Strategies to improve reprogramming, characterization & genomic excision of polycistronic reprogramming factors in induced pluripotent stem cells
Dr. Vi Chu
R&D Manager of Stem Cell/ Cell Biology department, Millipore Corporation, U.S.A.
Abstract
Induced pluripotent stem cells (iPS) offer great promise for the future of regenerative medicine. iPS cells possess similar properties to embryonic stem cells (ESC) but without the ethical and moral concerns associated with ESCs. iPS cells were originally generated using a combination of four retroviral vectors each carrying separately the transcription factors, Oct-4, Klf-4, Sox-2 and c-Myc. Recently, a single lentiviral vector was generated which enabled the expression of the four Yamanaka transcription factors from a single multicistronic transcript. Here we show that expression of this “stem cell cassette” (STEMCCA) was able to efficiently reprogram mouse embryonic fibroblast (MEF) and human foreskin fibroblasts (HFF) into iPS cells. Both human and mouse iPS cells display typical ES cell- like morphology, express the correct ES cell markers, can be rapidly expanded in either serum or serum-free, feeder-free cell culture conditions, and possess multilineage differentiation capacity. A number of enabling tools have been developed to further characterize these reprogrammed cells and will be discussed regarding (1) how to distinguish and select fully reprogrammed colonies from mixtures of partially reprogrammed cells, (2) how to quantify for the degree of viral transgene silencing in iPS cells and their differentiated progeny, (3) how to excise the transgene in a Cre-LOX STEMCCA system and (4) chemical strategies to increase reprogramming efficiency. The use of a single lentiviral vector rather than 4 separate vectors to achieve reprogramming significantly reduces the risk of insertional mutagenesis ad viral reactivation and is a step towards safer and more effective utilization of iPS technology for clinical therapies.
编辑: helen 作者:Dr. Vi Chu
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