is a devastating, lifelong condition that currently affects an estimated 550,000-1,100,000 Americans,41 including many children. It imposes a significant burden on the U.S. healthcare system and the economy as a whole, over and above the disabilities and impairments borne by individual sufferers. Recent estimates suggest that treatment of all forms of diabetes costs Americans a total of $132 billion per year.42 At 5-10 percent of all diabetes cases, the costs of Type-1 diabetes can be estimated as $6.5-$13 billion per year.
2. Current Therapy Choices and Outcomes.
The current treatment of Type-1 diabetes consists of insulin injections, given several times a day in response to repeatedly measured blood glucose levels. Although this treatment is life-prolonging, the procedures are painful and burdensome, and in many cases they do not adequately control blood glucose concentrations. Whole pancreas transplants can essentially cure Type-1 diabetes, but fewer than 2,000 donor pancreases become available for transplantation in the U.S. each year, and they are primarily used to treat patients who also need a kidney transplant. Like all recipients of donated organs, pancreas transplant recipients must continuously take powerful drugs to suppress the immunological rejection of the transplanted pancreas.In addition to treatment with whole pancreas transplantation, small numbers of Type-1 diabetes patients have been treated by transplantation of donor pancreatic islets into the liver of the patient coupled with a less intensive immunosuppressive treatment (the Edmonton protocol).43 Expanded clinical trials of this procedure are currently underway. Scientists are also evaluating methods of slowing the original autoimmune destruction of pancreatic beta cells that produces the disease in the first place.Whole pancreas and islet cell transplants ameliorate Type-1 diabetes, but there is nowhere near enough of these materials to treat all in need. To overcome this shortage, people hope that human stem cells can be induced—at will and in bulk—to differentiate in vitro into functional pancreatic beta cells, available for transplantation. Of course, it would still be crucial to prevent immunological destruction of the newly transplanted stem cell-derived beta cells.
indicate, cells derived from some human stem cells transplanted into specific strains of mice mimicking major aspects of Type-1 human diabetes51 were able to reverse high blood glucose concentrations. Although these results are encouraging, the transplant rejection question remains unanswered because the likely immune rejection of the transplanted human cells was prevented in these experiments by using special strains of immunodeficient mice that lack the capacity to recognize and attack foreign cells.No tumors were observed in the transplanted mice, but the experiments were terminated after about three months, an insufficient time for much tumor development to occur. Because many Type-1 diabetes patients are children and because a largely effective therapy (insulin injection) is currently available, the introduction of islet cell transplant therapy will need a high degree of certainty that the introduced cells or their derivatives will not become malignant over the course of the patient’s life. Stringent tests of the cancer-causing potential of candidate cell preparations will be required, including multi-year studies in animals that live longer than mice or rats. Long-term follow-up of children and adult patients who had received bone marrow transplants many years ago has revealed an increased risk of severe neurologic complications52 and a variety of types of cancer.53
C. Therapeutic Applications of Mesenchymal Stem Cells (MSCs)
Before stem cell based therapies are used to treat human diseases, they will have to gain approval through the Food and Drug Administration (FDA) regulatory process. The first step in this process is filing an Investigational New Drug (IND) application. As of July 2003, four IND applications have been filed for clinical applications of mesenchymal stem cells. The disease indications include: (1) providing MSC support for peripheral blood stem cell transplantation in cancer treatment, (2) providing MSC support for cord blood transplantation in cancer treatment, (3) using MSCs to stimulate regeneration of cardiac tissue after acute myocardial infarction (heart attack), and (4) using MSCs to stimulate regeneration of cardiac tissue in cases of congestive heart failure. The first two applications are currently in Phase II of the regulatory process, with pivotal Phase III trials scheduled to begin in 2004.54
D. Evaluating the Different Types of Stem Cells
A major unresolved issue at present involves the therapeutic potential of human adult stem cells compared with embryonic stem cells. The answer may well be different for different diseases and for patients of different ages. For example, in treating an elderly patient with Parkinson’s Disease, the use of adult stem cells may be appropriate even if these cells may have a more limited number of cell divisions remaining. On the other hand, treating a child with Type I Diabetes, one may want to use embryonic stem cells because of their potentially greater longevity, or other factors. The only valid way to resolve these questions is by instituting rigorous therapeutic trials which test the efficacy of the different types of stem cells in treating a variety of different diseases to determine their comparative efficacy. Clearly, such trials would be a long-term endeavor, since it would take years to obtain answers to these very critical questions.
