Homeobox (Hox) transcription factors are important regulators of hematopoietic cell proliferation and differentiation. Of them, HoxB4 is of particular interest because overexpression promotes rapid expansion of mouse hematopoietic stem cells (HSCs) without causing neoplastic transformation. Despite the effects of HoxB4 overexpression on HSCs, mice that are homozygous for HoxB4 gene deletion have only subtle defects in HSCs and progenitor cells. We hypothesized that other paralogs of HoxB4 may also be capable of inducing HSC expansion could thereby compensate for loss of HoxB4 function. To test this hypothesis, we have studied the effects of retroviral overexpression of a HoxC4 gene in murine progenitors and HSCs. The murine HoxC4 cDNA was cloned and inserted into an MSCV vector that co-expresses an IRES-YFP reporter gene. We transduced murine bone marrow cells with a MSCV-HoxC4-YFP vector and compared the secondary replating efficiency of myeloid colonies (CFU-Cs) to that seen using either a MSCV-HoxB4-GFP or an MSCV-GFP vector. This assay tests for progenitor cell self-renewal which is increased using HoxB4-expressing vectors. Cells transduced with the MSCV-HoxC4-YFP vector formed 20–40 times more secondary CFU-Cs than with cells transduced with the MSCV-GFP control vector. This increase in CFU-C replating efficiency was equivalent to that seen with the MSCV-HoxB4-IRES-GFP vector. To test the in vivo effects of the MSCV-HoxC4-YFP vector on self-renewal of HSCs, we transplanted lethally irradiated mice with a mixture of cells; 20% transduced with the MSCV-HoxC4-YFP vector and 80 % mock-transduced. Peripheral blood analysis of the transplanted recipients up to 28 weeks post-transplantation showed that the percentage of cells transduced with the MSCV-HoxC4-YFP vector was 70–85% in both lymphoid and myeloid cells in the peripheral blood. A similar degree of chimerism was noted in concurrent controls using the MSCV-HoxB4-GFP vector. In contrast, the percentages of peripheral blood cells transduced with the MSCV-GFP vector was only 15–25%, paralleling the input ratios of transplanted cells. Secondary transplantation experiments showed stable levels of chimerism in both HoxC4 and HoxB4 groups, indicating that the expansion seen with the MSCV-HoxC4-YFP vector occurred at the HSC level. These results indicate that retroviral-mediated expression of HoxC4, like HoxB4, can cause significant expansion of HSCs in vivo. Because several other Hox genes can cause hematopoietic abnormalities and leukemia when expressed from a retroviral vector, we transplanted lethally irradiated mice with 4x106 cells that were transduced with the MSCV-HoxC4-YFP vector and monitored the animals for survival and complete blood counts. Now, at 33 weeks post transplantation, no tumor formation was observed in mice expressing either the HoxB4 or the HoxC4 vector, and peripheral blood counts have remained normal. Our results show that retroviral overexpression of HoxC4 can induce a significant expansion of the HSCs in vivo, and suggest that expression of HoxC4 may compensate for the loss of HoxB4 in knockout mice. We are currently analyzing the effects of HoxA4 and HoxD4 to determine if they share the same functional characteristics, and are also determining whether HoxB4 and HoxC4 are modulating the same downstream genes using microarray analysis of transduced murine bone marrow cells.