Unknown,Transcriptomics,Genomics,Proteomics

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Generation of Autologous Pluripotent Stem Cells from Adult Testis Biopsy

ABSTRACT: In regenerative medicine, histocompatibility of pluripotent stem cells is required to solve the problem of immunorejection after therapeutic transplantation. In this study, we show that autologous germline stem cells (GSCs), often called spermatogonial stem cells, could be derived by testis biopsy from individual mice and that GSCs subsequently could be dedifferentiated into autologous germline-derived pluripotent stem (gPS) cells. The establishment of GSCs by testicular biopsy in the mouse model can prove the principle of human clinical application to derive autologous GSCs to generate patient-specific pluripotent cells for regenerative medicine. For transcriptome profiling, 400 ng of total DNA-free RNA was used as input for labelled cRNA synthesis (Illumina TotalPrep RNA Amplification Kit - Ambion) following the manufacturer's instructions (IVT: 12h). Quality-checked cRNA samples were hybridized as biological or technical duplicates for 18 h onto mouse-8 v2 expression BeadChips (Illumina), washed, stained, and scanned following guidelines and using materials / instrumentation supplied / suggested by the manufacturer 13 samples were analyzed, GSC1: Mouse Germ Stem Cells OG2, line 1 (1 replicate); GSC2: Mouse Germ Stem Cells OG2, line 2 (1 replicate); GSC3: Mouse Germ Stem Cells OG2, line 3 (1 replicate); GSCr1: Mouse Germ Stem Cells OG2-ROSA, line 1 (1 replicate); GSCr2: Mouse Germ Stem Cells OG2-ROSA, line 2 (2 replicate); GSCr3: Mouse Germ Stem Cells OG2-ROSA, line 3 (1 replicate); ESC: Mouse Embryonic Stem Cells (duplicate) gPS1: Mouse clonal germ Pluripotent Stem cells from OG2, line 1, (1 replicate); gPS2: Mouse clonal germ Pluripotent Stem cells from OG2, line 2, (1 replicate); gPS3: Mouse clonal germ Pluripotent Stem cells from OG2, line 3, (1 replicate); gPSr1: Mouse clonal germ Pluripotent Stem cells from OG2-ROSA, line 1, (1 replicate); gPSr2: Mouse clonal germ Pluripotent Stem cells from OG2-ROSA, line 2, (1 replicate); gPSr3: Mouse clonal germ Pluripotent Stem cells from OG2-ROSA, line 3, (1 replicate).

ORGANISM(S): Mus musculus

SUBMITTER: Marcos Arauzo-Bravo

PROVIDER: E-GEOD-20031 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Embryonic stem cells (ESCs), which are derived from the primitive ectoderm of pre-implantation blastocysts, are pluripotent cells and can thus contribute to the formation of all somatic cell lineages in chimeric animals. Similarly, epiblast stem cells (EpiSCs), which are derived from epiblast tissue of post-implantation embryos, are also pluripotent and can give rise to derivatives of all three germ layers in teratoma assays. Introduction of the four transcription factors Oct4/Sox2/Klf4/c-Myc into somatic cells has been shown to generate induced pluripotent stem cells (iPSCs). iPSCs exhibit ESC-like properties and are virtually identical to ESCs with respect to a number of charactertistics. However, generation of EpiSC-like cells by direct reprogramming of somatic cells using these transcription factors has not been shown to date. Here we show that Yamanaka’s four transcription factors can be used to directly generate EpiSC-like iPS cells (ePSCs) under EpiSC culture conditions. These ePSCs are identical to EpiSCs with respect to morphology, gene expression pattern, epigenetic status, and chimera formation. This is the first study to demonstrate that the culture environment in transcription factor-mediated reprogramming determines the cell fate of the reprogrammed cell. We could therefore envision that we eventually are able to shape the identity of a directly reprogrammed cell at will simply by modulating the culture conditions. RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 500 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates. 24 samples were analyzed: MEF: Female mouse embryonic fibroblast, 2 biological rep ESC OG2: OG2 female embryonic stem cell, 2 biological rep EpiSC OG2: OG2 female EpiSC grown as single cells with conditioned medium, 2 biological rep EpiSC GOF18: GOF18 EpiSC grown as single cells with conditioned medium, 2 biological rep EpiSC T9b: T9 EpiSC on MEFs in activin-containing CDM, harvested w/o feeders, subconfluent, 2 biological rep ePSC L4: EpiSC-like iPS Cells L4, MEF reprogrammed to EpiSCs with 4F, line L4, grown with LIF and activin, 2 biological rep ePSC L7: EpiSC-like iPS cells L7, MEF reprogrammed to EpiSCs with 4F, line L7, grown with LIF and activin, 2 biological rep ePSC C1: EpiSC-like iPS Cells C1, MEF reprogrammed to EpiSCs with 4F, line C1, grown with LIF and activin, 2 biological rep ePSC C3: EpiSC-like iPS Cells C3, MEF reprogrammed to EpiSCs with 4F, line C3, grown with LIF and activin, 2 biological rep ePSC-R: EpiSC-like iPS Cells reverted, L4 ePSC cells reverted to an ESC-like state using Klf4 virus, 2 biological rep ePSC-RC1: EpiSC-like iPS Cells reverted, L4 ePSR Cre1, Cre-treated L4 ePSR cells, line 1, 2 biological rep ePSC-RC2: EpiSC-like iPS Cells reverted, L4 ePSR Cre2, Cre-treated L4 ePSR cells, line 2, 2 biological rep

Project description:Pluripotent stem cell lines derived from embryos of different stages have distinct pluripotent ground states, but similar levels of the transcription factor Oct4. Epiblast-derived pluripotent stem cells (EpiSCs), in contrast to embryonic stem (ES) cells, cannot form chimeras. We show that EpiSCs express lower levels of the transcription factors Sox2 and Klf4 than ES cells and have limited reprogramming potential, as shown by cell fusion. Sox2 overexpression dramatically increases the reprogramming potential, chimera formation, and germline contribution of EpiSCs. Therefore, although Oct4 is essential for reprogramming, the level of Sox2 defines both the reprogramming capability and the pluripotent ground states. RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates. 12 sample types were analyzed, each of them in duplicate. ESCm: Mouse ESC male; ESCf: Mouse ESC OG2 female; F9 EC: F9 EC (mouse embryonic carcinoma cell); F9-Sox2: F9 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCf: Mouse EpiSC OG2 female; Epi-Sox2f: Mouse EpiSC Sox2 (OG2 female) overexpressing wild type Sox2; P19 EC: P19 EC (mouse embryonic carcinoma cell); P19-Sox2: P19 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCm: Mouse EpiSC (GOF18 male) (duplicates); EpiSox2mL2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in condition EpiSC medium (CM); EpiSox2mE1: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like1); EpiSox2mE2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like2).

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Generation of Autologous Pluripotent Stem Cells from Adult Testis Biopsy

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