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.

项目摘要/摘要 目前没有有效的疗法可用于表皮溶解Bullosa（EB），这是一组稀有的遗传性皮肤 起泡的疾病导致严重的起泡和疤痕。尽管最近在发展躯体方面取得了进展 EB患者的细胞疗法，表皮干细胞（EPSC）耗竭，尤其是随着年龄的增长和困难 扩展体细胞的制造代表成功实施的关键障碍 EB治疗的疗法。将体细胞重编程到诱导的多能干细胞（IPSC）中 解决这些障碍，并提供适合患者特异性细胞的无限且可扩展的来源 移植。包括我们在内的许多团体已经解决了许多成功的障碍 实施基于IPSC的EB疗法。但是，这种疗法的安全仍然取决于 定义明确和身份验证的IPSC衍生角质形成细胞（IPSC-KCS）的推导。当前表征 测定并不能完全解决IPSC-KC培养物可能不完全污染的主要问题 分化细胞，可能导致皮肤移植衰竭和移植后肿瘤形成。我们建议 使用非集成，修改的mRNA（mod- 基于mRNA）的microRNA（miRNA）开关（miR开关）。 mir开关技术以前是 用于纯化IPSC衍生的细胞，例如心肌细胞和肝细胞，但尚未适应 到IPSC-KCS。 mir开关是一个mod-mRNA分子，它包含特定miRNA的结合位点，该分子 被合并到5'UTR中，并编码报告基因或选择标记蛋白。如果此mir开关特定 miRNA存在于用miR开关转染的细胞中，报告者/选择标记的翻译将会 由于miRNA与其在miR开关中的结合位点结合而被抑制。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 编码报告基因/选择标记，从而可以使用IPSC-KC进行更有效的检测和纯化 干细胞特性。该提案的目标1将通过利用EPSC特定的miRNA候选者 基于荧光的miR开关将转染到一级角质形成细胞和IPSC-KC中。这 由miR开关编码的荧光报告基因的抑制作用将验证开关的存在 细胞中的特定miRNA。 AIM 2将开发单元分类 - 同时消除的独立策略 未分化的IPSC和表达EPSC特异性miRNA的细胞的选择性富集。消除 策略将通过选择性抑制由IPSC-编码的嘌呤霉素耐药性蛋白 特定的miR开关，而阳性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。该提案中概述的目标成功完成 将开发用于IPSC-KC纯化的安全，临床相关的方法，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