VI. Private Sector Activity
In the United States, much of the basic research on animal stem cells and human adult stem cells has been publicly funded. Yet before 2001, research in the U.S., using human ESCs could only be done in the private sector (the locus also of much research on animal and human adult stem cells). The current state of knowledge about human ESCs (and also about human MSCs) reflects pioneering and on-going stem cell research funded by the private sector in the U.S.54,55 For example, the work that led to the 1998 reports of the first isolation of both ESCs and EGCs, was funded by Geron Corporation. Embryonic and adult stem cell research is today vigorously pursued by many companies and supported by several private philanthropic foundations,56 and the results of some of this research have been published in peer-reviewed journals.57 Private sector organizations have pursued and been awarded patents on the stem cells themselves and methods for producing and using them to treat disease. As noted above, at least one company (Osiris Therapeutics) has protocols under review at the FDA for clinical trials with MSCs. It seems likely that private sector companies will continue to play large roles in the future development of stem cell based therapies.
VII. Preliminary Conclusions
While it might be argued that it is too soon to attempt to draw any conclusions about the state of a field that is changing as rapidly as stem cell research, we draw the following preliminary conclusions regarding the current state of the field.Human stem cells can be reproducibly isolated from a variety of embryonic, fetal, and adult tissue sources. Some human stem cell preparations (for example, human ESCs, EGCs, MSCs, and MAPCs) can be reproducibly expanded to substantially larger cell numbers in vitro, the cells can be stored frozen and recovered, and they can be characterized and compared by a variety of techniques. These cells are receiving a large share of the attention regarding possible future (non-hematopoietic) stem cell transplantation therapies.Preparations of ESCs, EGCs, MSCs, and MAPCs can be induced to differentiate in vitro into a variety of cells with properties similar to those found in differentiated tissues.Research using these human stem cell preparations holds promise for: (a) increased understanding of the basic molecular process underlying cell differentiation, (b) increased understanding of the early stages of genetic diseases (and possibly cancer), and (c) future cell transplantation therapies for human diseases.The case study of developing stem cell-based therapies for Type-1 diabetes illustrates that, although insulin-producing cells have been derived from human stem cell preparations, we could still have a long way to go before stem cell-based therapies can be developed and made available for this disease. This appears to be true irrespective of whether one starts from human embryonic stem cells or from human adult stem cells. The transplant rejection problem remains a major obstacle, but only one among many.Human mesenchymal stem cells are currently being evaluated in pre-clinical studies and clinical trials for several specific human diseases.Much basic and applied research remains to be done if human stem cells are to achieve their promise in regenerative medicine.58 This research is expensive and technically challenging, and requires scientists willing to take a long perspective in order to discover, through painstaking research, which combinations of techniques could turn out to be successful. Strong financial support, public and private, will be indispensable to achieving success.
IV. Basic Research Using Human Stem Cells
Human stem cells are proving useful in basic research in several ways. They are useful in unraveling the complex molecular pathways governing human differentiation. For example, because ESCs can be stimulated in vitro to produce more differentiated cells, this transition can be studied in greater detail and under better-controlled conditions than it can be in vivo. In the best circumstances, these differentiated cells can be grown as largely homogeneous cell populations, and their gene expression profiles can be compared in detail.Also, stem cell preparations can be used to produce populations of specialized cells that are not easily obtained in other ways. In one case, for example, this approach has provided large quantities of human trophoblast-like cells that have not been previously available.35 In addition, cultures of differentiated cells derived from stem cells could be used to test new drugs and chemical compounds for toxicity and mutagenicity.36As experience with these differentiated derivatives of human ESCs grows, it may become possible to reduce or eliminate the use of live animals in such testing protocols.In the near future, the differentiated state of various human cell types will be characterized not just by a few biological markers, but by the pattern and levels of expression of hundreds or thousands of genes. Integration of this knowledge with the catalog of all human genes produced during the Human Genome Project will gradually give us knowledge of which genes are key regulators of human development and which genes are central to maintaining the stem cell state.37 Increased understanding of the molecular pathways of human cell differentiation should eventually lead to the ability to direct in vitro differentiation along pathways that yield cells useful in medical treatment. In addition, when the normal range of gene expression patterns is known, researchers can then determine which genes are expressed abnormally in various diseases, thus increasing our understanding of and ability to treat these diseases.A group of stem cell researchers has recently outlined a set of important research questions that, once answered, will greatly enhance our understanding of human embryonic stem cells and their potential fates and possible uses.38 They include the following:
What is the most effective way to isolate and grow ESCs?
