Patient-specific and universal donor blood cell from protein-induced iPS cells

描述（由申请人提供）：红细胞（RBC）和血小板供应有限是一个严重的医学问题，对于输血依赖患者，特别是那些发生血小板同种异体免疫的患者，可能会造成危及生命的后果。O rh阴性“万能献血者”血型在一般人群中的低流行率（西方国家<8%，亚洲<0.3%）进一步加剧了血液供应和/或分型有限的紧急情况下血液短缺的后果。基于Yamanaka和Thomson的开创性工作，人类诱导多能干细胞（hiPSCs）提供了在不破坏胚胎的情况下产生患者特异性干细胞的可能性。然而，目前的iPSCs存在主要缺陷，包括多种病毒整合和在不同染色体位置保留转基因，其中任何一种都可能导致不可预测的遗传功能障碍和/或肿瘤形成（Yamanaka, 2009）。在这项提议中，基于我们最近的“概念验证”结果，我们假设适合临床翻译的hiPS细胞可以通过直接递送附着在细胞穿透肽上的重编程蛋白来产生。特别是，我们将通过直接蛋白质递送从健康和O（-）受试者中产生完全重编程的hiPS细胞系，并测试这些细胞是否可以在体外无限繁殖和扩增，从而提供潜在的取之不竭和无供体的血液谱系细胞来源。本研究将充分优化蛋白质重编程方法，建立一种无需基因操作的高效、安全的人体组织重编程方法，并将探讨这些hiPS细胞是否可以作为个性化血小板来源和/或通用红细胞来源。

DESCRIPTION (provided by applicant): The limited supply of red blood cells (RBC) and platelets is a serious medical issue that can have life-threatening consequences for transfusion-dependent patients, particularly those who develop platelet allo-immunity. The low prevalence of O Rh-negative 'universal donor' blood type in the general population (<8% in Western countries and <0.3% in Asia) further intensifies the consequences of blood shortages for emergency situations where blood supply and/or typing is limited. Based on the pioneering work of Yamanaka and Thomson, human induced pluripotent stem cells (hiPSCs) offer the possibility to generate patient-specific stem cells without destruction of embryos. However, current iPSCs suffer from major drawbacks including multiple viral integrations and remaining transgenes at various chromosomal locations, any of which may cause unpredictable genetic dysfunction and/or tumor formation (Yamanaka, 2009). In this proposal, based on our recent "proof-of-concept" results, we hypothesize that hiPS cells suitable for clinical translation can be generated by direct delivery of reprogramming proteins attached to a cell penetrating peptide. In particular, we will generate fully reprogrammed hiPS cell lines from both healthy and O(-) subjects by direct protein delivery and test whether these cells can be propagated and expanded in vitro indefinitely, thus providing a potentially inexhaustible and donor-less source of blood lineage cells. This proposal will fully optimize the protein reprogramming methods to establish a highly efficient and safe way of reprogramming human tissues without genetic manipulation, and will address whether these hiPS cells can be used as a personalized platelet source and/or universal RBC source. PUBLIC HEALTH RELEVANCE: Although still in its infancy, "induced pluripotent stem cell (iPSC)" technology has the potential to revolutionize biomedical research, disease mechanism studies, and customized cell-based therapies. To explore the potential of iPSC's as a source of universal red blood cells and personalized platelets, we propose to establish and characterize clinically viable iPSC lines from healthy O Rh-negative [O(-)] subjects by direct delivery of reprogramming proteins without the use of viruses or foreign-DNA vectors. Using these protein-induced iPS cells, we will address whether they can be propagated and expanded in vitro indefinitely into blood cell progenitor's lineages that can be used as a donor-less source of universal red blood cells and/or personalized platelets.