What is a stem cell?
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 How are pluripotent 
    stem cells derived?
  Potential Applications 
   of Pluripotent Stem Cells
 Stem cells — what lies 
    ahead?
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 Gene and Stem Cell   
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Stem cells — what lies ahead?
The following chapters explore some of the cutting edge research featuring stem cells. Disease and disorders with no therapies or at best, partially effective ones, are the lure of the pursuit of stem cell research. Described here are examples of significant progress that is a prologue to an era of medical discovery of cell-based therapies that will one day restore function to those whose lives are now challenged every day — but perhaps in the future, no longer.

Contents ( Read information in .pdf by Acrobat Reader)

	Preface
	Executive Summary
	Chapter 1: The Stem Cell
	Chapter 2: The Embryonic Stem Cell
	Chapter 3: The Human Embryonic Stem Cell and The Human Embryonic                           Germ Cell
	Chapter 4: The Adult Stem Cell
	Chapter 5: Hematopoietic Stem Cells
	Chapter 6: Autoimmune Diseases and the Promise of Stem Cell-Based                       Therapies
	Chapter 7: Stem Cells and Diabetes
	Chapter 8: Rebuilding the Nervous System with Stem Cells
	Chapter 9: Can Stem Cells Repair a Damaged Heart?
	Chapter 10: Assessing Human Stem Cell Safety
	Chapter 11: Use of Genetically Modified Stem Cells in Experimental Gene                         Therapies
	Appendix A: Early Development
	Appendix B: Mouse Embryonic Stem Cells
	Appendix C: Human Embryonic Stem Cells and Embryonic Germ Cells
	Appendix D: Stem Cell Tables
		i. Published Reports on Isolation and Differentiation of Mouse Stem Cells
		ii. Published Reports on Isolation and Differentiation of Human Fetal Tissue      Germ Cells
		iii. Published Reports on Isolation and Differentiation of Human Embryonic        Stem Cells
		iv. Published Reports on Isolation and Differentiation of Human Embryonic        Carcinoma Stem Cells
		v. Published Reports on Isolation and Differentiation of Human Adult Stem       Cells
		vi. References
	Appendix E: Stem Cell Markers
		i. Markers: How Do Researchers Use Them to Identify Stem Cells?
		ii. Commonly Used Markers to Identify Stem Cells and Characterize      Differentiated Cell Types
	Appendix F: Glossary and Terms
		i. Glossary
		ii. Terms
	Appendix G: Informational Resources
		i. Persons Interviewed
		ii. Special Contributions
		iii. Acknowledgments

Gene and stem cell therapies hold promise for the treatment of a wide variety of inherited and acquired human diseases. Identification of genes involved in human disease and development of novel vectors and devices for delivering therapeutic genes to different tissues in vivo have resulted in significant progress in the area of gene therapy. Isolation of stem cells from organs formerly thought to have no regenerative potential, the demonstration of stem cell plasticity, and the creation of human embryonic stem cells clearly demonstrate the feasibility of human stem cell therapy. Much additional work remains to be done in the areas of vector development and stem cell biology before the full therapeutic potential of these approaches can be realized. Of equal importance, the ethical issues surrounding gene- and cell-based therapies must be confronted.

JAMA. 2001;285:545-550

Individuals are born with a relatively fixed genetic status that in combination with environmental factors determines the propensities for a variety of disease states. Until recently, the concept that the genetic status of the individual is fixed and unalterable was widely accepted, and lifestyle, pharmacological, and surgical therapies were developed to treat patients in whom genetic and environmental influences combined to produce disease. For example, a patient with familial hypercholesterolemia due to a heterozygous mutation in the low-density lipoprotein (LDL) receptor gene may be advised to give up smoking and start treatment with HMG Co-A (3-hydroxy 3-methylglutaryl coenzyme A) reductase inhibitors (statins) to decrease cholesterol synthesis and undergo coronary artery bypass grafting to treat his/her coronary atherosclerosis. Although successful in ameliorating the symptoms and progression of this disorder, none of these therapies directly address the genetic cause of the disease in either the patient or the offspring.

