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AF4-MLL in human embryonic hematopoietic development
expression of early hematopoietic and endothelial master genes (Figure 5D). Thus, A4M cooperates with MA4 to promote hematopoietic and endothelial cell fate.
Genome-wide transcriptomic and H3K79 methylation profiles support the developmental cooperation between A4M and MA4
To identify patterns of gene expression that might pro- vide a molecular explanation of the functional cooperation between A4M and MA4 in hematopoietic specification,
A
we performed RNA-sequencing analysis on FACS-purified EV-, MA4-, A4M- and double fusion-expressing HEP from day 15 EB. Figure 6A shows a heatmap representation of the hierarchical clustering of the 335 genes differentially expressed between the four genotypes (Online Supplementary Table S2). There is a clear transcriptomic transition towards a hematopoietic/endothelial gene sig- nature from EV-HEP to double fusion-expressing HEP. Single fusion-expressing HEP clustered interspersed between EV and double-fusion HEP. The biological func-
BC
D
Figure 4. Enhanced endothelial cell fate from hemato-endothelial precursors co-expressing MA4 and A4M. (A) Scheme of hemato-endothelial precursor (HEP) endothelial differentiation and phenotypic characterization. (B) FACS-sorted HEP from day 9 human ESC-OP9 co-cultures were cultured in EGM2 medium for 5 days and analyzed by immunofluorescence for VE-cadherin, endothelial nitric oxide synthase and von Willebrand factor. (C) Top, Endothelial-like structures were identified and quantified based on VE-cadherin staining (white dotted-lined areas in B, top panel). Bottom, Frequency of CD45-CD31+CD144+ endothelial cells quantified by flow cytometry. (D) In vivo endothelial engraftment potential (HLA.ABC+CD31+CD144+CD45-) analyzed in bone marrow of NSG mice 8 weeks after transplantation of HEP. Data are presented as mean ± standard error of mean from five independent experiments. *P<0.05. bFGF: basic fibroblast growth factor; EV: empty vector; CB: cord blood.
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