Comment[ArrayExpressAccession] E-GEOD-43775 MAGE-TAB Version 1.1 Public Release Date 2013-08-04 Investigation Title Induction of the mouse germ cell fate by transcription factors in vitro [exp1] Comment[Submitted Name] Induction of the mouse germ cell fate by transcription factors in vitro [exp1] Experiment Description The germ cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have established a culture system that recapitulates the mouse germ-cell specification pathway: Using cytokines, embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) are induced into epiblast-like cells (EpiLCs) and then into primordial germ cell-like cells (PGCLCs) with capacity both for spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous over-expression of three transcription factors (TFs), Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), directs EpiLCs, but not ESCs, swiftly and highly efficiently into a PGC state with endogenous transcription circuitry. The induction of the PGC state on EpiLCs minimally requires Prdm14 but not Blimp1 or Tfap2c. The TF-induced PGC state reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC specification in vivo and in vitro by cytokines including BMP4. Importantly, the TF-induced PGC-like cells robustly contribute to spermatogenesis and fertile offspring. Our findings provide not only a novel insight into the transcriptional logic that creates a germ cell state, but also a foundation for the TF-based reconstitution and regulation of mammalian gametogenesis. Aim of this analysis is characterization of transcription factor-induced primordial germ cells (TF-PGCLCs) compared with cytokine-induced primordial germ cells (Ck-PGCLCs) (Hayashi et al., 2011, Cell), epiblast-like cells (EpiLCs) (Hayashi et al., 2011, Cell), and embryonic stem cells (ESCs) and identification of genes differentially expressed among them. TF-PGCLCs induced by multiple combinations of TFs (Blimp1 (B), Prdm14 (P14), and Tfap2c (A) (BP14A), BP14, P14A, P14) on day 2 and 4 (for BP14A cells) of the induction were also compared. Parental clone without exogenous TFs cultured with doxycycline, are also included as a negative control. Ck-PGCLCs day 2 and day 4 samples, which are previously unreported, EpiLCs and ESCs used in this study were also included. Overexpression of exogenous three TFs in ESCs yields stella-ECFP (SC) positive cells, which were sorted and included in the analysis. cDNA samples, prepared from approximately 20,000 cells, were amplified with a quantitative global PCR method (Kurimoto et al., 2006, Nucleic Acids Research). Two biological duplicates for each cell type were analyzed. Samples from GSE30056 were also included and reanalysed (GSM1070855-GSM1070864). Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name NAKAKI Nakaki Hayashi Ohta Kurimoto Yabuta Saitou Person First Name Fumio Fumio Katsuhiko Hiroshi Kazuki Yukihiro Mitinori Person Email nakaki@anat2.med.kyoto-u.ac.jp Person Affiliation Kyoto University Person Phone +81-75-753-4343 Person Fax +81-75-751-7286 Person Address Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, Japan Person Roles submitter Protocol Name P-GSE43775-1 P-GSE43775-5 P-GSE43775-6 P-GSE43775-2 P-GSE43775-3 P-GSE43775-4 P-GSE43775-7 Protocol Description CEL data were generated by GeneChip Operating Software and then subjected to the dCHIP software. Data were normalized together with the default settings. ID_REF = VALUE = The Model-Based Expression Indices (MBEI) were calculated using the PM/MM difference mode with log-2 transformation of signal intensity. ABS_CALL = Biotinylated cRNA were prepared from total RNA according to the standard Affymetrix protocol (Affymetrix GeneChip Expression Analysis Technical Manual, 2004, Affymetrix: Eukaryotic One-Cycle Target Labeling Assay) Hybridization was performed according to the standard Affymetrix protocol (Affymetrix GeneChip Expression Analysis Technical Manual, 2004, Affymetrix: Eukaryotic Target Hybridization) After 36 hrs of differentiation, cells were harvested and cultured in a Lipidure-Coat 96-well plate (NOF) to be aggregated (started with 2,000 cells/well) in GK15 with 1.5 μg/ml of Dox (Clonetech) to induce TF-PGCLCs. Ck-PGCLCs were induced by BMP4 (500 ng/ml), BMP8A (500 ng/ml), SCF (100 ng/ml), LIF (1000 U/ml) and EGF (50 ng/ml) as previously described (Hayashi et al., Cell, 2011) Transfected ESCs were maintained under 2i+LIF condition and adapted to a feeder-free condition prior to induction. EpiLC differentiation was performed as reported previously (Hayashi et al., Cell, 2011). total RNA was extracted using Qiagen RNeasy according to manufacturer's instruction The microarray image data were processed with the GeneChip Scanner 3000 (Affymetrix) Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TRANSGENE CELL TYPE GENETIC BACKGROUND SEX Experimental Factor Type transgene cell type genetic background sex Publication Title Induction of mouse germ-cell fate by transcription factors in vitro. Publication Author List Nakaki F, Hayashi K, Ohta H, Kurimoto K, Yabuta Y, Saitou M PubMed ID 23913270 Publication DOI 10.1038/nature12417 Comment[SecondaryAccession] GSE43775 Comment[GEOReleaseDate] 2013-08-04 Comment[ArrayExpressSubmissionDate] 2013-01-25 Comment[GEOLastUpdateDate] 2013-08-06 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-43775.sdrf.txt