Role of c-mpl in early hematopoiesis.

Recently, several lines of evidence have indicated an expanded role for thrombopoietin (TPO) and its receptor, c-mpl, in hematopoiesis. In addition to being the primary physiological regulator of platelet production, it is now apparent that TPO also acts during early hematopoiesis. To futher define the role of TPO in early hematopoiesis we have identified discrete murine and human stem cell populations with respect to c-mpl expression and evaluated their potential for hematopoietic engraftment. Fluorescence-activated cell sorter analysis of enriched stem cell populations showed the presence of c-mpl expressing subpopulations. Approximately 50% of the murine fetal liver stem cell-enriched population, AA4(+)Sca+c-kit+, expressed c-mpl. Analysis of the murine marrow stem cell population LinloSca+c-kit+ showed that 70% of this population expressed c-mpl. Expression of c-mpl was also detected within the human bone marrow CD34(+)CD38(-) stem cell progenitor pool and approximately 70% of that population expressed c-mpl. To rigorously evaluate the role of TPO/c-mpl in early hematopoiesis we compared the repopulation capacity of murine stem cell populations with respect to c-mpl expression in a competitive repopulation assay. When comparing the fetal liver progenitor populations, AA4(+)Sca+c-kit+c-mpl+ and AA4(+)Sca+c-kit+c-mpl-, we found that stem cell activity segregates with c-mpl expression. This result is complemented by the observation that the LinloSca+ population of c-mpl gene-deficient mice was sevenfold less potent than LinloSca+ cells from wild-type mice in repopulating activity. The engraftment potential of the human CD34(+)CD38(-)c-mpl+ population was evaluated in a severe combined immunodeficient-human bone model. In comparison to the CD34(+) CD38(-)c-mpl- population, the CD34(+)CD38(-)c-mpl+ cells showed significantly better engraftment. These results demonstrate a physiological role for TPO and its receptor, c-mpl, in regulating early hematopoiesis.

A role for the Wnt gene family in hematopoiesis: expansion of multilineage progenitor cells.

The microenvironment is a key regulator of hematopoietic stem cells (HSCs) and is a likely source of extracellular factors that control stem cell fate. A better understanding of these microenvironmental factors may come from investigations of developmental cell fate determination in which the critical roles of cell-cell interactions of multipotential cells have been shown. The Wnt gene family is known to regulate the cell fate and cell-cell interactions of multipotential cells in a variety of tissues. Expression of Wnts and of their putative receptors encoded by murine homologs of the Drosophila frizzled gene in hematopoietic tissues was examined by reverse transcriptase-polymerase chain reaction. Wnt-5a and Wnt-10b were expressed in day-11 murine yolk sac, day-14 fetal liver, and fetal liver AA4+ cells. The expression profiles of four murine frizzled homologs, Mfz3-7, were nearly identical to that of Wnt-5a and Wnt-10b. Notably, Wnt-10b was expressed in the fetal liver AA4+ Sca+ c-kit+ flASK) HSC population. A role for Wnts in HSC fate determination was studied by treatment of HSC populations in culture with soluble WNT proteins. The addition of conditioned media from cells transfected with Wnt-1, Wnt-5a, or Wnt-10b cDNAs to cultures of flASK cells stimulated a sevenfold, eightfold, and 11-fold expansion in cell number, respectively, relative to control media. Removal of WNT-5a from this media by immunodepletion depleted the stimulatory activity from the media, whereas addition of a partially purified WNT-5a stimulated a fivefold expansion relative to control cells. Transduction of flASK cells with a retrovirus bearing a Wnt-5a cDNA enhanced proliferation. We conclude that WNTs stimulate the survival/proliferation of hematopoietic progenitors, demonstrating that WNTs comprise a novel class of hematopoietic cell regulators.

A role for leptin and its cognate receptor in hematopoiesis.

BACKGROUND: Hematopoiesis entails the production of multiple blood cell lineages throughout the lifespan of the organism. This is accomplished by the regulated expansion and differentiation of hematopoietic precursors that originate from self-renewing hematopoietic stem cells. Studies of lineage commitment and proliferation have shown that the cytokine family of growth factors plays an important role in hematopoietic differentiation. However, in hematopoiesis, as in most self-renewing biological systems, the molecules that regulate the stem cells directly remain largely unknown. In this study, we have undertaken a search for novel cytokines that may influence the fate of hematopoietic stem cells. RESULTS: We have cloned three splice variants of a novel cytokine receptor from human hematopoietic stem cells expressing the CD34 antigen, one of which is identical to the leptin receptor. Expression analysis revealed that the leptin receptor is expressed in both human and murine hematopoietic stem cell populations, and that leptin is expressed by hematopoietic stroma. We show that leptin provides a proliferative signal in hematopoietic cells. Importantly, we demonstrate that leptin provides a proliferative signal in BAF-3 cells and increases the proliferation of hematopoietic stem cell populations. The proliferative effects of leptin seem to be at the level of a multilineage progenitor, as shown by increased myelopoiesis, erythropoiesis and lymphopoiesis. Analysis of db/db mice, in which the leptin receptor is truncated, revealed that the steady-state levels of peripheral blood B cells and CD4-expressing T cells were dramatically reduced, demonstrating that the leptin pathway plays an essential role in lymphopoiesis. Colony assays performed using marrow from db/db and wild-type mice indicated that db/db marrow has a deficit in lymphopoietic progenitors; furthermore, db/db mice are unable to fully recover the lymphopoietic population following irradiation insult, and although the levels of peripheral blood erythrocytes are normal in db/db mice, spleen erythrocyte production is severely compromized. CONCLUSIONS: We have discovered that leptin and its cognate receptor constitute a novel hematopoietic pathway that is required for normal lymphopoiesis. This pathway seems to act at the level of the hematopoietic stem/progenitor cell, and may well also impact upon erythropoiesis, particularly in anemic states that may require output from the spleen. These findings offer a new perspective on the role of the fat cell in hematopoiesis.