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Stem Cells: a Journey Into a New Frontier

Stem Cells: a Journey Into a New Frontier

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 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, “” 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 , until murine embryonic develop into every of the body. Indeed, ES cells were stem was established in 1981. Through recent the only nontransformed mammalian stem cells that could be progress made in 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 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 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 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 inhibitory factor (LIF) (6). 1046-6673/1208-1773 LIF belongs to the interleukin-6 (IL-6) 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, , 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 (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 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 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 and high 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 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). differentiated tissues in all three germ layers: ectoderm, meso- derm, and . In addition to these in vivo tests, ES cells Adult Stem Cells are able to differentiate into multiple cell types in vitro. Of the many different types of adult stem cells, the most The in vitro differentiation of ES cells is induced basically widely known and best studied are the hematopoietic stem cells by removing the ES cells from the feeder layer of mouse (HSC). HSC have been studied since the early 1960s with the embryonic fibroblast cells or by removing LIF from the culture ground-breaking work of several investigators, which showed medium. When differentiating ES cells are cultured in suspen- that a population of (BM) cells transplanted into sion on petri dishes, ES cells aggregate and form structures, lethally irradiated mice could colonize the spleen and rescue termed embryoid bodies, that spontaneously differentiate into the mice from death (37,38). These studies then were taken a various cell types, including cardiac myocytes, neuronal cells, step further by Siminovitch et al. (39), who showed that these erythrocytes, , and others (17–19). Numerous at- clonogenic cells could be transplanted into another irradiated tempts have been made to enrich and isolate specific tissue animal and rescue that animal as well and reconstitute its entire precursors from differentiating ES cells. Enrichment and/or BM system. Through the years, it was found that these clono- isolation of certain types of cells has been achieved in some genic cells could be enriched through various techniques by cases by addition of various growth/differentiation factors or either the physical or the surface characteristics that are present chemicals. For example, pure populations of mast cell precur- on these cells (40,41). With the development of clonal-based sors can be obtained easily from mouse ES cells using IL-3 and assays for all hematopoietic cell lineages and cell-sorter–based stem cell factor (c-kit ligand) (20). A combination of basic fluorescence-antibody staining of certain cell populations, spe- fibroblast growth factor, platelet-derived growth factor, and cific populations of BM cells could be isolated (42,43). The epidermal growth factor can enrich glial precursors in differ- isolation of these subsets of BM cells led to possible candidates entiating mouse ES cells (21). In other cases, tissue-specific for stem cells in the murine model (44–46). precursors can be sorted using fluorescence-activated cell In terms of research and understanding the molecular path- sorter based on expression of specific markers on the cell ways of stem cells, the hematopoietic field is far ahead of the surface. Flk1-positive cells from mouse ES cells were demon- rest of the stem cell world. The cytokine requirements, cell to strated to serve as vascular progenitors (22). Tissue-specific cell interactions, integrin expression patterns, and transcription promoter-derived drug selection has been used to purify other regulatory factors all have been well documented and defined cell types, including cardiac myocytes and insulin-secreting for the HSC (47,48). Current theory for stem cell research cells (23,24). ES cells also can be differentiated into specific professes a linear model of hierarchy, whereby naive stem cells lineages by co-culture with other cells. Differentiation into exposed to certain growth factors and/or will pro- hematopoietic cells and dopaminergic , for instance, gressively acquire specific intrinsic factors and differentiate was induced when mouse ES cells were replated on feeder into a hierarchy of progeny/progenitor cells. These progenitor layers of OP9 and PA6 cells, respectively (25,26). In addition cells are thought to be stem-like themselves but restricted to the to the cell types described above, mouse ES cells have been number of options (pathways) available to them for differen- demonstrated to have a potential to differentiate into chondro- tiation (47). cytes, dendritic cells, and in vitro (27–29). The Despite the attractiveness of this linear model for the hema- most obvious application of ES-derived tissue-specific precur- topoietic lineage selection, recent evidence has begun