SCIENCE WATCH J Am Soc Nephrol 12: 1773–1780, 2001 Stem Cells: A Journey into a New Frontier BRYON E. PETERSEN and NAOHIRO TERADA Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, and Stem Cell Program, University of Florida, Gainesville, Florida. Stem cells are cells that are capable of self-renewal and are embryonic origin first isolated from the inner cell mass of multipotent, meaning that they can differentiate into many developing mouse blastocysts (1,2). The distinguished features specific cell types. For a long time, “stem cell” had been a of ES cells are their capacity to be maintained in an undiffer- concept in mammalian biology but not a reality that could be entiated state indefinitely in culture and their potential to seen, touched, and expanded in vitro, until murine embryonic develop into every cell type of the body. Indeed, ES cells were stem cell culture was established in 1981. Through recent the only nontransformed mammalian stem cells that could be progress made in adult stem cell research, we now can isolate propagated continuously in vitro until recently, when culture various stem/progenitor cells derived from adult tissues, such methods of various adult stem cells were established. as neural stem cells, vascular endothelial progenitor cells, and Self-renewing, totipotent ES cells may provide a virtually hepatic oval cells. Of the many adult stem cells described in the unlimited donor source for transplantation and tissue genera- literature (hematopoietic, neural, hepatic, dermal, pancreatic, tion in vitro in the future. Mouse ES cell–derived hematopoi- and intestinal), it once was thought that these cells could etic precursors, cardiomyocytes, neural precursors, or insulin- differentiate only into the cell type from which they originated. producing cells, have been transplanted successfully into It now seems that the adult stem cell is more plastic, versatile, recipient animals. Because human ES cells (and embryonic and capable of becoming many different types of cells, e.g., germ cells) were isolated and shown recently to have a similar blood to brain/liver, brain to blood, liver to blood. These new potential for differentiation as the mouse ES cells (3,4), these findings give the once lonely adult stem cell a new lease on life techniques may be applied in the near future to patients with and perhaps a better chance in enabling both investigators and various diseases. Ultimately, in vitro–generated tissues from clinicians to fight life-threatening human diseases. Recent stem human ES cells may take the place at least in part of organ cell–based cell therapies have been shown to be successful in transplantation. ES-derived tissue-specific cells also will be an animal models for various diseases, such as Parkinson’s dis- ideal source for drug efficacy and toxicity testing (5). ease and insulin-dependent diabetes mellitus. In the coming Counterbalancing the promise of ES cells are serious ethical decade, the focus of research will be stem cell–based cell concerns for the use of human ES cells because it requires therapy (or a combination of cell and gene therapy techniques). fertilized human eggs to establish them. Because of recent In this article, we discuss the present status and the future of advances in methods that establish various stem cells from stem cell research via two major categories: embryonic stem adult tissues, enthusiasm for the direct application of human (ES) cells and adult stem cells. Self-renewal is discussed, ES cells clinically, once extremely high at the close of the 20th mostly from a point of view of gene regulation in the ES cell century, seems to be waning gradually. However, the signifi- and from a point of view of the microenvironment in the adult cance of studies in the ES cell as a prototype of pluripotent stem cell. In addition, the pluripotency of stem cells is ad- stem cells will go unchanged. The mechanism underlying dressed. Finally, we refer to the controversies arising in stem self-renewal activity of stem cells has been studied in depth cell research and the future applications of stem cells in using ES cells as described below. In terms of clinical appli- medicine. cation, the methods developed for generation of tissue-specific cells from ES cells would be applied easily to adult stem cells. ES Cells Hence, ES cells will remain important as a research tool in the Not many people would argue against the statement that study of mammalian development (see Figure 1). establishment of pluripotent murine ES cell culture is one of the great achievements in mammalian cell and developmental Self-Renewal of Pluripotent ES Cells biology. ES cells are continuously growing stem cell lines of Self-renewal is one of the most important characteristics of stem cells. Its molecular mechanism has been studied inten- sively in ES cells but is incompletely understood. Mouse ES Received April 16, 2001. Accepted May 4, 2001. cells can proliferate in vitro in an undifferentiated, pluripotent Correspondence to Dr. Bryon E. Petersen, P.O. Box 100275, Department of state on a feeder layer of mouse embryonic fibroblast cells or Pathology, University of Florida, Gainesville, FL 32610. Phone: 352-392- 6261; Fax: 352-392-6249; E-mail: [email protected] in a medium containing leukemia inhibitory factor (LIF) (6). 