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AF4-MLL in human embryonic hematopoietic development
culture (Figure 3C) or onto MS5 feeders (Figure 3D). After 10 days on OP9 stroma, double fusion-expressing hESC yielded a 10-fold higher number of CD45+ hematopoietic cells than did single fusion-expressing hESC (Figure 3B). Moreover, when HEP were FACS-sorted from day 9 OP9
co-cultures and allowed to differentiate into CD45+ blood cells, the yield of CD45+ cells was up to 60-fold higher in double fusion-expressing HEP than in single fusion- expressing HEP (Figure 3C,D). Encouraged by these results, we next investigated whether ectopic expression
A
B
CD
Figure 2. A4M cooperates with MA4 to accelerate human embryonic stem cell/erythroid body specification towards hemato-endothelial precursors and subse- quent hematopoietic differentiation. (A) Schematic of erythroid body hematopoietic differentiation of human embryonic stem cells (hESC) and end-point analyses. (B) Upper left, specification into hemato-endothelial precursors (HEP; CD31+CD34+CD45-) is accelerated in double fusion-expressing hESC. Subsequent differentia- tion of HEP into hematopoietic progenitors (upper right) and mature CD45+ blood cells (bottom left) is enhanced in double fusion-expressing HEP. Bottom right, Colony-forming unit read-out and scoring (pie charts) confirming accelerated and enhanced hematopoietic progenitor potential from double fusion-expressing blood derivatives. (C) Reverse transcriptase polymerase chain reaction analysis confirming stable expression of MA4 and A4M upon erythroid body differentiation. (D) Neither MA4- nor double fusion-expressing blood derivatives display in vivo hematopoietic engraftment potential in irradiated NSG mice. Data are presented as mean ± standard error of mean from at least three independent experiments. *P<0.05. EB: erythroid body; bFGF: basic fibroblast growth factor; BMP4: bone morphogenet- ic protein-4; SCF: stem cell factor; IL: interleukin; G-CSF: granulocyte colony-stimulating factor; CFU: colony-forming unit; EV: empty vector; IBMT; intra-bone marrow transplantation; C+: positive control; CB: cord blood.
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