Hematopoietic

stem-cell

differentiation

Gerald J. Spangrude Rocky Mountain Hematopoietic

Hamilton,

stem cells can be identified

tissues of mammalian stem cell activity hematopoietic described.

Laboratories,

USA

and isolated from hematopoietic

hosts. Assay systems that solely reflect hematopoietic

are being developed, stem-cell

Differentiation

proliferation pathways

hematopoiesis

Current

Montana,

Opinion

and new cytokines and that

that

differentiation

lead

to

lymphoid

influence

have

been

stages

of

have also been suggested.

in Immunology

Introduction In this review I have chosen to concentrate on progress made during the past 18 months in two aspects of hematopoiesis. First, I will consider several topics that relate to very primitive hematopoietic stem cells (HSC), defined as cells that can differentiate in erythroid, myelomonocytic, megakaryocytic, and lyrnphoid lineages and that possess the capacity to maintain hematopoiesis over long periods of time, i.e. the lifetime of an animal. I will discuss the concept of ‘self-renewal by these cells and review current thinking about appropriate assay systems that specifically detect HSCs. I will also review effects of various cytokines on HSCs, and summarize the identification of a new cytokine that plays a central role in the hematopoietic process. As a second topic, I have chosen to consider two as pects of the early stages of T-lymphocyte development: an interesting bit of evidence suggesting that the T-lym phoid developmental potential of HSCs changes during embryogenesis, and a brief look at the question of commitment to lymphoid development in the process of hematopoiesis. I have chosen not to review recent progress in understanding late stages of B-lymphocyte or intrathymic T-lymphocyte development, nor have I considered any developmental aspects of erythroid, myelomonocytic, or megakatyocytic lineages. These are topics that are either covered in more detail elsewhere in this section, or do not directly relate to lymphocyte development, the general theme of this section.

Assays for HSCs Over the past 18 months a great deal of attention has been focused on the development of assay systems that speciiically detect the activity of the most primitive of stem cells for hematopoiesis. The delinitive assay

1991, 3~171-178

for many years has been the ability of HSCs to generate macroscopic colonies of hematopoietic cells in the spleens of irradiated animals. With the advent of refinements in HSC characterization (reviewed in [ 1,2]), however, it has become evident that many types of hematopoietic cells that form spleen colonies lack the ability to reconstitute long-term hematopoiesis in irradiated animals [3]. Conversely, two groups have demonstrated a long-term potential for repopulation in the apparent absence of splenic colony-forming potential [4=*,5**]. Although it is not yet clear whether inhibitory cytokines [6*] or splenic seeding considerations [7*-l explain the failure of certain HSC preparations to form spleen colonies, it is very clear that spleen colony formation is not a unique characteristic of primitive HSCs. Furthermore, the inability to use such an assay in the investigation of human hematopoiesis, with the possible exception of chime& human-mouse model systems [8], dictates the need to develop specific in vitro assays for HSC. The process of hematopoiesis depends on the intimate association of HSCs with non-hematopoietic, fixed tissue cells in the medullary cavity of bone and, in some rodents, in the spleen. These tissue cells are generally termed ‘stromal cells’, a generic term that may be applied to any one of a wide variety of non-mobile cells (reviewed in [9]). Many recent attempts to develop specific in vitro assays for HSCs have relied upon the coculture of candidate HSC populations with feeder layers of bone marrow-derived stroma. Four studies have used this approach to analyze hematopoiesis in mouse and human models [ lo**,1 l*-13.1. In most cases, the assays involve harvesting the cells that differentiate in the cultures and testing these progeny cells for their ability to produce macroscopic colonies in semi-solid medium under the influence of a variety of cytokines (Fig. 1). Although colony formation is not an activity uniquely associated with HSCs, the differentiation of HSCs in these stromal cell co-cultures is thought to result in the production of,

Abbreviations HSC-hematopoietic

stem cell; IL-interleukin; M-CSF-macrophage colony-stimulating factor; SCID-severe combined immunodeficiency. @

Current Biology Ltd ISSN 0952-7915

171

172

lymphocyte development

and thus a net increase in, the number of colony-forming cells. If the co-culture is initiated under limiting dilution conditions [ 10**,12*,13*], then an estimate of the frequency of the HSCs in any given cell population is possible. Furthermore, a higher absolute number of colonyforming cells produced in any one culture is an indicator of initiation by a more primitive HSC.

