Embryonic vascular stem-progenitors for treatment of ischemic retinopathies

分支静脉阻塞（BVO）和糖尿病视网膜病变（DR）是新发失明的主要原因， 美方这些血管疾病导致继发于视网膜缺血性死亡的无细胞毛细血管。 血管内皮细胞（EC）和收缩性周细胞。如果脱细胞视网膜血管可以 用自体或细胞库的自我更新血管周细胞干祖细胞再生，缺血 在这些血管病变中，可以缓解和逆转或稳定终末期失明。我们集团 建立了移植患者特异性胚胎血管祖细胞（VP）的可行性， 从人诱导多能干细胞分化后，细胞周潜能直接进入眼睛 细胞（hiPSC）。我们还建立了一种新的基于端锚聚合酶/PARP底物的小分子鸡尾酒， 将常规的谱系致敏的hiPSC逆转为具有更高的免疫原性的“幼稚”hiPSC（N-hiPSC）。 具有较高功能多能性的原始上胚层状态。幼稚VP的再生潜力（N-VP） 从正常和患病的N-hiPSC中分化的N-hiPSC相对于致敏的显著更多产， 常规hiPSC。例如，幼稚糖尿病血管祖细胞（N-DVP）从 患者特异性的未治疗-逆转的糖尿病hiPSC（N-DhiPSC）具有更高的血管功能， 保持了更大的基因组稳定性，降低了谱系启动基因的表达， 更有效地迁移到缺血视网膜的深层神经层并使其再血管化， 来自常规的致敏DhiPSC的同基因糖尿病血管祖细胞（DVP）。在本提案中，我们 开发N-VP治疗缺血性视网膜病变的潜力。我们会雇佣一个人性化的 模拟视网膜缺血的动物模型[即，缺血/再灌注（I/R）损伤]来测试 人N-DVP形成通畅血管、挽救缺血视网膜和改善视网膜缺血的治疗能力 视觉功能我们将测试N-DVP在体内的发育潜力，以有效地分化为 内皮细胞和多能周细胞干祖细胞在缺血损伤的血管中长期植入后的变化 视网膜龛我们还将进一步改进我们的方法，以产生无限量的 表观遗传可塑性的、原始的、未患病的幼稚胚胎祖细胞， 探索N-hiPSC重编程如何消除功能失调的表观遗传供体细胞记忆和糖尿病- 相关的代谢畸变具有比常规hiPSC重编程更高的效率。这些 研究将概述一种有效同步生成幼稚血管和 来自相同N-hiPSC系的视网膜干细胞-祖细胞，用于更有效和全面的 患病视网膜的再生。更广泛地说，我们将开发这类新型药物的临床前效用， 具有高表观遗传可塑性、改善的功能性的人血管周细胞干细胞， 对眼部再生医学有潜在的巨大影响。

Branch vein occlusion (BVO) and diabetic retinopathy (DR) are major causes of new onset blindness in the US. These vascular disorders result in acellular capillaries secondary to ischemic death of retinal vascular endothelial cells (ECs) and contractile pericytes. If acellular retinal blood vessels could be regenerated with autologous or cell-banked self-renewing vascular-pericytic stem-progenitors, ischemia could be relieved, and end stage blindness reversed or stabilized in these vasculopathies. Our group established the feasibility of transplanting patient-specific embryonic vascular progenitors (VP) with pericytic potential directly into the eye, following differentiation from human induced pluripotent stem cells (hiPSC). We also established a novel tankyrase/PARP inhibitor-based small molecule cocktail for reversion of conventional, lineage-primed hiPSC to ‘naïve’ hiPSCs (N-hiPSCs) that possessed a more primitive epiblast state with higher functional pluripotency. The regenerative potential of naïve VP (N-VP) differentiated from normal and diseased N-hiPSC was significantly more prolific relative to primed, conventional hiPSC. For example, naive diabetic vascular progenitors (N-DVP) differentiated from patient-specific naïve-reverted 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) from conventional, primed DhiPSC. In this proposal, we develop the potential of N-VP for treatment of ischemic retinopathies. We will employ a humanized animal model that mimics retinal ischemia [i.e., ischemia/reperfusion (I/R) injury] for testing the therapeutic capacity of human N-DVP to form patent blood vessels, rescue ischemic retina, and improve visual function. We will test the in vivo developmental potential of N-DVP to efficiently differentiate to ECs and multipotent pericytic stem-progenitors following long-term engraftment in an ischemia-damaged retinal niche. We will also aim to further improve our approach for generating unlimited amounts of epigenetically-plastic, pristine, non-diseased naïve embryonic progenitors for cellular therapies by probing how N-hiPSC reprogramming erases dysfunctional epigenetic donor cell memory and diabetes- associated metabolic aberrations with greater efficiency than conventional hiPSC reprogramming. These studies will outline a future pathway for the efficient synchronous generation of naïve vascular and retinal stem-progenitors from the same N-hiPSC line for a more effective and comprehensive regeneration of diseased retina. More broadly, we will develop the pre-clinical utility of this novel class of human vascular-pericytic stem cells that possess high epigenetic plasticity, improved functionality, and potentially high impact for ocular regenerative medicine.