Gene expression of human chord blood hematopoietic progenitor cells and induced hemato-endothelial progenitor cells derived from human iPSC

Induced pluripotent stem cells (iPSC) offer a promising platform to model early embryonic developmental processes, to create disease models and proof-of-concept experiments for regenerative medicine. However, generation of iPSC derived hemato-endothelial and hematopoietic progenitor cells for these applications is challenging due to variable and limited cell numbers, which necessitates enormous up-scaling or development of demanding protocols. Here, we unravel the function of key transcriptional regulators SCL, LMO2, GATA2, ETV2 (SLGE) on early hemato-endothelial specification and establish a fully inducible and stepwise hemato-endothelial forward programming system, based on SLGE regulated overexpression. Regulated induction of SLGE in stable SLGE-iPSC lines drives very efficient generation of large numbers of hemato-endothelial progenitor cells (HEP) (CD144+/CD73-), which generate hematopoietic progenitor cells (CD45+/CD34+/CD38-/CD45RA-/CD90+/CD49f+) through a gradual process of endothelial-to-hematopoietic transition (EHT). A human fibroblast-derived iPSC line (H2E6C) was engineered to conditionally (doxycyclin-induction) express the transcription factors SCL (S), LMO2 (L), GATA2 (G) and ETV2 (E). On day 0, Phase I (hemato-endothelial programming; "SLGE-HEP") was started with a change to differentiation medium and an initial mesodermal priming boost by adding a GSK3 inhibitor (CHIR990221). After mesodermal priming, expression of SGLE was initiated by addition of Dox on day 1. Cells were differentiated towards the hemato-endothelial lineage via SLGE-expression and a mixture of supportive cytokines (SCF, TPO, IL-3, FGF2, VEGF). Phase II (generation of hematopoietic progenitor cells; "SLGE-HPC") was started on day 7 by Dox-withdrawal and cultivation of dissociated SLGE-HEP in STEMdiff APEL 2 medium supplemented SCF, TPO, Flt3L, IL-3 and FGF2. RNA-Seq was performed from iPS, day1, day 2, day 7 SLGE-HEP and compared to day 11 SLGE-HPC and human umbilical cord blood (CB) hematopoietic stem cells sorted for CD45+/CD34+/CD38-/CD45RA-/CD90high/CD49fhigh expressing cells.