Fig. 2

MSX2 deletion enhances the generation of HPCs from hESCs. a Scheme of sgRNA design and the sequences targeting exon2 of MSX2 mediated by CRISPR/Cas9. The lower panel shows DNA sequencing results of the targeted exon of MSX2 in H1 MSX2^−/− 1# and 2# cells. Numbers indicate the change of nucleotides. b The real-time PCR analysis of NANOG, SOX2, and OCT4 expression in undifferentiated H1 WT, H1 MSX2^−/− 1# and 2# cells. Expression is normalized to the level (= 1) of mRNA in WT H1 cells. c Representative immunofluorescence images of H1 WT and H1 MSX2^−/− cells showing the generation of CD43⁺ HPCs (red) at day 8 of hematopoietic differentiation. Nuclei were stained with DAPI (blue). d Flow cytometry analysis of the percentage of CD43⁺ HPCs from H1 WT and H1 MSX2^−/− cells at day 8 of hematopoietic differentiation. e Flow cytometry analysis showing the number of CD45⁺ hematopoietic cells from H1 WT and H1 MSX2^−/− cells at days 8 + 6 of hematopoietic differentiation. f Left panel: Total colony number generated from WT or MSX2^−/− H1-derived cells from chemical defined hematopoietic differentiation. Right panel: The distribution of different colony types generated from WT or MSX2^−/− H1-derived cells from chemical defined hematopoietic differentiation. CFU-GEMM (colony-forming unit-granulocyte/erythroid/macrophage/monocyte), CFU-GM (colony-forming unit-granulocyte/macrophage), BFU-E (burst-forming unit-erythroid), and CFU-E (colony-forming unit-erythrocyte) were documented and calculated. Results are shown as means ± SD (n = 3). NS, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001