Human iPSC-based model of DNMT3A-mutated clonal hematopoiesis

Debora Scheuermann (Freiburg im Breisgau)1, T.-S. Dederichs (Freiburg im Breisgau)1, V. Haacke (Freiburg im Breisgau)1, R. Hammad (Freiburg im Breisgau)2, J. Alzubi (Freiburg im Breisgau)2, R. Schäfer (Freiburg im Breisgau)2, C. Mussolino (Freiburg im Breisgau)2, T. Cathomen (Freiburg im Breisgau)2, S. Preissl (Freiburg im Breisgau)3, D. Westermann (Freiburg im Breisgau)4, I. Hilgendorf (Freiburg im Breisgau)1

1Universitäts-Herzzentrum Freiburg - Bad Krozingen Klinik für Kardiologie und Angiologie Freiburg im Breisgau, Deutschland; 2Universitätsklinikum Freiburg Institut für Transfusionsmedizin und Gentherapie Freiburg im Breisgau, Deutschland; 3Universitätsklinikum Freiburg Institut für Pharmakologie Freiburg im Breisgau, Deutschland; 4Universitäts-Herzzentrum Freiburg - Bad Krozingen Innere Medizin III, Kardiologie und Angiologie Freiburg im Breisgau, Deutschland



CHIP (clonal hematopoiesis of indeterminate potential) is an acquired, genetic risk factor of cardiovascular diseases, defined as the presence of a mutated clone ≥ 2% in the blood without overt hematologic abnormalities. The most common CHIP-driver genes are DNMT3A and TET2, which are epigenetic regulators. Studies in human and mice suggest that inflammatory myeloid cells mediate CHIP-associated cardiovascular risks. We aim to establish an in vitro system that models myelopoiesis in a milieu with CHIP clones, using human induced pluripotent stem cells (hiPSC).



We generated DNMT3A-mutated hiPSC lines using CRISPR/Cas9-mediated gene editing techniques to cut off the enzymatic domain of DNMT3A gene (exon 21-23). Moreover, we established an extrinsic factor-guided differentiation protocol to derive hiPSC into hematopoietic stem cells (HSC) and further into monocytes. At first, hiPSC self-aggregated into embryoid bodies under BMP4 stimulation. Subsequently, Activin A, fibroblast growth factors and a Wnt pathway activator were supplemented to regulate mesoderm differentiation and specify definitive hematopoiesis. Under the influence of hematopoiesis-specific growth factors and cytokines, HSC were derived from the hemogenic endothelial cells. Lastly, M-CSF was added to further differentiate HSC into monocytes. We collected the mutated and non-mutated hiPSC-derived HSC (CD34+) and monocytes (CD11b+CD14+) on day 14 and day 22, respectively, using fluorescence-activated cell sorting. Genomic DNA isolated from the sorted cells were subjected to bisulfite sequencing for the examination of genome-wide DNA methylation.



Our CRISPR/Cas9 system effectively edited 30% of hiPSC, from which we produced 24 single cell-derived hiPSC clones. Applying multiple quality control measures, we identified two pure hiPSC clones, one had truncated DNMT3A (the mutated) while the other had intact DNMT3A (the non-mutated). Both clones presented adequate pluripotency (SSEA4+, OCT4+) and had no cytogenetic abnormalities. Bisulfite sequencing of these two hiPSC lines and the derived HSC and monocytes revealed a distinctive genome-wide DNA methylation between the mutated and non-mutated cells. In hiPSC, 335 loci were differentially undermethylated and 13 overmethylated in mutated cells. Moreover, 773 undermethylated loci and 9 overmethylated ones were detected in mutant monocytes. Undermethylated genes, such as IL6 and PIM1, may indicate a higher expression of proinflammatory cytokines and proto-oncogenes in the mutated cells upon exposure to stimulants. 



Our in vitro model demonstrated a genome-wide hypomethylation of DNMT3A-defective myelopoiesis. This model serves as a foundation for studying hematopoiesis and will support deciphering how CHIP mutations lead to cell function changes, on the basis of which therapeutic strategies can be developed against CHIP-aggravated cardiovascular diseases.

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