DNA methylation changes during hematopoietic differentiation of human induced pluripotent stem cells

• DNA Methylierungsänderungen während der hämatopoetischen Differenzierung von humanen induzierten pluripotenten Stammzellen

Cypris, Olivia; Wagner, Wolfgang (Thesis advisor); Zenke, Martin (Thesis advisor)

Aachen : RWTH Aachen University (2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2023

Abstract

Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling, drug screening and cellular therapy. They can self-renew indefinitely and can be differentiated into various cell types, such as hematopoietic lineages, recreating characteristics of their primary counterparts like morphology and immunophenotype. During cellular differentiation, DNA methylation is tightly regulated. This epigenetic modification is involved in the orchestration of gene expression and can be used for tracking developmental processes. DNA methyltransferase 3A (DNMT3A) is a key player in establishing de novo methylation patterns and is often mutated in clonal hematopoiesis and acute myeloid leukemia (AML). In this thesis, the DNA methylome of iPSC-derived hematopoietic progenitor cells (iHPCs) was compared with primary hematopoietic lineages. Methylation changes were especially observed in genes relevant for hematopoiesis but iHPCs remained epigenetically distinct to primary hematopoietic cells. Additional co-culture with mesenchymal stromal cells did not support further epigenetic maturation towards natural hematopoietic cells. After generation of iPSC lines with knockout of exon 2, 19, or 23 of DNMT3A, its impact on mesenchymal and hematopoietic differentiation was analyzed. An almost entire de novo methylation loss was measured for exon 19-/- and 23-/- lines for both lineages. Yet, differentiation efficiency was enhanced in exon 23-/- clones, while it was slightly reduced in exon 19-/- cells. Despite profound impact on DNA methylation, no changes in gene expression of iHPCs were measured. Finally, differentiation in competitive syngeneic co-culture with multicolor barcoding resulted in growth advantage of exon 23-/- iHPCs. Altogether, it was demonstrated that current differentiation regimen require further adjustment to create iHPCs completely resembling primary cells and DNA methylation is a suitable tool for tracking this process. Furthermore, it was shown that knockout of different DNMT3A exons still enable hematopoietic differentiation despite loss of de novo methylation and that our knockout iPSCs can be used as model systems for clonal hematopoiesis.

Institutions

• Department of Biology [160000]
• [811002-3]