During the last several years, advances in human genetics, cell biology, and gene therapy have resulted in a fundamental change in this therapeutic paradigm. Physicians in the new millennium will not only use therapies to help patients live better with their genetic constitutions, but also will use novel therapies to alter the genetic makeup of the patient. Somatic gene therapy and stem cell transplantation are 2 of the most promising of these novel treatment modalities.

This article summarizes recent advances in gene and stem cell therapies with particular emphasis on the therapeutic potentials and the significant hurdles that must be overcome for effective treatment of diseases. Some important ethical questions raised by such novel treatments are also discussed. The scope and the extent of progress in these areas preclude a comprehensive review; the reader is referred to several excellent and comprehensive reviews for additional information.^1-5

Gene Therapy

Gene therapy can be most simply defined as the genetic modification of cells to produce a therapeutic effect.¹ Such genetic modifications can be carried out in cultured cells that are subsequently administered to the patient (ex vivo approaches) or involve in vivo modifications of cells (in vivo approaches) (Figure 1). Most early studies of gene therapy involved attempts to replace a defective gene with a normal copy of that gene in patients with single-gene genetic disorders. Examples include replacement of the cystic fibrosis transmembrane regulator (CFTR) gene in the respiratory epithelium of patients with cystic fibrosis,⁶ the replacement of the dystrophin gene in the muscle of patients with Duchenne muscular dystrophy,⁷ and the replacement of the LDL receptor gene in the livers of patients with familial hypercholesterolemia.⁸

Other work, however, suggests that the major use of gene therapy will involve the genetic modification of cells to produce a therapeutic effect in complex or acquired diseases in which the genetic bases are not completely understood. Examples include the use of cytotoxic gene therapies such as the herpes simplex virus thymidine kinase gene and ganciclovir for the treatment of cancer,⁹ cytostatic therapies such as the introduction of the retinoblastoma gene into vascular smooth muscle cells for the treatment of restenosis following balloon angioplasty or stenting,¹⁰ and the use of angiogenic genes such as the vascular endothelial growth factor (VEGF) gene for the treatment of ischemic cardiomyopathy.¹¹

Most approaches to gene therapy involve 3 interacting components: a therapeutic gene or other nucleic acid (eg, RNA molecule or a synthetic oligonucleotide), a vector that allows delivery of the therapeutic nucleic acid to the appropriate cell, and a device (eg, a catheter, syringe, or stent) to deliver the gene/vector combination to the appropriate tissue in vivo.

Progress has been made in each of these 3 areas. Human geneticists and genomic scientists have identified the genes involved in many single gene disorders ranging from the muscular dystrophies¹² to the hyperlipidemias¹³ and some cancers.^{14, 15} Similar approaches are being used to elucidate the genetic bases of complex multigenic disorders such as diabetes mellitus and Alzheimer disease. Completion of the Human Genome Project promises to accelerate this progress and to provide an expanding list of potential therapeutic genes. Thus, the availability of therapeutic genes will likely not limit the future of genetic therapies.

Progress has also been made in vector development for gene therapy. However, problems in this area continue to limit most gene therapy approaches. The ideal vector would be easily produced in pure form at high titers, would efficiently and stably transduce nonproliferating cells in vivo, and would enable long-term transgene expression without producing cytotoxic effects, inflammation, or immune responses. Such a vector also might be capable of tissue-specific targeting and transgene expression and allow for pharmacologically or physiologically regulated transgene expression.

