DNA methylation changes in hematopoietic development and iPSC-derived model systems

  • DNA Methylierungs-Veränderungen in der hämatopoetischen Entwicklung und in aus iPS-Zellen abgeleiteten Modellsystemen

Frobel, Joana; Wagner, Wolfgang (Thesis advisor); Elling, Lothar (Thesis advisor); Zenke, Martin (Thesis advisor)

Aachen (2018)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2018


Human induced pluripotent stem cells (iPSCs) represent an attractive cell source for in vitro generation of various cell types and thus raise high hopes for application in regenerative and personalized medicine. De novo DNA methylation (DNAm) is a key epigenetic mechanism involved in cellular differentiation processes, which is orchestrated by DNA methyltransferase 3A (DNMT3A). DNMT3A is spliced in a tissue- and disease-specific manner and modulation of DNMT3A is suggested to play pivotal roles for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the development of hematological malignancies. So far, it has hardly been analyzed whether cell type-specific DNAm patterns are recapitulated in iPSC-derived cells and how the modulation of DNMT3A transcripts impacts on hematopoietic differentiation. In this thesis, DNAm changes upon differentiation of iPSCs into mesenchymal stromal cells (MSCs) were analyzed and substantial differences in the DNAm patterns of iPS-derived MSCs (iPS-MSCs) and their in vivo counterparts were observed. To address the relevance of different DNMT3A splice variants, shRNA-mediated knockdown of individual DNMT3A transcripts was performed in cord blood-derived HSPCs. Particularly, knockdown of transcripts 2 and 4 reduced the proliferation of HSPCs significantly, and transcript 2 appears to be the main transcript responsible for downregulation of CD34 upon culture expansion of HSPCs. In colony-forming unit (CFU) assays downregulation of transcript 4 resulted in a clear bias towards erythroid colonies. Notably, analysis of genome-wide gene expression and DNAm patterns suggests that individual isoforms target distinct genomic regions. Furthermore, CRISPR/Cas9-mediated removal of a coding exon for the methyltransferase domain indicated a possible impact of DNMT3A on the differentiation potential of iPSC-derived HSPCs (iPS-HSPCs) in a CFU assay. In contrast, the spontaneous differentiation potential of iPSCs and their directed differentiation into iPS-HSPCs was not impaired. Effects of a xenogeneic environment on epigenetic patterns of human HSPCs were then investigated in a murine transplantation model. Genome-wide analysis of DNAm patterns revealed that stably engrafted human hematopoietic cells are epigenetically primed toward the B cell lineage, which is in line with the immunophenotypic characterization. Thus, the murine environment recapitulates epigenetic changes that are observed in human hematopoietic development, while the faster aging environment does not impact on epigenetic aging of the engrafted cells. It was furthermore investigated if the cell type-specific DNAm patterns can be used to determine blood counts in a clinical setting. To this end, individual CG dinucleotides that facilitate site-specific analysis of the cellular composition of blood were identified. This Epi-Blood-Count provides a tool to robustly determine blood counts of stored and frozen blood samples based on cell type-specific DNAm. The results of this thesis underline the importance of precisely orchestrated DNAm in cellular differentiation. DNMT3A variants can be used to modulate these patterns and a perspective on how epigenetic characteristics can be used for diagnostics is provided.