Figure 5.

Universal immune cell differentiation platform based on the induction of an HPC intermediate state. (A) 2D protocol for the induction of HPCs by days 14-17. (B) W = BMP and WNT signaling stimulation; V = VEGFA; S, X = target cell-dependent factors such as SCF (see Methods). After 1 week, the cells resembled hemogenic endothelium-like precursors (HE). (B) Expression profiling of the day 7 cells indicates a hybrid signature of endothelial and hematopoietic precursor cells. (C) Flow cytometry data of day 7 cells suggesting near-homogeneous differentiation into HE-like cells (n = 3). (D) Cellular morphology in experimental 12-well format and illustration of EHT process. (E) Representative time course data (flow cytometry) highlighting a homogeneous transition into hematopoietic suspension cells after day 7 (CD34⁺/CD45⁺), at the expense of endothelial commitment marked by VE-cadherin. (F) Methylcellulose assays using independent iPSC lines indicate robust formation of hematopoietic colonies from days 14 to 17 cells, comparable to primary HSCs. (G) Expression signature of iPSC-derived HPCs depends on basic medium used, with defined conditions giving rise to naïve CD34⁺/CD43⁺/CD117⁺/CD38⁻ cells. (H) Proof-of-concept experiment demonstrating directed differentiation competence of day 14 cells into monocytes (myeloid lineage). (I) Proof-of-concept experiment demonstrating directed differentiation competence of iPSC-derived HPCs into immature T cells marked by CD4 and 8a. A small fraction also showed mature alpha beta T-cell receptor expression (bottom right panel).