HOXB4-induced self-renewal of hematopoietic stem cells is significantly enhanced by p21 deficiency

¹ Molecular Medicine and Gene Therapy and Lund Strategic Center for Stem Cell Biology and Cell Therapy Lund University Hospital, 221 84 Lund, Sweden
² Center for Regenerative Medicine and Technology, AIDS Research Center and Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

^* To whom correspondence should be addressed. E-mail: Ann.Brun{at}med.lu.se.

	Abstract

Enforced expression of the HOXB4 transcription factor and downregulation of p21^Cip1/Waf (p21) can each independently increase proliferation of murine hematopoietic stem cells (HSCs). We asked whether the increase in HSC self-renewal generated by overexpression of HOXB4 is enhanced in p21 deficient HSC. HOXB4 was overexpressed in hematopoietic cells from wild type (wt) and p21^-/- mice. Bone marrow (BM) cells were transduced with a retroviral vector expressing HOXB4 together with GFP (MIGB4), or a control vector containing GFP alone (MIG) and maintained in liquid culture for up to 11 days. At day 11 of the expansion culture, the number of primary CFU-GM colonies and the repopulating ability were significantly increased in MIGB4 p21^-/- BM (p21B4) cells compared to MIGB4 transduced wt BM (wtB4) cells. In order to test proliferation of HSC in vivo we performed competitive repopulation experiments and obtained significantly higher long-term engraftment of expanded p21B4 cells compared to wtB4 cells. The 5-day expansion of p21B4 HSCs generated 100-fold higher numbers of CRU compared to wtMIG, and 3-fold higher numbers compared to wtB4. The findings demonstrate that increased expression of HOXB4, in combination with suppression of p21 expression, could be a useful strategy for effective and robust expansion of HSCs.

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