to ex- sors is in cell-replacement therapies (5). To date, mouse ES pose weaknesses in such a tight and neatly packaged theory. cell–derived hematopoietic precursors (30), cardiomyocytes The emergence of both transgenic and knockout mouse models (22), neural precursors (20,25,31), insulin-producing cells (23), in conjunction with a better understanding of molecular anal- and mast cells (32) have been transplanted successfully into ysis of HSC and their progeny has led to the realization that recipient animals. Human ES cells also demonstrated a poten- these stem cells are very dynamic. The expression of certain tial to differentiate into cell types of all three germ layers key transcription factors does not necessarily mean commit- (33,34). In contrast to previous studies using mouse ES cells, ment to one lineage versus another lineage (49,50), making which were performed mostly within small laboratories, more lineage selection considerably more difficult to understand systematic approaches now are being undertaken to generate than once thought. The complexity grows even larger when human ES cell–derived tissue-specific cells at the industrial one takes into account other factors, such as the microenviron- level or as a larger collaborative research effort. ment (cell to cell contact and surrounding matrix), the positive In addition to its clinical application, in vitro differentiation and negative regulators of motility versus division, and factors of ES cells has been used in basic science to study gene that control differentiation (growth factors and chemokines). expression during development of specific cell types. Because When one looks at stem cell biology, it is important to encom- gene modulation techniques, including gene targeting, are well pass a view that is flexible and takes into account that nature established in ES cells, it is relatively easy to identify a role of has provided back-ups to back-ups and that lineage commit- a specific gene in the development of a certain cell lineage ment can happen in a variety of ways. 1776 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 1773–1780, 2001

Overall, with the number of recent studies demonstrating Nature Versus Nurture blood to brain (51), to muscle (52), to liver (53), and perhaps A formidable task in stem cell biology will be defining even to kidney, the HSC may be more plastic than once which key component(s) within the niche is important and thought, because they can give rise to a number of different cell transmits to the stem cells the many properties that they display types (Figure 2). If these HSC possess the entire genome, then while in that particular environment. The BM is a perfect they should be capable of becoming any cell type if given the example of the complexities found in one niche. Within the right set of signals. Even though the HSC seem to be very BM exists a vast number of different cell types (stromal cells, different from other cell types at first glance, they rely on the macrophages, ), all of which produce a variety of interactions with their niches and regularly use similar mech- cytokines and growth factors that will influence the HSC and anisms in doing so. HSC rely heavily on the use of integrins its inevitable differentiation pathway (55). Although great suc- not only for migration and mobility but also for maintaining cess has been seen over the years in the culture of HSC, it still their residence within the BM (54). As seen in other adult remains to be seen whether HSC can be cultured in vitro in stem-cell systems, the interaction of such away that they remain stem cells. Once HSC are removed (ECM) and integrins is used not only for adhesiveness but also from their niche and placed into culture, they begin to differ- for both survival and proliferation of stem cells within the BM entiate into a variety of hematopoietic cell types. HSC seem to (47). Thus, integrins may act as a common link to keep stem be a very social cell, meaning that they survive better in culture cells in place while maintaining their proliferative potential. In when placed in a co-culture system with BM stroma. The performing these chores, integrins carry a certain versatility reasons for this phenomenon are still unknown, but it seems that makes them essential in stem cell physiology. They can that the intrinsic properties of the niche are extremely impor- both respond to the ECM surrounding them and transmit in- tant in maintaining stem cell survival and proliferation. HSC ternal signals to activate various signaling pathways as well as are not the only cell type that requires co-culturing; epidermal receive internal signals from activated growth factor pathways keratinocytes also require to maintain optimal and respond by rearranging the ECM on the surface of the stem growth and survival (56). cell (47). To add more complexity to the picture of the microenviron-

