Stem Cells
Authors: BRYON E. PETERSEN, NAOHIRO TERADA
Journal: Journal of the American Society of Nephrology (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 subsets of BM cells led to possible candidates for stem cells in the murine model (44,45,46). In terms of research and understanding the molecular pathways of stem cells, the hematopoietic field is far ahead of the rest of the stem cell world. The cytokine requirements, cell to cell interactions, integrin expression patterns, and transcription regulatory factors all have been well documented and defined for the HSC (47, 48). Current theory for stem cell research professes a linear model of hierarchy, whereby naive stem cells exposed to certain growth factors and/or cytokines will progressively acquire specific intrinsic factors and differentiate into a hierarchy of progeny/progenitor cells. These progenitor cells are thought to be stem-like themselves but restricted to the number of options (pathways) available to them for differentiation (47). Despite the attractiveness of this linear model for the hematopoietic lineage selection, recent evidence has begun to expose weaknesses in such a tight and neatly packaged theory. The emergence of both transgenic and knockout mouse models in conjunction with a better understanding of molecular analysis of HSC and their progeny has led to the realization that these stem cells are very dynamic. The expression of certain key transcription factors does not necessarily mean commitment to one lineage versus another lineage (49, 50), making lineage selection considerably more difficult to understand than once thought. The complexity grows even larger when one takes into account other factors, such as the microenvironment (cell to cell contact and surrounding matrix), the positive and negative regulators of motility versus division, and factors that control differentiation (growth factors and chemokines). When one looks at stem cell biology, it is important to encompass a view that is flexible and takes into account that nature has provided back-ups to back-ups and that lineage commitment can happen in a variety of ways. Overall, with the number of recent studies demonstrating blood to brain (51), to muscle (52), to liver (53), and perhaps even to kidney, the HSC may be more plastic than once thought, because they can give rise to a number of different cell types (Figure 2). If these HSC possess the entire genome, then they should be capable of becoming any cell type if given the right set of signals. Even though the HSC seem to be very different from other cell types at first glance, they rely on the interactions with their niches and regularly use similar mechanisms in doing so. HSC rely heavily on the use of integrins not only for migration and mobility but also for maintaining their residence within the BM (54). As seen in other adult stem-cell systems, the interaction of extracellular matrix (ECM) and integrins is used not only for adhesiveness but also for both survival and proliferation of stem cells within the BM (47). Thus, integrins may act as a common link to keep stem cells in place while maintaining their proliferative potential. In performing these chores, integrins carry a certain versatility that makes them essential in stem cell physiology. They can both respond to the ECM surrounding them and transmit internal signals to activate various signaling pathways as well as receive internal signals from activated growth factor pathways and respond by rearranging the ECM on the surface of the stem cell (47).Figure 2.: In vivo and in vitro adult stem cell plasticity. Bone marrow (BM) stem cells (hematopoietic stem cell [HSC] or mesenchymal stem cell [MSC]) have been shown to give rise to endothelial cells of the vascular system (72) and muscle (52) as well as hepatocytes (53 , 65 , 66) in vivo. In addition, BM stem cells have been shown to participate in neural development and vice versa (51 , 73). In vitro stem cells have been shown to produce bone, connective tissue, and cartilage (57). Last, neural stem cells from the adult mouse brain can contribute to the formation of chimeric chick and mouse embryos and give rise to cells of all germ layers (64). All of this demonstrates that adult stem cells have a very broad developmental capacity. Someday, these stem cells may be used in a variety of ways in the treatment of different human diseases.NatureVersusNurture A formidable task in stem cell biology will be defining which key component(s) within the niche is important and transmits to the stem cells the many properties that they display while in that particular environment. The BM is a perfect example of the complexities found in one niche. Within the BM exists a vast number of different cell types (stromal cells, macrophages, adipocytes), all of which produce a variety of cytokines and growth factors that will influence the HSC and its inevitable differentiation pathway (55). Although great success has been seen over the years in the culture of HSC, it still remains to be seen whether HSC can be cultured in vitro in such away that they remain stem cells. Once HSC are removed from their niche and placed into culture, they begin to differentiate into a variety of hematopoietic cell types. HSC seem to be a very social cell, meaning that they survive better in culture when placed in a co-culture system with BM stroma. The reasons for this phenomenon are still unknown, but it seems that the intrinsic properties of the niche are extremely important in maintaining stem cell survival and proliferation. HSC are not the only cell type that requires co-culturing; epidermal keratinocytes also require fibroblasts to maintain optimal growth and survival (56). To add more complexity to the picture of the microenvironment, the BM also may contain more than one type of stem cell. It seems that the mesenchymal stem cells (MSC) reside in the BM as well. These MSC are responsible for differentiating into a variety of