How is the self-renewal of ESCs regulated?
Are all ESC lines the same?
How can ESCs be genetically altered?
What controls the processes of ESC differentiation?
What new tools are needed to measure ESC differentiation in vitro and in vivo?
What is the most effective way to isolate and grow ESCs?
How is the self-renewal of ESCs regulated?
Are all ESC lines the same?
How can ESCs be genetically altered?
What controls the processes of ESC differentiation?
What new tools are needed to measure ESC differentiation in vitro and in vivo?
III. Major Examples of Human Stem Cells
In this section we discuss major examples of human stem cells that meet many of the criteria listed above. Among human adult stem cells, we focus on mesenchymal stem cells (MSCs),4 multipotent adult progenitor cells (MAPCs),3 and neural stem cells, and among human embryonic stem cells, on ESC2 and EGC1 cells. For information on the wide variety of other human stem cell preparations isolated from adult tissues, see reference (4) (Appendix K).Further research on some of these other adult stem cell preparations may demonstrate that they can also be “single cell cloned,” expanded considerably by growth in vitro with retention of normal chromosome structure and number, and preserved by freezing and storage at low temperatures. At that point, it would be very important to compare the properties of these other adult stem cells, and the more differentiated cells that can be derived from them, with the already characterized human embryonic and adult stem cell preparations.
A. Human Adult Stem Cells
1. Human Mesenchymal Stem Cells.
Bone marrow contains at least two major kinds of stem cells: hematopoietic stem cells,10 which give rise to the red cells and white cells of the blood, and mesenchymal stem cells,viii which can be reproducibly isolated and expanded in vitro and that can differentiate in vitro into cells with properties of cartilage, bone, adipose (fat), and muscle cells.14The characteristics (morphology, expressed proteins, and biological properties) of these cells have been somewhat difficult to specify, because they appear to vary depending upon the in vitro culture conditions and the specific cell preparation.15 However, there is a recent report indicating that MSCs, if isolated using three somewhat different methods, give rise to stem cell preparations whose properties are very similar to one another.16 Using dual antibody staining and fluorescence-activated cell sorting, Gronthos and colleagues17 isolated human MSCs in almost pure form and expanded them substantially in vitro. Thus, human MSC preparations isolated in different laboratories by different methods may have similar but not identical properties.A molecular analysis of genes expressed in a single-cell-derived colony of MSCs provided evidence for the activity of genes also turned on in bone, cartilage, adipose, muscle, hematopoiesis-supporting stromal, endothelial, and neuronal cells.15 These results are surprising in that MSCs derived from a single cell appear to be expressing genes associated with multiple major cell lineages. It is possible that different cells within the colony had already entered into distinct differentiation pathways, resulting in a developmentally heterogeneous population composed of several different cell types.Mesenchymal stem cells are important for research and therapy for several reasons. First, because they can be differentiated in vitro into multiple cell types, they make possible detailed research on the molecular events underlying differentiation into bone,18 cartilage, and fat cell lineages. Second, they have recently been shown to support the in vitro growth of human embryonic stem cells.19 Thus, they could replace the mouse feeder cells used previously, obviating the need to satisfy FDA requirements for xenotransplantation, should the ESCs or their derivatives ever be used in human clinical research or transplantation therapy. Third, clinical studies are already underway in which MSCs are co-transplanted with autologous hematopoietic stem cells into cancer patients to replace their blood cell-forming system, destroyed by radiation or high dose chemotherapy.20 It is believed that the MSCs will support the repopulation of the bone marrow by the injected hematopoietic stem cells.In addition, injecting allogeneic MSCs (MSCs from a genetically different human donor) may also prove valuable in modulating the immune system to make it more accepting of foreign tissue grafts [see Itescu review, reference (5)]. Finally, MSCs have the potential for cell-replacement therapies in injuries involving bone, tendon, or cartilage and possibly other diseases. They are, in fact, already being tested as experimental therapies for osteogenesis imperfecta,21 metachromatic leukodystrophy, and Hurler syndrome.22 These last two studies are of great interest, since allogeneic MSCs were used and no serious adverse immune reactions were noted.
