Fig 2: Purified hemogenic endothelium (HE) can differentiate into all haematopoietic lineage cells. (A) Cell morphology in HE before and after CD34 sorting. Purified and homogenous HE underwent EHT on Day 8 after sorting. Magnification, 20×. (B) CD34 purification was confirmed and mRNA expression of representative haematopoietic lineage committed factors in CD34+ and CD34- cells measured by qRT-PCR. (C) Giemsa staining in differentiated cells from purified HE. Scale bar, 20 µM. All haematopoietic lineage cells were generated from CD34-sorted HE within 32 days. Lineage cell differentiation from HE was confirmed using specific lineage markers. Black arrows, shedding platelets, MK-III types. Magnification, 20×. (D) Flow cytometry data show abundant haematopoietic progenitors, implying full commitment toward blood lineages. CD45+CD34+CD3+ cells also emerged from purified HE from Day 10 (n = 4–6). Markers of maturation also increased in differentiated cells by Day 40. Data are derived from at least two independent experiments with duplicates (n = 4–7). ***p < 0.001

Fig 3: Bone morphogenetic protein 4 (BMP4) (25 ng/ml) induced hemogenic endothelium (HE) optimization from pluripotent stem cells (PSCs) and sustained definitive HE properties. (A) Representative morphology of PSC-derived HE according to BMP4 concentrations. Magnification 20×. (B) Proportions of CD34+, CD34dim and CD34bright cells in differentiated HE cells according to BMP4 concentrations. Red and blue plots, CD34bright and CD34dim populations, respectively. Images are representative of three independent experiments (*p < 0.05; **p < 0.01; n = 3 per group). (C) Proportions of CXCR4-CD73- cells in CD34dim and CD34bright cells based on BMP4 concentration. Red and blue plots, CD34bright and CD34dim populations, respectively. Representative images from three independent experiments (*p < 0.05; **p < 0.01; n = 3 per group).

Fig 4: The bipotential capability of hemogenic endothelium (HE) was evident in CD34+ purified HE. (A) Endothelial-to-haematopoietic transition (EHT) from single HE and HE proliferation with erythroblasts generated in HE. White arrows, EHT from HE. Cumulative cell numbers show robust HE expansion for up to 20 days after differentiation. HE proliferation was arrested by Day 30, suggesting that the cell growth gradually decreases after 20 days. Red dots, HE cells with arrested growth. Data are derived from at least three independent experiments (n = 13–18 per group). (B) Immunocytochemistry findings show CD34dim and CD34bright cells in pluripotent stem cell-derived HE. CD34dim cells remained adherent, whereas CD34bright cells differentiated into haematopoietic lineages. Magnification, 20×. (C) Immunochemistry findings of RUNX1, Brachyury (T), KDR and CD34 expression in early HE on Day 5. From Day 6, CD31, CD34 and TIE2 were co-expressed in HE and abundantly expressed over time. Magnification 40×, 10×. (D) Representative images of tube formation assay in CHA52 and H1 ESCs. The hESC-derived CD34+ HEs were cultured in Matrigel-coated 24-well plates with EGM media for 9 h. The ability of HE to differentiate into endothelial lineages determined by tube formation in early HE. Stemline® 2-based media was proper to encourage endothelial lineages. (E) Immunocytochemistry results showed endothelial properties in tube formed HE from hESCs. CD31 (green), Von Willebrand Factor (red), and nuclei stained with DAPI (blue). Scale bar: 10 µm. (F) The ability of HE to differentiate into haematopoietic lineages determined by CFU assays in early HE. CFU-colonies, especially CFU-M, -G and -E was significantly increased in APEL™ 2-based media, compared to that of Stemline® 2-based media. Data are shown as means ± SEM from at least three independent experiments (n = 7–13 per group; *p < 0.05; **p < 0.01; ***p < 0.001).

Fig 5: Bipotential hemogenic endothelium (HE) generated from pluripotent stem cells (PSCs) and conventional HE markers expressed on differentiation Day 6. (A) Schema for generating HE cells from PSCs via mesodermal lineages. (B) Immunocytochemistry findings of qualitatively expressed OCT4, NANOG and SSEA-4 in PSCs and mesodermal markers ETV2 and Brachyury in mesodermal lineage cells. (C) Morphological changes indicate the bipotential of HE cells to differentiate into haematopoietic and endothelial lineages. Magnification 20×. (D) Expression of CD34, TIE2 and CDH5 markers in HE cells at Day 6 after differentiation. Cells were analysed using FACS analysis. Data are shown as means ± SEM of at least three experiments. (E) Proportions of undifferentiated CXCR4-CD73- and differentiated CXCR4+CD73+ cells in HE expressing markers. CD34 cells contained the most CXCR4-CD73- cells. Data are shown as means ± SEM from at least three experiments. (F) Unlike TIE2+ and CDH5+ cells, CD34+ cells comprised CD34dim and CD34bright types. High proportions of TIE2 and CDH5 were detected in CD34bright and CD34dim populations, and high proportions of CXCR4-CD73- cells expressed low levels of TIE2 and CDH5. Data are shown as means ± SEM of at least three experiments.