Stem Cells (2001)

Stem cells are cells that are capable of self-renewal and are multipotent, meaning that they can differentiate into many specific cell types. For a long time, “stem cell” had been a concept in mammalian biology but not a reality that could be seen, touched, and expanded in vitro, until murine embryonic stem cell culture was established in 1981. Through recent progress made in adult stem cell research, we now can isolate various stem/progenitor cells derived from adult tissues, such as neural stem cells, vascular endothelial progenitor cells, and hepatic oval cells. Of the many adult stem cells described in the literature (hematopoietic, neural, hepatic, dermal, pancreatic, and intestinal), it once was thought that these cells could differentiate only into the cell type from which they originated. It now seems that the adult stem cell is more plastic, versatile, and capable of becoming many different types of cells, e.g., blood to brain/liver, brain to blood, liver to blood. These new findings give the once lonely adult stem cell a new lease on life and perhaps a better chance in enabling both investigators and clinicians to fight life-threatening human diseases. Recent stem cell—based cell therapies have been shown to be successful in animal models for various diseases, such as Parkinson's disease and insulin-dependent diabetes mellitus. In the coming decade, the focus of research will be stem cell—based cell therapy (or a combination of cell and gene therapy techniques). In this article, we discuss the present status and the future of stem cell research via two major categories: embryonic stem (ES) cells and adult stem cells. Self-renewal is discussed, mostly from a point of view of gene regulation in the ES cell and from a point of view of the microenvironment in the adult stem cell. In addition, the pluripotency of stem cells is addressed. Finally, we refer to the controversies arising in stem cell research and the future applications of stem cells in medicine. ES Cells Not many people would argue against the statement that establishment of pluripotent murine ES cell culture is one of the great achievements in mammalian cell and developmental biology. ES cells are continuously growing stem cell lines of embryonic origin first isolated from the inner cell mass of developing mouse blastocysts (1, 2). The distinguished features of ES cells are their capacity to be maintained in an undifferentiated state indefinitely in culture and their potential to develop into every cell type of the body. Indeed, ES cells were the only nontransformed mammalian stem cells that could be propagated continuously in vitro until recently, when culture methods of various adult stem cells were established. Self-renewing, totipotent ES cells may provide a virtually unlimited donor source for transplantation and tissue generation in vitro in the future. Mouse ES cell—derived hematopoietic precursors, cardiomyocytes, neural precursors, or insulin-producing cells, have been transplanted successfully into recipient animals. Because human ES cells (and embryonic germ cells) were isolated and shown recently to have a similar potential for differentiation as the mouse ES cells (3, 4), these techniques may be applied in the near future to patients with various diseases. Ultimately, in vitro—generated tissues from human ES cells may take the place at least in part of organ transplantation. ES-derived tissue-specific cells also will be an ideal source for drug efficacy and toxicity testing (5). Counterbalancing the promise of ES cells are serious ethical concerns for the use of human ES cells because it requires fertilized human eggs to establish them. Because of recent advances in methods that establish various stem cells from adult tissues, enthusiasm for the direct application of human ES cells clinically, once extremely high at the close of the 20th century, seems to be waning gradually. However, the significance of studies in the ES cell as a prototype of pluripotent stem cells will go unchanged. The mechanism underlying self-renewal activity of stem cells has been studied in depth using ES cells as described below. In terms of clinical application, the methods developed for generation of tissue-specific cells from ES cells would be applied easily to adult stem cells. Hence, ES cells will remain important as a research tool in the study of mammalian development (see Figure 1).Figure 1.: Promise of embryonic stem (ES) cells. Self-renewable ES cells (mouse or human) can differentiate into multiple lineages in vitro. This in vitro differentiation system has been used to study gene regulation in mammal development and roles of genes in specific tissues. Furthermore, ES cell—derived tissues are now expected for cell transplantation therapy, tissue engineering, and drug efficacy/toxicity tests. Counterbalancing the promise of ES cells are ethical concerns for the use of human ES cells because it requires fertilized human eggs to establish them.Self-Renewal of Pluripotent ES Cells Self-renewal is one of the most important characteristics of stem cells. Its molecular mechanism has been studied intensively in ES cells but is incompletely understood. Mouse ES cells can proliferate in vitro in an undifferentiated, pluripotent state on a feeder layer of mouse embryonic fibroblast cells or in a medium containing leukemia inhibitory factor (LIF) (6). LIF belongs to the interleukin-6 (IL-6) cytokine family, which includes IL-6, IL-11, oncostatin M, ciliary neurotropic factor, and cardiotropin-1 (7). The effect of LIF is mediated through a cell surface receptor (LIFRβ) and gp130, a common receptor subunit of the IL-6 cytokine family. LIF binds with LIFRβ, which then forms a high-affinity heterodimer complex with gp130. Among the signals transduced by the LIFR complex, activation of STAT3 was determined to be indispensable for the maintenance of undifferentiated ES cells (8,9,10). Expression of a dominant negative mutant of STAT3 abrogated self-renewal of ES cells and induced differentiation in the presence of LIF (8). Furthermore, activation of STAT3 by forced dimerization of the molecule was sufficient for the self-renewal of ES cells in the absence of LIF (9). Because STAT3 is a transcription factor, self-renewal of ES cells likely is maintained by the molecule(s) of which expression is regulated directly or indirectly by STAT3. It should be noted that primate ES cells, including human ES cells, differentiate or die in the absence of fibroblast feeder layers, even in the presence of LIF (3, 11). The molecular basis of undifferentiating factors remains unknown in human ES cells. It also is unclear whether human ES cells require STAT3 activation for self-renewal. Oct3/4 is a POU-domain transcription factor expressed exclusively in early embryonic cells and germ cells (12). Both mouse and human ES cells highly express Oct3/4 when they are maintained undifferentiated and lose