2. Multipotent Adult Progenitor Cells (MAPCs).
Verfaillie and coworkers recently described the isolation of MAPCs from rat, mouse, and human bone marrow [see (3) and references cited therein]. Like MSCs, MAPCs can also be differentiated in vitro into cells with the properties of cartilage, bone, adipose, and muscle cells. In addition, there is evidence for the in vitro differentiation of human MAPCs into functional, hepatocyte-like cells,23 a potential that has not so far been shown for MSCs. There is increasing interest in MAPCs, both as potential precursors of multiple differentiated tissues and, ultimately, for possible autologous transplantation therapy.The relationship between human MSCs and the human MAPCs described by Verfaillie and coworkers [see (3)] needs to be clarified by further research. Both kinds of cells are isolated from bone marrow aspirates as cells that adhere to plastic. Each can be differentiated in vitro into cells with cartilage, bone, and fat cell properties. They express several of the same cell antigens, but are reported to differ in a few others.3 MAPCs have to be maintained at specific, low cell densities when grown in vitro, otherwise they tend to differentiate into MSCs.3 It remains important that the isolation and properties of MAPCs be reproduced in additional laboratories.
3. Human Neural Stem Cells.
The nervous system is made up of three major types of cells, neurons or nerve cells proper, and two kinds of supporting or glial cells (oligodendrocyte, astrocyte). Stem cells capable of differentiating into one or more of these neural cell lineages can be isolated from brain tissue (particularly the olfactory bulb and lining of the ventricles)24,25 and grown in vitro. In the presence of purified growth-factor proteins, the population of cells can be expanded by growth in vitro as round clumps of cells called neurospheres. However, many neurospheres grown in culture are developmentally heterogeneous in that they contain more than one neural cell type, and the number of self-renewing cells is frequently low (less than five percent).26Although neural stem cells are still insufficiently understood, they are already proving valuable in basic research on neural development. The ability to grow reproducible neural stem cells in vitro has facilitated identification of important neural stem cell growth factors and their cellular receptors. For example, human neural stem cells from the developing human brain cortex, expanded in culture in the presence of leukemia inhibitory factor (LIF), allowed growth of a self-renewing neural stem cell preparation for up to 110 population doublings. Withdrawal of LIF led to decreased expression of about 200 genes,27 which were specifically identified through use of “gene chips” manufactured by Affymetrix. These genes are presumably involved in promoting or preserving the stem cell’s capacity for self-renewal in the undifferentiated state. The number and specificity of the molecular changes characterized in these experiments powerfully illustrate the usefulness of neural and other stem cell preparations in basic biomedical research.Human neural stem cells are also being injected into animals to test their effects on animal models of human neurological disease. To track the fate of the introduced human cells, they must first be modified or “marked” in ways that permit their specific detection.ix Marked human neural stem cells are easily tracked after they are injected into experimental animals, making it possible to determine whether they survive and migrate following injection. Studies of this type have provided evidence that human neural cells can migrate extensively in the brain after injection.28 In addition, such cells can be injected into animal models of human diseases such as intracerebral hemorrhage and Parkinson Disease (PD) to study their effect on the progression of the disease.29 Although human neural stem cells may not yet be as well characterized as MSCs or ESCs, they are being actively studied with the hope that they can be used in future treatments for devastating neurological diseases such as Alzheimer Disease and PD.
4. Adult Stem Cells from Other Sources.
Prentice [see (4)] has summarized a large amount of recent information on preparations of stem cells isolated from amniotic fluid, peripheral blood, umbilical cord blood, umbilical cord, brain tissue, muscle, liver, pancreas, cornea, salivary gland, skin, tendon, heart, cartilage, thymus, dental pulp, and adipose tissue. Studies of many of the stem cell preparations from these sources are just getting started, and further work is needed to determine their biological properties and their relatedness to other stem cell types. In some cases, the long-term expandability in vitro of these stem cells has not been demonstrated. Yet, the demonstration that they can be isolated from such tissue compartments in animals should spur the search for similar human stem cell types.As Prentice also reports,4 many attempts have already been made using various preparations of adult stem cells to influence or alter the course of diseases in animal models. Despite the fact that the stem cell preparations used are not well characterized, and reproducible results have yet to be obtained, preliminary findings are sometimes encouraging. It is of course not yet clear whether the injected cells are functioning as stem cells, fusing with existing host cells, or stimulating the influx of the host’s own stem cells into the target tissue.x But, if reproduced, these preliminary findings may point the way to future therapies, even in the absence of precise knowledge of the mechanism(s) of cellular action.
