Additional file 1 of Standardized generation of human iPSC-derived hematopoietic organoids and macrophages utilizing a benchtop bioreactor platform under fully defined conditions

Additional file 1: Fig. S1. Organoid-based production of iPSC-Mac in intermediate scale bioreactors recapitulates embryonic hematopoietic development. A Analysis of KDR/VEGFR and CD45/PPTRC at different stages of differentiation as well as in iPSC-derived macrophages (iPSC-Mac) by qRT-PCR. Values are represented as relative RNA expression to GAPDH (housekeeping gene) (individual values with mean ± SD, iPSC line#1: blue square, and iPSC line#2: purple dots, n = 2–3 per line, n.d. indicates detection limit of the target gene) B and C Flow cytometric analysis of CD34, CD144, CD43 and CD45 expression during early hematopoietic differentiation. Hemanoids were dissociated and analyzed on day 4, 7 and 10 of mesoderm priming as well as during hematopoietic differentiation after they initiated production of iPSC-derived Mac. B Representative FACs plots CD34+/CD144+/CD45− Hemato-endothelial progenitors, CD34+/CD144-/CD43+ early hematopoietic progenitors and CD34+/CD144-/CD45+ hematopoetic progenitors during mesoderm priming and early hematopoietic differentiation (representative data shown for iPSC line#1). C Representative gating strategy: Populations were pre-gated for viable cells (FSC/SSC), single cells (FSC-A/FSC-H), CD34+ cells (CD34-FITC/autofluorescence), viability staining (Zombie-Aqua/PB450-A). D Immunohistochemical analysis of VE-Cadherin/CD144, VEGFR2 and CD45 expression in hemanoids derived from day 4, 7 and 10 of mesoderm priming as well as from hemanoids during hematopoietic differentiation after they initiated production of iPSC-derived Mac (day 7–10). Arrows indicate characteristic regions (scale bar = 200 µm, data shown for iPSC line#2, representative of n = 2).