Novel Hyperactive Transposons for Genetic Modification of Induced Pluripotent and Adult Stem Cells: A Nonviral Paradigm for Coaxed Differentiation
Adult stem cells and induced pluripotent stem cells (iPS) hold great promise for regenerative medicine. The development of robust nonviral approaches for stem cell gene transfer would facilitate functional studies and potential clinical applications. We have previously generated hyperactive transpos...
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| Published in: | Stem cells (Dayton, Ohio) Vol. 28; no. 10; pp. 1760 - 1771 |
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| Main Authors: | , , , , , , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Hoboken
Wiley Subscription Services, Inc., A Wiley Company
01-10-2010
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | Adult stem cells and induced pluripotent stem cells (iPS) hold great promise for regenerative medicine. The development of robust nonviral approaches for stem cell gene transfer would facilitate functional studies and potential clinical applications. We have previously generated hyperactive transposases derived from Sleeping Beauty, using an in vitro molecular evolution and selection paradigm. We now demonstrate that these hyperactive transposases resulted in superior gene transfer efficiencies and expression in mesenchymal and muscle stem/progenitor cells, consistent with higher expression levels of therapeutically relevant proteins including coagulation factor IX. Their differentiation potential and karyotype was not affected. Moreover, stable transposition could also be achieved in iPS, which retained their ability to differentiate along neuronal, cardiac, and hepatic lineages without causing cytogenetic abnormalities. Most importantly, transposon‐mediated delivery of the myogenic PAX3 transcription factor into iPS coaxed their differentiation into MYOD+ myogenic progenitors and multinucleated myofibers, suggesting that PAX3 may serve as a myogenic “molecular switch” in iPS. Hence, this hyperactive transposon system represents an attractive nonviral gene transfer platform with broad implications for regenerative medicine, cell and gene therapy. STEM CELLS 2010;28:1760–1771 |
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| Bibliography: | Author contributions: E.B.: designed the experiments, characterized the hyperactive transposases by transfection into iPS and adult stem cells, conducted the G‐banding analysis, analyzed the data and wrote part of the manuscript; J.M.: performed immunohistochemistry staining, microscopy, FIX expression analysis, analyzed the data and wrote part of the manuscript; A.A.‐S.: performed the molecular biology experiments and analyzed the data; L. Ma: designed the experiments and characterized the hyperactive transposases in myoblasts and MSC; M.Q.: designed and performed the Pax3‐coaxed myogenic differentiation of iPS; L. Mátés: designed and generated the hyperactive transposases and transposons; P.S.‐B.: designed and performed the hepatic and cardiomyogenic differentiation of iPS; M.G.: designed and performed the neural and glial differentiation of iPS; B.Y.: performed the adipogenic and myogenic differentiation experiments from MSC and myoblasts, respectively; J.V.: conducted the cytogenetic analysis and the array comparative genomic hybridization (aCGH) analysis; M.Y.R.: contributed to the cardiac differentiation experiments of iPS; E.S.‐K.: performed molecular biology and cell culture experiments; Z.I.: designed and supervised the generation of the hyperactive transposases and transposons, interpreted the data; C.V.: designed and supervised the hepatic, cardiomyogenic, neural and glial differentiation experiments, interpreted the data; M.S.: designed and supervised the Pax3‐coaxed myogenic differentiation experiments, interpreted the data; Z.I.: designed and supervised the generation of the hyperactive transposases and transposons, interpreted the data; T.V.: designed and supervised the experiments, interpreted the data and wrote the manuscript; M.K.L.C.: designed, supervised, and managed the experiments, interpreted the data, wrote the manuscript and coordinated the overall research effort. E.B. and J.M. contributed equally to this article. First published online in STEM CELLS Disclosure of potential conflicts of interest is found at the end of this article. August 16, 2010. EXPRESS Telephone: 32‐16‐330557; Fax: 32‐16‐345990 Telephone: 32‐16‐330558; Fax: 32‐16‐345990 Telephone: 49‐30‐9406‐3510 or ‐2546; Fax: 49‐30‐9406‐2547 or ‐3382 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 1066-5099 1549-4918 |
| DOI: | 10.1002/stem.501 |