Figure 4: Haematopoietic differentiation of hESCs induced by GATA2 and TAL1.

(a) Cell morphology and flow cytometric analysis of H1 hESCs differentiated by expression of GATA2 and TAL1 on day 7 post transduction. (b) Types of haematopoietic colonies formed by GATA2 and TAL1 differentiated cells on day 7 post transduction. Erythroid colonies (E); macrophage colonies (M); megakaryocytic colonies (Mk). Scale bar for CFC assay, 250 μm; for cytospins, 20 μm. (c) Cell morphology and flow cytometric analysis of H1 hESCs differentiated by expression of GATA2/TAL1/LMO2 on day 7 post transduction. (d) Types of haematopoietic colonies formed by GATA2/TAL1/LMO2 differentiated cells on day 7 post transduction. Scale bar for CFC assay, 250 μm; for cytospins, 20 μm. (e) Phenotypic characterization of GATA2/TAL1/LMO2-induced haematopoietic cells grown in SFEM serum-free medium supplemented with 100 ng ml⁻¹ SCF, 50 ng ml⁻¹ TPO, 3 u ml⁻¹ EPO and 20 ng ml⁻¹ bFGF for 14 days. (f) CFC potential of hESCs transduced with GATA2-based combinations. Error bars represent s.e.m. from three experiments. (g) Kinetic analysis of VE-cadherin and CD43 expression by flow cytometry following transduction of H1 hESCs with indicated combinations of TFs. (h) VE-cadherin and CD43 immunofluorescent staining of hESCs transduced with indicated TFs at different time points after transduction. Scale bars, 100 μm. (i) Expression of markers of associated with haemogenic and non-haemogenic endothelium by VE-cadherin⁺ cells emerging on day 3 post transduction with GATA2/TAL1 (upper panels) and GATA2/TAL1/LMO2 (lower panels). Histograms show expression of CD226 and CD73 by VE-cadherin-gated cells. Endothelial cells induced by GATA2/TAL1 and GATA2/TAL1/LMO2 shows CD226⁺CD73⁻ phenotype associated with haemogenic endothelium¹⁰. (j) Haematopoietic potential of VE-cadherin⁺CD43⁻CD73⁻ endothelial cells isolated from GATA2/TAL1 programing cultures. On day 3 after transduction with GATA2/TAL1 or ETV2/GATA2, VE-cadherin⁺CD43⁻CD73⁻ cells were isolated by sorting and cultured on OP9 to assess the haematopoietic potential by counting CD43⁺ haematopoietic colonies using immunofluorescent staining. Blue and red rectangles show position of gates used for cell sorting. Scale bar, 100 μm.