# MicroRNA-based purification of keratinocytes derived from pluripotent stem cells for the treatment of skin diseases > **NIH NIH R21** · UNIVERSITY OF COLORADO DENVER · 2020 · $171,050 ## Abstract 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 completi... ## Key facts - **NIH application ID:** 9882955 - **Project number:** 5R21AR074642-02 - **Recipient organization:** UNIVERSITY OF COLORADO DENVER - **Principal Investigator:** Ganna Bilousova - **Activity code:** R21 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2020 - **Award amount:** $171,050 - **Award type:** 5 - **Project period:** 2019-03-01 → 2022-02-28 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/9882955 ## Citation > US National Institutes of Health, RePORTER application 9882955, MicroRNA-based purification of keratinocytes derived from pluripotent stem cells for the treatment of skin diseases (5R21AR074642-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9882955. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*