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Uniform, integration-free iPS cells with a safeguard system using human artificial chromosome vectors

ABSTRACT: Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vector, e.g., episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown construct (see Kazuki et al 2011). The iHAC1 partially reprogrammed MEFs, and the iHAC2 efficiently reprogrammed MEFs. Global gene expression showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. We established iHAC-free iPS cells by isolating cells that spontaneously lost the iHAC2. Analyses of pluripotent markers, teratomas, and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex Virus Thymidine Kinase were eliminated by Ganciclovir treatment; therefore, the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a safeguard system.

ORGANISM(S): Mus musculus

PROVIDER: GSE29441 | GEO | 2011/10/16

SECONDARY ACCESSION(S): PRJNA141519

REPOSITORIES: GEO

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Uniform, integration-free iPS cells with a safeguard system using human artificial chromosome vectors

Project description:Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vector, e.g., episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown construct (see Kazuki et al 2011). The iHAC1 partially reprogrammed MEFs, and the iHAC2 efficiently reprogrammed MEFs. Global gene expression showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. We established iHAC-free iPS cells by isolating cells that spontaneously lost the iHAC2. Analyses of pluripotent markers, teratomas, and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex Virus Thymidine Kinase were eliminated by Ganciclovir treatment; therefore, the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a safeguard system. Total RNA from mouse embryonic fibroblasts, two control ES cells (TT2 and B6ES), retrovirus vector-derived iPS cells (20D17), and twelve HAC-derived iPS clones were hybridized to the Agilent Whole Mouse Genome microarrays.

2011-10-15 | E-GEOD-29441 | biostudies-arrayexpress

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Project description:Mouse B cells, upon ectopic expression of the transcription factor C/EBPalpha for 18h, can be reprogrammed to induced pluripotent stem (iPS) cells with extremely high efficiency. To understand the molecular control of this phenomenon we performed a time course proteomic analysis by label-free protein quantification of proteins during B cell reprogramming to iPS cells.

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Transcription profiling of human induced pluripotent stem cells free of exogenous DNA are derived with episomal vectors (super series)

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Global gene-expression analyses of the Esrrb reprogrammed cells

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2009-01-15 | GSE13211 | GEO

Generation of induced pluripotent stem cells by efficient reprogramming of adult bone marrow cells

Project description:Reprogramming of somatic cells provides potential for the generation of specific cell types, which could be a key step in the study and treatment of human diseases. In vitro reprogramming of somatic cells into a pluripotent embryonic stem (ES) cellM-bM-^@M-^Slike state has been reported by retroviral transduction of murine fibroblasts using four embryonic transcription factors or through cell fusion of somatic and pluripotent stem cells. The generation of reprogrammed pluripotent cells using a somatic cell donor source such as bone marrow (BM) or peripheral blood is of particular therapeutic interest because of the relative ease of harvesting these cell types. Here we show that mouse adult BM mononuclear cellsM-oM-<M-^HBM MNCsM-oM-<M-^Iare competent as donor cells and can be reprogrammed into pluripotent ES cell-like cells. We isolated BM MNCs and embryonic fibroblasts (MEFs) from Oct4-EGFP transgenic mice, fused them with ES cells and infected them with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc. Fused BM cells formed more ES-like colonies than did MEFs. Infected BM cells gave rise to iPS cells, although transduction efficiencies were not high. It was more efficient to pick up iPS colonies as compared with MEFs. BM-derived iPS (BM iPS) cells expressed embryonic stem cell markers, formed teratomas, and contributed to chimera mice with germline development. Clonal analysis revealed that BM iPS clones had diversity, although some clones were found to be genetically identical with different phenotypes. Here we demonstrate, for the first time, the induction of pluripotent cells directly from hematopoietic tissue. Gene expression profiling was performed in mouse BMMNCs, ES and BMMNC derived iPS cell lines.

2009-07-02 | E-GEOD-15775 | biostudies-arrayexpress

Generation of induced pluripotent stem cells by efficient reprogramming of adult bone marrow cells

Project description:Reprogramming of somatic cells provides potential for the generation of specific cell types, which could be a key step in the study and treatment of human diseases. In vitro reprogramming of somatic cells into a pluripotent embryonic stem (ES) cell–like state has been reported by retroviral transduction of murine fibroblasts using four embryonic transcription factors or through cell fusion of somatic and pluripotent stem cells. The generation of reprogrammed pluripotent cells using a somatic cell donor source such as bone marrow (BM) or peripheral blood is of particular therapeutic interest because of the relative ease of harvesting these cell types. Here we show that mouse adult BM mononuclear cells（BM MNCs）are competent as donor cells and can be reprogrammed into pluripotent ES cell-like cells. We isolated BM MNCs and embryonic fibroblasts (MEFs) from Oct4-EGFP transgenic mice, fused them with ES cells and infected them with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc. Fused BM cells formed more ES-like colonies than did MEFs. Infected BM cells gave rise to iPS cells, although transduction efficiencies were not high. It was more efficient to pick up iPS colonies as compared with MEFs. BM-derived iPS (BM iPS) cells expressed embryonic stem cell markers, formed teratomas, and contributed to chimera mice with germline development. Clonal analysis revealed that BM iPS clones had diversity, although some clones were found to be genetically identical with different phenotypes. Here we demonstrate, for the first time, the induction of pluripotent cells directly from hematopoietic tissue.

2009-07-02 | GSE15775 | GEO

Comparisons between fully and partially reprogrammed iPS cells induced by pMX-Klf4, pMX-Oct4 and pMX-Sox2 retroviruses

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Human induced pluripotent stem cells free of exogenous DNA are derived with episomal vectors (fig 1.c)

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Human induced pluripotent stem cells free of exogenous DNA are derived with episomal vectors (fig 4.a)

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