Cell Fate Determination from Stem Cells

AJ Wagers1, JL Christensen1 and IL Weissman1,2 1Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; and 2Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA

In the adult, tissue-speciﬁc stem cells are thought to be that inﬂuence stem cell fate, taking as a primary example responsible for the replacement of differentiated cells within the cell fate determination of hematopoietic stem cells. continuously regenerating tissues, such as the liver, skin, Gene Therapy (2002) 9, 606–612. DOI: 10.1038/sj/gt/3301717 and blood system. In this review, we will consider the factors

Keywords: stem cells; progenitors; hematopoiesis; self-renewal; differentiation; migration

Introduction to hematopoietic stem and and characterized in the fetal liver (FL).9–11 FL HSC differ progenitor cells from adult HSC both phenotypically and functionally. Unlike adult LT-HSC, FL HSC express the cell surface The study of stem cells within the hematopoietic system markers Mac-1 and AA4.1, are cycling rapidly, and give began nearly 40 years ago, with the demonstration by Till a more robust and rapid reconstitution of irradiated and McCulloch that a single precursor cell exists in the hosts.9–15 While adult HSC readout in CFU-S assays at bone marrow (BM) of adult animals that is capable of day 12, FL HSC form visible colonies at day 8.16 Finally, both extensive self-renewal and multi-lineage differen- while the adult stem cell pool is composed mostly of tiatiation.1–3 This finding gave rise to the hypothesis that, short-term and multipotent progenitors,6 most stem cells during hematopoiesis, a small population of multipotent, in FL are thought to be LT-HSC.15 self-renewing, clonogenic stem cells divides to generate The oligopotent progenitors downstream of FL HSC progeny cells, each progressively more restricted in also differ slightly from their adult counterparts (Figure developmental potential, until ultimately mature effector 1). Unlike adult CMP, FL CMP, but not GMP or MEP, cells are formed. Careful regulation of cell fate decisions give rise to B cells (but never T cells) both in vivo and in in the stem cell pool profoundly influences the pro- vitro.16 Likewise, while adult CLP have no macrophage duction of mature blood cells, and is required both to activity in vivo or in vitro, FL CLP form macrophage (but maintain homeostasis and to enable rapid and robust never granulocyte, erythrocyte, or megakaryocyte) colon- responses to physiologic stresses, including blood loss, ies in methylcellulose and stromal cell cultures.17 In infection and injury. addition to these differences in the lineage relationships Hematopoietic stem cells (HSC) are rare cells within of fetal progenitors, several blood cells that cannot be the BM or fetal liver which can both self-renew and dif- generated by adult HSC, including B-1a B cells and V␥3+ ferentiate to form all of the major blood cell lineages. The and V␥4+ T cells, arise during fetal hematopoiesis from HSC pool has been characterized phenotypically and is fetal HSC.18–20 separable into distinct subpopulations based on both The prospective isolation of HSC and progenitor cells phenotype and function (Figure 1).4,5 Long-term reconsti- in mice has provided key insights into the molecular and tuting HSC (LT-HSC) have the greatest self-renewal cellular regulators of hematopoiesis. Careful control of capacity and give rise to all hematopoietic lineages HSC developmental decisions is clearly essential for throughout life.4,6 The immediate progeny of LT-HSC are blood cell homeostasis. As discussed below, the develop- short-term (ST-) HSC, which also generate all hematopo- mental options of HSC include not only self-renewal and ietic lineages, but do so for only 8–10 weeks. Further differentiation, but also apoptosis and migration. The downstream are lineage-restricted oligopotent progenitor synthesis of signals from diverse hematopoietic regu- cells, including both lymphoid (common lymphoid pro- lators ultimately determines the fate of dividing HSC. genitors, CLP) and myeloid (common myeloid progenitors, CMP; granulocyte–monocyte progenitors, GMP; and Self-renewal versus differentiation in the megakaryocyte–erythrocyte progenitors, MEP) restricted precursors.7,8 control of HSC fate Multipotent, clonogenic HSC also have been identified Most studies of HSC cell fate determination focus on the choice between stem cell self-renewal, in which the daughter(s) of a dividing HSC retains the properties of Correspondence: IL Weissman, Department of Pathology, Beckman B257, the parent cell, and differentiation, in which the daughter Stanford University School of Medicine, Stanford, CA 94305, USA cell(s) becomes restricted in its potential and diminished The first two authors contributed equally to this work in its capacity for self-renewal. Both stochastic and Cell fate determination from stem cells AJ Wagers et al 607

