Stem Cells, Vol. 14, No. 4, 388-395,
July 1996
© 1996 AlphaMed Press
Regulatory Roles of the Ligand for Flk2/Flt3 Tyrosine Kinase Receptor on Human Hematopoiesis
Reiko Namikawa,
Marcus O. Muench,
Maria-Grazia Roncarolo
DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, California, USA
Key Words. Flk2/Flt3 ligand • Tyrosine kinase receptor • Hematopoiesis • Growth factor • Progenitor cells • Kit ligand
Dr. Reiko Namikawa, DNAX Research Institute of Molecular and Cellular Biology, Inc., 901 California Avenue, Palo Alto, CA 94304, USA.
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Abstract
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The biological activities of the ligand for the Flk2/Flt3 receptor tyrosine kinase (FL) on human hematopoietic cells are reviewed. In in vitro studies, FL shows relatively few effects by itself on the proliferation and differentiation of hematopoietic cells, but exhibits a potent costimulatory activity in enhancing the proliferation of progenitor cells of multiple lineages. FL promotes the growth of clonogenic myeloid progenitor cells in the presence of other cytokines known to be active on myeloid progenitors, including GM-CSF, interleukin 3 (IL-3), kit ligand (KL), M-CSF and G-CSF. In addition, FL synergizes with IL-7 in inducing the proliferation of pro-B cells, whereas FL has little effect on the growth of clonogenic erythroid progenitors. Furthermore, FL induces the in vitro expansion of the high proliferative potential colony-forming cells (HPP-CFC) and stimulates the proliferation of long-term culture-initiating cells (LTC-IC), suggesting an activity on the proliferation of putative stem cells. Thus, FL plays important roles in regulating the proliferation of hematopoietic progenitor cells and, therefore, may have therapeutic applications.
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Introduction
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Receptor tyrosine kinases and their ligands play an important role in regulating the proliferation and differentiation of hematopoietic cells. M-CSF, the ligand for the receptor c-fms, regulates the proliferation of monocytic progenitors [1, 2]. The kit ligand (KL), which is the ligand for c-kit, and is also called Steel factor, mast cell growth factor or stem cell factor (SCF) [3–5], is a potent growth factor for hematopoietic progenitors of multiple lineages and for mast cells [6, 7].
A new member of this receptor tyrosine kinase family, the murine Flt3 or Flk2, was independently cloned by two groups of investigators. Flt3 (fms-like tyrosine kinase-3) was cloned from murine placenta based upon its sequence homology to c-fms [8]. Flk2 (fetal liver kinase-2) was cloned from highly enriched murine fetal liver progenitor cells [9]. The murine Flk2/Flt3 gene was shown to share sequence homology with c-kit and c-fms and was found to be expressed in hematopoietic progenitor cells [9]. The human homolog of murine Flk2/Flt3 gene was also cloned [10, 11] and found to be expressed in CD34+ progenitor cells [11] and in some leukemic cells [10, 12]. These observations suggested that the Flk2/Flt3 receptor tyrosine kinase may be involved in regulating the early events of hematopoiesis.
The murine and human ligands for the Flk2/Flt3 receptor (FL) were recently cloned, and demonstrated to share structural similarities with KL and M-CSF [13–16]. Here we review recent studies on the effects of FL on human hematopoiesis in vitro. In particular, we will focus on our recent findings on the effects of FL on fetal hematopoietic cells.