(a) HSC

two cell types. The mobile HSC interdigitates between and beneath the stromal monolayer, resulting in HSCs with a characteristic non-refractile appearance, observed by phase-contrast microscopy. These stromal-covered cells proliferate to form clusters of tightly packed cells, which have been referred to as cobblestone areas [lo**] (Fig. 1). The frequency of formation of cobblestone areas can be correlated with hematopoietic repopulating activity in vivo, and the kinetics of cobblestone area formation reflect the relative maturation stage of the initiating cells - more primitive cells requiring a longer period of time to establish a cobblestone area [lo**].

stromal cells

Colony-stimulating

(b)

Fig 1. A general approach to an in vitro assay for hematopoietic stem cells (HSC). HSCs are added at limiting dilution to an established stromal-cell culture. HSCs, in close association with stromal cells, proliferate to form clusters of cells that look dark by phase contrast microscopy. These foci are termed ‘cobblestone areas’. Upon further proliferation and differentiation, the HSCs in the cobblestone areas release committed progenitors into the culture medium. These progeny cells are collected and quantitated by a separate culture in semi-solid medium containing cytokines (not shown).

Co-cultures of HSCs and supportive bone-marrow stroma evolve to generate unique associations between the

factors and HSC regulation

Stromal cell co-cultures have proved to be useful models for studying the development of HSCs in vitro, but ideally one would prefer to identify and characterize the stromal cell-derived cytokines that are responsible for driving each stage of hematopoiesis. Although many hematopoietic cytokines have been identilied, purllied, and have had their genebcloned, on their own these molecules are primarily involved in later stages of myeloid and lymphoid differentiation. Early hematopoietic progenitors require as many as three cytokines to induce colony formation in vitro [ 14**], and it is not yet clear ðer the most primitive HSCs respond to known cytokines at all. One experimental system [ 15.1 has approached this question in a manner analogous to the stromal cell-HSC co-cultures described above. Bone marrow cells cultured for 4days in the presence of interleukin(IL)-1 and IL-3 ditferentiate to produce an expanded population of cells capable of forming macroscopic, multi&age colonies ln methyl cellulose medium. The bone marrow culture can be lnitiated at limltlng dilution to provide an estimate of the frequency of primitive cells, and the initiating cells can be partially separated from more mature progenitors by density centrlfugation. Although a variety of known cytokines are unable to substitute for IL-1 and IL-3 in the primary culture, the possibility remains that the interleukins stimulate the production of unknown mediators in the bulk bone marrow culture. IL-l and IL-3 in combination with colony-stimulating factor-l (macrophage colony-stimulating factor or M-CSF), are sulhcient to drive in vitro colony formation by highly enriched populations of HSCs [ 14**]. A similar combination of cytokines, but not single cytokines, was shown to stimulate highly enriched HSCs at a clonal level, to differentiate into osteoclasts as well as mature blood cells [16*].

~-6, a cytokine produced by a variety of cellular sources, has been identilied as a co-stimulator or primitive HSCs when used in combination with IL-3 [17*]. The combination of IL-3 and IL-~ provides the stimulation necessary to achieve retroviral infection of primitive HSCs in vitro [XI*]. Furthermore, IL-~ can induce adult or fetal hematopoietic progenitors to activate cell cycling, as assessed by thymidine suicide studies [ 19*]. A wider spectrum of action of IL-~was noted when human fetal (cord blood) progenitors served as target cells, because in vitro

Hematopoietic

colonies of various morphologies were induced by IL6 in combination with granulocyte macrophage [ 191. Similar activity was not seen in adult HSC preparations. When considered with numerous reports of negative reg ulatoty influences mediated by macrophage inflammatory protein-la [6*,20*] and transforming growth factor-g [21,221, these studies reinforce the concept of synergism and antagonism between the effects of colony-stimulating factors on primitive HSCs.

