Hematopoietic differentiation persists in human iPSCs defective in de novo DNA methylation

BACKGROUND: DNA methylation is involved in the epigenetic regulation of gene expression during developmental processes and is primarily established by the DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B). DNMT3A is one of the most frequently mutated genes in clonal hematopoiesis and leukemia, indicating that it plays a crucial role for hematopoietic differentiation. However, the functional relevance of Dnmt3a for hematopoietic differentiation and hematological malignancies has mostly been analyzed in mice, with the specific role for human hematopoiesis remaining elusive. In this study, we therefore investigated if DNMT3A is essential for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs). RESULTS: We generated iPSC lines with knockout of either exon 2, 19, or 23 and analyzed the impact of different DNMT3A exon knockouts on directed differentiation toward mesenchymal and hematopoietic lineages. Exon 19(−/−) and 23(−/−) lines displayed an almost entire absence of de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation efficiency was only slightly reduced in exon 19(−/−) and rather increased in exon 23(−/−) lines, while there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A(−/−) iHPCs recapitulate some DNA methylation patterns of acute myeloid leukemia (AML) with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed growth advantage of exon 23(−/−) iHPCs in a syngeneic competitive differentiation assay. CONCLUSIONS: Our results demonstrate that iPSCs with homozygous knockout of different exons of DNMT3A remain capable of mesenchymal and hematopoietic differentiation—and exon 23(−/−) iHPCs even gained growth advantage—despite loss of almost the entire de novo DNA methylation. Partial recapitulation of DNA methylation patterns of AML with DNMT3A mutations by our DNMT3A knockout iHPCs indicates that our model system can help to elucidate mechanisms of clonal hematopoiesis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-022-01343-x.