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FL Stimulates the Proliferation of Myeloid Progenitor Cells
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Initial studies showed that FL, similarly to KL, synergizes with IL-3 or GM-CSF in inducing colony formation by human fetal liver [14], adult bone marrow or cord blood CD34+ progenitors [13, 15]. In subsequent studies, the effects of FL on the growth of different subsets of fetal liver progenitor cells were investigated and compared with the effects of KL [17]. It has been demonstrated that the most primitive progenitor cells in the fetal liver express the highest levels of the CD34 antigen, while expressing very low levels of CD38 antigen [18]. As progenitor cells differentiate, CD34 expression is decreased, whereas the levels of CD38 expression increase. Therefore, fetal liver progenitor cells were depleted of mature cells and B cell progenitors expressing the lineage (Lin) markers, CD3, CD8, CD10, CD4, CD16, CD19, CD20 and CD56, and were separated into three different subsets based on the expression of the CD34 and CD38 antigens. The CD34++CD38–Lin– population contained the most primitive progenitors, the intermediate CD34++CD38+Lin– population was enriched for committed progenitor cells, and the CD34+Lin– cells, most of which are CD38+, represented the most mature population. Results from both short-term proliferation assays and colony-forming assays in serum-deprived conditions demonstrated that FL stimulates the proliferation of the CD34++CD38–Lin– and CD34++CD38+Lin– cells, but not of the CD34+Lin– cells, when combined with either interleukin 3 (IL-3), GM-CSF, or KL. Similarly, FL alone showed a modest activity on the proliferation of the CD34++CD38–Lin– and CD34++CD38+Lin– populations, but not on the CD34+Lin– population. In contrast, KL was active on all three populations, either alone or in synergy with other cytokines. Taken together these data show that KL and FL share stimulatory activities on myeloid progenitors, but FL specifically supports the growth of early (CD34++) progenitors.
Several studies have suggested that one of the mechanisms by which KL supports hematopoiesis is that it reduces the concentrations of other cytokines required for optimal stimulation of progenitors [19, 20]. However, our results do not support this hypothesis. In studies in which the responsiveness of progenitor cells to GM-CSF in the presence or absence of FL or KL was measured, neither FL nor KL was shown to sensitize progenitors to the actions of a second cytokine [17]. In proliferation assays, the doses of GM-CSF required for the half-maximal proliferation of CD34++CD38+Lin– cells were similar in cultures stimulated by either GM-CSF alone, GM-CSF + FL or GM-CSF + KL. Similar results were obtained when FL or KL was titrated in the presence of a constant concentration of GM-CSF. The discrepancy between these data and those of other investigators may be due to the different assays employed. The short-term proliferation assays used in our studies are more sensitive indicators of proliferation by progenitors, as compared with colony assays used in other studies.
It has been demonstrated that multiple cytokines are required to induce proliferation of primitive progenitor cells [21, 22]. Individual cytokines, however, can support the survival of quiescent progenitor cells [23–25]. Our studies show that both FL and KL can promote the survival of the most primitive CD34++CD38–Lin– cells [17]. In delayed-cytokine addition experiments, progenitors were cultured in the presence or absence of an individual cytokine for up to 96 h, and the number of clonogenic progenitors that survived was compared with the number present at time 0. In the absence of any growth factors, the number of progenitors was found to decline by more than 50% after 24 h of culture, and nearly all progenitor activity was lost after 96 h. The addition of a single cytokine, either FL, KL, GM-CSF or IL-3, prior to the addition of a cocktail of cytokines which induces clonal growth, was found to support the survival of primitive progenitors for up to 48 h of culture. These results provide evidence for a direct action of FL, KL, GM-CSF and IL-3 in supporting the short-term survival of early fetal liver hematopoietic progenitor cells. It has been shown that hematopoietic stem cells are predominantly in a quiescent state in vivo [26, 27], with only a few stem cell clones actively proliferating [28–30]. However, the mechanisms by which quiescent stem cells are maintained are unknown. Our results suggest that these cytokines may contribute to support the survival of quiescent stem cells, but they also suggest that other elements of the bone marrow (BM) microenvironment must be responsible for the long-term maintenance of quiescent progenitors.
Recently the costimulatory activities of FL with a wide range of cytokines have been further confirmed using progenitor cells isolated from different sources. In addition to GM-CSF, IL-3 and KL, FL has been found to synergize with M-CSF in inducing colony formation by progenitor cells in peripheral blood [31] and cord blood [32]. Furthermore, FL enhanced colony formation by cord blood progenitors induced by G-CSF [32]. Collectively, these results indicate that FL is a potent costimulatory molecule on the proliferation and differentiation of myeloid progenitor cells.
