Human Pluripotential Stem Cells

Abstract

After completing this article, readers should be able to:The dream of one day being able to provide an unlimited supply of human tissues for transplantation came one step closer 2 years ago when two teams of scientists from Johns Hopkins University and the University of Wisconsin announced the successful derivation of human pluripotential stem cells (PSCs). This research immediately caught the public’s eye because of its enormous impact on transplantation therapies and the sources of tissues. Human stem cells are renewable in culture and are capable of differentiating into a wide variety of tissue types. The unlimited ability to divide and the capability to form into almost every cell type provide the source of replacement cells for transplantation and raise the hopes of numerous patients who have debilitating conditions, such as Parkinson disease, Alzheimer disease, stroke, and type I diabetes. Human stem cells will be important for in vitro studies of human gene discovery, for pharmaceutical research such as drug toxicology studies for screening and testing, and as a renewable source of cells for tissue transplantation and gene therapies. In addition to clinical applications, human stem cells provide a powerful tool for biomedical research into human embryogenesis, specific gene functions, and lineage development.PSCs, primarily embryonic stem (ES) cells, have been used extensively in studies of embryogenesis, gene function, and development in the mouse. Present in the early stages of embryo development, PSCs can generate all of the cell types in a fetus and an adult and are capable of self-renewal. However, they cannot form an embryo because they are unable to give rise to the placental tissues necessary for development in the uterus. In other words, PSCs do not have the capacity to develop into a conceptus (ie, an embryo) and its extraembryonic membranes (those forming placental tissues). In the mouse, PSCs can be derived from: 1) blastocysts, a preimplantation stage embryo; 2) primordial germ cells, cells of the early embryo that eventually differentiate into sperm and oocytes; or 3) teratocarcinomas, tumors that have the capacity to produce many types of tissues, such as cartilage, bone, nervous tissue, epithelia, teeth, and smooth and cardiac muscle.Mouse ES cells are derived from the inner cell mass (ICM) of blastocysts. In culture, they can replicate indefinitely in an undifferentiated state when maintained on feeder cells. When introduced into the ICM of a host blastocyst, they can integrate into the embryo and contribute to all tissue types, including germ line. Teratomas, tumors that consist of derivatives of all three definitive germ layers, can be obtained when ES cells are injected in ectopic sites of mice. In vitro, these cells can differentiate spontaneously into many cell types under appropriate culture conditions.Embryonic germ (EG) cells can be derived from germ cell precursors, primordial germ cells (PGCs). In mice, EG cells can be derived efficiently from PGCs on feeder layers with leukemia inhibitory factor, stem cell factor, and basic fibroblast growth factor. Like ES cells, EG cells can contribute to various lineages in chimera, including germ cells. Teratomas can be obtained on injection into mice as well. In vitro, EG cells can differentiate spontaneously into many cell types, including muscle cells, neurons, and hematopoietic cells.Embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas. They can be derived from teratocarcinomas, induced by the surgical transplantation of early-stage postimplantation embryos or genital ridges to extrauterine sites, or derived from spontaneous tumors usually seen in the gonads of a few inbred strains of mice, particularly in strain 129. Similar to ES and EG cells, they can differentiate into a variety of tissue types in vitro. EC cells are also capable of contributing to somatic tissues followed by blastocyst injection. However, their capacity to passage to the germ line is somehow limited. In addition, most of the EC cell lines are hyperdiploid, with nonrandom chromosomal rearrangement. To date, several EC cell lines have been derived from germ cell tumors in humans. NTERA2, or NT2, a human teratocarcinoma cell line, can differentiate into postmitotic neurons after retinoic acid treatment. It has been reported that transplanted NT2-derived neurons could integrate into the host central nervous systems of immunocompromised mice, extend axon-like and dendrite-like processes, and retain a postmitotic phenotype for more than 1 year. A clinical trial of neurotransplantation in stroke patients using these neural precursors differentiated from EC cells has been conducted at University of Pittsburgh. Due to their pluripotency, ES cells have been widely used in experimental biology. ES cells are particularly valuable for the generation of “knock-out” mice by gene targeting via homologous recombination followed by injection into blastocysts. In vitro, as ES cells differentiate in culture, they give rise to structures resembling early postimplantation embryos called embryoid bodies (EBs). In mice, EBs consist of extraembryonic endoderm, which surrounds the outer layers, and ectoderm, which occupies the inner layer of the EBs. Within the EBs, cell-cell interactions result in the initial events of cell differentiation, perhaps recapitulating the processes that occur in normal embryogenesis. Thus, the in vitro differentiation of ES cells could, to some degree, mimic the differentiation pathway of certain cell types in early embryogenesis, which enables further understanding of early embryonic development, gene functions, and molecular mechanisms related to cell type determination and differentiation.For the past few years, many culture regimens have been established to direct, select, or enhance ES cell differentiation into specialized cell types, including hematopoietic cells, neural cells, and cardiomyocytes. Of all the culture systems, hematopoietic differentiation from ES cells is probably the best characterized. In most cases, different lineages of hematopoietic cells can be obtained by the formation of EBs followed by dissociation and seeding in a semisolid medium that contains cytokines, including