Identification of genes specific to mouse primordial germ cells through dynamic global gene expression
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE23322 | GEO | 2010/11/16
SECONDARY ACCESSION(S): PRJNA131231
REPOSITORIES: GEO
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Project description:The molecular mechanisms underlying the commitment of cells to the germ cell lineage and the subsequent formation of germ cells during mammalian development remain poorly understood due to an inability to obtain sufficient amounts of cellular materials to conduct thorough in-vitro analyses. Although primordial germ cells (PGCs) have been generated and isolated from embryonic stem cells (ESCs) in vitro, concurrent expression of several cell surface receptors and transcription factors, at various levels, confounds the identification of these cells. To date, only a few genes that mark the onset of germ cell commitment to the epiblast, including tissue nonspecific alkaline phosphatase (TNAP), Blimp1, Stella, and Fragilis, have been used with some degree of success to detect PGC formation in in-vitro model systems. In light of the current literature, the generation of an unlimited supply of germ cells and gametes from pluripotent stem cells would greatly facilitate studies into reproductive development. To accomplish this, we would need to discover novel markers that are specifically expressed in germ cells. Here we identified four genes that are specifically expressed in male and female germ cells, both in vivo and in vitro, but are not expressed in somatic tissues or ESCs. Expression of these genes therefore allows us to distinguish committed germ cells from undifferentiated pluripotent cell populations, a prerequisite for the successful derivation of germ cells and gametes in vitro. For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using RNeasy Micro kits with on-column DNase digestion as per the manufacturer's instructions. Integrity of RNA samples was quality-checked using a 2100 Bioanalyzer (Agilent). If possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA amplification kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hrs of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality-checked on a 2100 Bioanalyser, and hybridized as biological or technical duplicates for 18 hrs onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader and accompanying software. 44 samples were analyzed. ESCm: OG2 male Embryonic Stem Cell, +/+ Oct4-GFP ES cells, 1 biological rep 11.5: 11.5 embryonic day PGC (Primordial Germ Cell), 2 biological reps 12.5F: 12.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 13.5F: 13.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 14.5F: 14.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 15.5F: 15.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 16.5F: 16.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 17.5F: 17.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 18.5F: 18.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 12.5M: 12.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 13.5M: 13.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 14.5M: 14.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 15.5M: 15.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 16.5M: 16.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 17.5M: 17.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 18.5M: 18.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 8M: 8 day after birth male PGC (Primordial Germ Cell), 2 biological reps 10M: 10 day after birth male PGC (Primordial Germ Cell), 2 biological reps 12M: 12 day after birth male PGC (Primordial Germ Cell), 2 biological reps 14M: 14 day after birth male PGC (Primordial Germ Cell), 1 biological rep 16M: 16 day after birth male PGC (Primordial Germ Cell), 2 biological reps 18M: 18 day after birth male PGC (Primordial Germ Cell), 2 biological reps 20M: 20 day after birth male PGC (Primordial Germ Cell), 2 biological reps
2010-11-16 | E-GEOD-23322 | biostudies-arrayexpress
Project description:Transgenic StellaGFP ESCs were used to derive primordial germ cells during embryoid body (EB) differentiation, and microarry analysis used to compared FACS sorted Stella-positive cells of day 7 Ebs with the parental ESCs and Stella-negative cells of day 7 Ebs. Keywords: in vitro, primordial germ cells, embryonic stem cells, stella
2009-06-22 | GSE7948 | GEO
Project description:Transgenic StellaGFP ESCs were used to derive primordial germ cells during embryoid body (EB) differentiation, and microarry analysis used to compared FACS sorted Stella-positive cells of day 7 Ebs with the parental ESCs and Stella-negative cells of day 7 Ebs. Experiment Overall Design: Four replicates of Stella-positive day 7 cells were compared to two replicates of stella-negative day 7 cells. Also, there were three replicated of FACS sorted Stella-negative ESCs, two replicated of FACS-sorted Stella-postive ESCs, and two replicates of unsorted StellaGFP ESCs.
2009-07-02 | E-GEOD-7948 | biostudies-arrayexpress
Project description:The molecular mechanisms of human primordial germ cell (PGC) specification are poorly understood due to the inaccessibility of cell materials and the lack of an alternative in vitro model that enables tracking of the earliest stages of germ cell development. Here, we introduce a defined and efficient differentiation system for the induction of pre-migratory PGC-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). By step-wise differentiation, we generated an OCT4+/ T+/BLIMP1+ cell population that transitioned into STELLA expressing PGC-like cells that exhibited a similar key gene expression as mouse PGCs as well as global epigenetic reprogramming. Even though, these PGC-like cells expressed PRDM14 at very low levels, they underwent activation of pluripotency/PGC genes, suppression of neural induction and suppression of de novo DNA methylation, events that are regulated by Prdm14 during mouse PGC specification. This study demonstrates that human PGC commitment shares many key features with mouse PGC specification, but harbors unique and so far unknown mechanisms that, point to a novel human transcriptional regulation. 7 samples were analyzed. ESC: Human Embryonic Stem Cells, 1 biological rep iPSC: Human induced Pluripotent Stem Cells, 1 biological rep d2: Human induced Pluripotent Stem Cells, 2 days differentiation treatment , 2 biological rep d4PGCLC: Human induced Pluripotent Stem Cells, 4 days differentiation treatment towards Primordial Germ Cells Like Cells, 1 biological rep d6PGCLC: Human induced Pluripotent Stem Cells, 6 days differentiation treatment towards Primordial Germ Cells Like Cells, 2 biological rep
2015-04-06 | E-GEOD-53498 | biostudies-arrayexpress
Project description:Reconstitution of female germ-cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in mice are induced into primordial germ cell-like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, develop pre-meiotic germ cell characteristics, including X-reactivation, imprint erasure, and cyst formation. Upon transplantation under ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which, through in vitro maturation and fertilization, contribute to fertile offspring. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ-cell development in vitro. We performed expression analysis of female PGC-like cells [PGCLCs; day 3 (d3), day 6 (d6), and day 3 followed by day 6 in aggregation with female embryonic day (E) 12.5 gonadal somatic cells (d3ag6)] in comparison to in vivo embryonic PGCs (E12.5 female PGCs and E9.5 PGCs) and male d6 PGCLCs. PGCLCs were marked with fluorescence of Blimp1-mVenus, stella-ECFP transgenic reporters (BVSC). PGCs were marked with fluorescence of the stella-EGFP transgenic reporter.