A new hematopoietic

growth

factor

The separation of the functions of HSCs and of the microenvironments that support them are illustrated by two mutant mouse strains, that result from mutations at the white spotting (W) and steel (9) loci on chromosomes 5 and 10, respectively. Both mutations cause similar deficiencies in the development of melanocytes, reproductive capacity and hemopoietic function. Interestingly, hematopoietic abnormalities in Wlocus mutants result from defective HSCs, while SI-locus mutants provide a defective microenvironment for normal HSCs. In both cases, the result is characterized by a severe macrocytic anemia and increased sensitivity to radiation. Three years ago the W locus was shown to encode the cellular homolog (c-kit) of the viral oncogene, vkit [23,24]. The proto-oncogene, c-kit, encodes a transmembrane receptor protein that includes an internal tyrosine-kinase domain and is highly homologous to the cellular receptors for the cytokines M-CSF and plateletderived growth factor. Although the natural ligand for the c-kit-encoded cell-surface receptor was not identified, the complementaty phenotypic relationship between the W and SZmutations suggested that the SZlocus may encode a protein that interacts with the c-kit product of the W locus [23]. A remarkable series of eight papers published in the journal Celllast year coniirm this hypothesis. Using mast cells [25-,26-l or enriched preparations of HSCs [27**] as target cells, three groups deiined a hematopoietic growth factor that maps to the SZlocus [28e,29-] and binds to the c-kit product [ 25**,26**,29-1. Predicted amino acid sequences of cDNA clones suggest that this factor is a membrane-bound cell-surface molecule [26*-l, and soluble forms of the same molecule were found as products of a liver-cell line [30-l and a bone marrow stromal-cell line [25-,31-l. In addition to its action as a growth factor for mast cells, the SIlocus factor synergizes with other cytokines to stimulate hematopoietic colony formation in v&-o [27**,29-,30*-,31-l, and in vivo administration of the growth factor leads to a reversal of the macrocytic anemia and mast-cell deficiency obselved in mice with mutations at the Sl gene locus [ 29.1. Embry onic expression of SZgene transcripts is consistent with the observations that defects at either the Sl or W loci result in defective development of neural crest-derived melanocytes, primordial germ cells, and HSCs [32-l. Thus, these studies provide compelling evidence that the gene product of the Sllocus is an important participant in

stem-cell differentiation Spangrude

the development and maintenance of hematopoiesis and other embryonic and adult organ systems. The availability of the Slgrowth factor in soluble and stromal cell-associated forms will certainly accelerate our investigations of the hematopoietic process.

Self-renewal

of HSCs

The concept of self-renewal in hematopoiesis may be interpreted in several ways: on the one hand, the notion of self-renewal of individual HSCs leads to the conclusion that one stem cell may contribute indefinitely to hematopoiesis. On the other hand, if one envisions a heterogeneous compartment of HSCs that share the ability to initiate development in multiple hematopoietic lineages, but which differ in their ability to give rise to more multipotent cells (Fig. 21, the conclusion is consistent with the clonal succession model as proposed by Kay 25 years ago [ 331. An intermediate situation consisting of essential elements from both extremes produces a further variation. It will be diflicult to prove or disprove whether one HSC can divide to produce progeny of identical proliferative and developmental potential, but manyexperiments have demonstrated clonal succession and the heterogeneous nature of the HSC compartment. Sequential activation of stem-cell clones leads to clonal or quasi-clonal contributions to hematopoiesis, as demonstrated by transplantation experiments carried out on animals differing at various isoenzyme loci [34,35] or by transplants of bone marrow cells cartying unique retrovirally-induced genetic markers [ 36*,37*,38]. Serial transplantation of bone marrow, which eventually leads to a loss of repopulating activity [39*,40], demonstrates two distinct phases of engraftment in recipient animals [41]. The first unsustained phase occurs apparently because more committed members of the HSC compartment are unable to maintain hematopoiesis in the long term, while the second sustained phase is a result of very primitive HSC 1411. These observations are compatible with clonal succession and with the generation-age hypothesis [ 421, which extends the clonal succession model to predict that the number of generations by which a stem cell is removed from its initial progenitor is inversely proportional to its proliferation potential (and hence hematopoietic repopulating potential) and directly proportional to its state of activation (Fig. 2b). Compelling evidence for this concept has been provided at a population level by Bertoncello and colleagues [43] and was recently extended to the clonal level [7**] by the demonstration that cells with both myeloid and lymphoid differentiation potential are heterogeneous with regard to their long-term (sustained) repopulating potential. Application of transplantation pressure in hematopoiesis results in exhaustion of HSCs [39’,40]. A very intriguing study using allophenic chimeras, made by aggregating embryos of two inbred mouse strains, has shown that HSC exhaustion may be observed under normal devel-

173

Fig 2. Two views of self-renewal by hematopoietic stem cells (HSCs). (a) The HSC compartment may consist of a homogeneous group of cells, each able to divide without differentiating to reproduce itself, or to divide with differentiation to produce committed progenitors. Self-renewal occurs at a cellular level.(b)The HSC compartment may be heterogeneous, and cell division may always be accompanied by differentiation to a more ‘activated HSC. The most activated HSCs remain multipotent, but will give rise to committed progenitors on the next division. The HSC compartment self-renews, because one HSC generates many more, although individual cells do not selfrenew. Strong experimental evidence exists for the model shown in (b), but these experiments do not exclude the possibility that cellular self-renewal may also occur.