cell types, including bone, muscle, cartilage, adipocytes, and marrow stroma (57). The MSC also seem to be different from HSC because they can replicate as an undifferentiated cell. Furthermore, it seems that these MSC also can differentiate into cells of the epithelial lineages, such as hepatic and neural, but were unable to differentiate into cells of the hematopoietic lineage (58). Until recently, it was thought that tissue-specific stem cells could differentiate only into cells of the original source (e.g., hepatic oval cells into hepatocytes and bile ductular epithelium). However, a number of recent studies suggest that adult organ-specific stem/progenitor cells may be capable of differentiating into cells that are different from the original cell type. Two groups showed that stem cells of ectodermal origin can differentiate into blood cells (51, 59, 60), whereas others showed that MSC transplanted into mice can differentiate into astrocytes, neurons, and oligodendrocytes (61, 62). A recent study by Miyazaki et al. (63) showed that BM-derived cells migrate to the kidney and may play a role in the progression of renal injury. Although unexpected, this can be considered within the realm of possibility. The next step in renal research should be to determine whether HSC and/or MSC can participate in the repair of the kidney after injury. Finally, Clarke et al. (64) showed that neural stem cells injected into mouse blastocysts can give rise to all tissue types in the newborn mouse. Theise et al. (65) and Alison et al. (66) both provided evidence that this phenomenon is not isolated to the rodent model. They showed that this pathway of blood to liver exists in humans as well, thus providing preliminary evidence that this pathway might be a highly conserved defense mechanism within the evolution of mammals as a whole. In addition, Zanjani et al. (67) showed that human stem cells transplanted into the preimmune fetus of sheep can contribute to the architecture of the developing liver and other organs as well. This suggests that stem cells may actually be recruited to sites of injury where they receive key signals from the microenvironment, which allows them to differentiate into the desired cell type. The reports by Theise et al. and Alison et al. seem to demonstrate this theory in which host cells were able to repopulate partially an orthoptically transplanted liver where liver damage had occurred (59, 65, 66). BM is a very complex tissue that performs many different functions in the body and contains a variety of cell types. Both HSC and MSC are rare cells found within the BM. It has been shown that the cells move out of the yolk sac to the aortagonad-mesonephros region of the fetus and from there move into the embryonic liver. Around the time of birth, the cells migrate out of the liver to the BM environment; this movement is controlled through the SDF-1/CXCR4 homing interaction of the hematopoietic and stromal cells (68). Data presented by Petersen et al. (69) showed that SDF-1 and CXCR4 could be a plausible mechanism by which BM-derived cells are recruited to the injured liver. This raises some interesting questions regrading the prospects that different stem cells can reside in the same place and respond to different stimuli and perchance even have influence over one another. Altogether, these findings add additional hurdles in defining the molecular characteristics of the stem cell and its niche. Time and further experiments in the phenotypic characterization of these stem/progenitor cells eventually will lead to exciting and novel therapeutic approaches for a number of life-threatening diseases. Controversies of Stem Cell Research The battle lines have been drawn over the use of stem cells in research, particularly over the use of human ES and human fetal stem cells. The issue at the center of this controversy is the use of federal dollars to be spent on cell/tissue derived from the destruction of human life and the ethical concerns of what these cells are going to be used for. Seventy members of Congress signed a letter of objection to the use of human embryonic stem cells in research and published it in the journal Science (70), whereas more than 70 scientists, 67 of them Nobel laureates, voiced their strong support for the stance taken by the National Institutes of Health in support of funding for human ES cell research (71). In August 2000, the National Institutes of Health released guidelines for the use of federal money to support human stem cell research much to the dismay of the conservative right. A recent article by Charles Karuthammer printed in the February 12, 2001, issue of TIME magazine (71) warns of the impending monsters that we will be capable of creating if allowed to continue this line of investigation. Many people express these concerns and believe that stem cell research is immoral, illegal, and unnecessary. Stem cell scientists do not know where the major breakthroughs will come because embryonic, fetal, and adult stem cells all hold scientific potential. Application of findings obtained from nonhuman research must await subsequent adjudication, and until the story is complete, it is highly appropriate that research be allowed to move forward. Understandably, rigorous guidelines must be followed just as they are for organ donation. Lessons learned from the recombinant DNA debate were useful in considering policies for the use of human ES and fetal stem cells. The process of policy development was public, and committees consisted of individuals with very diverse backgrounds of expertise and opinions. Although public debate was and still is contentious, a careful and well-thought-out analysis of the issues prevailed, which allows researchers to be supported by federal dollars. The results illustrate that government officials, scientists, and the public can work together and draft guidelines that all can abide by in this sensitive and controversial area of investigation. The