ABSTRACT
Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.
ABSTRACT
Human-induced pluripotent stem cell-derived endothelial cells (iECs) provide opportunities to study vascular development and regeneration, develop cardiovascular therapeutics, and engineer model systems for drug screening. The differentiation and characterization of iECs are well established; however, the mechanisms governing their angiogenic phenotype remain unknown. Here, we aimed to determine the angiogenic phenotype of iECs and the regulatory mechanism controlling their regenerative capacity. In a comparative study with HUVECs, we show that iECs increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) mediates their highly angiogenic phenotype via regulation of glycolysis enzymes, filopodia formation, VEGF mediated migration, and robust sprouting. We find that the elevated expression of VEGFR2 is epigenetically regulated via intrinsic acetylation of histone 3 at lysine 27 by histone acetyltransferase P300. Utilizing a zebrafish xenograft model, we demonstrate that the ability of iECs to promote the regeneration of the amputated fin can be modulated by P300 activity. These findings demonstrate how the innate epigenetic status of iECs regulates their phenotype with implications for their therapeutic potential.
ABSTRACT
Ischemic retinopathies are major causes of blindness worldwide. Local hypoxia created by loss of vascular supply leads to tissue injury and aberrant neovascularization in the retina. There is a great need for therapies that enhance revascularization of hypoxic neuroretinal tissue. To test the therapeutic feasibility of human-induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) for the treatment of ischemic retinopathies, we compared the angiogenic potential of hiPSC-ECs with mature human retinal endothelial cells (HRECs) in response to hypoxia. hiPSC-ECs formed more robust and complex vascular networks in collagen gels, whereas HRECs displayed minimal sprouting. The cells were further tested in the mouse oxygen-induced retinopathy (OIR) model. Retinas with hiPSC-EC injection showed colocalization with host vessels, whereas HRECs lacked such responses. hiPSC-ECs markedly reduced vaso-obliteration and pathological neovascularization. This beneficial effect of hiPSC-ECs was explained by the stromal cell-derived factor-1a (SDF1a)/CXCR4 axis; hiPSC-ECs exhibited much higher cell-surface expression of CXCR4 than HRECs and greater chemotaxis toward SDF1a-embedded 3D collagen hydrogel. Furthermore, treatment with neutralizing antibody to CXCR4 abolished recruitment of hiPSCs in the OIR model. These findings suggest superior angiogenic potential of hiPSC-ECs under hypoxia and underscore the importance of SDF1a/CXCR4 in the reparative function of hiPSC-ECs in ischemic diseases.
