Introduction:
CHIP (clonal hematopoiesis of indeterminate potential) is an acquired, genetic risk factor of cardiovascular diseases. The most common CHIP-driver gene is DNMT3A, an epigenetic regulator. 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 DNMT3A-mutated cells, using human induced pluripotent stem cells (hiPSC).
Methods:
We generated DNMT3A-mutated hiPSC lines using CRISPR/Cas9-mediated gene editing techniques to eliminate the enzymatic domain of the DNMT3A gene (exon 21-23). Next, we differentiated hiPSC into hematopoietic stem cells (HSC) and further into monocytes with a newly established extrinsic factor-guided differentiation protocol. We collected the mutated and non-mutated hiPSC-derived HSC and monocytes using fluorescence-activated cell sorting. Genomic DNA isolated from the sorted cells was subjected to bisulfite sequencing for the examination of genome-wide DNA methylation. RNA sequencing was performed to profile transcriptome. We plan to co-culture mutated and non-mutated hiPSC-derived HSC during monocyte differentiation for modelling the clonal chimerism in vivo.
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). 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. DNMT3A-mutated cells are significantly hypomethylated compared to non-mutated cells. Hypomethylated promoters or enhancers of genes, such as IL6 and TNF receptors, however, may indicate a higher expression of proinflammatory cytokines in the mutated cells. Transcriptomes showed a more inflammatory and proliferative phenotype in mutated cells, and thus correlated with the results of the methylation analyses.
Conclusion:
Our in vitro model demonstrated a genome-wide hypomethylation during DNMT3A-deficient myelopoiesis, leading to a more inflammatory and proliferative phenotype. 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.