Unfortunately, such an ideal vector has not yet been developed. Briefly, plasmid DNA vectors are easy to produce and manipulate and are capable of stably transducing cells both in vitro and in vivo. However, these vectors are inefficient in delivering trangenes to nonproliferating cells and can cause immune responses directed against CpG repeat sequences.¹⁶ In contrast, adenovirus vectors are easy to produce in high titers and can transduce most cell types with high efficiencies but produce significant local tissue inflammation and potent immune responses that limit the duration of transgene expression. The fact that most retroviral vectors require cell proliferation for efficient transduction limits their usefulness for in vivo gene therapies, but they efficiently and stably transduce proliferating cells in vitro and are relatively nonimmunogenic, making them useful for ex vivo approaches. Indeed, 2 children with X-linked immunodeficiency have been cured following ex vivo infection of their hematopoietic stem cells with a retroviral vector expressing the common chain of the interleukin 2 receptor.¹⁷

Adeno-associated virus¹⁸ (AAV) vectors and the lentivirus¹⁹ (based on human immunodeficiency virus [HIV]) vectors are the most promising gene therapy vectors. Like adenoviruses, these vectors can stably and efficiently transduce nonproliferating cells and can be rendered less immunogenic and less inflammatory. Consequently, both of these vector systems can induce long-term, high-level transgene expression. Ongoing clinical trials of AAV vectors that express clotting factor IX in patients with hemophilia B should help to define the utility of this system.²⁰ Human trials of lentivirus vectors are likely several years away.

Despite their promise, current versions of both AAV and lentivirus vectors have disadvantages. Adeno-associated virus vectors can only accommodate transgenes of less than 4.5 kilobases and are difficult to produce in large quantitities. Lentiviruses contain some residual HIV genes and also are difficult to produce.

Many applications of gene therapy will require targeting transgene expression to the appropriate cell type in vivo and the regulation of transgene expression by drugs or physiological cues. Research in these areas is just beginning, but preliminary results in cultured cells and animals are promising. For example, envelope or capsid proteins on the surface of retrovirus²¹ and adenovirus²² vectors can be modified to enhance gene delivery to specific cell types. Similarly, by using tissue-specific transcriptional regulatory elements (eg, muscle- or liver-specific transcriptional enhancers and promoters), adenovirus and AAV vectors can be modified to program transgene expression in specific cell types in vivo. Also, by using synthetic promoter systems, transgene expression in animals can be regulated by rapamycin²³ or tetracycline.²⁴ Similarly, the incorporation of hypoxia-inducible elements into gene therapy vectors can be used to produce hypoxia inducible gene expression both in vitro and in vivo.²⁵

In contrast to the progress in gene discovery and vector development, the development of gene delivery devices is just beginning. The importance of this area is underscored by the report that most commercially available gene delivery catheters rapidly and efficiently inactivate adenovirus vectors.²⁶ Therefore, it is critical to produce and rigorously test the compatibility of gene delivery devices with each gene therapy vector prior to initiating clinical trials.

In summary, progress in the areas of gene discovery, vector development, and transgene regulation have accelerated the pace of progress of gene therapy. Despite the negative publicity surrounding the tragic complications associated with some of the gene therapy clinical trials,²⁷ successful gene therapies have now been reported in humans.¹⁷ Nevertheless, much work remains to be done before human gene therapy is safe and effective. In particular, vectors are needed that can be easily produced at high titers and in large quantities, that can be safely targeted to specific cell types, and that can produce regulated transgene expression. Devices also are needed for efficient and targeted delivery of these vectors to the appropriate tissues in vivo. Finally, a better understanding is needed of both normal cell biology and the biochemical and genetic bases of human disease pathways to facilitate the design of novel genetic therapies for common human diseases.

Stem Cell Therapy

During normal human embryogenesis, the totipotent fertilized egg differentiates into a wide variety of cell types that form the adult organs. Many mature organs, including the bone marrow (hematopoietic system), skin, and small intestine, maintain a pool of undifferentiated stem cells that are capable of both self-renewal and of differentiating into at least 1 or more mature cell types. Such stem cells make it possible to regenerate damaged or senescent cells throughout life.

