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
Introduction:
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).
Methods:
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.
Results:
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.
Conclusion:
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.