opmental pressure [44**]. In these experiments, one of the two mouse strains, DBA/Z, has spleen colony-form ing cells of which 24% are normally in cell cycle, and the other, C57BL/G, has spleen colony-forming cells of which only 2.6% are in cycle. In allophenic chimeras and in F, recipients of bone-marrow transplants from these animals, the DBA/2 HSC population was eventually eclipsed by the C57BZ/Gpopulation. Early in the time course after transplantation the D&3/2 HSC population predominated, but was later overtaken by the C57BI/G genotype. These experiments bear out basic predictions of the generation-age hypothesis and point to intrinsic dif ferences in HSCs as factors governing the longevity of hematopoiesis as well as the life span of the animal. Do

HSC also self-renew at a cellular level? The final answer will have to wait for more sophisticated techniques of analysis.

Characterization

of embryonic HSC

Hematopoiesis develops in an essentially identical process in all mammalian species, with blood islands in the yolk sac providing hematopoietic precursors to the fetal liver, and later, to the fetal spleen and bone marrow. Two groups have recently investigated cellular and developmental properties of HSCs derived from fetal liver. Jordan

Hematopoietic stem-ceil differentiation Spangrude

and colleagues used monoclonal antibodies to enrich HSCs from fetal liver populations that had been marked by unique retroviral integration sites [45**]. By using a competitive repopulation strategy in populations marked by two retroviral genomes distinguishable by Southern blotting, they demonstrated that the monoclonal antibody AA4.1 marks all HSCs contained in fetal liver. Further fractionation using a variety of methods defined a rare subset of fetal liver cells (0.1-0.2 % of the total) that includes very primitive HSCs. Interestingly, separation of AA4.1+ fetal liver cells on the basis of expression of hematopoietic lineage markers (Lin) demonstrated a lowlevel expression of at least one of these markers, in contrast to previous characterizations of HSCs in adult bone marrow [46] that reported HSC activity in the Lin- fraction. This discrepancy may reflect a difference between fetal and adult HSCs. Ikuta and colleagues [ 47**] have documented a developmental distinction between fetal and adult HSCs, reflected by maturation potential in the thyrnus. Fetal HSCs, isolated by antibody staining and fluorescence-activated cell sorting, from gestational day 14 liver developed as T lym phocytes expressing the Vr3 T-cell receptor when organcultured in vitrowith fetal thymic lobes. Adult HSCs were incapable of developing in a similar manner, as were fetal HSCs when placed in adult thymic lobes. This is a clear demonstration that the developmental potential of HSCs changes during ontogeny. The wave of Vr3 T lymphocytes that arises in the fetal thymus between day 14 and day 17 in vivo depends upon a primitive class of HSCs that develop in a fetal environment.

Lymphoid commitment

by bone marrow

HSC

The demonstration by Ikuta and colleagues [47**] of a restriction for T-cell development at the level of the HSC raises other questions regarding lymphoid development by HSCs. For example, does the commitment to develop in the T lineage occur in the HSC compartment in the bone marrow, producing cells that specifically home to the thymus, or do multipotent cells that enter the thymus respond to the thymic microenvironment by a commitment to the T lineage? If the commitment occurs in the bone marrow, does a restricted lymphoid progenitor arise that can develop as B lymphocytes in the bone marrow or as T lymphocytes in the thymus? Although the latter situation has been implied by hematology textbooks for decades, no dehnitive evidence has been published to establish this pathway. Highly enriched populations of bone marrow HSCs, when microinjected at limiting dilution into irradiated thymic lobes in vivo, differentiate into both T-lymphoid and myeloid progeny [7**]. Injection of large numbers of cells also results in B-lymphocyte development [7**,48*]. Although these observations establish that HSCs require no further environmental conditioning from the marrow prior to development in the thymus, they do not necessarily prove that a commitment to the T lineage does not

