Mouse embryonic germ (EG) cell lines: transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3197-3204 ◽  
Author(s):  
P.A. Labosky ◽  
D.P. Barlow ◽  
B.L. Hogan
Keyword(s):  
Growth Factor ◽  
Germ Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Coat Color ◽  
Primordial Germ Cells ◽  
Methylation Status ◽  
Embryonic Stem ◽  
Insulin Like Growth Factor ◽  
Significant Difference

Primordial germ cells of the mouse cultured on feeder layers with leukemia inhibitory factor, Steel factor and basic fibroblast growth factor give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells can be induced to differentiate extensively in culture, form teratocarcinomas when injected into nude mice and contribute to chimeras when injected into host blastocysts. Here, we report the derivation of multiple embryonic germ cell lines from 8.5 days post coitum embryos of C57BL/6 inbred mice. Four independent embryonic germ cell lines with normal male karyotypes have formed chimeras when injected into BALB/c host blastocysts and two of these lines have transmitted coat color markers through the germline. We also show that pluripotent cell lines capable of forming teratocarcinomas and coat color chimeras can be established from primordial germ cells of 8.0 days p.c. embryos and 12.5 days p.c. genital ridges. We have examined the methylation status of the putative imprinting box of the insulin-like growth factor type 2 receptor gene (Igf2r) in these embryonic germ cell lines. No correlation was found between methylation pattern and germline competence. A significant difference was observed between embryonic stem cell and embryonic germ cell lines in their ability to maintain the methylation imprint of the Igf2r gene in culture. This may illustrate a fundamental difference between these two cell types.

scholarly journals Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines

2018 ◽  
Vol 2018 ◽  
pp. 1-17 ◽  
Author(s):  
Bence Lázár ◽  
Mahek Anand ◽  
Roland Tóth ◽  
Eszter Patakiné Várkonyi ◽  
Krisztina Liptói ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Expression Profiles ◽  
Dominant Role ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Proliferation Rate ◽  
Wide Range ◽  
Significant Difference

Primordial germ cells (PGCs) are the precursors of adult germ cells, and among the embryonic stem-like cells in the bird embryo, only they can transmit the genetic information to the next generation. Despite the wide range of applications, very little is known about the mechanism that governs primordial germ cell self-renewal and differentiation. As a first step, we compared 12 newly established chicken PGC lines derived from two different chicken breeds, performing CCK-8 proliferation assay. All of the lines were derived from individual embryos. A significant difference was found among the lines. As microRNAs have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, we continued with a complex miRNA analysis. We could discover miRNAs expressing differently in PGC lines with high proliferation rate, compared to PGC lines with low proliferation rate. We found that gga-miR-2127 expresses differently in female and male cell lines. The microarray analysis also revealed high expression level of the gga-miR-302b-3p strand (member of the miR-302/367 cluster) in slowly proliferating PGC lines compared to the gga-miR-302b-5p strand. We confirmed that the inhibition of miR-302b-5p significantly increases the doubling time of the examined PGC lines. In conclusion, we found that gga-miR-181-5p, gga-miR-2127, and members of the gga-miR-302/367 cluster have a dominant role in the regulation of avian primordial germ cell proliferation.

154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 207
Author(s):  
J. Galiguis ◽  
C. E. Pope ◽  
C. Dumas ◽  
G. Wang ◽  
R. A. MacLean ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Transcript Abundance ◽  
Domestic Cat ◽  
Sample Type

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

Immunomagnetic Isolation of Primordial Germ Cells and the Establishment of Embryonic Germ Cell Lines in the Mouse

Cloning ◽  
1999 ◽  
Vol 1 (4) ◽  
pp. 217-224 ◽  
Author(s):  
Gabriela Durcova-Hills ◽  
Tomoyuki Tokunaga ◽  
Satoshi Kurosaka ◽  
Manabu Yamaguchi ◽  
Seiya Takahashi ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Immunomagnetic Isolation

An Evidence That Murine Marrow-Derived CXCR4+ SSEA-1+ Oct-4+ Very Small Embryonic-Like (VSEL) Stem Cells Are Pluripotent and Express Several Primordial Germ Cell (PGC) Markers - Hypotesis for Developmental Deposition of PGC in Various Organs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1676-1676 ◽  
Author(s):  
Magda Kucia ◽  
Ewa Zuba-Surma ◽  
Ryan Reca ◽  
Janina Ratajczak ◽  
Mariusz Ratajczak
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Fetal Liver ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
C2c12 Cells

