Fig. 2

TPO knock-in enhances hematopoietic differentiation of hESCs. a Schematic of hematopoietic differentiation from hESCs to HPCs by coculturing with mAGM-S3 stromal cells. b Phase-contrast images of derived HPCs from Ctr or TPO-KI-1/-2 H1 hESCs at day 10 of differentiation. Typical cobblestone-like HPCs marked with white arrow. Scale bar = 40 μm. c Representative immunofluorescence images displaying generation of CD43⁺ HPCs from Ctr or TPO-KI-1/-2 H1 hESCs at day 7 of differentiation. Scale bar = 40 μm. d Flow cytometry analysis showing percentage of CD43⁺ HPCs in Ctr or TPO-KI-1/-2 H1 hESCs at day 7. Data shown as mean ± SEM (n = 3). **P < 0.01. e Representative immunofluorescence images of CD45⁺ HPCs generated from Ctr or TPO-KI-1/-2 H1 hESCs at day 10. Scale bar = 40 μm. f Flow cytometry analysis showing percentage of CD45⁺ HPCs in Ctr or TPO-KI-1/-2 H1 hESCs at day 10. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. g, h Hematopoietic colony-forming potential of CD43⁺ HPCs generated from Ctr or TPO-KI-1/-2 H1 hESCs. Total CFU number (g) or individual BFU-E, CFU-E, CFU-GM, or CFU-GEMM number (h) in Ctr or TPO-KI-1/-2 H1 hESCs calculated. Data shown as mean ± SEM (n = 3). *P < 0.05; NS, not significant. bFGF basic fibroblast growth factor, BFU-E burst-forming unit erythroid, BMP4 bone morphogenetic protein 4, CFU colony-forming unit, CFU-E colony-forming unit erythrocyte, CFU-GEMM colony-forming unit granulocyte/erythroid/macrophage/megakaryocyte, CFU-GM colony-forming unit granulocyte/macrophage, Ctr control, DAPI 4′,6-diamidino-2-phenylindole, hESC human embryonic stem cell, HPC hematopoietic progenitor cell, TPO thrombopoietin, VEGF vascular endothelial growth factor