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C. Bueno et al.
of both A4M and MA4 confers in vivo engraftment capac- ity to hESC-derived hematopoietic derivatives. To do this, we transplanted 5×105 hESC hematopoietic derivatives from each genotype into myeloablated NSG mice,4,43,47 finding that, despite regulating hematopoietic develop- ment in vitro, double fusion-expression did not confer in vivo engraftment to hESC hematopoietic derivatives (Figure 2D).
The increased hematopoietic output of double fusion- expressing hESC might be the consequence of transgene- mediated proliferation/survival of the emerging HEP or CD45+ cells. To address this, we analyzed cell cycle distri- bution (Online Supplementary Figure S2B) and apoptosis (Online Supplementary Figure S2C) within both HEP and the CD45+ cell population. No differences in the proportions of either cycling HEP or CD45+ cells were detected between genotypes (25–36% for HEP and 35–41% for CD45+ cells) (Online Supplementary Figure S2B). Apoptotic levels were similarly low in the different genotypes of HEP (6–8%) and CD45+ cells (5–7%) (Online Supplementary Figure S2C). Collectively, these results show that A4M cooperates with MA4 to induce HEP specification and blood commitment, without hijacking proliferation or sur- vival of HEP.
A4M and MA4 cooperate to enhance endothelial cell fate from hemato-endothelial precursors
We next addressed the developmental impact of A4M in endothelial maturation from HEP.10,47 We hypothesized that co-expression of A4M and MA4 in HEP may con- comitantly promote subsequent endothelial and hematopoietic commitment or skew the hemato- endothelial commitment in favor of hematopoiesis. To test this, we analyzed the ability of HEP to differentiate
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Figure 3. Co-expression of MA4 and A4M enhances hematopoietic differentiation of human embryonic stem cells in OP9 co-culture. (A) Experimental design of OP9-based human embryonic stem cell (hESC) differentiation towards hemato-endothelial precursors (HEP) and further hematopoietic commitment of HEP main- tained in either liquid culture for 16 days or in MS5 co-culture for 30 days. (B) Frequency of total CD45+ blood cells after 9 days in OP9 co-culture. (C,D) CD45- CD31+CD34+ HEP were FACS-purified at day 9 of OP9 co-culture and allowed to differentiate into CD45+ cells in liquid culture (C) or in MS5 co-culture (D). Data are represented as the mean ± standard error of mean from independent experiments. bFGF: basic fibroblast growth factor; MTG: monothioglycerol; SCF: stem cell factor; IL: interleukin; G-CSF: granulocyte colony-stimulating factor; EV: empty vector.
into mature endothelial cells. OP9-hESC co-cultures were dissociated on day 9 of development and HEP were FACS- sorted and cultured for 1 week in endothelial-promoting conditions (Figure 4A). The expression of the mature endothelial markers VE-cadherin (CD144), von Willebrand factor, endothelial nitric oxide synthase and CD31 was then analyzed. Irrespective of the genotype, HEP cultured in endothelial conditions attached, became spindle-shaped, and formed VE-cadherin+ endothelial-like structures co-expressing endothelial nitric oxide synthase, von Willebrand factor and CD31 (Figure 4B,C, top panel). However, double fusion-expressing HEP were more prone to differentiate into mature endothelial cells than were single fusion-expressing HEP. Accordingly, they yielded a 20-fold higher number of VE-cadherin+ endothelial-like structures (Figure 4C, top panel) and CD144+CD31+ endothelial cells (Figure 4C, bottom panel). Interestingly, endothelial cells (HLA.ABC+CD31+CD34+CD144+CD45- CD43-) were found in the bone marrow of mice trans- planted with double fusion-expressing hESC blood deriv- atives at levels ~4-fold higher than those in mice trans- planted with single fusion-expressing cells (Figure 4D).
Within CD34+CD31+CD45- HEP, two subpopulations of phenotypically and functionally distinct HEP can be dis- tinguished based on the expression of CD34 and CD43: hemogenic HEP (CD34low/+CD43+CD45-) and endothelial HEP (CD34++CD43-CD45-) (Figure 5A).40,48,51 We thus ana- lyzed the contribution of both HEP populations to the superior hematopoietic and endothelial differentiation observed in double fusion-expressing HEP. Co-expression of A4M and MA4, but not single fusions, robustly enhanced the emergence of both endothelial and hemogenic HEP (Figure 5B,C). The identity of hemogenic and endothelial HEP was confirmed by the specific
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haematologica | 2019; 104(6)