occur in the bone marrow. Palacios and colleagues [49”] have reported isolation of a bone-marrow population in mice that reconstitutes T- but not B-lymphocyte differentiation following intravenous transfer into irradiated mice with severe combined immunodeficiency (SCID). An additional population was reported that reconstituted both T and B lineages, but no data were presented to indicate whether this population contains a lymphoid-committed progenitor or whether the cells are also capable of myeloid or erythroid differentiation [49**]. Whether these observations can be reproduced in normal recipient animals remains to be seen. In another in vivo system, Huby and colleagues [50] used cyclosporln-A to induce inhibition of medullary, but not cortical, thymic development following bone-marrow transplantation. If the bone marrow was depleted of either Thy-l + or CD5+ cells prior to the transplant, thymus regeneration was inhibited in both anatomical compartments in a reversible manner. These results were interpreted to suggest that cyclosporin-A inhibits the development of a Thy- 1 +/CD5 + thymic-repopulating descendant of HSCs in bone marrow. Several in vitro experiments have hinted at a T-cell commitment in the bone marrow. Benveniste and colleagues [ 511 have characterized cells present in the marrow of congenitally athymic (nude) mice by in vitrorulture with defined growth factors and have presented evidence for some degree of extrathymic T-cell development. Investigation of human T-cell development [52] has provided evidence of a clonogenic pre-T-cell population that differentiates in vitro to form colonies of cells expressing mature T-cell markers. Whereas virtually every cultured cell of a population enriched from normal human bone marrow responded to a complex culture that included feeder cells and an undefined mixture of growth factors to produce colonies expressing T-cell markers, only l/35 cells responded to IL-3 to generate myeloid colonies in vitro. Most putative T-cell colonies expressed up but not y6 T-cell receptors. However, in the absence of more rigorous criteria to establish the presence of specific T-cell functions among the colonies, the possibility of aberrant expression of T-cell antigens because of excessive stimulation in the cultures remains. In spite of this caveat, the very high efficiency of cloning in this system and the absence of myeloid or B-lymphoid cells in the colonies, coupled with the comparatively low frequency of myeloid differentiation after IL-3 stimulation, is very intriguing and suggests the presence of committed T-lymphoid progenitors in human bone marrow. Kurtzberg and colleagues [53] have described a unique leukemic syndrome in humans, characterized by cells that are capable of multilineage differentiation in vitro. These leukemias are characterized by expression of a marker (CD7) that is associated with early human T-cell development. Therefore, the acute leukemia syndrome may represent transformation of a multipotent hematopoietic cell that is at the stage of thyrnic homing, thus providing evidence against the concept of a strict T-cell commitment in the bone marrow. As additional evidence, these workers have shown that immature human thymocytes

175

176

Lymphocyte development include not only clonogenic pre-T cells, but also populations of cells that can differentiate into myeloid or erythroid lineages following appropriate stimulation [54*]. However, no demonstration that individual fetal thymus cells possess both lymphoid and non-lymphoid diRerentiation potential was reported. In spite of this, the presence of myeloerythroid progenitors in developing thpus is substantial evidence that progenitors other than committed pre-T cells are able to home to and survive within the thymus. This leaves the door open to the possibility that multipotent HSCs are responsible for initiating at least some degree of T-cell development in the thymus. Evidence for a common progenitor for B and T lineages has been difficult to obtain. Although unique chromosomal markers induced by radiation can sometimes be observed exclusively in lymphoid cells, the existence of a lymphoid-restricted stem cell has not been demonstrated unequivocally. One hurdle has been the absence of an in vitro model system in which both T- and B-lymphoid development can occur simultaneously. This would allow limiting dilution studies to determine the common origin of the two lineages; although even in this case one would also need to show the absence of differentiation potential in other lineages to argue strongly for lymphoid commitment. Fulop and Phillips [55*] have used data from reconstitution of SCID mice at limiting dilution to argue the existence of lymphoid-restricted stem cells. Cells harvested from long-term bone marrow cultures and transferred at limiting dilution into lightly irradiated SCID mice were able to reconstitute both T- and B-cell function in the recipient animals. Further, secondary transplantation experiments showed that the progenitors had been able to establish sufficiently in the primary animals to mediate transfer of B-cell function into a second generation of irradiated animals. Although the authors did not evaluate myeloid repopulating activity by the transferred cells, they propose that the frequency of lymphoid-restricted stem cells in their cultures is much higher than the frequency of multipotent stem cells. However, the possibility remains that the cells that reconstitute T- and Bfunction in their experiments are also capable of differentiation in the myeloid lineage, even in the absence of long-term repopulating (HSC) activity; cells fitting this description have recently been demonstrated in normal mouse bone marrow [7**]. The existence of lymphoidrestricted stem cells has still not been unequivocally proved. Further characterization of the lymphoid-reconstituting cells in long-term bone marrow culture and introduction of unique genetic markers by retroviral vectors are approaches that may lead to an unequivocal demonstration of this stage of hematopoiesis.

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