1046-6673/1208-1773 LIF belongs to the interleukin-6 (IL-6) cytokine family, which Journal of the American Society of Nephrology includes IL-6, IL-11, oncostatin M, ciliary neurotropic factor, Copyright © 2001 by the American Society of Nephrology and cardiotropin-1 (7). The effect of LIF is mediated through a 1774 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 1773–1780, 2001 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. cell surface receptor (LIFR␤) and gp130, a common receptor trophectodermal-like differentiation of the cells, respectively. subunit of the IL-6 cytokine family. LIF binds with LIFR␤, Although Oct3/4-deficient mouse embryos develop to the blas- which then forms a high-affinity heterodimer complex with tocyst stage, the inner cell mass cells are not pluripotent but gp130. Among the signals transduced by the LIFR complex, restricted to differentiation along the extraembryonic tropho- activation of STAT3 was determined to be indispensable for blast lineage (14). These studies indicate that Oct3/4 is an the maintenance of undifferentiated ES cells (8–10). Expres- essential factor for maintenance of undifferentiated ES cells. sion of a dominant negative mutant of STAT3 abrogated self- Oct3/4 seems not to be a direct target of STAT3 (9). The renewal of ES cells and induced differentiation in the presence STAT3 pathway and the Oct3/4 pathway rather are considered of LIF (8). Furthermore, activation of STAT3 by forced dimer- to be two separate pathways, both of which are working ization of the molecule was sufficient for the self-renewal of coordinately for self-renewal of ES cells (13). ES cells in the absence of LIF (9). Because STAT3 is a Maintenance of telomere length is another feature observed transcription factor, self-renewal of ES cells likely is main- and required in self-renewal of stem cells. Both mouse and tained by the molecule(s) of which expression is regulated human ES cells have prolonged telomeres and high telomerase directly or indirectly by STAT3. It should be noted that primate activity. The molecular mechanism by which ES cells maintain ES cells, including human ES cells, differentiate or die in the telomere length, however, is unknown. It is interesting that absence of fibroblast feeder layers, even in the presence of LIF recent studies have revealed that telomere shortening is not a (3,11). The molecular basis of undifferentiating factors remains major reason for cell senescence in rodent cells, whereas it is unknown in human ES cells. It also is unclear whether human the major limiting factor in survival of human cells (15,16). ES cells require STAT3 activation for self-renewal. Oxidative stress or other factors may play more critical roles in Oct3/4 is a POU-domain transcription factor expressed ex- senescence of primary rodent cells. In this sense, mouse ES clusively in early embryonic cells and germ cells (12). Both cells should have a feature to escape from this senescence mouse and human ES cells highly express Oct3/4 when they mechanism as well. are maintained undifferentiated and lose expression when dif- ferentiated. It was demonstrated that expression of the appro- In Vitro Differentiation of ES Cells priate amount of Oct3/4 was critical for maintenance of undif- The most rigorous test of the developmental potential of ferentiated murine ES cells (13). According to the study, mouse ES cells is their ability to contribute to all cell lineages, increase and decrease in Oct3/4 resulted in mesodermal- and including the germ line of chimeric animals. This ability, in J Am Soc Nephrol 12: 1773–1780, 2001 Stem Cells 1775 conjunction with effective homologous recombination of DNA (35). Furthermore, in combination with the techniques to purify in ES cells, enabled us to generate knockout mice. In addition, tissue-specific cells described above, we can determine the when injected into immunocompromised animals, both murine function of genes in a specific tissue solely by in vitro assay and human ES cells form teratomas composed of multiple (19,30,36).