plasticity of adult stem cells seems to be bright and possibly a better choice over ES or fetal stem cells for the treatment of patients based on immune response mechanisms. The use of one's own stem cells for treatment would reduce the need for immunosuppressive drugs (self to self-transplantation) and would end the ethical issues surrounding the use of ES and fetal stem cells. Nonetheless, ES and fetal stem cell research should continue because the invaluable information gained from these cell systems will provide a better understanding on the manner in which to manipulate and manage adult stem cells in a more proficient manner. Future of Stem Cells and the New Field of Regenerative Medicine Stem cell biologists will have to overcome two very different obstacles to succeed in making stem cells a viable tool in the treatment of humans. The first hurdle will be to maintain stem cells as undifferentiated cells in culture. The second hurdle will be to differentiate cells, such as ES cells, down lineage commitment pathways. It may be unrealistic to think that the cell biologist will be able to reproduce the precise set of signals that a cell receives in vivo that permits a cell to achieve full commitment. What comes first? Is it the cell-to-cell contact, the ECM, or perhaps the cytokine/growth factor signaling? So far, the first switch that sends the cell down the path of commitment has been elusive. There are far too many signals being sent to the cell for us to decipher. It makes sense to try to develop culture systems that allow stem cells to proliferate and survive as stem cells and use these cells as a source for gene/cell therapy techniques. Transplanting these stem-like cells into the site of preference and allowing the microenvironment of the body to set into motion the proper signals seems to be a logical approach to developing the desired cell type. However, in the case of ES cells, it will be essential to initiate the pathway of commitment before transplanting them into humans. When ES cells are transplanted into mice, the cells develop into teratoma. Thus, the problem will not be to grow them in a stem-like state but to direct them down various lineage pathways without taking them so far that they become useless for transplantation. If this occurs in the next few years—and by all indications, it seems that it is likely to succeed—a great deal of new therapeutic uses can be foreseen. Among these new therapies will be the generation of different types of neural cells for the treatment of degenerative diseases such as Alzheimer's and Parkinson's. It would be within the realm of possibility that spinal cord injury patients may regain full function of their body. Even some genetic disorders could be cured, not just treated. For example, stem cells from a patient with hemophilia could be isolated and grown in culture, then transfected with the clotting factor(s) gene(s) and transplanted back into the patient's liver. These stem cells also could be used as building blocks in the making of artificial organs, such as the liver or kidney or perhaps even heart muscle. Clearly, there are major hurdles to overcome, including the problem of differentiation discussed previously and the ability to devise three-dimensional structures in which the matrix will disintegrate while the cells build there own matrix. Bioengineers and material scientists are already addressing these matrix problems. Medical centers all over the world have been using artificial skin (dermal cells grown in culture) for more than two decades and with great success in helping severely burned patients recover from their injuries. Clearly, more work needs to be accomplished to overcome the complexities of organ development, but being able to grow somatic cells in culture over a long period of time (hepatocyte long-term cultures) and to take some stem cell types (e.g., MSC) and begin their differentiation down distinct lineages are critical first steps for making the once seemingly unrealistic into reality. In conclusion, the possibilities for stem cell—based therapies seem limitless. The German philosopher Nietzsche once said, “Many a man fails as an original thinker simply because his memory is too good.” It is this type of mentality that has kept the blinders on many very prominent scientists, which may have slowed the pace of stem cell research. This may or may not have been a bad thing. Fortunately, it now seems that the blinders have been taken off and stem cell research is now proceeding at a very rapid pace; through the use of stem cells, a host of human ailments possibly may be eliminated in the not-too-distant future. It took billions of years of evolution to produce cells that carry the awesome power to develop independently through a very precise set of instructions enabling them to differentiate into anything from a liver cell to a kidney cell to a fully grown organism. The stem cell dogmas of yesterday are not withstanding the research findings of today, and many investigators are discovering that what once was, is no longer. Now that we stand at the crossroads, do we try to harness this power for the good of humankind despite a plethora of potential problems, or do we turn our back on the potential opportunity to cure a vast number of sick people throughout the world who require organ replacement? It is imperative that we, the stem cell scientists, work with the public officials to address the difficult questions that lies ahead. We have already begun the process with the new National Institutes of Health guidelines, but the process must continue. To gain the support of the public, we must keep them informed and include them in the decision- making process as well. As scientists, we cannot and must not forge recklessly ahead without gaining public support. What should give all of us reason to stop and contemplate is not from where the cells originated but the sheer power that these cells possess, which is the essence of life itself.