Functional Vascular Progenitors from Naive Human iPSC

描述(申请人提供)：来自Na�ve人类IPSC干细胞的功能性血管前体细胞治疗儿童和成人缺血性疾病，如脑血管中风、镰状细胞疾病和糖尿病视网膜病变，最终不仅需要受损心肌、脑、造血和视网膜组织的再生，而且还需要重建最初引发疾病的有缺陷的血管生态位。人类的血管系统通常起源于高度繁殖的胚胎血管母细胞或血管前体细胞(VP)，在早期发育过程中分化为血管内皮细胞和周细胞。这种多产的胚胎型VP在成人中很少见或根本不存在。此外，尽管循环内皮祖细胞(EPC)已被提出用于血管细胞治疗，但这种EPC不仅在多能性和扩增方面受到限制，而且在糖尿病等疾病中也存在功能缺陷。如果能够用自体或人类白细胞抗原相合的胚胎血管/周细胞祖细胞有效地修复缺血的无细胞毛细血管，终末期血管疾病如糖尿病和心肌缺血可能被逆转。一种解决方案是将人诱导多能干细胞(HiPSC)分化为VP，它具有高度繁殖的胚胎样内皮-间充质-周细胞再生疾病组织的潜力。然而，这一目标目前受到低效率和可变性的限制。 定向血管分化为hiPSC。有必要首先通过更安全的非整合、非病毒衍生方法产生高质量的临床级HiPSC。在这个项目中，我们将通过有效地将患者自身的血细胞重新编程为一种新的高质量的多能性状态来实现这一目标，这种状态被称为“NA�ve基态”，类似于小鼠胚胎干细胞(Esc)。NA�ve hIPSC(N-hiPSC)比传统的人胚胎干细胞和hIPSC具有更多的功能，包括更快的培养扩增，更大的分化潜能，以及更高的基因打靶和基于质粒的同源重组的基因操纵能力。我们将产生和分化N-hiPSC，并专门测试它们接受基因靶向的能力，以及产生功能性胚胎VP的能力，这些VP功能地整合到缺血组织中，用于未来的细胞治疗。最后，我们将对NA�ve和传统的hPSC及其分化的VP后代进行详细的表观遗传学比较分析，以阐明不同的CpG甲基化和组蛋白结构。我们认为，针对患者的NA�ve hiPSC最终将为儿童和成人再生医学的细胞和基因治疗提供更多的多功能性。

DESCRIPTION (provided by applicant): Functional Vascular Progenitors from Na�ve Human iPSC Stem cell treatments for pediatric and adult ischemic disorders such as cerebro-vascular stroke, sickle cell disease, and diabetic retinopathy ultimately require not only the regeneration of damaged myocardium, brain, hematopoietic, and retinal tissues, but also the reconstruction of the defective vascular niche that instigated the initial disease to begin with. The human vasculature normally arises from highly prolific embryonic angioblasts or vascular progenitors (VP) that differentiate into vascular endothelial cells and pericytes during early development. Such prolific embryo-like VP are rare or non-existent in the adult. Furthermore, although circulating adult endothelial progenitor cells (EPC) have been proposed for vascular cellular therapies, such EPC are not only limited in multipotency and expansion, but also functionally defective in diseases such as diabetes. If ischemic acellular capillaries could be efficiently repaired with autologous or HLA- matched embryonic vascular/pericytic progenitors, end stage vascular diseases such as diabetes and cardiac ischemia could be reversed. One solution would be to differentiate human induced pluripotent stem cells (hiPSC) into VP that possess highly prolific embryo-like endothelial- mesenchymal-pericytic potential for regenerating diseased tissues. However, this goal is currently limited by the poor efficiency and variability of directed vascular differentiation from hiPSC. It will be necessary to first generate high-quality clinical grade hiPSC via safer non-integrating, nonviral derivation methods. In this project, we will accomplish this goal by efficiently reprogramming a patient's own blood cells to a novel high quality state of pluripotency called the "na�ve ground state" that resembles mouse embryonic stem cells (ESC). Na�ve human iPSC (N-hiPSC) possess more versatile abilities than conventional human ESC and hiPSC including more rapid expansion in culture, greater differentiation potencies, and a higher capacity to be genetically manipulated by gene targeting with plasmid-based homologous recombination. We will generate and differentiate N-hiPSC, and specifically test for their capacity to undergo gene targeting and to produce functional embryonic VP that functionally integrate into ischemic tissues for future cell therapies. Finally, we will conduct detailed comparative epigenetic analyses of na�ve and conventional hiPSC and their differentiated VP progeny to elucidate differential CpG methylation and histone architectures. We propose that patient-specific na�ve hiPSC will ultimately provide greater versatility for cellular and gene therapies for pediatric and adult regenerative medicine.