B. Human Embryonic Stem Cells
1. Human Embryonic Stem Cells (ESCs).
Human embryonic stem cells have been isolated from the inner cell masses of blastocyst-stage human embryos in multiple laboratories around the world.xi There is great interest in understanding the properties of these cells because they hold out the promise of being able to be differentiated into a large number of different cell types for possible cell therapies, as contrasted with the more limited number of cell types available by differentiation of specific adult stem cell preparations. As of July 2003, 12 ESC preparations (up from 2 such preparations a year earlier) out of a total of 78 “eligible” preparations of human ESCs were available for shipment to recipients of U.S. federal research grants.xii The review by Ludwig and Thomson2 lists more than 40 peer-reviewed human ESC primary research papers that have been published since the initial publication in 1998.Although isolated from different blastocyst-stage human embryos in laboratories in different parts of the world, ESCs have a number of properties in common. These include the presence of common cell surface antigens (recognized by binding of specific antibodies), expression of the enzymes alkaline phosphatase and telomerase, and production of a common gene-regulating transcription factor known as Oct-4. At least 12 different preparations of ESCs have been expanded by growth in vitro, frozen and stored at low temperature, and at least partially characterized.13 Some of these ESC preparations have been “single-cell cloned.”Human ESCs have been differentiated in vitro into neural (neurons, astrocytes, and oligodendrocytes), cardiac (synchronously contracting cardiomyocytes), endothelial (blood vessels), hematopoietic (multiple blood cell lineages), hepatocyte (liver cell), and trophoblast (placenta) lineages.2 In the case of neural and cardiac lineages, similar results have been obtained in different laboratories using different preparations of ESCs, thus fulfilling the “reproducible results” criterion described above. For other lineages, the results described have not yet been reproduced in another laboratory.
2. Embryonic Germ Cells.
Human embryonic germ cells are isolated from the primordial germ tissues of aborted fetuses. Gearhart1 has summarized the results of recent research with human and mouse EG cells. One study focused on regulation of imprinted genes in EG cells: it showed “that general dysregulation of imprinted genes will not be a barrier to their (EG cell) use in transplantation studies.”30 xiii In addition, Kerr and coworkers31 showed that cells derived from human EG cells, when introduced into the cerebrospinal fluid of rats, became extensively distributed over the length of the spinal cord and expressed markers of various nerve cell types. Rats paralyzed by virus-induced nerve-cell loss recovered partial motor function after transplantation with the human cells. The authors suggested that this could be due to the secretion of transforming growth factor-a and brain-derived growth factor by the transplanted cells and subsequent enhancement of rat neuron survival and function.Until recently, work with human EG cells came primarily from one laboratory. Recently the isolation and properties of human EG cells have been independently confirmed.32 Because human EG cells share many (but not all) properties with ESCs, these cells offer another important avenue of inquiry.
3. Embryonic Stem Cells from Cloned Embryos (Cloned ESCs).
Although it has yet to be accomplished in practice, somatic cell nuclear transfer (SCNT) could create cloned human embryos from which embryonic stem cells could be isolated that would be genetically virtually identical to the person who donated the nucleus for SCNT: hence cloned ESCs [see (7)]. In theory, using such cloned embryonic stem cells from individual patients might provide a way around possible immune rejection (see below), though in practice this could require individual cloned embryos for each prospective patient—a daunting task. And clinical uses might require a separate FDA approval for every single cloned stem cell line or its derivatives.The ability to produce cloned mouse stem cells and genetically modify them in vitro has made possible an experiment demonstrating the potential of cloned human embryonic stem cells in the possible future treatment of human genetic diseases. Rideout et al.33 used a mutant mouse strain that was deficient in immune system function. They produced a cloned mouse embryonic stem cell line carrying the mutation, and then specifically repaired that gene mutation in vitro. The repaired cloned stem cell preparation was then differentiated in vitro into bone marrow precursor cells. When these precursor cells were injected back into the genetically mutant mice, they produced partial restoration of immune system function.Production of cloned human embryonic stem cell preparations remains technically very difficult and ethically controversial. Recently however, Chen and coworkers34 have reported that fusion of human fibroblasts with enucleated rabbit oocytes in vitro leads to the development of embryo-like structures from which cell preparations with properties similar to human embryonic stem cells can be isolated. This work needs to be confirmed by repetition in other laboratories. In addition, further work is needed to decisively settle the question of whether rabbit (or human egg donor) mitochondrial DNA and rabbit (or human egg donor) mitochondrial proteins persist in the embryonic stem cell preparations. Persistence of these foreign mitochondrial proteins in these human ESC-like preparations could possibly increase the probability of immune rejection of the cloned cells, thus limiting their clinical application, although the immune reaction might not be as severe as that to foreign proteins produced under the direction of chromosomal genes. The presence of foreign or aberrant mitochondria also carries the risk of transmitting mitochondrial disease (caused by defects in mitochondrial DNA) that could be detrimental to the cells and to the recipient into whom they might eventually be transplanted.
