Forced HoxB4 Sustains CD45-c-kit+ Pre-Hematopoieitc Stem Cells (HSCs) Derived from Murine Induced-Pluripotent Stem Cells, Which Develop Long-Term and Short-Term Repopulating HSCs According to GATA2 Expression Level

Abstract

Background: Hematopoietic stem cells (HSCs) constitute a rare population of bone marrow cells (BMCs) and their ex vivo expansion with stemness for a prolonged period is quite unlikely. It is also difficult to efficiently produce HSCs from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). HoxB4 is a positive regulator of murine HSC self-renewal when ectopically expressed. Moreover, it was reported that HoxB4 overexpression could promote differentiation of ESCs to definitive HSCs. We established iPSCs (GGH-iPSCs) from BMCs of a C57BL/6J-Ly5.2 GATA2-GFP knock-in mouse (PNAS 103:2202, 2006) and transduced them with a 4-hydroxytamoxyfen (4-HT)-inducible HoxB4 construct. Using GGH-iPSCs, we showed that enforced HoxB4 activity throughout their culture period resulted in the long-term maintenance of iPSC-derived HSCs, which could repopulate in recipient mice over 30 weeks after transplantation, in vitro (Blood 122:2418, 2013). In the present study, we tried to elucidate the precise stage of hematopoietic differentiation governed by enforced HoxB4 in this iPSC model, especially in association with GATA2 expression.Experimental procedures and Results: GGH-iPSCs were induced toward hematopoietic differentiatin under enforced HoxB4, and after 2 months of culture, almost all the resulting cells expressed c-kit but not CD45, and approximately half of them were highly positive for GFP (GATA2). We also found that these CD45- cell populations expressed HSC marker genes such as SCL and LMO2, and that they produced CD45+ blood cells after HoxB4 was switched off. These results suggest that CD45- pre-HSCs, which can develop LT-HSCs, may be included in HoxB4-sustained iPSC-derived cell populations. Next, according to the GFP (GATA2) expression level, we divided CD45-c-kit+ cells on HoxB4 into two fractions; CD45-c-kit+GATA2high and CD45-c-kit+GATA2low/- cells, and cultured FACS-sorted cells over an OP9 monolayer with cytokines and without 4-HT to switch off HoxB4. Then, CD45+ blood cells emerged from both cell fractions over time, and CD45-c-kit+GATA2low/- cells produced much more CD45+ cells than CD45-c-kit+GATA2high cells one month later (Figure Left). Furthermore, to test a long-term repopulating activity in mice, each of cell fractions was cultured without 4-HT for 4 days after sorting, and then subjected to transplanttion into sublethally-irradiated C57BL/6-Ly5.1 recipient mice. Two weeks after transplantation, peripheral blood Ly5.2+ donor cells were much more frequent in mice transplanted with CD45-c-kit+GATA2low/- cells, compared to those with CD45-c-kit+GATA2high cells, whereas the CD45-c-kit+GATA2low/- fraction-derived donor cells rapidly decreased and disappeared around 2 months later. In contrast, CD45-c-kit+GATA2high donor cells could repopulate in recipient mice over 20 weeks after transplantation (Figure Right). These results suggest that CD45-c-kit+GATA2low/- and CD45-c-kit+GATA2high cell fractions include short-term (ST) and long-term (LT) -repopulating HSCs, respectively.Conclusion: Taken together, in a hematopoietic differentiation model of murine GGH-iPSCs, long-term repopulating hematopoietic stem cells (LT-HSCs) emerge from CD45-c-kit+GATA2high pre-HSC fraction which can be sustained in vitro by enforced HoxB4. In this experimental setting, enforced HoxB4 is likely to hinder differentiation from CD45- pre-HSCs to CD45+ HSCs. [Display omitted] [Display omitted] DisclosuresTojo:Novartis: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; Chugai: Research Funding, Speakers Bureau; Dainippon Sumitomo: Research Funding; Pfizer: Research Funding; Celgene: Consultancy.