Figure 1 Hematopoietic cell ontogeny. This illustration describes the development of mature blood cells through the sequential restriction of the cell fate potential of oligopotent progenitor cells derived from multipotent HSC. The stem cell pool is comprised of both LT- and ST-HSC. LT-HSC have a significantly greater potential for self-renewal than ST-HSC. Oligopotent progenitor cells do not self-renew appreciably. Dashed lines indicate developmental pathways that operate only during fetal hematopoiesis (see text for details). instructive models have been proposed to describe this genitor cells has demonstrated that HSC constitutively choice, and while both models invoke a key role for cyto- express low levels of many lineage-specific cytokine kines, different functions are assigned to these factors. receptors and transcription factors.7,24,28 Among progeni- Under the stochastic model, HSC randomly commit to tors, lineage commitment is accompanied by loss of either self-renew or differentiate. Cytokines present in the expression of genes associated with unrelated lineages,7 BM milieu do not direct this choice per se, but do allow indicating that hematopoietic maturation may be survival and proliferation of the cells that ultimately mediated by sequential restriction of gene expression. develop into mature lineages. Under the instructive Promiscuous gene expression by HSC may provide a model, the particular cytokines to which HSC are framework for stochastic fluctuations in expression of exposed directly determine the outcome of each mitosis, signaling or transcriptional complexes, which ultimately whether self-renewing or differentiating. are amplified or repressed to cement lineage choice.26 While several studies support an instructive role for Specific molecular mediators that influence HSC self- cytokines in the maturation of terminally differentiated renewal versus differentiation decisions have only cells from progenitor and precursor cells,21–24 the initial recently been identified. Increased HSC self-renewal divisions of HSC are likely to involve stochastic fate appears to be associated with HSC-intrinsic over- choices.25 In fact, it has been postulated that stochastic expression of HoxB4,29,30 and induction of the Wnt (Reya determination of HSC cell fate may be important for HSC et al, submitted), Notch,31,32 and Sonic hedgehog (Shh)33 function, in that this scheme allows for the maintenance signaling pathways. Extrinsic signals that promote HSC of production of all blood cell lineages even in the face self-renewal have been more difficult to identify, as of substantial demand for one particular lineage.26 Sup- shown by the notorious lack of effective means of porting the stochastic model for HSC differentiation, in expanding HSC in vitro, while maintaining their self- vitro culture of Bcl-2 transgenic HSC with steel factor renewing properties. Currently published literature34–38 (SLF) alone induces these cells to proliferate, but does not points to several cytokines, including SLF, Flt3L, throm- preserve their primitive status.27 This induction of HSC bopoietin, IL-3, and IL-6, as working individually or syn- differentiation in the absence of any overt differentiating ergistically to permit one to two self-renewing divisions signals, suggests that lineage choice need not be initiated in liquid culture. However, long-term maintenance of by particular cytokine signals. Similarly, lineage-restric- HSC function clearly requires more complex systems and ted progenitors can develop from HSC at normal fre- may rely on as yet uncharacterized factors provided by quency, even in the absence of lineage-specific cytokines. stromal support cells (see below). For example, although maturation of lymphocytes from A central issue in HSC fate choices is the consideration CLP requires IL-7, and IL-7 induces CLP proliferation in of symmetry within stem cell divisions. Are the individ- vitro, CLP develop at normal frequency in mice deficient ual mitoses of HSC obligately symmetric (always produc- ␥ in expression of the common signaling receptor ( c)of ing two daughter cells with identical fates), obligately the IL-7 receptor.8 asymmetric (always producing two daughters with dis- RT-PCR analysis of rigorously purified stem and pro- tinct fates), or variably symmetric and asymmetric?