expression when differentiated. It was demonstrated that expression of the appropriate amount of Oct3/4 was critical for maintenance of undifferentiated murine ES cells (13). According to the study, increase and decrease in Oct3/4 resulted in mesodermal- and trophectodermal-like differentiation of the cells, respectively. Although Oct3/4-deficient mouse embryos develop to the blastocyst stage, the inner cell mass cells are not pluripotent but restricted to differentiation along the extraembryonic trophoblast lineage (14). These studies indicate that Oct3/4 is an essential factor for maintenance of undifferentiated ES cells. Oct3/4 seems not to be a direct target of STAT3 (9). The STAT3 pathway and the Oct3/4 pathway rather are considered to be two separate pathways, both of which are working coordinately for self-renewal of ES cells (13). Maintenance of telomere length is another feature observed and required in self-renewal of stem cells. Both mouse and human ES cells have prolonged telomeres and high telomerase activity. The molecular mechanism by which ES cells maintain telomere length, however, is unknown. It is interesting that recent studies have revealed that telomere shortening is not a major reason for cell senescence in rodent cells, whereas it is the major limiting factor in survival of human cells (15, 16). Oxidative stress or other factors may play more critical roles in senescence of primary rodent cells. In this sense, mouse ES cells should have a feature to escape from this senescence mechanism as well. In VitroDifferentiation of ES Cells The most rigorous test of the developmental potential of mouse ES cells is their ability to contribute to all cell lineages, including the germ line of chimeric animals. This ability, in conjunction with effective homologous recombination of DNA in ES cells, enabled us to generate knockout mice. In addition, when injected into immunocompromised animals, both murine and human ES cells form teratomas composed of multiple differentiated tissues in all three germ layers: ectoderm, mesoderm, and endoderm. In addition to these in vivo tests, ES cells are able to differentiate into multiple cell types in vitro. The in vitro differentiation of ES cells is induced basically by removing the ES cells from the feeder layer of mouse embryonic fibroblast cells or by removing LIF from the culture medium. When differentiating ES cells are cultured in suspension on petri dishes, ES cells aggregate and form structures, termed embryoid bodies, that spontaneously differentiate into various cell types, including cardiac myocytes, neuronal cells, erythrocytes, melanocytes, and others (17,18,19). Numerous attempts have been made to enrich and isolate specific tissue precursors from differentiating ES cells. Enrichment and/or isolation of certain types of cells has been achieved in some cases by addition of various growth/differentiation factors or chemicals. For example, pure populations of mast cells precursors can be obtained easily from mouse ES cells using IL-3 and stem cell factor (c-kit ligand) (20). A combination of basic fibroblast growth factor, platelet-derived growth factor, and epidermal growth factor can enrich glial precursors in differentiating mouse ES cells (21). In other cases, tissue-specific precursors can be sorted using fluorescence-activated cell sorter based on expression of specific markers on the cell surface. Flk1-positive cells from mouse ES cells were demonstrated to serve as vascular progenitors (22). Tissue-specific promoter-derived drug selection has been used to purify other cell types, including cardiac myocytes and insulin-secreting cells (23, 24). ES cells also can be differentiated into specific lineages by co-culture with other cells. Differentiation into hematopoietic cells and dopaminergic neurons, for instance, was induced when mouse ES cells were replated on feeder layers of OP9 and PA6 cells, respectively (25, 26). In addition to the cell types described above, mouse ES cells have been demonstrated to have a potential to differentiate into chondrocytes, dendritic cells, and hepatocytes in vitro (27,28,29). The most obvious applications of ES-derived tissue-specific precursors is in cell-replacement therapies (5). To date, mouse ES cell—derived hematopoietic precursors (30), cardiomyocytes (22), neural precursors (20, 25, 31), insulin-producing cells (23), and mast cells (32) have been transplanted successfully into recipient animals. Human ES cells also demonstrated a potential to differentiate into cell types of all three germ layers (33, 34). In contrast to previous studies using mouse ES cells, which were performed mostly within small laboratories, more systematic approaches now are being undertaken to generate human ES cell—derived tissue-specific cells at the industrial level or as a larger collaborative research effort. In addition to its clinical application, in vitro differentiation of ES cells has been used in basic science to study gene expression during development of specific cell types. Because gene modulation techniques, including gene targeting, are well established in ES cells, it is relatively easy to identify a role of a specific gene in the development of a certain cell lineage (35). Furthermore, in combination with the techniques to purify tissue-specific cells described above, we can determine the function of genes in a specific tissue solely by in vitro assay (19, 30, 36). Adult Stem Cells Of the many different types of adult stem cells, the most widely known and best studied are the hematopoietic stem cells (HSC). HSC have been studied since the early 1960s with the ground-breaking work of several investigators, which showed that a population of bone marrow (BM) cells transplanted into lethally irradiated mice could colonize the spleen and rescue the mice from death (37, 38). These studies then were taken a step further by Siminovitch et al. (39), who showed that these clonogenic cells could be transplanted into another irradiated animal and rescue that animal as well and reconstitute its entire BM system. Through the years, it was found that these clonogenic cells could be enriched through various techniques by either the physical or the surface characteristics that are present on these cells (40, 41). With the development of clonal-based assays for all hematopoietic cell lineages and cell-sorter—based fluorescence-antibody staining of certain cell populations, specific populations of BM cells could be isolated (42, 43). The isolation of these of BM cells to for stem cells in the murine In terms of research and the molecular of stem cells, the hematopoietic is of the of the stem cell The cytokine cell to cell expression and transcription factors all have been well and for the HSC for stem cell research a of stem cells to certain growth factors and/or will specific factors and differentiate into a of cells. 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