Abstract
Induced pluripotent stem cells (iPSCs) are a promising source of mesenchymal stem cells (MSCs) for clinical applications. In this study, we transformed human iPSCs using a non-viral vector carrying the IL24 transgene pHrn-IL24. PCR and southern blotting confirmed IL24 integration into the rDNA loci in four of 68 iPSC clones. We then differentiated a high expressing IL24-iPSC clone into MSCs (IL24-iMSCs) that showed higher expression of IL24 in culture supernatants and in cell lysates than control iMSCs. IL24-iMSCs efficiently differentiated into osteoblasts, chondrocytes and adipocytes. Functionally, IL24-iMSCs induced in vitro apoptosis in B16-F10 melanoma cells more efficiently than control iMSCs when co-cultured in Transwell assays. In vivo tumor xenograft studies in mice demonstrated that IL24-iMSCs inhibited melanoma growth more than control iMSCs did. Immunofluorescence and histochemical analysis showed larger necrotic areas and cell nuclear aggregation in tumors with IL24-iMSCs than control iMSCs, indicating that IL24-iMSCs inhibited tumor growth by inducing apoptosis. These findings demonstrate efficient transformation of iPSCs through gene targeting with non-viral vectors into a rDNA locus. The ability of these genetically modified MSCs to inhibit in vivo melanoma growth is suggestive of the clinical potential of autologous cell therapy in cancer.
Highlights
Human mesenchymal stem cells (MSCs) are adherent adult stem cells with an ability to differentiate into various mesenchymal cell types like osteoblasts, adipocytes, and chondrocytes [1]
Immunofluorescence and histochemical analysis showed larger necrotic areas and cell nuclear aggregation in tumors with Interleukin 24 (IL24)-iMSCs than control iMSCs, indicating that IL24-iMSCs inhibited tumor growth by inducing apoptosis. These findings demonstrate efficient transformation of Induced pluripotent stem cells (iPSCs) through gene targeting with non-viral vectors into a ribosomal DNA (rDNA) locus
The ribosomal DNA locus that consists of nearly 400 copies of the 45S pre-RNA gene clustered on the short arms of all five acrocentric chromosomes 13, 14, 15, 21, and 22 with high transcriptional activity has been explores as a candidate site for transgene integration and long term expression [15]
Summary
Human mesenchymal stem cells (MSCs) are adherent adult stem cells with an ability to differentiate into various mesenchymal cell types like osteoblasts, adipocytes, and chondrocytes [1]. While most studies have used viral vectors to modify MSCs, it is preferable to use non-viral methods to engineer therapeutic cells for safety considerations. Their lack of integration into host genome has prevented long term expression that is necessary for clinical applications. We successfully targeted a non-viral vector into the rDNA locus of different human cell types including hepatocyte cell lines, MSCs and embryonic stem cells (ESCs) and demonstrated efficient expression of transgenes [16,17,18]
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