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. 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 purified the c-Kit-neg population based on Sca-1 expression and found that the Sca-1-positive cells develop more rapidly than the Sca-1-negative subset in vivo. We also identified, for the first time, an in vitro system that is permissive for the maturation of active c-Kit-pos cells stem cells from c-Kit-neg cells. We have determined that c-Kit-neg PHSC can be induced to proliferate and differentiate on modified 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 LTMBC, and that 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 hematopoietic progenitors that lack C/EBP alpha show increased erythroid growth in vitro suggesting that C/EBP alpha may act as a switch to negatively regulate erythroid differentiation. We found that C/EBP alpha inhibited an in vitro model of erythroid differentiation. In addition, activation of C/EBP alpha induced inhibitor of differentiation 1 (Id1) protein expression that is known to prevent erythroid differentiation. Thus, we propose that C/EBP alpha is required for the differentiation of common myeloid progenitors (CMP) to more committed granulocyte macrophage progenitors and, negatively regulates the differentiation of CMP to erythroid progenitors. We have discovered that Bcl11a deficient embryos lack B cells and have developmental defects in several T cell subsets. Both B and T cell maturation are arrested in mice transplanted with Bcl11a KO fetal liver cells demonstrating that this defect is intrinsic to the hematopoietic compartment. Phenotypic and expression studies places Bcl11a upstream of Ebf and Pax5 transcription factors in the B cell pathway. Mice that receive Bcl11a KO FL also develop a host and donor CD-4+ CD-8+ leukemia after 3-5 months suggesting that Bcl11a may function as a tumor suppressor. The human homologue of this gene has been recently cloned and may be involved in lymphoid malignancies through chromosomal translocation or amplification. We have discovered that the interferon inducible p205 (IFI-205), and another family member, IFI-204, inhibit the growth of hematopoietic stem cells and stem cell lines suggesting these family members may play important functional roles in the negative regulation of stem cell growth. To investigate the molecular mechanism(s) by which IFI-205 regulates cell growth we used a yeast two-hybrid screen and have identified a number of candidate proteins including Miz-1 and p53BP. We have also found that IFI-205 inhibits cell growth in cells that lack RB and p53. We have discovered that Id1 is not expressed in hematopoietic stem cell (HSC) and common lymphoid progenitors (CLP) but is expressed in more committed common myeloid progenitor cells (CMP). Id1 is expressed in progenitor-enriched populations and is decreased in granulocytes, shows little or no expression in erythroid cells, B and T cells. Id1 is up-regulated by in HSC, CMP, and Linlo populations by culturing cells in interleukin-3 and other cytokines that promote myeloid development, but not by interleukin-7 and Flt-3 Ligand, which promotes lymphoid development. Id-1 expression in transgenic animals is known to inhibit B-cell development. Therefore, IL-3 may instruct hematopoietic stem cells towarda myeloid versus lymphoid cell fate through the induction of the HLH protein Id1.
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