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 further purified this stem cell population. First, we determined its cell surface phenotype using antibodies that recognize lineage specific and other cell surface antigens by flow cytometry. We found that the freshly isolated c-Kit-neg cells lack lineage specific markers for granulocytes (GR-1) and macrophages (F4/80) but contain cells that express Ter119 (erythroid cells) and B220/CD-19 (B cells). Therefore, the c-Kit-neg cells were FACS separated into B220-Ter119- and B220+Ter119+ cell populations. The c-Kit-neg B220-Ter119- cells contain the PHSC activity when transplanted into irradiated mouse recipients while the c-Kit-neg B220+Ter119+ cells do not. We further defined the phenotype of the c-Kit-neg B220-Ter119- cells by 3-color flow cytometry and showed that roughly 22% of the cells express Sca-1(also expressed on c-Kit-pos PHSC), 30% expressed CD-34, while they lacked the expression of Flt-3 and IL-6? receptors. When c-Kit-neg B220-Ter119- cells were sorted into Sca-1+ Sca-1- populations, the c-Kit-neg B220-Ter119-Sca-1+ cells gave rise to macroscopic spleen colonies in secondary recipients when transplanted in vivo, suggesting that this population contained PHSC. Finally, >99% of the c-Kit-neg cells are in G1/G0 . We are currently investigating the mechanisms, which regulate the c-Kit maturation pathway in bone marrow cell development. In other studies, we have continued our efforts to develop more efficient vectors to transfer genes into primitive hematopoietic stem cells. To this end, we have redirected Adenoviral infection to hematopoietic cells by covalently linking biotin to the surface of recombinant Adenovirus and coupling biotinylated SCF through an avidin bridge. Thus, we have developed an Adenoviral vector with expanded tropism such that infection can occur through c-Kit receptors expressed on PHSC. While we have redirected adenoviral infection to hematopoietic progenitor cell lines using biotinylated ligands and antibodies, we have also redirected infection of adenovirus vectors to stimulated and unstimulated primary T cells using biotinylated IL-2 and biotinylated antibodies that recognize CD-44. We have successfully purified biotinylated adeno-associated virus to redirect viral infection since this virus can stably infect target cell populations. We are testing whether this vector can stably introduce reporter genes into hematopoietic progenitor cell lines. The molecular events that regulate lineage commitment and terminal differentiation of pluripotential hematopoietic stem cells (PHSC) are largely unknown. To identify potentially important genes involved in regulating PHSC growth and differentiation we used differential display (dd)-RT-PCR. Several cDNA clones were isolated and differential expression was confirmed by northern blot analysis. One clone showed DNA sequence identity to p205, a previously described gene with unknown function, that is a member of the interferon-inducible 200 (IFI-200) gene family. We characterized the expression of this gene during hematopoietic cell differentiation and have found that p205 is induced in normal PHSC populations under conditions that promote myelomonocytic cell differentiation (SCF plus interleukin-3 {IL-3}). p205 inhibits the proliferation of IL-3-dependent BaF3 cells in transient transfection assays (3H-Thymidine incorporation and total viable cell numbers). In addition, a closely related family member p204 also inhibits BaF3 cell proliferation under the same assay conditions. Further, microinjection of p205 expression vectors into serum starved NIH-3T3 cells inhibits serum-induced proliferation. Current studies are directed at better understanding the potential tumor suppressive activities of this family of proteins in hematopoietic cells, and their role in regulating differentiation and apoptosis.
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