Ectopic HOXB4 Expression Expands the Pool of Hemogenic Endothelium-Initiating Progenitor Cells during Pluripotent Stem Cell Differentiation

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for future somatic gene and cell therapy. So far, HSCs capable of long-term multilineage reconstitution in mice have only been obtained when the homeodomain transcription factor HOXB4 was ectopically expressed during pluripotent stem cell differentiation (Kyba et al. Cell 109(1): 29-37, 2002; Pilat et al. Proc Natl Acad Sci USA 102(34): 12101-12106, 2005; Lesinski et al. Stem Cells Transl Med 1(8): 581-591, 2012). However, the primary "target" cell of HOXB4 during hematopoietic development, in vitro, is not yet known. Its identification is a prerequisite for unambiguously identifying the molecular circuits driving HSC development, at least in vitro.

To pin down this cell, we retrovirally expressed HOXB4 or a Tamoxifen-inducible HOXB4-ERT2 fusion protein in different reporter and knock-out mouse embryonic stem cell (ESC) lines. For these experiments, ESCs were differentiated for 6 days as embryoid bodies (EBs), dissociated and subsequently cocultured on OP9 stroma cells in medium supplemented with 100 ng/ml mSCF, 40 ng/ml mTPO, 100 ng/ml hFlt3L and 40 ng/ml hVEGF (STFV) for further 3 days. Use of a Runx1^(-/-) ESC-line containing a doxycycline-inducible Runx1 coding sequence (“iRunx1”; kindly provided by G. Lacaud, Manchester) uncovered that HOXB4 acts during formation of the hemogenic endothelium (HE) from which HSCs arise. Without Runx1 induction, which arrests hematopoietic development at the HE-stage, ectopic HOXB4 expression mediated an approximately 30-fold increase in the number of circular endothelial, bona fide HE-sheets being Flk1⁺VE-Cadherin⁺Tie2⁺ (mean values: control: 11+/-4.8 n=7; HOXB4: 301+/-47 n=7; P<0.0001, unpaired, 2-sided Student´s t-test) and expressing Sox17 and Lmo2. This observation suggested an expansion of HE progenitors, detectable from day 5 of EB differentiation on. Determination of their frequencies within the VE-Cadherin⁺ population revealed a HOXB4-mediated increase from 1:360 cells (control) up to 1:15 cells (HOXB4; 95% C.I. = 1:12-1:21). After additional Runx1 induction, the endothelial cells morphologically underwent an Endothelial-to-Hematopoietic Transition (EHT) as verified at the single cell level by time-lapse microscopy. Concomitantly, they upregulated transcription of Gfi1, Gfi1b and Pu.1, initiated surface expression of the pan-hematopoietic marker CD45 and generated hematopoietic colony forming cells (CFC), thus proving their identity as real hemogenic endothelial cells.

Taken together, our results strongly suggest that HOXB4 first and foremost promotes hematopoiesis by substantially increasing the number of hemogenic endothelium progenitors during mouse pluripotent stem cell differentiation.