Abstract
Purpose :
Human iPSC (hiPSC) could provide unlimited amounts of embryonic-like vascular progenitors (VP) to repair ischemic diabetic tissue. However, clinical translation would require safe, efficient and reproducible reprogramming and differentiation into functional VP. We recently defined a human naïve pluripotent state using LIF and chemical inhibitors of WNT, MEK and tankyrase (LIF-3i). LIF-3i has been validated in >30 genetic backgrounds and recapitulates transcriptomic, epigenetic, and signaling signatures of the pre-implantation epiblast. LIF-3i cells tolerate direct differentiation without re-priming, with reduced interline variability and lineage bias, and improved multi-lineage capacity. The purpose of this study was to test the therapeutic potential of VP generated from patient-specific naïve hiPSC.
Methods :
Type-I diabetic skin fibroblasts (D-hiPSC) were reprogrammed using a non-integrative episomal 7-factor system and conventional E8 primed culture medium. Exposure to a GSK3β inhibitor and ascorbic acid facilitated reprogramming kinetics. Three D-hiPSC lines were reverted in LIF-3i and differentiated into VP (i.e, APEL medium) in parallel with isogenic primed controls. Functional capacity of D-hiPSC-derived VP was assessed by cell cycle, acetylated-Dil-LDL uptake, Matrigel-tube and -plug assays, pH2AX foci quantification after neocarzinostatin exposure, and vascular engraftment in an ischemic retinopathy (IR) mouse model.
Results :
Naive D-hiPSC rapidly acquired expressions associated with naïve pluripotency (e.g., pSTAT3, DNMT3L, NR5A2, and DPPA3) and produced CD31+CD146+ VP cells at higher efficiencies than isogenic primed controls. Long-term cultures of naïve-D-hiPSC-derived VP displayed superior proliferation, reduced senescence and higher resistance to DNA damage. Greater numbers of naïve D-hiPSC-VP were detected in the retinas of an IR mouse model up to 4 weeks after intravitreal injection. At 1-week post-transplantation, human naïve D-iPSC-VP migrated efficiently in superficial and deep retinal blood vessels. In contrast, isogenic primed D-iPSC-VP remained in superficial layers of retina.
Conclusions :
Our results suggest that the LIF-3i naïve reversion dramatically improves the differentiation efficiency, functionality, and therapeutic potential of VP generated from diabetic patient hiPSC.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.