By modulating levels of Pbx-1, a homeodomain protein and DNA binding cofactor of Hoxb4, it is possible to generate pluripotent, ultracompetitive in vivo repopulating Hoxb4hi and Pbx1lo hematopoietic stem cells (HSCs) (Immunity, 2003, Krosl et al). Despite the tremendous regenerative potential demonstrated by these cells, the total in vivo HSC pool in recipients remained within physiological limits (~20 000 HSC/mouse), implying that environmental factors (niche availability?) restricted their expansion. These studies thus implied that bypassing the in vivo constraints of niche availability by culturing the transduced HSCs in vitro might reveal the intrinsic expansion potential of these cells. To study this hypothesis, Hoxb4hiPbx1lo transduced HSCs were generated by co-infecting primary mouse bone marrow (BM) cells with retroviruses encoding antisense Pbx1 cDNA plus YFP, and Hoxb4 plus GFP. At the end of the co-culture with retroviral producers, double gene transfer (Hoxb4hiPbx1lo ) was ~20% as determined by flow cytometric analysis of GFP and YFP co-expression. These cells were then cultured in the presence of serum and cytokines for additional 12–16 days, and the numbers of total cells, clonogenic progenitors and HSCs were determined at regular intervals. To quantify the magnitude of the in vitro HSC expansion, CRU assays for determination of cells with long-term lympho-myeloid repopulation potential were performed after removal of BM cells from co-culture with retroviral producers, and then at various time points. The increase in HSC numbers over time in culture was calculated as the ratio between absolute numbers of Hoxb4hiPbx1lo HSCs at a given time point, and their numbers at initiation of culture. In our initial experiment, numbers of Hoxb4hiPbx1lo CRU increased from 1000 at day 0 to 1.2 x 107 at day 12, for a net 10 000-fold expansion. After transplantation into irradiated mice, these cells underwent an additional 360-fold in vivo expansion to regenerate HSC pools of recipients up to, but not above, normal levels. Southern blot analyses of proviral integrations in DNA isolated from sorted Mac-1+, B-220+ and CD4+CD8+ cells derived from primary and secondary recipients demonstrated that cultured Hoxb4hiPbx1lo HSCs retained their ability to differentiate into all hematopoietic lineages examined. To estimate the numbers of distinct Hoxb4hiPbx1lo HSCs in cultures, BM cells from primary recipients of 1 x 106 cells from a 16-day expansion culture were plated in methylcellulose, and the clonal origin of individual myeloid colonies was determined by Southern blot analysis. These experiments showed that several distinct HSC clones reconstituted each recipient, and that some clones reconstituted at least 2 mice, illustrating the polyclonal nature and self-renewal activity of the cultured HSCs. Together, our experiments show that after unprecedented levels of in vitro expansion, Hoxb4hiPbx1lo HSCs remained capable of reconstituting myeloid and lymphoid systems of primary and secondary recipients, and yet responsive to the in vivo regulatory mechanisms that limit total stem cell pool size, reflecting the interplay between autonomous and non-cell autonomous control of HSC self-renewal. Decreasing Pbx1 levels in Hoxb4 overexpressing HSCs could thus be used as a tool for studying mechanisms of self-renewal and replicative senescence, and may lead to clinically relevant protocols for HSC expansion.

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