A. Human Adult Stem Cells
1. Human Mesenchymal Stem Cells.
Bone marrow contains at least two major kinds of stem cells: hematopoietic stem cells,10 which give rise to the red cells and white cells of the blood, and mesenchymal stem cells,viii which can be reproducibly isolated and expanded in vitro and that can differentiate in vitro into cells with properties of cartilage, bone, adipose (fat), and muscle cells.14The characteristics (morphology, expressed proteins, and biological properties) of these cells have been somewhat difficult to specify, because they appear to vary depending upon the in vitro culture conditions and the specific cell preparation.15 However, there is a recent report indicating that MSCs, if isolated using three somewhat different methods, give rise to stem cell preparations whose properties are very similar to one another.16 Using dual antibody staining and fluorescence-activated cell sorting, Gronthos and colleagues17 isolated human MSCs in almost pure form and expanded them substantially in vitro. Thus, human MSC preparations isolated in different laboratories by different methods may have similar but not identical properties.A molecular analysis of genes expressed in a single-cell-derived colony of MSCs provided evidence for the activity of genes also turned on in bone, cartilage, adipose, muscle, hematopoiesis-supporting stromal, endothelial, and neuronal cells.15 These results are surprising in that MSCs derived from a single cell appear to be expressing genes associated with multiple major cell lineages. It is possible that different cells within the colony had already entered into distinct differentiation pathways, resulting in a developmentally heterogeneous population composed of several different cell types.Mesenchymal stem cells are important for research and therapy for several reasons. First, because they can be differentiated in vitro into multiple cell types, they make possible detailed research on the molecular events underlying differentiation into bone,18 cartilage, and fat cell lineages. Second, they have recently been shown to support the in vitro growth of human embryonic stem cells.19 Thus, they could replace the mouse feeder cells used previously, obviating the need to satisfy FDA requirements for xenotransplantation, should the ESCs or their derivatives ever be used in human clinical research or transplantation therapy. Third, clinical studies are already underway in which MSCs are co-transplanted with autologous hematopoietic stem cells into cancer patients to replace their blood cell-forming system, destroyed by radiation or high dose chemotherapy.20 It is believed that the MSCs will support the repopulation of the bone marrow by the injected hematopoietic stem cells.In addition, injecting allogeneic MSCs (MSCs from a genetically different human donor) may also prove valuable in modulating the immune system to make it more accepting of foreign tissue grafts [see Itescu review, reference (5)]. Finally, MSCs have the potential for cell-replacement therapies in injuries involving bone, tendon, or cartilage and possibly other diseases. They are, in fact, already being tested as experimental therapies for osteogenesis imperfecta,21 metachromatic leukodystrophy, and Hurler syndrome.22 These last two studies are of great interest, since allogeneic MSCs were used and no serious adverse immune reactions were noted.
2. Multipotent Adult Progenitor Cells (MAPCs).
Verfaillie and coworkers recently described the isolation of MAPCs from rat, mouse, and human bone marrow [see (3) and references cited therein]. Like MSCs, MAPCs can also be differentiated in vitro into cells with the properties of cartilage, bone, adipose, and muscle cells. In addition, there is evidence for the in vitro differentiation of human MAPCs into functional, hepatocyte-like cells,23 a potential that has not so far been shown for MSCs. There is increasing interest in MAPCs, both as potential precursors of multiple differentiated tissues and, ultimately, for possible autologous transplantation therapy.The relationship between human MSCs and the human MAPCs described by Verfaillie and coworkers [see (3)] needs to be clarified by further research. Both kinds of cells are isolated from bone marrow aspirates as cells that adhere to plastic. Each can be differentiated in vitro into cells with cartilage, bone, and fat cell properties. They express several of the same cell antigens, but are reported to differ in a few others.3 MAPCs have to be maintained at specific, low cell densities when grown in vitro, otherwise they tend to differentiate into MSCs.3 It remains important that the isolation and properties of MAPCs be reproduced in additional laboratories.