Figure 2. In vivo and in vitro adult stem cell plasticity. Bone marrow (BM) stem cells ( [HSC] or [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. J Am Soc Nephrol 12: 1773–1780, 2001 Stem Cells 1777 ment, the BM also may contain more than one type of stem regrading the prospects that different stem cells can reside in cell. It seems that the mesenchymal stem cells (MSC) reside in the same place and respond to different stimuli and perchance the BM as well. These MSC are responsible for differentiating even have influence over one another. Altogether, these find- into a variety of cell types, including bone, muscle, cartilage, ings add additional hurdles in defining the molecular charac- adipocytes, and marrow stroma (57). The MSC also seem to be teristics of the stem cell and its niche. Time and further different from HSC because they can replicate as an undiffer- experiments in the phenotypic characterization of these stem/ entiated cell. Furthermore, it seems that these MSC also can progenitor cells eventually will lead to exciting and novel differentiate into cells of the epithelial lineages, such as hepatic therapeutic approaches for a number of life-threatening and neural, but were unable to differentiate into cells of the diseases. hematopoietic lineage (58). Until recently, it was thought that tissue-specific stem cells Controversies of Stem Cell Research could differentiate only into cells of the original source (e.g., The battle lines have been drawn over the use of stem cells hepatic oval cells into hepatocytes and bile ductular epitheli- in research, particularly over the use of human ES and human um). However, a number of recent studies suggest that adult fetal stem cells. The issue at the center of this controversy is organ-specific stem/progenitor cells may be capable of differ- the use of federal dollars to be spent on cell/tissue derived from entiating into cells that are different from the original cell type. the destruction of human life and the ethical concerns of what Two groups showed that stem cells of ectodermal origin can these cells are going to be used for. Seventy members of differentiate into blood cells (51,59,60), whereas others Congress signed a letter of objection to the use of human showed that MSC transplanted into mice can differentiate into embryonic stem cells in research and published it in the journal astrocytes, neurons, and oligodendrocytes (61,62). A recent Science (70), whereas more than 70 scientists, 67 of them study by Miyazaki et al. (63) showed that BM-derived cells Nobel laureates, voiced their strong support for the stance migrate to the kidney and may play a role in the progression of taken by the National Institutes of Health in support of funding renal injury. Although unexpected, this can be considered for human ES cell research (71). In August 2000, the National within the realm of possibility. The next step in renal research Institutes of Health released guidelines for the use of federal should be to determine whether HSC and/or MSC can partic- money to support human stem cell research much to the dismay ipate in the repair of the kidney after injury. Finally, Clarke et of the conservative right. al. (64) showed that neural stem cells injected into mouse A recent article by Charles Karuthammer printed in the blastocysts can give rise to all tissue types in the newborn February 12, 2001, issue of TIME magazine (71) warns of the mouse. impending monsters that we will be capable of creating if Theise et al. (65) and Alison et al. (66) both provided allowed to continue this line of investigation. Many people evidence that this phenomenon is not isolated to the rodent express these concerns and believe that stem cell research is model. They showed that this pathway of blood to liver exists immoral, illegal, and unnecessary. Stem cell scientists do not in humans as well, thus providing preliminary evidence that know where the major breakthroughs will come because em- this pathway might be a highly conserved defense mechanism bryonic, fetal, and adult stem cells all hold scientific potential. within the evolution of mammals as a whole. In addition, Application of findings obtained from nonhuman research Zanjani et al. (67) showed that human stem cells transplanted must await subsequent adjudication, and until the story is into the preimmune fetus of sheep can contribute to the archi- complete, it is highly appropriate that research be allowed to tecture of the developing liver and other organs as well. This move forward. Understandably, rigorous guidelines must be suggests that stem cells may actually be recruited to sites of followed just as they are for organ donation. injury where they receive key signals from the microenviron- Lessons learned from the recombinant DNA debate were ment, which allows them to differentiate into the desired cell useful in considering policies for the use of human ES and fetal type. The reports by Theise et al. and Alison et al. seem to stem cells. The process of policy development was public, and demonstrate this theory in which host cells were able to repop- committees consisted of individuals with very diverse back- ulate partially an orthoptically transplanted liver where liver grounds of expertise and opinions. Although public debate was damage had occurred (59,65,66). and still is contentious, a careful and well-thought-out analysis BM is a very complex tissue that performs many different of the issues prevailed, which allows researchers to be sup- functions in the body and contains a variety of cell types. Both ported by federal dollars. The results illustrate that government HSC and MSC are rare cells found within the BM. It has been officials, scientists, and the public can work together and draft shown that the cells move out of the yolk sac to the aorta- guidelines that all can abide by in this sensitive and contro- gonad-mesonephros