Abstract Recently we identified in murine BM a homogenous population of rare (~0.01% of BMMNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia2006;20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for embryonic stem cells such as i) small size (2–4 um in diameter), ii) large nuclei surrounded by a narrow rim of cytoplasm, and iii) open-type chromatin (euchromatin). We also found that VSELs may be released from BM and circulate in peripheral blood during tissue/organ injuries (e.g., heart infarct, stroke). Recently we noticed that ~5–10% of purified VSELs if plated over a C2C12 murine sarcoma cell feeder layer are able to form spheres that resemble embryoid bodies. Cells from these VSEL-derived spheres (VSEL-DS) are composed of immature cells with large nuclei containing euchromatin, and similarly as purified VSELs are CXCR4+SSEA-1+Oct-4+. Furthermore, VSEL-DS after replating over C2C12 cells may again (up to 5–7 passages) grow new spheres or if plated into cultures promoting tissue differentiation expand into cells from all three germ-cell layers. The formation of VSEL-DS was observed in a presence of C2C12 cells obtained from different sources. Furthermore, VSELs isolated from GFP+ mice grew GFP+ VSEL-DS which show a diploid content of DNA. This suggests that VSEL-DS are in fact derived from VSELs and not from the supportive C2C12 cell line as well as excludes the possibility of cell fusion to the observed phenomenon. Similar spheres were also formed by VSELs isolated from murine fetal liver, spleen and thymus. Interestingly formation of VSEL-DS was associated with a young age, and no VSEL-DS were observed by cells isolated from old mice (> 2 years). We also found that cells isolated from VSEL-DS similarly as embryonic stem cells grow tumors after injection into immunodeficient NOD/SCID mice (51/52 inoculated mice). Since VSELs isolated by us express several markers of primordial germ cells (fetal-type alkaline phosphatase, Oct-4, SSEA-1, CXCR4, Mvh, Stella, Fragilis, Nobox, Hdac6) we hypothesize that VSELs are closely related to a population of primordial germ cells. These cells are specified during early gastrulation in the proximal epiblast and subsequently migrate in a CXCR4-SDF-1 dependent manner through the embryo proper to their final destination in genital ridges. It is possible that some of these cells or a population of cells closely related to them migrate astray being chemoattracted by SDF-1 to fetal liver and subsequently, during the third trimester of gestation seed together with hematopoietic stem cells in bone marrow and perhaps other organs as well. In conclusion, we postulate that VSELs identified by us and purified at the single cell level could become an important source of pluripotent stem cells for regeneration.

Germ cells and pluripotent stem cells in the mouse

2001 ◽  
Vol 13 (8) ◽  
pp. 661 ◽  
Author(s):  
Anne McLaren ◽  
Gabriela Durcova-Hills
Keyword(s):  
Growth Factors ◽  
Cell Lines ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Individual Growth ◽  
Developmental Potential

For many years, attempts to achieve long-term culture of mouse primordial germ cells (PGCs) proved unsuccessful, even when feeder layers were used and individual growth factors were added to the medium. However, when three growth factors were added simultaneously to the medium, some of the cells continued to proliferate indefinitely. Similar to embryonic stem cell lines, these embryonic germ (EG) cell lines were capable of giving rise to embryoid bodies in vitro, and colonizing all cell lineages in chimeras, including the germline. Initially, EG cells were made from PGCs before migration, 8.5 days post coitum (dpc), and after entry into the genital ridge, 11.5 and 12.5 dpc. New EG cell lines from 9.5 dpc (migrating) and 11.5 dpc PGCs, carrying either a LacZ or GFP transgene, are described here. The developmental potential of the new EG cell lines in vitro, in vivoin chimeras, and in tissue aggregates in organ culture was studied. The EG cells were compared with PGCs at the stage from which the EG cells were derived. The two cell types show several similarities, but also some differences in gene expression and cell behaviour, which require further exploration.

scholarly journals In vivo and in vitro differentiation of male germ cells in the mouse

Reproduction ◽  
2004 ◽  
Vol 128 (2) ◽  
pp. 147-152 ◽  
Author(s):  
Orly Lacham-Kaplan
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Cellular Interactions ◽  
Germ Cell Differentiation

Primordial germ cells appear in the embryo at about day 7 after coitum. They proliferate and migrate towards the genital ridge. Once there, they undergo differentiation into germ stem cells, known as ‘A spermatogonia’. These cells are the foundation of spermatogenesis. A spermatogonia commit to spermatogenesis, stay undifferentiated or degenerate. The differentiation of primordial germ cells to migratory, postmigratory and germ stem cells is dependent on gene expression and cellular interactions. Some of the genes that play a crucial role in germ cell differentiation are Steel, c-Kit, VASA, DAZL, fragilis, miwi, mili, mil1 and mil2. Their expression is stage specific, therefore allowing solid identification of germ cells at different developmental phases. In addition to the expression of these genes, other markers associated with germ cell development are nonspecific alkaline phosphatase activity, the stage specific embryonic antigen, the transcription factor Oct3/4 and β1- and α6-integrins. Commitment of cells to primordial germ cells and to A spermatogonia is also dependent on induction by the bone morphogenetic protein (BMP)-4. With this knowledge, researchers were able to isolate germ stem cells from embryonic stem cell-derived embryoid bodies, and drive these into gametes either in vivo or in vitro. Although no viable embryos were obtained from these gametes, the prospects are that this goal is not too far from being accomplished.

