MicroRNA-based purification of keratinocytes derived from pluripotent stem cells for the treatment of skin diseases

项目摘要/摘要 大疱性表皮松解症(EB)是一种罕见的遗传性皮肤，目前尚无有效的治疗方法 导致严重起泡和结疤的起泡障碍。尽管最近在体细胞发育方面取得了进展 细胞治疗，EB患者的表皮干细胞(EpSC)耗竭，特别是随着他们年龄的增长，以及 体细胞的规模化生产是成功实现这些目标的关键障碍 EB治疗的疗法。将体细胞重新编程为诱导多能干细胞(IPSCs)将 解决这些障碍，并提供适合患者的无限且可扩展的细胞来源 移植。许多团体，包括我们在内，已经克服了成功的一些障碍 以IPSC为基础的EB治疗的实施。然而，这种疗法的安全性仍取决于 明确定义和鉴定的IPSC来源的角质形成细胞(IPSC-KCs)的来源。电流表征 检测不能完全解决IPSC-KC培养可能被不完全污染的主要问题 分化的细胞，这可能会导致皮肤移植失败和移植后形成肿瘤。我们建议 用非整合修饰的信使核糖核酸(mod-mRNAs)从分化的IPSC-KCs培养物中纯化EPSC 基于miRNA(MiRNA)的开关(miR开关)。MiR交换机技术以前已经 用于提纯IPSC来源的细胞，如心肌细胞和肝细胞，但尚未被适应 敬IPSC-KCS。MiR开关是一种mod-mRNA分子，它包含特定miRNA的结合位点，这 被整合到5‘非编码区，并编码一个报告蛋白或选择标记蛋白。如果此miR开关特定 MiRNA存在于转染miR开关的细胞中，报告/选择标记的翻译将 由于miRNA与其在miR开关中的结合部位结合而被抑制。我们假设EPSC的特异性 MiRNAs将能够促进基于mod-mRNA的EpSC特异性miR开关的翻译抑制 编码报告/选择标记，从而能够更有效地检测和纯化IPSC-KCs 干细胞的特性。该提案的目标1将通过利用以下方法确定EpSC特定的miRNA候选 基于荧光的miR开关将被导入原代角质形成细胞和IPSC-KCs。这个 抑制由miR开关编码的荧光报告将验证开关的存在- 细胞中特定的miRNA。AIM 2将开发一种不依赖细胞分选的策略，以同时消除 未分化的IPSCs和表达EpSC特异性miRNAs的细胞的选择性浓缩。淘汰赛 该策略将通过选择性抑制由IPSC-1编码的扑霉素抗性蛋白来实现。 特定的miR开关，而正选择策略将依赖于促凋亡蛋白的抑制， BIM，由EPSC特定的miR开关编码。成功地完成这项提案中概述的目标 将导致开发一种安全的、临床相关的IPSC-KC纯化方法，并可能加快 批准一项基于IPSC的EB治疗的临床试验。

Project Summary/Abstract No effective treatments are currently available for epidermolysis bullosa (EB), a group of rare inherited skin blistering disorders that result in severe blistering and scarring. Despite recent progress in developing somatic cell therapies, epidermal stem cell (EpSC) depletion in EB patients, especially as they age, and difficulties in scaling the manufacture of somatic cells represent key roadblocks in the successful implementation of these therapies for EB treatment. Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) would address these roadblocks and provide an unlimited and scalable source of patient-specific cells suitable for transplantation. Many groups, including ours, have already solved a number of hurdles for successful implementation of an iPSC-based therapy for EB. However, the safety of this therapy still depends on the derivation of well-defined and authenticated iPSC-derived keratinocytes (iPSC-KCs). Current characterization assays do not fully address the major concern that iPSC-KC cultures may be contaminated with incompletely differentiated cells, which can cause skin graft failure and tumor formation upon transplantation. We propose to purify EpSCs from the cultures of differentiated iPSC-KCs using non-integrating, modified mRNA (mod- mRNA)-based microRNA (miRNA) switches (miR switches). The miR switch technology has been previously used for purification of iPSC-derived cells, such as cardiomyocytes and hepatocytes, but has not been adapted to iPSC-KCs. A miR switch is a mod-mRNA molecule that contains a binding site for a specific miRNA, which is incorporated into the 5'UTR, and encodes a reporter or selection marker protein. If this miR switch specific miRNA is present in cells transfected with the miR switch, the translation of the reporter/selection marker will be repressed due to miRNA binding to its binding site in the miR switch. We hypothesize that EpSC-specific miRNAs will be able to promote translational repression of mod-mRNA-based EpSC-specific miR switches encoding reporter/selection markers, thus enabling more efficient detection and purification of iPSC-KCs with stem cell properties. Aim 1 of this proposal will identify EpSC-specific miRNA candidates by utilizing fluorescence-based miR switches that will be transfected into primary keratinocytes and iPSC-KCs. The repression of a fluorescence reporter encoded by the miR switch will validate the presence of the switch- specific miRNA in cells. Aim 2 will develop a cell sorting - independent strategy for simultaneous elimination of undifferentiated iPSCs and selective enrichment of cells expressing EpSC-specific miRNAs. The elimination strategy will be achieved by selective repression of the puromycin resistance protein encoded by a iPSC- specific miR switch, while the positive selection strategy will rely on the repression of the proapoptotic protein, BIM, encoded by an EpSC-specific miR switch. The successful completion of the aims outlined in this proposal will result in the development of a safe, clinically relevant approach for iPSC-KC purification and may expedite approval of a clinical trial for an iPSC-based therapy for EB.