region of the fetus and from there move versial area of investigation. into the embryonic liver. Around the time of birth, the cells The plasticity of adult stem cells seems to be bright and migrate out of the liver to the BM environment; this movement possibly a better choice over ES or fetal stem cells for the is controlled through the SDF-1/CXCR4 homing interaction of treatment of patients based on immune response mechanisms. the hematopoietic and stromal cells (68). Data presented by The use of one’s own stem cells for treatment would reduce the Petersen et al. (69) showed that SDF-1 and CXCR4 could be a need for immunosuppressive drugs (self to self-transplantation) plausible mechanism by which BM-derived cells are recruited and would end the ethical issues surrounding the use of ES and to the injured liver. This raises some interesting questions fetal stem cells. Nonetheless, ES and fetal stem cell research 1778 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 1773–1780, 2001 should continue because the invaluable information gained injuries. Clearly, more work needs to be accomplished to from these cell systems will provide a better understanding on overcome the complexities of organ development, but being the manner in which to manipulate and manage adult stem cells able to grow somatic cells in culture over a long period of time in a more proficient manner. ( 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 Future of Stem Cells and the New Field of unrealistic into reality. In conclusion, the possibilities for stem cell–based therapies Stem cell biologists will have to overcome two very differ- seem limitless. The German philosopher Nietzsche once said, ent obstacles to succeed in making stem cells a viable tool in “Many a man fails as an original thinker simply because his the treatment of humans. The first hurdle will be to maintain memory is too good.” It is this type of mentality that has kept stem cells as undifferentiated cells in culture. The second the blinders on many very prominent scientists, which may hurdle will be to differentiate cells, such as ES cells, down have slowed the pace of stem cell research. This may or may lineage commitment pathways. It may be unrealistic to think not have been a bad thing. Fortunately, it now seems that the that the cell biologist will be able to reproduce the precise set blinders have been taken off and stem cell research is now of signals that a cell receives in vivo that permits a cell to proceeding at a very rapid pace; through the use of stem cells, achieve full commitment. What comes first? Is it the cell-to- a host of human ailments possibly may be eliminated in the cell contact, the ECM, or perhaps the cytokine/growth factor not-too-distant future. It took billions of years of evolution to signaling? So far, the first switch that sends the cell down the produce cells that carry the awesome power to develop inde- path of commitment has been elusive. There are far too many pendently through a very precise set of instructions enabling signals being sent to the cell for us to decipher. It makes sense them to differentiate into anything from a liver cell to a kidney to try to develop culture systems that allow stem cells to cell to a fully grown organism. The stem cell dogmas of proliferate and survive as stem cells and use these cells as a yesterday are not withstanding the research findings of today, source for gene/cell therapy techniques. Transplanting these and many investigators are discovering that what once was, is stem-like cells into the site of preference and allowing the no longer. Now that we stand at the crossroads, do we try to microenvironment of the body to set into motion the proper harness this power for the good of humankind despite a pleth- signals seems to be a logical approach to developing the ora of potential problems, or do we turn our back on the desired cell type. However, in the case of ES cells, it will be potential opportunity to cure a vast number of sick people essential to initiate the pathway of commitment before trans- throughout the world who require organ replacement? It is planting them into humans. When ES cells are transplanted imperative that we, the stem cell scientists, work with the into mice, the cells develop into teratoma. Thus, the problem public officials to address the difficult questions that lies will not be to grow them in a stem-like state but to direct them ahead. We have already begun the process with the new down various lineage pathways without taking them so far that National Institutes of Health guidelines, but the process must they become useless for transplantation. continue. To gain the support of the public, we must keep them If this occurs in the next few years—and by all indications, informed and include them in the decision- making process as it seems that it is likely to succeed—a great deal of new well. As scientists, we cannot and must not forge recklessly therapeutic uses can be foreseen. Among these new therapies ahead without gaining public support. What should give all of will be the generation of different types of neural cells for the us reason to stop and contemplate is not from where the cells treatment of degenerative diseases such as Alzheimer’s and originated but the sheer power that these cells possess, which Parkinson’s. 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