interleukin-3 (IL-3), IL-6, erythropoietin, stem cell factor, and vascular endothelial growth factor. Under appropriate culture conditions, erythroid cells, macrophages, neutrophils, mast cells, megakaryocytes, and to a lesser extent, lymphoid lineages can be derived from ES cells in a highly reproducible fashion. Furthermore, the sequence of events leading to lineage commitment in culture roughly parallels that seen in vivo. Thus, this culture system provides a valuable tool for studies of early hematopoiesis. In fact, growth factors and cytokines involved in hematopoiesis have been examined in great detail by a large number of investigators. Gene expression and molecular mechanisms related to the differentiation process are also well characterized using this culture system.Although different lineages of hematopoietic cells can be derived from ES cells, their long-term repopulating capacity remains unanswered. Palacio and coworkers differentiated ES cells by coculturing with a marrow stromal cell line and a combination of cytokines. The ES-derived cells from this procedure were able to reconstitute the lymphoid-, myeloid-, and erythroid-cell lineages of primary and secondary irradiated mice. The success of this experiment could be due to the microenvironment provided by stromal cells, which contains critical growth factors or matrices lacking in other culture systems.Neural differentiation from ES cells has also been studied extensively for the past few years. In most studies, inducing agents such as retinoic acid, dimethyl sulfoxide, and methoxybenzamide are used in the differentiation protocol. ES cells are usually differentiated after retinoic acid treatment and followed by dissociation and plating on permissive substrates precoated with cell matrix. Both neurons and glia lineages can be obtained from this procedure, as confirmed by a panel of antibodies against proteins specific to neuronal and glial cells, such as neurofilament and glial fibrillary acidic protein, respectively. In addition, gene expression and electrophysiologic analysis were performed to characterize these ES-derived cells further. Due to the pluripotency of ES cells, it is not surprising to see other cell types in these cultures in addition to neural cells. Some culture systems using serum-free medium in the presence of growth factors also have been established, which permit a more efficient differentiation of neural precursors from ES cells. In culture medium controlled by basic fibroblast growth factor, the majority of the ES-derived cells were stained with the neuroepithelial precursor cell marker nestin. In addition, ES-derived cells have been successfully transplanted into ventricles of embryonic rats and have demonstrated reconstitution of neuronal and glial lineages in the central nervous system.Most recently, McKay and colleagues demonstrated that precursors of oligodendrocytes and astrocytes could be differentiated efficiently from ES cells using defined factors. These precursors then were transplanted into the spinal cords of 1-week-old myelin-deficient rats, which carry mutations in the X-linked gene encoding myelin proteolipid protein (PLP). The ES cell-derived precursors interacted with host neurons and efficiently myelinated axons in brain and spinal cord. In addition, the researchers performed intraventricular transplants in embryonic day 17 (E17) hosts. PLP-positive myelin sheaths were observed in a variety of brain regions, including cortex and corpus callosum, anterior commissure, hippocampus, tectum, thalamus, and hypothalamus.McDonald and colleagues used EBs differentiated from ES cells after retinoic acid treatment for transplantation. They transplanted these ES-derived cells into rat spinal cord 9 days after contusion injury. Histologic analysis showed that the transplants successfully integrated and differentiated into astrocytes, oligodendrocytes, and neurons. Furthermore, the transplanted rats showed hindlimb weight support and partial hindlimb coordination. These studies indicate that not only can highly differentiated cells be derived from these PSCs, but such cells can integrate into the host and function.Rhythmic contraction, which can be observed regularly during ES cell differentiation, indicates the development of cardiac muscle. It was reported that myosin heavy- and light-chain genes, including myosin light chain-2, which are exclusively expressed in cardiac muscle, are highly expressed in ES-derived cardiomyocytes. Furthermore, a heterogeneous population of cardiomyocytes as early pacemaker-like, Purkinje-like, and atrial-/ventricular-like cells, with different overall shape and myofibrillar organization stage, also could be differentiated from ES cells. Genetic manipulation of ES cells carrying a fusion gene consisting of the alpha-cardiac myosin heavy-chain promoter and a sequence encoding aminoglycoside phosphotransferase was constructed to select pure cultures of cardiomyocytes differentiated from ES cells.Endothelium also can be differentiated from ES cells. Blood islands found in some EBs are lined by endothelial cells, and these endothelial cells subsequently were found to line interconnected channels that resemble blood vessels. Vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, was observed in cystic EBs by injecting ES cells into the peritoneal cavities of mice. In addition, transplanting cystic EBs onto the quail chorioallantoic membrane can induce angiogenesis, the sprouting of capillaries from pre-existing vessels. Therefore, ES cells possess the ability to undergo vasculogenesis and induce angiogenesis when differentiated in vitro.Recently, a unique culture system was developed to differentiate ES cells into endothelial cells without the formation of EBs. Endothelial cell progenitors were first sorted with a tyrosine kinase receptor, Flk-1. The maturation of these precursors into endothelium in vitro was followed by using a sequence of markers that included vascular endothelial-cadherin, platelet/endothelial cell adhesion molecule-1, and clusters of differentiation 34.Less is know about the in vitro differentiation capacity of EG cells than ES cells. EG cells can differentiate into cardiac, skeletal muscle, and neuronal cells. Hematopoietic lineages