Deterministic Trigger of Self-Renewal in Expanded HSCs Expressing Hoxb4 + Antisense Pbx1.

We previously reported the generation of pluripotent and ultracompetitive HSCs through modulation of Hoxb4 and Pbx1 levels. These Hoxb4hiPbx1lo HSCs display a tremendous regenerative potential, yet they are still fully responsive to in vivo regulatory signals that control stem cell pool size (20 000 HSCmouse) and differentiation pathways. Further work in our laboratory attempted to circumvent these physiological constraints by expanding Hoxb4hiPbx1lo transduced HSCs in vitro, and hence revealing their intrinsic expansion potential. Independent experiments were performed where primary mouse BM cells were co-infected with retroviruses encoding antisense Pbx1 cDNA plus YFP, and Hoxb4 plus GFP (double gene transfer ranged between 20–50%). Hoxb4hiPbx1lo HSCs measured using the CRU assay expanded by 105-fold during a 12 day in vitro culture. Following serial transplantations, these cells displayed an additional 4–5 log expansion in vivo. Total stem cell content per animal remained within normal limits. Southern blot analyses of proviral integrations showed that the expansion was polyclonal, and analyses of individually expanded clones provided a molecular proof of in vitro self-renewal (SR). This unprecedented level of HSC expansion in such a short time course (105-fold in 12 days) implies an absolute HSC doubling time of approximately 17 hours in our culture, raising the possibility that virtually all dividing HSCs undergo self-renewal. This analysis prompted us to dissect the impact of Hoxb4 on cell proliferation versus cell fate (SR?). When analyzed during the period of maximal HSC expansion, the cell cycle distribution of Sca+ or Sca+Lin− cells were comparable between the cultures initiated with neo control versus Hoxb4 BM cells (CTL vs Hoxb4: G0/G1: 66% vs 83%; S: 15% vs 9%; G2/M: 18% vs 7%). Correspondingly, CFSE tracking studies confirmed the identical, or even lower, number of cellular divisions in Sca+ cells isolated from cultures initiated with Hoxb4 versus neo transduced cells. Annexin V studies precluded protection from apoptosis as the major mechanism to increase HSC numbers since similar results (3–10% positive cells) were observed in the Hoxb4 versus neo-transduced cells. In summary, our studies support the emerging concept that distinct molecular pathways regulate cell proliferation and self-renewal, suggesting that Hoxb4 + antisense Pbx1 predominantly triggers self-renewal over HSC proliferation.