A recent transcriptome analysis of human umbilical cord blood and bone marrow (BM) CD34+CD33CD38ckit+Rholo (Rholo) vs. CD34+CD33CD38ckit+Rhohi (Rhohi) cells identified the gene RASSF8, to be more highly expressed in the hematopoietic stem cell (HSC) rich compartment (Rholo) versus committed progenitor cells (Rhohi). In a subsequent screen for the function of differentially expressed genes in hematopoiesis, the knockdown of RASSF8 using morpholinos in zebrafish embryos resulted in decreased blood formation and reduced expression of early (scl and gata1) and late (hbae1 and lcp1) hematopoietic markers. To test the function of RASSF8 in mammalian hematopoiesis, we overexpressed RASSF8 cDNA in lineage depleted murine bone marrow cells (Lin) by retroviral transduction using the MSCV-RASSF8-IRES-GFP vector (rMIG-RASSF8). Sorted transduced GFP+ cells were cultured in serum-free medium (supplemented with SCF, TPO, Flt3L and Il-3) for three to five days. No significant differences were seen in overall cell expansion, cell death (7-AAD staining) or cell proliferation (thymidine incorporation assay and propidium iodide staining) between rMIG-RASSF8 transduced cells and cells transduced with the control vector (rMIG). However, we noted a significant accumulation of the stem cell enriched cKit+LinSca1+ (KLS) population in rMIG-RASSF8 transduced cells (43+/−9%) compared to control cells transduced with rMIG (13+/−8%, p=0.003; n=4) after five days. The total number of colony forming cells (CFCs) produced by 750 rMIG-RASSF8 transduced Lin cells (24+/−7) was significantly lower than in rMIG transduced cells (54+/−8, p=0.008; n=3). CFCs from rMIG-RASSF8 transduced cells were morphologically smaller and more compact, consistent with the morphology of primitive blast-forming colonies. Competitive repopulation assays using 25 000 rMIG-RASSF8 or rMIG transduced C57/Bl6 CD45.1 Lin- cells versus 100 000 C57/Bl6 CD45.2 mononuclear BM cells were performed to evaluate HSC engraftment potential. Four weeks after transplantation only 1+/−0.96% CD45.1 cells were found in the peripheral blood (PB) of the animals receiving rMIG-RASSF8 transduced cells vs. 23+/−12% CD45.1 cells in animals that had received rMIG transduced cells (p< 0.05; n=3). Likewise, PB analysis at 3 months demonstrated nearly no reconstitution of rMIG-RASSF8 transduced CD45.1 cells (1+/−1.7%) vs 30+/−23% rMIG transduced CD45.1 cells (p=0.02). In conclusion, constitutive overexpression of RASSF8 increases the retention/expansion of KLS cells in vitro, blocks differentiation of progenitors giving rise to CFC and inhibits engraftment of HSC. These studies thus suggest that short term overexpression of RASSF8 might ultimately lead to new methods of HSC expansion.

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