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FL Stimulates the Proliferation of Lymphoid Progenitor Cells
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In studies on human B lymphopoiesis it has been shown that FL alone does not induce the proliferation of B cell progenitors and precursors. However, it acts as a strong synergistic factor in combination with IL-7 in promoting the expansion of the early B lymphoid progenitors [33]. Highly purified human fetal BM pro-B cells, defined as CD34+CD19+, were sorted and cultured under serum-deprived conditions in the presence of various cytokines. In contrast to IL-7, FL, KL, IL-3 or GM-CSF alone did not induce the proliferation of human pro-B cells in short-term (10-day) proliferation assays. However, FL or IL-3, but not KL or GM-CSF, showed significant synergistic effects when combined with IL-7. These results indicate that FL differs from KL in its activity on B lymphopoiesis. It is interesting to note that GM-CSF did not affect IL-7-induced pro-B cell proliferation, despite the fact that GM-CSF and IL-3 share the ß-chain of their receptors, indicating that the effects of these cytokines are strictly regulated by the expression of the 
-chain of the receptors. Optimal proliferation of pro-B cells was observed in the presence of a combination of IL-7, IL-3 and FL. Under this condition, 15- to 30-fold increases in cellularity were achieved after two to three weeks of culture. Furthermore, differentiation of CD34+CD19+cIgM– pro-B cells into CD34–CD19+cIgM+ pre-B cells was observed. These data show that human fetal BM pro-B cells can be cultured in the absence of stromal cells when FL is present in combination with IL-7 and IL-3. Therefore, FL may be one of the stromal cell-derived molecules required for long-term B lymphopoiesis in vitro.
In addition to its effects on CD34+CD19+ pro-B cells, FL acts on the generation of B cells from the more immature CD19– progenitor cells (Namikawa et al., unpublished observation). Fetal BM cells which expressed the CD34 antigen brightly but were negative for CD19 (CD34++CD19–) were sorted and cultured under stromal-cell-free conditions in the presence or absence of FL, IL-7 or IL-3. After two to four weeks of culture in the presence of IL-7 and FL, CD19+ B cells were generated from CD34++CD19– progenitors in vitro. IL-7 alone or FL alone was not effective, indicating synergism between FL and IL-7. These results show that FL may play an important role on early B lymphopoiesis by promoting the generation of B cells from progenitors more immature than pro-B cells. Interestingly, the addition of IL-3 into cultures stimulated by IL-7 and FL induced vigorous increases in myeloid cell number but was associated with the loss of CD19+ B cell generation. Thus, FL in combination with IL-7 stimulates the growth of B lineage cells from the primitive progenitor stage through the pro-B cell stage, while IL-3 acts as a synergistic factor mainly at the pro-B cell stage.
FL is known to be produced by a variety of organs including hematopoietic tissues [14, 15] while the production of IL-3 is strictly limited to activated T cells [34]. Therefore, it is conceivable that FL plays a role in regulating physiological B lymphopoiesis in the marrow, while IL-3 induces a rapid expansion of B cell precursors under pathological conditions. Although it was reported that KL augmented the proliferation of IL-7-induced B cell proliferation in the murine system, the role of KL on human B lymphopoiesis is still unclear. We did not observe any significant effect of KL on the growth of human pro-B cells. Further studies are, however, required to elucidate the role of KL in the growth of B cell precursors among CD34++CD19– cells, since the majority of these cells express c-kit [18].
Although FL augmented IL-7-induced proliferation of murine thymocytes [14], no significant effects of FL alone, or in combination with other cytokines, including IL-7, IL-2 and IL-4, have been observed on human T cell progenitors isolated from fetal thymus. Since both murine and human thymi express Flk2/Flt3 mRNA, as detected by Northern blotting or RNA polymerase chain reaction (PCR) [8, 9], it remains unclear why FL acts on murine but not on human thymocytes. Further studies using purified subsets of immature human and mouse T cell progenitors are needed to rule out a selective effect of FL on some T cell subsets.
