Vascular progenitors generated from tankyrase inhibitor-regulated naïve diabetic human iPSC potentiate efficient revascularization of ischemic retina

Abstract

Here, we report that the functionality of vascular progenitors (VP) generated from normal and disease-primed conventional human induced pluripotent stem cells (hiPSC) can be significantly improved by reversion to a tankyrase inhibitor-regulated human naïve epiblastlike pluripotent state. Naïve diabetic vascular progenitors (N-DVP) differentiated from patient-specific naïve diabetic hiPSC (N-DhiPSC) possessed higher vascular functionality, maintained greater genomic stability, harbored decreased lineage-primed gene expression, and were more efficient in migrating to and re-vascularizing the deep neural layers of the ischemic retina than isogenic diabetic vascular progenitors (DVP). These findings suggest that reprogramming to a stable naïve human pluripotent stem cell state may effectively erase dysfunctional epigenetic donor cell memory or disease-associated aberrations in patientspecific hiPSC. More broadly, tankyrase inhibitor-regulated naïve hiPSC (N-hiPSC) represent a class of human stem cells with high epigenetic plasticity, improved multi-lineage functionality, and potentially high impact for regenerative medicine.

Running the full human developmental clock in interspecies chimeras using alternative human stem cells with expanded embryonic potential

“…c Western blot analysis of primed (P) and TIRN (N) CB-hiPSC (E5C3). TIRN-CB-hiPSC markedly upregulated naïve epiblast-specific DNMT3L whilst maintaining DNMT1 protein expressions following TIRN reversion 109 , 110 . d Infinium CpG methylation heatmaps of genomic Imprinted promoter regions of a panel of primed and TIRN-reverted isogenic hPSC samples revealed that TIRN-hPSC retains normal somatic epigenomic imprint configurations, similar to their isogenic primed states.…”

“…5a, b ). Tankyrase/PARP inhibitor regulated naïve hPSC (TIRN-hPSC) possessed multiple naïve epiblast-like ICM characteristics that included MEK-ERK/bFGF signaling independence, activated phosphorylated JAK/STAT3 signaling, distal OCT4 enhancer usage, global DNA CpG hypomethylation, and increased expression of activated beta-catenin 109 , 110 . Importantly, in contrast to other naïve reversion methods 84 , 94 – 97 , Zimmerlin et al 109 reported that supplementation of the core LIF-2i cocktail with only this tankyrase / PARP inhibitor (XAV939) protected a wide repertoire of genetically independent TIRN-hPSC lines against the erosion of CpG methylated genomic imprinted regions.…”

“…The human TIRN system is the only human naïve reversion method described thus far that generates naïve epiblast-like hPSC with expanded multi-lineage differentiation potency, normal epigenomic imprints, and improved in vivo survival of differentiated progenitors 109 – 112 . TIRN-hPSC was recently shown to generate fully differentiated vascular progenitors with improved functional and long-term engraftment capacities in vivo that were superior vascular progenitors generated by normal or diseased primed, conventional hiPSC 110 .

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“…This tankyrase/PARP inhibitor-mediated modification of the classical LIF-2i method has been validated to stably revert over 30 hPSC lines, and is independent of genetic donor background 109 , 111 . a (Left panels) Three representative hPSC lines 109 , 110 (RUES1 hESC, cord blood (CB)-hiPSC 6.2, and fibroblast (fib)-hiPSC C.2 are shown in their starting primed (i.e., E8 medium; PRIMED) conditions, and 6–10 passages post culture in continuous TIRN-reverted (NAÏVE) conditions 109 , 111 . Monolayer bFGF-dependent hPSC colonies in primed conditions became tolerant to bulk single-cell passaging and acquired a typical dome-shape morphology following TIRN reversion.…”

“…Collectively, these studies promote the concept, originally proposed by Zimmerlin et al, that tankyrase/PARP enzymatic inhibition likely promotes global cellular processes in stem cells that expand their functional pluripotency and protect against epigenomic instability 112 . For example, tankyrase/PARP inhibitor-based culture promoted erasure of dysfunctional epigenetic donor cell memory, and possibly disease-associated aberrations in patient-specific TIRN-hiPSC 110 . Thus, TIRN-hPSC may represent a new class of stem cells with improved epigenetic plasticity and multi-lineage differentiation potential, that may have high impact for both regenerative therapies and the generation of human–animal chimeras 110 , 112 .…”