2012-11-05 | E-GEOD-40716 | biostudies-arrayexpress
Project 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).
2013-08-04 | E-GEOD-43775 | biostudies-arrayexpress
Project description:The molecular mechanisms of human primordial germ cell (PGC) specification are poorly understood due to the inaccessibility of cell materials and the lack of an alternative in vitro model that enables tracking of the earliest stages of germ cell development. Here, we introduce a defined and efficient differentiation system for the induction of pre-migratory PGC-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). By step-wise differentiation, we generated an OCT4+/ T+/BLIMP1+ cell population that transitioned into STELLA expressing PGC-like cells that exhibited a similar key gene expression as mouse PGCs as well as global epigenetic reprogramming. Even though, these PGC-like cells expressed PRDM14 at very low levels, they underwent activation of pluripotency/PGC genes, suppression of neural induction and suppression of de novo DNA methylation, events that are regulated by Prdm14 during mouse PGC specification. This study demonstrates that human PGC commitment shares many key features with mouse PGC specification, but harbors unique and so far unknown mechanisms that, point to a novel human transcriptional regulation.
2015-04-06 | GSE53498 | GEO
Project description:Primordial germ cells (PGCs) are the embryonic precursors to egg and sperm. When removed from the embryonic gonad, PGCs can give rise to embryonic germ cell lines (EGs), pluripotent stem cells that display most of the characteristics of embryonic stem cells (ESCs) including the ability to form teratomas and to contribute to chimera formation. In mice, EG cells can be derived between E8.5 up to E12.5 of embryonic development, at which point the PGCs undergo sexual differentiation and in the male transition into unipotent gonocytes. Dazl, a germ cell-specific RNA-binding protein, is specifically expressed in developing PGCs and is required for proper germ cell development. Dazl knockout mice are infertile, but the molecular mechanisms underlying this phenotype are still unknown. Here we demonstrate that Dazl localizes in granular structures in mammalian PGCs but not in ESCs. We demonstrate Dazl plays a central role in a large mRNA/protein interactive network that includes members of Fragile-X family RNA-binding proteins. We demonstrate that Dazl and Fxr1 play a central role in these granules and directly regulate the translation of specific core pluripotency factors, including Sox2 and Suz12. Global gene expression changes during in vitro germ cell differentiation from murine ES cells and comparison to in vivo germ cells. In vitro primordial germ cells differentiated from Stella-GFP ES cells and Dazl-GFP ES cells. The GFP-positive cells were isolated by FACS analysis. Dazl-GFP+ cells were isolated from transgenic E13.5 embryonic gonads and P7-P11 juvenile mouse testes. The global gene expression profiles were analyzed by Agilent Mouse Whole Genome 4X44K one-color microarrays. Two replicates per condition.
2013-12-31 | E-GEOD-39546 | biostudies-arrayexpress
Project description:Pluripotent Embryonic Stem Cells (ESCs) can be captured in vitro in different states, ranging from unrestricted ‘naïve’ to more developmentally constrained ‘primed’ pluripotency. Complexes involved in epigenetic regulation and key transcription factors have been shown to be involved in specifying these distinct states. In this study, we use proteomic profiling of the chromatin landscape in naive pluripotent ESCs, Epistem cells (EpiSCs) and early differentiated ESCs to survey the chromatin in naïve and primed pluripotency and during differentiation. We provide a comprehensive overview of epigenetic complexes situated on the chromatin and identify proteins associated with the maintenance and loss of pluripotency. The findings presented here indicate major compositional alterations of epigenetic complexes starting from ESC priming onwards. Our results contribute to the understanding of ESC differentiation and provide a framework for future studies of lineage commitment of ESCs.
2019-07-04 | PXD011782 | Pride
Project description:Primordial germ cells (PGCs) are fate restricted to differentiate into gametes in vivo. However when removed from their embryonic niche PGCs undergo reversion to generate pluripotent embryonic germ cells (EGCs) in vitro. One of the major differences between EGCs and embryonic stem cells (ESCs) involves variable methylation at imprinting control centers (ICCs), a phenomenon that is poorly understood. In the current study we show that reverting PGCs to EGCs involves ICC methylation erasure, which remain stably hypomethylated at Snrpn, Igf2r and Kcnqot1. In contrast, the H19/Igf2 ICC undergoes almost complete de novo remethylation. Using the same approach for PGCs differentiated in vitro from ESCs we show that the Snrpn ICC is erased however the hypomethylated state is highly unstable. We also discovered that when the H19/Igf2 ICC is abnormally hypermethylated in ESCs, ICC methylation is not erased with differentiation into PGCs. This highlights the importance of not only launching germline differentiation with correctly methylated ESC lines but also the need to better stabilize the hypomethylated state in the in vitro derivatives following ICC erasure.
2015-06-01 | GSE69354 | GEO