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

基于 MicroRNA 的多能干细胞角质形成细胞纯化用于治疗皮肤病

• 批准号: 9882955
• 负责人: Ganna Bilousova
• 金额: $ 17.11万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9882955
• 关键词: 5' Untranslated Regions; Address; Age; Autologous; BCL2L11 gene; Back; Binding; Binding Sites; Biological Assay; Biopsy; Bulla; Cardiac Myocytes; Cell Culture Techniques; Cell Differentiation process; Cell Separation; Cell Therapy; Cells; Cicatrix; Clinic; Clinical; Clinical Research; Clinical Trials; Code; Collagen Type VII; Derivation procedure; Detection; Development; Disease; Endothelial Cells; Epidermolysis Bullosa; Epidermolysis Bullosa Dystrophica; Epithelial; Epithelial Cells; Epithelium; Fluorescence; Gene Expression; Gene Expression Profiling; Hepatocyte; Human; Inherited; Integrins; Junctional Epidermolysis Bullosa; Laminin; Lead; Medicine; Messenger RNA; MicroRNAs; Mus; Patients; Pluripotent Stem Cells; Population; Process; Production; Property; Proteins; Puromycin; Reporter; Repression; Residual state; Resistance; Safety; Skin; Skin graft; Somatic Cell; Source; Stem cell transplant; Surface; Technology; Time; Tissues; Translational Repression; Translations; Transplantation; Undifferentiated; Viral Vector; base; candidate identification; cell type; clinical translation; clinically relevant; effective therapy; epidermal stem cell; graft failure; improved; in vivo; induced pluripotent stem cell; keratinocyte; pluripotency; progenitor; protein biomarkers; public health relevance; reconstitution; skin disorder; stem cell differentiation; stem cell technology; stem cell therapy; stem cells; tumor

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.

项目概要/摘要 大疱性表皮松解症（EB）是一种罕见的遗传性皮肤病，目前尚无有效的治疗方法 导致严重水泡和疤痕的水疱性疾病。尽管最近在开发躯体细胞方面取得了进展 细胞疗法、EB 患者的表皮干细胞 (EpSC) 耗竭（尤其是随着年龄的增长）以及治疗困难 体细胞制造规模的扩大是成功实施这些计划的关键障碍 EB 治疗的疗法。将体细胞重编程为诱导多能干细胞（iPSC） 解决这些障碍并提供无限且可扩展的患者特异性细胞来源，适合 移植。许多团体，包括我们的团体，已经解决了许多成功的障碍 实施基于 iPSC 的 EB 疗法。然而，该疗法的安全性仍取决于 衍生出明确且经过验证的 iPSC 衍生角质形成细胞 (iPSC-KC)。电流特性 检测并没有完全解决 iPSC-KC 培养物可能被不完全污染的主要问题 分化的细胞，可能导致皮肤移植失败和移植后形成肿瘤。我们建议 使用非整合、修饰的 mRNA（mod- 基于 mRNA）的 microRNA (miRNA) 开关（miR 开关）。 miR switch技术此前已被 用于纯化 iPSC 衍生细胞，例如心肌细胞和肝细胞，但尚未经过改造 iPSC-KC。 miR 开关是一种 mod-mRNA 分子，包含特定 miRNA 的结合位点， 被整合到 5'UTR 中，并编码报告蛋白或选择标记蛋白。如果这个 miR 开关特定 miRNA 存在于用 miR 开关转染的细胞中，报告基因/选择标记的翻译将 由于 miRNA 与 miR 开关中的结合位点结合而受到抑制。我们假设 EpSC 特异性 miRNA 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-KC 干细胞特性。该提案的目标 1 将利用以下方法识别 EpSC 特异性 miRNA 候选者： 基于荧光的 miR 开关将被转染至原代角质形成细胞和 iPSC-KC 中。这 抑制 miR 开关编码的荧光报告基因将验证该开关的存在 细胞中特定的 miRNA。目标 2 将开发一种独立于细胞分选的策略，同时消除 未分化的 iPSC 和表达 EpSC 特异性 miRNA 的细胞的选择性富集。消除 该策略将通过选择性抑制 iPSC 编码的嘌呤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 特异性 miR 开关编码。成功完成本提案中概述的目标 将导致开发出一种安全的、临床相关的 iPSC-KC 纯化方法，并可能加快 批准基于 iPSC 的 EB 疗法的临床试验。

项目成果

A High-Efficiency Method for the Production of Endothelial Cells from Human Induced Pluripotent Stem Cells.

• DOI: 10.1007/7651_2021_377
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Pavlova M;McGarvey SS;Bilousova G;Kogut I
• 通讯作者: Kogut I