Tracking of epigenetic changes during hematopoietic differentiation of induced pluripotent stem cells

Differentiation of induced pluripotent stem cells (iPSCs) toward hematopoietic progenitor cells (HPCs) raises high hopes for disease modelling, drug screening, and cellular therapy. Various differentiation protocols have been established to generate iPSC-derived HPCs (iHPCs) that resemble their primary counterparts in morphology and immunophenotype, whereas a systematic epigenetic comparison was yet elusive. In this study, we compared genome wide DNA methylation (DNAm) patterns of iHPCs with various different hematopoietic subsets. Furthermore, we analyzed if additional co-culture for two weeks with syngenic primary mesenchymal stromal cells (MSCs) or iPSC-derived MSCs (iMSCs) further supports epigenetic maturation toward hematopoietic lineage. After 20 days of in vitro differentiation cells revealed typical hematopoietic morphology, CD45 expression and colony forming unit (CFU) potential. DNAm changes were particularly observed in genes that are associated with hematopoietic differentiation. On the other hand, the epigenetic profiles of iHPCs remained overall very distinct from normal hematopoiesis. Co-culture with MSCs or iMSCs enhanced proliferation of iHPCs and maintenance of CFU potential. However, morphology, immunophenotype, and DNAm profiles did not indicate that additional culture expansion with stromal support increases hematopoietic differentiation. In conclusion, differentiation of iPSCs towards hematopoietic lineage remains epigenetically incomplete. These results substantiate the need to elaborate advanced differentiation regimen while DNAm profiles provide a suitable measure to track this process. DNA from iPSC-derived hematopoietic progenitor cells was isolated after 20 days of differentiation or after additional 14 days of culture expansion on either tissue culture plastic or in co-culture with bone marrow derived MSCs or iPSC-derived MSCs. DNA was bisulfite converted and hybridized to the Illumina Infinium MethylationEPIC BeadChip.