183 In Vitro Generation and Characterization of Putative Primordial Germ Cells Derived from Induced Pluripotent Stem Cells in Cattle

2018 ◽  
Vol 30 (1) ◽  
pp. 231
Author(s):  
F. F. Bressan ◽  
M. A. Lima ◽  
L. S. Machado ◽  
N. C. G. Pieiri ◽  
P. Fantinato-Neto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Growth Factor ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Farm Animals ◽  
Induced Pluripotent

Embryonic pluripotent stem cells (ESC) and induced pluripotent stem cells (iPSC) were reported capable of differentiating into primordial germ cell-like (PGCL) and functional gametes in vitro in the murine model (Hikabe et al. 2016 Nature 539, 299-303). The in vitro generation of primordial germ cells (PGC) and gametes from farm animals would greatly contribute to enhance animal production technologies and to the creation of adequate models for several disorders. The present study aimed at the generation of PGC in vitro (iPGC) from iPSC in cattle and their characterisation through pluripotency and germ cell markers. For that, bovine iPSC previously generated and characterised (Bressan et al. 2015 Reprod. Fertil. Dev. 27, 254) were submitted to in vitro differentiation into epiblast-like cells (EpiLC) and iPGC by the protocol adapted from mice (Hayashi et al. 2011 Cell 146, 519-532). The biPS cells were induced into EpiLC by culture in fibronectin-coated (16.7 µg mL−1) 6-well plates in N2B27 culture medium supplemented with 20 ng mL−1 activin A, 12 ng mL−1 basic fibroblast growth factor (bFGF), and 1% knockout serum replacement (KSR) for 48 h and further differentiated into iPGC by non-adherent culture (Agreewell plates, StemCell Technologies, Vancouver, BC, Canada) with GK15 medium (GMEM supplemented with 15% KSR, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 2 mm l-glutamine, and 1% antibiotics) in the presence of 500 ng mL−1 BMP4, 100 ng mL−1 SCF, 500 ng mL−1 BMP8b, and 50 ng mL−1 epidermal growth factor for 4 days. The cells were then characterised regarding morphology, detection of alkaline phosphatase, immunofluorescence for OCT4, DDX4, VASA, and c-Kit proteins, and transcripts of pluripotency-related genes OCT4 and SOX2, as well as of imprinted genes (H19, SNRPN) and imprinted-related (DNMT1, DNMT3B) genes were analysed through RT-qPCR and compared with constitutive genes GAPDH, NAT1, and ACTB. Alkaline phosphatase and immunofluorescence analysis were positive for all specific markers. Interestingly, although OCT4 and SOX2 expression was present in iPS, EpiLC, and iPGC, this last group presented greater OCT4 and lesser SOX2 transcript amounts compared with other groups, suggesting, as expected, that PGC are still pluripotent but may already be differentiating into germ-cell lineages. The expression of H19 was increased in iPGC, whereas the expression of SNRPN was decreased only in the fibroblast group, potentially indicating epigenetic reprogramming process in these cells. Expression of DNMT1 and DNMT3B was not different between pluripotent groups but subtly increased when compared with that in fibroblasts. The results obtained herein represent an important first step in the in vitro generation of PGC and gametes from domestic farm animals, an unprecedented and desirable tool for enhancing new reproductive technologies and providing new understanding of cellular reprogramming and pluripotent germ cell biology. Financially supported by FAPESP grants 2013/08135-2, 2013/13686-8, 2015/26818-5; CNPq 482163/2013-5.

scholarly journals Transcription factors protect from DNA re-methylation during reprograming of primordial germ cells and pre-implantation embryos

2019 ◽  
Author(s):  
Isaac Kremsky ◽  
Victor G. Corces
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Methylation Status ◽  
Primordial Germ Cell ◽  
Cell Mass ◽  
Phenotypic Traits ◽  
Epigenetic Information

AbstractA growing body of evidence suggests that certain phenotypic traits of epigenetic origin can be passed across generations via both the male and female germlines of mammals. These observations have been difficult to explain owing to a global loss of the majority of known epigenetic marks present in parental chromosomes during primordial germ cell development and after fertilization. By integrating previously published BS-seq, DNase-seq, ATAC-seq, and RNA-seq data collected during multiple stages of primordial germ cell and preimplantation development, we find that the methylation status of the majority of CpGs genome-wide is restored after global reprogramming, despite the fact that global CpG methylation drops to 10% in primordial germ cells and 20% in the inner cell mass of the blastocyst. We estimate the proportion of such CpGs with preserved methylation status to be 78%. Further, we find that CpGs at sites bound by transcription factors during the global re-methylation phases of germ line and embryonic development remain hypomethylated across all developmental stages observed. On the other hand, CpGs at sites not bound by transcription factors during the global re-methylation phase have high methylation levels prior to global de-methylation, become de-methylated during global de-methylation, and then become re-methylated. The results suggest that transcription factors can act as carriers of epigenetic information during germ cell and pre-implantation development by ensuring that the methylation status of CpGs is maintained after reprogramming of DNA methylation. Based on our findings, we propose a model in which transcription factor binding during the re-methylation phases of primordial germ cell and pre-implantation development allow epigenetic information to be maintained trans-generationally even at sites where DNA methylation is lost during global de-methylation.

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