Physicians have exploited stem cells for therapeutic purposes for more than 40 years. For example, hematopoietic stem cell transplantation (ie, bone marrow transplantation) is life-saving for patients with certain types of bone marrow diseases and malignancies. However, the usefulness of stem cell transplantation has been limited by the fact that many organs (brain, spinal cord, heart, kidney) were thought to lack detectable stem cells. It was also believed that cells from these organs could not be reprogrammed to differentiate into different cell lineages during adulthood.

Three recent discoveries have revolutionized stem cell biology and have demonstrated the clinical potential of these cells in a wide range of human diseases (Figure 2). First, stem cells have been detected in organs, such as brain and muscle, previously thought to lack stem cells and regenerative potential. For example, several areas of the brain contain stem cells that maintain the ability to proliferate and to mature into different neural cell types in vitro and in vivo.^28-30 Animal studies have suggest that proliferating cells in the central nervous system play a role in learning and memory.³¹ Moreover, such cells can be cultured and transplanted into the central nervous systems of recipients where they differentiate into mature neurons. Similarly, skeletal muscle stem cells (myoblasts) can be cultured in vitro and transplanted into recipient muscle where they differentiate into myotubes and fuse with endogenous muscle fibers to repopulate damaged muscle.^{32, 33}

Second, organ-specific adult stem cells appear to display much more plasticity than originally thought. Stem cells isolated from one tissue can differentiate into a variety of unrelated cell types and tissues. For example, recent animal experiments have demonstrated that neural stem cells can differentiate into hematopoietic lineages.³⁴ Similarly, bone marrow–derived stem cells can differentiate into several nonhematopoietic cell types, including skeletal muscle,^{32, 35} microglia and astroglia in the brain,^{36, 37} and hepatocytes.³⁸ These findings raise the exciting possibility of using bone marrow transplantation to treat a wide variety of disorders, such as muscular dystrophies, Parkinson disease, stroke, and hepatic failure.

Perhaps the most remarkable demonstration of cell plasticity has come from animal cloning experiments. In 1997, researchers in England reported the cloning of a sheep (the now famous "Dolly") by transferring a mammary gland cell nucleus into an oocyte.³⁹ Mice, cows, and monkeys have been cloned subsequently using similar techniques. These experiments demonstrate that nuclei from terminally differentiated cells can be reprogrammed to totipotency. Thus, it might be possible to generate specific types of therapeutic stem cells in vitro starting with a small number of differentiated cells from the patient to be treated (eg, a skin or muscle biopsy specimen), thereby avoiding immune responses to the transplanted cells.

Third, human embryonic stem cells^{40, 41} can be isolated from early fetuses and made to differentiate in vitro into a wide variety of cell types. Embryonic stem cells are totipotent cells⁴² derived from the inner cell mass of an early stage fertilized embryo. Under appropriate tissue culture conditions, embryonic stem cells have the capacity for unlimited replication while maintaining totipotency, and when reimplanted into a blastocyst, such cultured embryonic stem cells can contribute to all of the organs of the resulting adult animal.

Moreover, cultured embryonic stem cells can differentiate into a wide variety of cell types in vitro, including hematopoietic cells, skeletal and cardiac myocytes, and adipocytes. Such embryonic stem cells also may have important therapeutic potential. For example, in a rat model of a hereditary human demyelinating disorder (Pelizaeus-Merzbacher disease), rodent embryonic stem cells that were differentiated in vitro into oligodendrocytes and astrocytes were successfully transplanted to generate myelin in various areas of the brain.⁴³ These results, in conjunction with the isolation of human embryonic stem cells, have significant implications for patients with this rare hereditary myelin deficiency. More important, the differentiation of human embryonic stem cells into different types of homogeneous precursor populations holds promise for the treatment of a variety of diseases requiring tissue repair or reconstitution, such as stroke, neurodegenerative diseases, myocardial infarction, and hepatic failure.

