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Generation of induced pluripotent stem cells using site-specific integration with phage integrase.

ABSTRACT: To date, a large number of reports have described reprogramming many somatic cell types into induced pluripotent stem (iPS) cells, using different numbers of transcription factors and devising alternate methods of introducing the transcription factor genes or proteins into the somatic cells. Here, we describe a method using bacteriophage ?C31 integrase to reprogram mouse embryonic fibroblasts and human amniotic fluid cells into iPS cells. These iPS cells showed morphology, surface antigens, gene expression, and epigenetic states similar to ES cells and formed teratomas with three germ layers in nonobese diabetic/severely compromised immunodeficient mice. Importantly, these iPS cells have only a single integration site in each cell line. The locations of integration favor the intergenic regions, and their distances from the adjacent genes extended from several hundred to >1 million bp. The effect of the insertion on the expression of these genes can be studied by RT-PCR. No insertion into microRNA gene loci was detected. Hence, it is possible to select cells in which adjacent gene functions are not affected, or the inserts can be removed if necessary. We conclude that phage integrase-mediated site-specific recombination can produce iPS cells that have undisturbed endogenous gene function and could be safe for future human therapeutic application.

SUBMITTER: Ye L

PROVIDER: S-EPMC2984165 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Generation of induced pluripotent stem cells using site-specific integration with phage integrase.

Ye Lin L Chang Judy C JC Lin Chin C Qi Zhongxia Z Yu Jingwei J Kan Yuet Wai YW

Proceedings of the National Academy of Sciences of the United States of America 20101025 45

To date, a large number of reports have described reprogramming many somatic cell types into induced pluripotent stem (iPS) cells, using different numbers of transcription factors and devising alternate methods of introducing the transcription factor genes or proteins into the somatic cells. Here, we describe a method using bacteriophage ΦC31 integrase to reprogram mouse embryonic fibroblasts and human amniotic fluid cells into iPS cells. These iPS cells showed morphology, surface antigens, gene ...[more]

PMID: 20974949

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Project description:BackgroundPigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.MethodsiPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.ResultsIn this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.ConclusionsOur findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

Dataset Information

Generation of induced pluripotent stem cells using site-specific integration with phage integrase.

Publications

Generation of induced pluripotent stem cells using site-specific integration with phage integrase.

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