Gene Therapy Cell fate determination from stem cells AJ Wagers et al 608 Because in the steady state the total number of BM HSC and a unique cloned marrow stromal line (ATF024), remains constant,4 half of all HSC divisions, at the popu- derived from murine FL, can maintain murine HSC lation level, must be self-renewing. However, HSC must activity in culture for weeks.42,43 On the other hand, while also have the capacity to expand their numbers, by freshly purified marrow myofibroblasts facilitate hemato- increasing the frequency of self-renewal, in response to poietic cell proliferation in vitro, marrow endothelial cells hematopoietic stress. Thus, HSC divisions cannot be obli- may inhibit hematopoiesis.51 gately asymmetric, as this would prohibit HSC expan- Whether direct contact of HSC with stromal cells is sion. Experiments in vitro have provided evidence for required for their effects on HSC is somewhat contro- both symmetric and asymmetric divisions of cultured versial. ATF024 cells fail to support long-term mainte- HSC,34,39 suggesting that the outcomes of HSC mitoses nance of primitive human progenitor cells in non-contact are variable, but the relevance of these findings to HSC cultures,42 but can maintain SCID-repopulating activity divisions in vivo remains to be determined. of umbilical cord blood CD34+ cells in transwell assays.52 Some stromal cell cultures support hematopoiesis in noncontact conditions, but inhibit it in direct co-cultures.53 Programmed cell death as a determinant of The differences in outcome of these in vitro cultures may HSC fate relate to differences in the source or type of HSC and/or stromal elements studied, or may depend on other differ- The hematopoietic compartment must produce large ences, such as the presence or absence of cytokines, numbers of cells daily (~2.4 x 108 in the mouse40), and serum, or other factors, in these culture systems. must further scale up this effort following injury or hem- The emergence during development of distinct blood atopoietic stress. Programmed cell death (apoptosis) of cell lineages, dependent on particular stromal microen- HSC has recently been shown to play an important role vironments, supports the importance of stromal cell in regulating HSC number. Transgenic mice with interactions to HSC fate choices in vivo. For example, the enforced overexpression of the proto-oncogene Bcl-2 + + + first TCR thymocytes are V␥3 and V␥4 .20 These T cells exhibit increased steady-state HSC frequency and arise only from FL HSC and absolutely require the fetal increased competitive repopulation potential.41 When thymic environment for their generation.19 The involve- transplanted into irradiated wild-type recipients, Bcl-2 ment of stromal cells in HSC function in vivo is further transgenic HSC give increasing hematopoietic readout evident from studies of naturally occurring mutations of over time, and in vitro culture of Bcl-2 transgenic HSC the Steel (Sl) locus in mice. BM transplantation has dem- with SF alone is sufficient to prevent HSC death.27 These onstrated that defective hematopoiesis in these mice data demonstrate that regular turnover of BM HSC results from a specificdeficiency (loss or impairment of through programmed cell death is an important regulator SLF expression) within the BM microenvironment.54,55 of HSC cell fate and hematopoietic homeostasis. The HSC from Sl mutant mice reconstitute the ablated BM of inability of Bcl-2 transgenic HSC to be removed from BM wild-type animals, while wild-type HSC fail to reconsti- niches by apoptosis may underlie the competitive advan- tute hematopoiesis in transplanted Sl mice and BM stro- tage of these HSC in wild-type transplant recipients. mal cells from these mice fail to support hematopoiesis in vitro.54–58 Finally, in recent studies, co-transplantation Adhesion and migration as regulators of of stromal elements with either adult or fetal BM HSC HSC fate significantly enhanced engraftment of preimmune fetal sheep,59 providing further evidence for support of HSC In the BM, hematopoietic cell development is thought to function by marrow stromal cells. occur in various specialized microenvironments, called Extensive and complex interactions within the BM ‘niches’. At present, support for the niche hypothesis microenvironment clearly influence the cell fate decisions remains indirect, and these specialized areas of BM have of HSC; however, these interactions do not necessarily never been described at the ultrastructural level. Evi- imply that HSC remain permanently lodged in specific dence for the existence of HSC-supporting niches derives BM niches. On the contrary, recent data suggests signifi- from several lines of investigation, including the lack of cant dynamism among HSC niches, and supports the in vitro conditions that can fully support HSC self- notion that migration through the bloodstream to distinct renewal, the infrequency of isolation of hematopoiesis BM sites or to other peripheral tissues is an additional supporting stromal cell lines,42,43 and the low level of BM developmental alternative for HSC (Figure 2). The impor- engraftment that can be achieved following intravenous tance of HSC migration is clearly evident during fetal injection of HSC, unless those HSC are given in extraordi- hematopoiesis. HSC arise first in the embryonic yolk narily high numbers or unless the BM is ablated by sac60–65 and aorta/gonad/mesonephros (AGM) region,66–69 irradiation or cytotoxic drugs.44,45 The hypothetical func- and are thought to migrate to and seed the fetal tion of the HSC niche is to provide HSC survival and liver.63,70,71 FL HSC subsequently engraft the newly self-renewal factors, either through direct contact with developing fetal BM. In the adult, the ability of hemato- HSC or through secreted factors. HSC niches are likely poietic cells to use the blood as a transit mechanism for formed from a subset of bone marrow stromal cells, migration from one organ to another was demonstrated which may play a further role in ‘translating’ external more than 40 years ago,72–74 but the realization that such signals to influence HSC developmental decisions.46 migration in the bloodstream is a property not only of BM stromal cells can play either supportive or inhibi- mature blood cells, but also of hematopoietic stem and tory roles in the maintenance of primitive hematopoietic progenitor cells, occurred more recently. HSC and pro- progenitors. Co-culture of human CD34+CD38Ϫ/lo human genitor cells are constantly present at low frequency in progenitor cells with marrow-derived stromal cells the blood of normal animals,75,76 and these blood-borne appears to promote maintenance of the primitive state,47–50 HSC can, at least, re-engraft BM to functionally contrib-

