Abstract
Abstract
Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. Since it was shown, that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, we sought to define the expression profile and function of the human homologue VENTX2 in hematopoietic development. When VENTX2 expression was assessed by TaqMan qRT-PCR in highly purified human cord blood subpopulations, expression of VENTX2 was detected in CD33 and CD15 positive myeloid cells as well as in B and T cells. In addition VENTX2 expression was observed in the primitive CD34+38- human subset as well as in 34+38+ compartment. Furthermore, VENTX2 expression was recurrently detected in bone marrow samples from AML patients at diagnosis as determined by TaqMan qRT-PCR (n=3). In an attempt to characterize the functional relevance of VENTX2 expression for hematopoietic development we retrovirally engineered murine hematopoietic progenitor cells to constitutively express the gene using a MSCV-based retroviral construct with an IRES-EGFP cassette. Successfully transduced cells were injected into lethally irradiated mice or used for in vitro experiments. At the level of the clonogenic progenitor VENTX2 induced myeloid and blocked erythroid differentiation with a significant 1.5 fold increase in the number of CFU-G (n=10; p<0.01) compared to the GFP control and a 5.4 fold decrease in the number of erythroid colonies (n=10; p<0.005) without a change of the total number of colonies produced indicating that VENTX2 promoted granulocytic differentiation in vitro. Re-plating assays showed an over 2 fold increase in the number of secondary CFU-G. In MS-5 murine assay VENTX2 transduced cord blood cells increase CD15+ cells 10.4 fold compared to the control (n=3)(p<0,01). When the effect of VENTX2 was determined on the frequency of LTC-IC by limiting dilution assay (n=2), no major differences were detected between the homeobox gene and the control arm. In NOD/SCID mice VENTX2 induced a 2.9 fold increase in the proportion of CD15+ mature myeloid cells as compared to the GFP control (VENTX2 mean 4 x 106 + 1 x 106 n=7; GFP mean 2x106 cells + 6 x 105 n=9; p<0.02). Using competitive repopulation unit assay (CRU) in NOD/SCID mice we could demonstrate that VENTX2 is able to increase the frequency of CRU cells by 1.8 over GFP control (n=4). These findings characterize VENTX2 as a novel regulatory protein of early human hematopoiesis and extend our knowledge on the regulatory role of non-clustered homeobox genes in hematopoietic development.