In summary, the discovery of stem cells in adult tissues, the unexpected plasticity of both adult stem cells and differentiated cells, and the isolation of human embryonic stem cells have expanded the potential therapeutic utility of cell-based therapies. Stem cell therapy, like gene therapy, is in its infancy. Increased understanding of how to isolate and culture human stem cells and how to regulate their survival and differentiation (and dedifferentiation) in vitro and in vivo is needed. Nevertheless, cell-based therapies using autologous donor cells hold tremendous promise for the treatment of both acquired and inherited diseases involving tissue degeneration and cellular dysfunction.

Combination Therapies

Gene and stem cell therapies by themselves hold promise for the treatment of a variety of human disease, but combinations of these approaches may be even more useful for certain disorders (Figure 2E). For example, implantation of skeletal muscle stem cells that have been modified genetically with vectors that program the expression and secretion of therapeutic proteins, such as erythropoietin or growth hormone, results in the stable delivery of recombinant proteins to the systemic circulation.⁴⁴ Similarly, the genetic reconstitution of myocytic or hepatic stem cells lacking specific gene products with a normal copy of the defective gene might be useful in the treatment of patients with inherited single gene mutation, such as hemophilia and muscular dystrophy. (Figure 3)

Ethical Considerations

Like many novel therapeutic approaches, gene and stem cell therapies raise a number of difficult and important ethical issues and concerns. Some are common to any new therapy involving human experimentation, whereas others are more unique to the specific genetic and cellular methods used in gene and stem cell therapeutics. These ethical debates will continue to be an important determinant of the progress and future of these therapies.

As is true of all areas of experimental therapeutics, translating basic scientific advances into clinical efficacy is a difficult and risky challenge that requires time, trust, and patience on the part of both physicians and patients. Given the uncertainties of clinical trials, open, honest and timely communication is critical to build trust between physicians, patients, and the general public. Unbridled enthusiasm as to the long-term potential of these therapies has led investigators to make unrealistic short-term promises regarding both efficacy and safety. Researchers and clinicians must communicate clearly that these therapies are in early development and therefore unlikely to produce widespread cures over the next decade. Likewise, the risks and benefits of experimental gene therapy approaches must be honestly presented to patients and their families, and adverse events from gene therapy trials must be reported to patients, families, and appropriate regulatory bodies.

Gene therapy, like many other fields of biomedical research, is affected by real and perceived conflicts of interest among some of its leading investigators, many of whom have failed to disclose financial interests in companies with which they are conducting clinical trials. The American Society of Gene Therapy suggested that clinical investigators involved in gene therapy trials should not have personal financial relationships (of any magnitude) with companies that may benefit from the results of these trials.

Several important and unique ethical concerns have been raised about gene and stem cell therapies. First, many members of the public are troubled by perceived and actual problems associated with altering the genetic composition of humans. Specific concerns have been raised about the appropriate traits to be selected for genetic modification. For example, few would disagree that gene therapy (if safe and efficacious) would be an appropriate therapy for most cancers. The use of this technology, however, to modify height, weight, or memory is problematic and controversial. Second, there is concern about the potential for inadvertently (or purposely) altering the genetic composition of germ cells, thereby resulting in germ line transmission of a gene therapy vector to the progeny of the treated patient. Similarly, the area of stem cell therapy has been plagued by concerns surrounding the source of stem cellsmore specifically, the use of human fetal tissue for stem cell isolation.^{45, 46} Balancing the therapeutic potential of fetal tissue with ethical concerns about abortion and fetal tissue experimentation remains one of the significant ethical challenges ahead.

Summary

Gene and stem cell research hold great promise for the development of novel therapies for important and prevalent human diseases. Much work remains before the therapeutic potential of these approaches will be fully understood. Like most medical therapies, these approaches can also be used in irresponsible and unethical ways, resulting in harm to patients and society. Accordingly, it is essential that clinical trials of gene and stem cell therapies be based on a solid foundation of basic scientific and animal experimentation and carried out with the highest medical and ethical standards. Of equal importance, vigorous and open ethical debates based on scientific and medical facts and the timely and honest communication between physicians, patients, and the public are essential to realize the full potential of these therapeutic approaches.