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Effects of FL on Erythropoiesis
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FL failed to affect erythropoietin (EPO)-induced BFU-E formation in most of the studies reported using progenitor cells derived from fetal liver [14], BM [15], cord blood [15] or peripheral blood [31]. However, other investigators reported that FL enhanced BFU-E formation by progenitors from cord blood or BM [32]. Our recent studies indicate that FL synergized modestly on the most primitive CD34++CD38–Lin– fetal liver cells in inducing colony-forming units-mixture (CFU-MIX) which gave rise to colonies containing both erythroid and myeloid cells, thereby demonstrating that the effects of FL are limited to primitive multipotent progenitors (Muench et al., manuscript in preparation). These data are in line with the recent observation that the expression of Flk2/Flt3 receptor on peripheral blood CD34+ cells is rapidly downregulated upon erythroid differentiation [31]. Thus, FL affects erythropoiesis only at the level of pluripotent stem cells which can give rise to erythroid progenitors. In contrast to FL, KL is a potent costimulator of erythropoiesis across the entire spectrum of CD34-expressing progenitors.
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The Role of FL on the Growth and Differentiation of Stem Cells
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One of the key questions is whether FL is capable of promoting the self-renewal of pluripotential stem cells. Although KL, which is also called SCF, was anticipated as a growth factor acting on stem cells, numerous studies have suggested that the maintenance of stem cells in vivo does not depend on KL [35–37]. In our studies we showed that FL stimulates the proliferation of the most primitive CD34++CD38–Lin– fetal liver progenitor population [17]. In addition, we demonstrated an activity of FL in potentiating the expansion of early progenitor cells. When CD34++CD38–Lin– fetal liver cells were cultured for one week in liquid cultures in the presence of FL and GM-CSF, the numbers of clonogenic progenitors increased as compared to those present on day 0. The growth of both high-proliferative potential (HPP) colony-forming cells (CFC) and low-proliferative potential (LPP)-CFC was induced. The expansion of HPP-CFC under this condition was, however, modest, and was lower than that induced by KL and GM-CSF.
Additional studies suggesting an activity of FL on stem cell proliferation have also been reported. Antisense oligonucleotides to human Flk2/Flt3 gene were shown to suppress colony formation by CD34+ BM progenitors in semisolid cultures [11]. Inhibition of colony-forming unit, granulocyte/macrophage (CFU-GM) and BFU-E was 40%-60%, and colony-forming units-granulocyte/erythrocyte/monocyte/megakaryocyte (CFU-GEMM), a more primitive subset of progenitors, showed inhibition of 60%-80%. More interestingly, addition of antisense oligonucleotides strongly suppressed the generation of CFU-GM in long-term BM cultures, indicating that signaling through the Flk2/Flt3 receptor is critically important in very early hematopoietic cells. Recent studies using CD34+ cells derived from peripheral blood of normal donors provided further evidence that FL induces the expansion of primitive progenitor cells [31]. FL stimulated the formation of blast cell colonies and HPP-CFC, which maintained a significantly higher recloning capacity than those induced by KL. In addition, FL stimulated the proliferation of long-term culture-initiating cells (LTC-IC), an activity not shared by KL. Finally, it has been shown that the cultures of murine stem cells in the presence of FL and IL-6 induced a significant expansion of day-12 colony-forming units-spleen (CFU-S) [38].
Collectively, these results support the hypothesis that FL plays a role in regulating stem cell proliferation. Further studies are required to elucidate the biological activity of progenitor cells expanded in vitro in the presence of FL.
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Conclusions and Future Prospects
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The results of the in vitro experiments with human hematopoietic cells summarized in this review indicate that FL is a potent costimulatory growth factor for early hematopoietic progenitor cells, including the primitive progenitors, progenitors for myeloid cells, and those for B cells. These results are concordant with those found in murine studies [38–41], indicating that the regulatory role of FL is well-conserved between humans and mice.
FL and KL are likely to be part of a network of cytokines which regulates the proliferation of hematopoietic stem and progenitor cells, as anticipated from the structural as well as functional similarities found between these two molecules. They have apparently common or redundant activities on the growth of primitive progenitors and myeloid progenitors, while playing unique roles in other hematopoietic compartments with different lineage specificity. FL stimulates the proliferation of B cell progenitors, but not progenitors of the erythroid lineage. In contrast, KL is active on erythroid progenitors, but does not stimulate pro-B cell growth. KL also stimulates mast cell growth as a single factor. These activities of FL and KL observed in in vitro studies correlate well to the hematopoietic defects found in mice which lack the expression of these molecules or their receptors. Mice with mutations either in c-kit gene (W/W) or Steel gene (Sl/Sl) show defects predominantly in the erythroid and mast cell compartments [42–44], while Flk2/Flt3 receptor-deficient mice have an impaired activity in the B cell progenitor compartment [45]. Furthermore, mice with mutations in both of these receptor genes have severely reduced cellularities in both the lymphoid and myeloid compartments [45], suggesting that FL and KL function together in the primitive progenitor cell compartment, insuring the production of normal numbers of hematopoietic cells.
