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

Abstract

BackgroundDifferentiation of induced pluripotent stem cells (iPSCs) toward hematopoietic progenitor cells (HPCs) raises high hopes for disease modeling, 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.ResultsIn this study, we compared genome-wide DNA methylation (DNAm) patterns of iHPCs with various different hematopoietic subsets. 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 distinct from natural HPCs. Furthermore, we analyzed if additional co-culture for 2 weeks with syngenic primary mesenchymal stromal cells (MSCs) or iPSC-derived MSCs (iMSCs) further supports epigenetic maturation toward the hematopoietic lineage. Proliferation of iHPCs and maintenance of CFU potential was enhanced upon co-culture. However, DNAm profiles support the notion that additional culture expansion with stromal support did not increase epigenetic maturation of iHPCs toward natural HPCs.ConclusionDifferentiation of iPSCs toward the 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.Electronic supplementary materialThe online version of this article (10.1186/s13148-019-0617-1) contains supplementary material, which is available to authorized users.

DNMT3A knockouts in human iPSCs prevent de novo DNA methylation and reveal growth advantage during hematopoietic differentiation

“…3g). DNAm profiles of wildtype iHPCs indicated multilineage differentiation, similar to previously published iHPC profiles (Cypris et al, 2019; Nishizawa et al, 2016). Despite the impact of exon 19 -/- and exon 23 -/- on de novo DNAm, estimates for cellular composition were similar between wildtype and knockout iHPCs.…”

“…CpG sites associated with promotor regions are shown without transparency. (g) A deconvolution algorithm (Houseman et al ., 2012) was used to estimate the composition of different mature hematopoietic cell types based on the DNAm profiles of our iHPCs, those of Nishizawa et al (Nishizawa et al ., 2016) and of our previously published iHPC data (Cypris et al ., 2019). h) Gene expression changes compared between WT iHPCs and exon 19 -/- or exon 23 -/- iHPCs depicted as log 2 fold change against the mean of normalized counts of all samples regarding sequencing depth (=base mean).…”

Hematopoietic Differentiation of Human Pluripotent Stem Cells: HOX and GATA Transcription Factors as Master Regulators

“…A recent report demonstrated that directed hematopoietic differentiation of hiPSCs using morphogens yields CD45 + cells that are epigenetically distinct from wild type human HSCs, when comparing their genome-wide DNA methylation patterns. Additionally, prolonged co-culture with stromal cells did not induce further epigenetic maturation of these iPSC-derived cells [150]. Taken together, with current differentiation protocols, iPSC-derived hematopoietic stem/progenitors seem to be both functionally and epigenetically immature, which brings into question their suitability for clinical application.…”

DNA methylation in blood—Potential to provide new insights into cell biology

“…It is well established that epigenetics contributes significantly to the developmental fate of cells and tissues [ 8 ]. For instance, epigenetic mechanisms contribute to the differentiation of hematopoietic stem cells from bone marrow [ 12 , 13 ]. Importantly, DNA methylation appears to play a crucial role at specific stages along the separation of blood cell lineages (myeloid, lymphoid) and contributes to the establishment and functionality of the final differentiated cell type [ 14 ].…”