and neural cells can also be derived from EG cells, consistent with previous findings in ES cells. These studies strongly suggest that EG cells are closely related to ES cells in terms of differentiation capacity. Human PSC lines, like their mouse counterparts, are established from blastocysts and primordial germ cells. Human ES cells are derived from preimplantation stage embryos produced by in vitro fertilization. The ICM of the embryos was isolated by immunosurgery and plated on irradiated mouse embryonic fibroblasts. Human EG cells are derived from PGCs of 5 to 9 weeks postfertilization on thioguanine- and ouabain-resistant subline of SIM mouse fibroblasts in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Both types of culture retain a normal karyotype during extensive passage in vitro. The blastocyst-derived cells produced teratomas containing derivatives of all three germ layers after injection into severe combined immunodeficient mice. These cells also express markers characteristic of trophoblast and endoderm formation. In the case of the germ cell-derived cultures, EBs that formed spontaneously in culture tested positive for markers representative of mesodermal, ectodermal, and endodermal lineages. These data strongly suggest the pluripotency of these human stem cells. The derivation of human PSCs immediately drew public attention and raised expectations because of their enormous potential in both biomedical research and clinical applications. If human PSCs are similar to their mouse counterparts in terms of pluripotency, they will have a huge impact on the study of human embryogenesis, gene functions involved in differentiation, drug and teratogen testing, and most importantly, in providing unlimited sources of cells for transplantation and tissue replacement. The immediate and obvious clinical targets include neurodegenerative disorders such as Parkinson disease, diabetes, spinal cord injury, and stroke. Based on the mice experiments of transplants of ES-derived cell integrating and functioning well in the recipients, it is encouraging and exciting to speculate about the potential of human PSCs in tissue transplantation therapies.In addition to providing an unlimited source of tissues for transplantation, human stem cells could be genetically engineered or modified to minimize immunologic rejection after transplantation. The alteration of major histocompatibility complex genes in stem cells to create “universal donor lines” is one way to avoid graft rejection. An alternative is to produce PSC lines from the somatic cells of prospective recipients. This concept, sometimes referred to as “therapeutic cloning,” involves replacing the nucleus of the human oocyte with the nucleus of the adult cell. However, instead of placing the resulting embryo in the uterus, it would be cultured in vitro until the blastocyst stage, and ES cells then would be derived from the ICM of the blastocyst. The desired cell types differentiated from the ES cells would contain the genome of the recipient, and immunorejection due to engraftment could be eliminated. Another possibility is to use ES cell cytoplasm instead of oocytes to reprogram the nucleus of adult cells. It has been reported that mouse EG cells can reprogram adult nuclei after cell hybrid formation. Thus, it is a reasonable speculation that the nuclei of a recipient’s somatic cells could be reprogrammed after nuclear transfers of the patient’s somatic cells into PSCs. The cell line derived from recipient’s somatic cells then could be expanded and differentiated for therapeutic purpose.There is no doubt that human PSCs possess enormous potential for transplantation therapies. However, many practical issues must be resolved and many questions addressed before these cells can be used in therapies. First, the culture conditions required for derivation of human stem cells efficiently and reliably must be defined further. Strategies must be developed for the easy expansion of human stem cells. Second, the differentiation pathway should be regulated and controlled. The conditions for directed, lineage-restricted differentiation of human stem cells must be defined, and the differentiated cells must be fully characterized to ensure their identities and functions. Furthermore, extreme care should be used to determine that differentiating derivatives are nontumorigenic. Teratomas were observed when blastocyst-derived human stem cells were injected into immunosupressed mice. Both mouse ES and EG cells are known to be tumorigenic after injection in ectopic sites of adult mice. Therefore, strategies such as cell sorting or tissue-specific promoters for selection of desired cell types should be used, and genetically controllable systems that allow the destruction of transplanted cells if they become tumorigenic are desired.In addition to complex biologic problems, there are ethical issues surrounding the derivation and use of human PSCs. The federal regulation and funding of this research has become immersed in ethical/political considerations because of the embryonic and fetal origins of the cells. The derivation of human EG cells, as well as any subsequent research, can be funded by the National Institutes of Health (NIH) as fetal tissue research. There is currently a ban on the federal funding of human embryo research. However, a ruling in January 1999 by the United States Department of Health and Human Services (DHHS) states that NIH can fund research on already derived human ES cells. NIH has released draft guidelines that seek to regulate the funding of the allowable research, as determined by DHHS. The National Bioethics Advisory Commission has also released its report on the ethics of human stem cell research in which it supports federal funding for the derivation of both human EG cells and human ES cells, given certain provisions.Other, tangential issues, such as human reproductive cloning, germline gene modification, and human-animal chimera formation, further complicate the debates. It is clear that many challenges remain, and the road to making this a reality is a long one. Full support and unequivocal regulation from the government and collaboration among experts in different fields is needed to explore fully the potential of human PSCs.