Although there is no direct evidence yet supporting the hypothesis that FL stimulates the self-renewal of hematopoietic stem cells, FL is a candidate cytokine to be included in cytokine combinations for the ex vivo expansion of early progenitor cells, since it stimulates the growth of the primitive progenitor cells. It is also possible that FL can improve the efficiency of gene transduction through retroviral vectors by specifically recruiting these early progenitors into cell cycle. One of the important issues regarding ex vivo expansion of progenitors and in vitro gene transfer into progenitors is the maintenance of multilineage potential during the culture period. It was demonstrated that murine early progenitors stimulated in vitro in the presence of IL-3 lost B cell potential [46], suggesting that the exposure of uncommitted early progenitors to certain cytokines may result in the lineage commitment of these cells. Further studies will elucidate the effects of FL in maintaining the multilineage potential of primitive progenitor cells in vitro. In this regard, the severe combined immunodeficiency-human (SCID-hu) mouse model [47–49] will be useful since it provides a unique in vivo tool to determine both myeloerythroid and lymphoid potentials of cells which have been expanded in vitro [50, 51].
The Flk2/Flt3 receptor is expressed on various types of human leukemia cells, in particular on acute myelogenous leukemia (AML) and pre-B acute lymphoblastic leukemia cells [10, 12]. The distribution of Flk2/Flt3 receptor expression on leukemia cells thus correlates with the lineages which are found to respond to FL in vitro, namely early myeloid and B lymphoid cells. These findings suggest that, in addition to a physiological role in regulating hematopoiesis, Flk2/Flt3 and FL may be involved in the leukemogenic processes of progenitor cells and in the subsequent regulation of leukemic cell growth. It is possible that genetic alteration of the receptor gene may cause unregulated proliferation of primitive cells, as suggested for receptors for EPO [52, 53], M-CSF [54–56] and G-CSF [57]. An aberrant expression of the ligand by transformed cells may also support autocrine proliferation. Investigation in this area would be of importance not only to further understand the role of FL in hematopoiesis, but also to develop new therapeutic modalities, especially for AMLs with poor prognoses.
The in vitro activities of FL suggest that it may have clinical potential. FL may support the hematopoietic recovery after myeloablative chemo- and/or radiotherapy or after BM transplantation. The unique activity of FL in stimulating B lymphopoiesis, in addition to its myeloid-stimulating activity, may also lead to accelerated B cell reconstitution, in contrast with other myelopoietic growth factors presently used in the clinic. FL may also induce the mobilization of BM progenitor cells into the periphery as do other early-acting hematopoietic growth factors [58–62]. Furthermore, the preferential expression of Flk2/Flt3 on AML cells suggests that FL can be one of the cytokines which recruit clonogenic AML leukemic blast cells in quiescent phase into S-phase, and thereby it may increase the sensitivity of these cells to cell-cycle-specific chemotherapeutic agents [63–66]. Finally, it is of importance to note that FL does not appear to affect mast cell growth in vitro [16, 38], and therefore, it will not have the undesired anaphylactoid responses of KL observed in clinical trials [67, 68]. Studies designed to investigate the in vivo effects of FL on human hematopoiesis are currently ongoing in our laboratory using the preclinical SCID-hu mouse model [47, 49, 69]. These studies, as well as the in vivo studies with experimental animals, will provide further indications on the potential clinical utility of FL.
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Acknowledgments
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The authors would like to acknowledge Drs. S. Menon, S. Zurawski, G. Zurawski, and C. Hannum for purification of the FL, and D. Rennick, S. Hudak, and J. E. de Vries for scientific discussions and suggestions. DNAX Research Institute of Molecular and Cellular Biology, Inc. is supported by the Schering-Plough Corporation.
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accepted for publication January 30, 1996.
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Abstract
Introduction
