3106 – DIRECT INDUCTION OF HEMOGENIC ENDOTHELIUM AND MYELOID CELLS FROM HUMAN IPSCS USING ETV2 MODIFIED RNA

Abstract

Previously, we demonstrated that overexpressing transcription factors ETV2 and GATA2 is sufficient to induce a pan-myeloid program in human induced pluripotent stem cells (hiPSCs), which proceeds through a hemogenic endothelium (HE) stage (Elcheva et al., 2014). Although we have found that constitutive overexpression of ETV2 using lentiviral vectors induces predominantly non-hemogenic endothelium. We also noted that ETV2 induces GATA2 expression in human pluripotent stem cells (hPSCs) and very few HE with macrophage potential. In addition, our recent studies suggest that molecular mechanisms upstream of GATA2 are sufficient to specify hematoendothelial program in hPSCs, while GATA2 is required for endothelial-to-hematopoietic transition (EHT) (Kang et al., 2018). Given these findings and studies demonstrating the critical role of ETV2 threshold for hematoendothelial commitment (Zhao and Choi, 2017) and obligatory downregulation of ETV2 during subsequent stages of hematopoietic development in the embryo (Hayashi et al., 2012), we explored whether transitional expression of ETV2 with modified mRNA (mmRNA) alone is sufficient for hematoendothelial programming in hiPSCs. We found that overexpression of modified ETV2 mmRNA (mmETV2) following culture of transfected hiPSCs in StemLine II serum-free medium with FGF2, rapidly induces CD144+ expressing endothelial cells that, upon addition of GM-CSF, form floating CD43+ blood cells most of which co-express CD45. Culture of ETV2-induced hematoendothelial progenitors in the presence of GM-CSF, FGF2 and UM171 led to production of CD34+CD33+ myeloid progenitors which could be harvested every 8-10 days for up to 30 days of culture. Subsequently, myeloid progenitors were differentiated into functional neutrophils in the presence of G-CSF and the retinoic acid agonist Am580 or macrophages in presence of M-CSF and IL3 or IL-1β and M-CSF. Overall, this technology is suitable for generating functional neutrophils and macrophages from iPSCs to interrogate the role of genes in a myeloid cell development and function when coupled with genetic engineering technologies.