Supplementary Material for: Emergence of CD43-Expressing Hematopoietic Progenitors from Human Induced Pluripotent Stem Cells

Background: The ex vivo generation of human hematopoietic stem cells (HSCs) with long-term repopulating capacity and multi-lineage differentiation potential represents the holy grail of hematopoiesis research. In principle, human induced pluripotent stem cells (hiPSCs) provide the tool for both studying molecular mechanisms of hematopoietic development and the ex vivo production of ‘true’ HSCs for transplantation purposes and lineagespecific cells, e.g. red blood cells, for transfusion purposes. CD43-expressing cells have been reported as the first hematopoietic cells during development, but whether or not these possess multilineage differentiation and long-term engraftment potential is incompletely understood. Methods: We performed ex vivo generation of hematopoietic stem cells from hiPSCs using an embryoid body(EB)-based, xeno-product-free differentiation protocol. We investigated the multilineage differentiation potential of different FACS-sorted CD43-expressing cell subsets by colony-forming assays in semisolid media. Further, erythroid differentiation was investigated in more detail using established protocols. Results: By using CD43 as a marker, we are able to measure hematopoietic induction efficiency during hiPSC-derived EB differentiation. Further, we determined CD43+ cells as the cell population of origin for in vitro erythropoiesis. CD43hi CD45+ cells represent hematopoietic stem and progenitor cells with multilineage differentiation potential with a bias towards the erythroid lineage.