We have continued to purify and characterize a novel class of pluripotential hematopoietic stem cells (PHSC) that lack c-Kit (c-Kit-neg), the receptor for stem cell factor (SCF). This is in contrast to previously characterized PHSC populations which express c-Kit (c-Kit-pos). We have demonstrated that the c-Kit-neg PHSC have delayed reconstituting activity and can give rise to c-Kit-pos PHSC when transplanted into irradiated mouse recipients. Taken together, we have hypothesized that steady state hematopoiesis is supported by PHSC that express c-Kit, and that c-Kit-neg PHSC represent a quiescent population of PHSC that are recruited into the actively contributing pool of c-Kit-pos stem cells. To better understand the mechanisms which regulate this maturation pathway, we have further purified this stem cell population and evaluated its growth and differentiation in vivo and in vitro. We have determined that c-Kit-neg PHSC can be induced to proliferate and differentiate on the M2-10B4 stromal cell line in vitro and on modified Tpo long term bone marrow LTBMC cultures. We have found that we can induce the expression of c-Kit on c-Kit-neg PHSC by co-culture on Tpo LTMBC. We are currently testing whether the c-kit-pos cells have PHSC activity. It is hoped that these studies will allow us to define the mechanism(s) which regulate the c-Kit maturation pathway. We have discovered that growth factor dependent-myelomonocytic progenitor cell lines can be directly established from C/EBP alpha KO fetal livers (FL) without crisis (C/EBP alpha is a bZIP transcription factor). IL-3 and SCF-dependent cell lines can be induced to undergo further differentation into macrophages when cultured in GM-CSF. These cell lines represent novel models to evaluate the molecular mechanism(s) that C/EBP alpha KO cells escape growth arrest and differentation. We have also found that FL progenitor cells give rise to leukemias after a long latency when transplanted in vitro suggesting that C/EBP alpha may act as a tumor suppresor gene and represent a potential therapeutic target for myelomonocytic leukemias. We have discovered that B cell maturation is arrested in the fetal liver (FL) and T cell maturation is impaired in the fetal thymus of Evi-9 KO mice (collaboration with Neal Copeland). In comparision, B and T cell maturation are arrested in mice transplanted with Evi-9 KO FL. Mice that receive Evi-9 KO FL also develop a CD-4+ leukemia after 2-5 months suggesting that Evi-9 may function as a tumor suppressor. The human homologue of this gene, BCL11A, has been recently cloned and may be involved in lymphoid malignancies through chromosomal translocation or amplification. We have discovered that p205 (a nuclear protein and member of the interferon-inducible-200 family) inhibits hematopoietic progenitor cell growth and may be important for regulating cell growth and differentiation. We have generated transgenic mouse strains that express p205 and a closely related family member p204 to evaluate their function in vivo. These transgenic mouse strains express p205 and p204 specifically in myeloid cells (MRP-8 promoter) and tymocytes (CD-2 promoter). Effects on myeloid and lymphoid development are currently being evaluated. We have discovered that interleukin-3 can induce the expression of inhibitor of DNA binding protein-1 (Id1) (helix-loop-helix transcription factor) in a stem cell line model during myeloid cell growth and differentiation. Id-1 expression in transgenic animals is known to inhibit B-cell development We are testing the hypothesis that Il-3 may play an instructive role in stem cell fate decisions via Id1.
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