3. Human Neural Stem Cells.
The nervous system is made up of three major types of cells, neurons or nerve cells proper, and two kinds of supporting or glial cells (oligodendrocyte, astrocyte). Stem cells capable of differentiating into one or more of these neural cell lineages can be isolated from brain tissue (particularly the olfactory bulb and lining of the ventricles)24,25 and grown in vitro. In the presence of purified growth-factor proteins, the population of cells can be expanded by growth in vitro as round clumps of cells called neurospheres. However, many neurospheres grown in culture are developmentally heterogeneous in that they contain more than one neural cell type, and the number of self-renewing cells is frequently low (less than five percent).26Although neural stem cells are still insufficiently understood, they are already proving valuable in basic research on neural development. The ability to grow reproducible neural stem cells in vitro has facilitated identification of important neural stem cell growth factors and their cellular receptors. For example, human neural stem cells from the developing human brain cortex, expanded in culture in the presence of leukemia inhibitory factor (LIF), allowed growth of a self-renewing neural stem cell preparation for up to 110 population doublings. Withdrawal of LIF led to decreased expression of about 200 genes,27 which were specifically identified through use of “gene chips” manufactured by Affymetrix. These genes are presumably involved in promoting or preserving the stem cell’s capacity for self-renewal in the undifferentiated state. The number and specificity of the molecular changes characterized in these experiments powerfully illustrate the usefulness of neural and other stem cell preparations in basic biomedical research.Human neural stem cells are also being injected into animals to test their effects on animal models of human neurological disease. To track the fate of the introduced human cells, they must first be modified or “marked” in ways that permit their specific detection.ix Marked human neural stem cells are easily tracked after they are injected into experimental animals, making it possible to determine whether they survive and migrate following injection. Studies of this type have provided evidence that human neural cells can migrate extensively in the brain after injection.28 In addition, such cells can be injected into animal models of human diseases such as intracerebral hemorrhage and Parkinson Disease (PD) to study their effect on the progression of the disease.29 Although human neural stem cells may not yet be as well characterized as MSCs or ESCs, they are being actively studied with the hope that they can be used in future treatments for devastating neurological diseases such as Alzheimer Disease and PD.
4. Adult Stem Cells from Other Sources.
Prentice [see (4)] has summarized a large amount of recent information on preparations of stem cells isolated from amniotic fluid, peripheral blood, umbilical cord blood, umbilical cord, brain tissue, muscle, liver, pancreas, cornea, salivary gland, skin, tendon, heart, cartilage, thymus, dental pulp, and adipose tissue. Studies of many of the stem cell preparations from these sources are just getting started, and further work is needed to determine their biological properties and their relatedness to other stem cell types. In some cases, the long-term expandability in vitro of these stem cells has not been demonstrated. Yet, the demonstration that they can be isolated from such tissue compartments in animals should spur the search for similar human stem cell types.As Prentice also reports,4 many attempts have already been made using various preparations of adult stem cells to influence or alter the course of diseases in animal models. Despite the fact that the stem cell preparations used are not well characterized, and reproducible results have yet to be obtained, preliminary findings are sometimes encouraging. It is of course not yet clear whether the injected cells are functioning as stem cells, fusing with existing host cells, or stimulating the influx of the host’s own stem cells into the target tissue.x But, if reproduced, these preliminary findings may point the way to future therapies, even in the absence of precise knowledge of the mechanism(s) of cellular action.
