Figure 7.

In vitro and in vivo characterization of hPSC-derived AGM-like hematopoietic cells. (A–C) Day 12 hPSC-derived hematopoietic cells were co-cultured with OP9-DLL4 for immune T and natural killer (NK) cell differentiation. At different time points, expression of CD4/CD8 (A) and CD45/CD56 (B) was assessed by flow cytometry. (C) hPSC-NK cells were co-cultured with different cancer cells at the indicated effector-to-target ratios, and the corresponding percentages of lysis were shown. (D–H) eGFP+ hPSC-derived hematopoietic stem and progenitor cells (HSPCs) were transplanted into femur of irradiated immunodeficient NRG mice for the assessment of engraftment and repopulation potentials. Engrafted human HSPCs were recorded at the indicated weeks (D) and representative flow plots of eGFP+ cells were shown in (E). The expressions of multi-lineage markers, including CD45 (F), CD4, CD8, CD33, GLY-A, and CD19 (G) in eGFP+ cells isolated from recipient peripheral blood (PB) and bone marrow (BM) were assessed by flow cytometry and quantified in (H). E: erythroid cells; M: myeloid cells; B: B cells; T: T cells. (I) Limiting dilution assay of hPSC-derived HSPCs during secondary transplantation. 103, 104, and 105 eGFP + cells isolated from bone marrow of primary recipients (#4 and #5) were transplanted to secondary recipients. Confidence intervals of 1/(stem cell frequency) were calculated by ELDA according to Poisson distribution. (J) A schematic model highlighting the specification of hPSCs to AGM-like hematopoietic cells by stage-specific modulation of Wnt with or without TGFβ signaling pathway. VEGF is required for endothelial specification of female hPSC lines.