Gene Therapy Cell fate determination from stem cells AJ Wagers et al 609

Figure 2 Transit of HSC and progenitor cells through the bloodstream. BM-derived HSC and progenitor cells rapidly and constitutively enter the bloodstream and may traffic to distinct hematopoietic and non-hematopoietic tissues, as well as back to BM. Thus, HSC are constantly present in most, if not all, tissues of the body, and may represent a source of pluripotent cells that participate in the repair of damaged or degenerating tissues. These cells also may be a source of contaminating hematopoietic activity in ‘transdifferentiation’ assays. ute to on-going hematopoiesis.76,110 In genetically marked cell transdifferentiation, some caution is warranted in parabiotic mice, which share a common blood circulation, considering these findings. First, it is important to significant cross-engraftment of BM HSC is observed in remember that BM contains several stem cell popu- the absence of BM ablation following as little as 2–3 lations, including mesenchymal and hematopoietic stem weeks of parabiosis (A. Wagers, unpublished cells, and that mesenchymal stem cells are defined by observation). This data suggests that constitutive, low- their potential to give rise to muscle, cartilage, bone, fat level HSC mobilization occurs in normal, unconditioned and stroma.85 Furthermore, because circulating tissue- animals. Surprisingly, the normal flux of HSC and pro- specific stem cells, including HSC, have been described genitor cells in the bloodstream of normal animals for several lineages, such blood-borne stem cells rep- appears to be quite high, as predicted by their extremely resent significant potential sources of contamination in short residence time in the blood, such that the effective transdifferentiation assays.75,76,105–107,110 Finally, some pool of circulating cells may include more than 30 000 claims of transdifferentiation involve cells grown in cul- stem and progenitor cells.110 While the full effects of ture, using high concentrations of growth factors, which constitutive migration on HSC developmental decisions may impart a more primitive potential to the cultured remain to be determined, the possibility that HSC may cells.103,104 For example, fetal murine primordial germ find themselves in unique microenvironments in tissues cells grown in basic fibroblast growth factor (bFGF) and outside of the BM raises the interesting possibility that leukemia inhibitory factor (LIF) take on embryonic stem itinerant HSC or their progeny may contribute to the (ES) cell qualities.108 Thus, to demonstrate true transdif- regeneration of non-hematopoietic, as well as hematopo- ferentiation, it is necessary that the stem cells in question ietic cells (see below). Furthermore, this finding provides are prospectively isolated and characterized, able to self- a physiological framework for understanding the renew and differentiate into all relevant cell types at the efficiency of bone marrow transplantation and the exist- clonal level, and that the transdifferentiated cells gener- ence of induced HSC mobilization, which occurs follow- ated by these stem cells are demonstrated to be fully ing myeloablation and/or increases in circulating functional in the target tissue. cytokine/chemokine levels,77 as these transplanted or Despite the caveats discussed above, good evidence for mobilized HSC likely exploit similar mechanisms of a greater than expected plasticity of adult stem cells is migration as those employed by constitutively circulating emerging, and this research has exciting implications for HSC in normal animals. potential clinical application to the repair of damaged or degenerating tissues. Highly purified HSC have been shown able to differentiate into colonies of hepatocytes Plasticity of stem cell fate Ϫ Ϫ when introduced into fumarylacetoacetate hydrolase / Recently much attention has been paid to stem cells in the (FAHϪ/Ϫ) mice.109 HSC-derived hepatocytes restored adult organism, and the possibility that stem cells may liver function in transplanted FAHϪ/Ϫ mice, and all hep- transdifferentiate into cell types of unrelated tissues. atic engrafted recipients also showed hematopoietic Stem cells or cells with stem cell-like qualities have been reconstitution. Whether hepatic regeneration is a normal isolated or described in several tissues, including neural role of HSC, or whether this function is induced by the (CNS78–80 and PNS81), pancreatic,82 epidermal,83 mes- mutant background of the recipient mice, remains to be enchymal,84-87 hepatic,88,89 bone,86 muscle,90 and endo- determined, but these findings certainly demand further thelial91,92 tissues. In recent years, published reports have inquiry into the possibility of HSC mediated therapy for indicated that cells found in BM are capable of giving liver degenerative disorders. rise to endothelial precursors,93 brain microglia and mac- 94–96 97–99 98,100 roglia, skeletal muscle, cardiac muscle, hep- Concluding remarks atic cells,101,102 and mesenchymal progeny.86,87 Other groups have reported that cultured stem cells of neural103 Hematopoiesis is regulated by the developmental or muscle104 origin can give rise in vivo to hematopoietic decisions of HSC, which include self-renewal, differen- cells. While these results may constitute evidence of stem tiation, death, and migration. Much has been learned

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