B. Human Embryonic Stem Cells
1. Human Embryonic Stem Cells (ESCs).
Human embryonic stem cells have been isolated from the inner cell masses of blastocyst-stage human embryos in multiple laboratories around the world.xi There is great interest in understanding the properties of these cells because they hold out the promise of being able to be differentiated into a large number of different cell types for possible cell therapies, as contrasted with the more limited number of cell types available by differentiation of specific adult stem cell preparations. As of July 2003, 12 ESC preparations (up from 2 such preparations a year earlier) out of a total of 78 “eligible” preparations of human ESCs were available for shipment to recipients of U.S. federal research grants.xii The review by Ludwig and Thomson2 lists more than 40 peer-reviewed human ESC primary research papers that have been published since the initial publication in 1998.Although isolated from different blastocyst-stage human embryos in laboratories in different parts of the world, ESCs have a number of properties in common. These include the presence of common cell surface antigens (recognized by binding of specific antibodies), expression of the enzymes alkaline phosphatase and telomerase, and production of a common gene-regulating transcription factor known as Oct-4. At least 12 different preparations of ESCs have been expanded by growth in vitro, frozen and stored at low temperature, and at least partially characterized.13 Some of these ESC preparations have been “single-cell cloned.”Human ESCs have been differentiated in vitro into neural (neurons, astrocytes, and oligodendrocytes), cardiac (synchronously contracting cardiomyocytes), endothelial (blood vessels), hematopoietic (multiple blood cell lineages), hepatocyte (liver cell), and trophoblast (placenta) lineages.2 In the case of neural and cardiac lineages, similar results have been obtained in different laboratories using different preparations of ESCs, thus fulfilling the “reproducible results” criterion described above. For other lineages, the results described have not yet been reproduced in another laboratory.
2. Embryonic Germ Cells.
Human embryonic germ cells are isolated from the primordial germ tissues of aborted fetuses. Gearhart1 has summarized the results of recent research with human and mouse EG cells. One study focused on regulation of imprinted genes in EG cells: it showed “that general dysregulation of imprinted genes will not be a barrier to their (EG cell) use in transplantation studies.”30 xiii In addition, Kerr and coworkers31 showed that cells derived from human EG cells, when introduced into the cerebrospinal fluid of rats, became extensively distributed over the length of the spinal cord and expressed markers of various nerve cell types. Rats paralyzed by virus-induced nerve-cell loss recovered partial motor function after transplantation with the human cells. The authors suggested that this could be due to the secretion of transforming growth factor-a and brain-derived growth factor by the transplanted cells and subsequent enhancement of rat neuron survival and function.Until recently, work with human EG cells came primarily from one laboratory. Recently the isolation and properties of human EG cells have been independently confirmed.32 Because human EG cells share many (but not all) properties with ESCs, these cells offer another important avenue of inquiry.
3. Embryonic Stem Cells from Cloned Embryos (Cloned ESCs).
Although it has yet to be accomplished in practice, somatic cell nuclear transfer (SCNT) could create cloned human embryos from which embryonic stem cells could be isolated that would be genetically virtually identical to the person who donated the nucleus for SCNT: hence cloned ESCs [see (7)]. In theory, using such cloned embryonic stem cells from individual patients might provide a way around possible immune rejection (see below), though in practice this could require individual cloned embryos for each prospective patient—a daunting task. And clinical uses might require a separate FDA approval for every single cloned stem cell line or its derivatives.The ability to produce cloned mouse stem cells and genetically modify them in vitro has made possible an experiment demonstrating the potential of cloned human embryonic stem cells in the possible future treatment of human genetic diseases. Rideout et al.33 used a mutant mouse strain that was deficient in immune system function. They produced a cloned mouse embryonic stem cell line carrying the mutation, and then specifically repaired that gene mutation in vitro. The repaired cloned stem cell preparation was then differentiated in vitro into bone marrow precursor cells. When these precursor cells were injected back into the genetically mutant mice, they produced partial restoration of immune system function.Production of cloned human embryonic stem cell preparations remains technically very difficult and ethically controversial. Recently however, Chen and coworkers34 have reported that fusion of human fibroblasts with enucleated rabbit oocytes in vitro leads to the development of embryo-like structures from which cell preparations with properties similar to human embryonic stem cells can be isolated. This work needs to be confirmed by repetition in other laboratories. In addition, further work is needed to decisively settle the question of whether rabbit (or human egg donor) mitochondrial DNA and rabbit (or human egg donor) mitochondrial proteins persist in the embryonic stem cell preparations. Persistence of these foreign mitochondrial proteins in these human ESC-like preparations could possibly increase the probability of immune rejection of the cloned cells, thus limiting their clinical application, although the immune reaction might not be as severe as that to foreign proteins produced under the direction of chromosomal genes. The presence of foreign or aberrant mitochondria also carries the risk of transmitting mitochondrial disease (caused by defects in mitochondrial DNA) that could be detrimental to the cells and to the recipient into whom they might eventually be transplanted.
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