207 ESTABILSHMENT OF CYNOMOLGUS MONKEY EMBRYONIC STEM CELL LINES AND CONFIRMATION OF THE POSSIBILITY FOR GERMINAL COMPETENCY

2006 ◽  
Vol 18 (2) ◽  
pp. 211
Author(s):  
T. Teramura ◽  
N. Kawata ◽  
N. Fujinami ◽  
M. Takenoshita ◽  
N. Sagawa ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Lines ◽  
Cynomolgus Monkey ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
Embryonic Stem ◽  
Alp Activity ◽  
Weak Expression ◽  
Germ Cell Formation

Embryonic stem cells (ESCs) of nonhuman primate are important tools for human gametogenesis research. Generally, ESCs, embryos, and fetuses of nonhuman primates are similar to these of human. Recently, germ cell formation of mouse ESCs in vitro has been reported. In this study, we established new cynomolgus monkey ES (cyES) lines and determined germinal competency by assessing expression of mRNA markers. CyES lines were established using blastocysts produced by intracytoplasmic sperm injection (ICSI). For inducing super-ovulation, females were treated with 25 IU/kg pregnant mare serum gonadotropin (PMSG) once a day for 9 days, followed by 400 IU/kg hCG. Oocytes were collected 40 h after injection of hCG. After sperm injection, embryos were cultured in mCMRL medium to the blastocyst stage. For ES line establishment, inner cell masses (ICMs) were isolated by immunosurgery. ESC colonies emerged at about 10 days after ICM plating; three cyES cell lines were successfully obtained (3/11; 27.3%). We characterized these lines by immunocytochemistry for Oct-3/4, SSEA-3, and SSEA-4, which are diagnostic markers for primate ESCs, and by assay for alkaline phosphatase (ALP) activity. All cell lines expressed Oct-3/4, SSEA-4 and ALP activity. The previously reported SSEA-3 weak expression in cyES cells was not observed. These lines differentiated spontaneously when they were replaced in non-adherent culture (embryoid body: EB) or injected into SCID mice subcutaneously. To assess germ cell competency in vitro, we analyzed for the presence of vasa mRNA which shows a restricted expression pattern to germ cell formation, and DMC1 and SYCP1 which show specific existence on synaptonema complex in meiosis. Detection of these germ cell markers was performed by RT-PCR with total cDNA from ESCs and EBs. Nanog mRNA was detected only in ESCs. Oct-4 was detected in gonadal tissue of both sexes, ESCs, and EBs. Vasa was expressed in testis, but not in ESCs or somatic cells. Interestingly, we recognized weak expression of Vasa in Day 12-16 EBs. DMC1 and SYCP1 as meiosis markers were not detected. Because Oct-4 and Vasa mRNA are transcribed simultaneously, similar to that in the early part of gametogenesis such as the latter period of primordial germ cell (PGC) migration, PGC formation in cynomolgus EBs could occurr as in some cases of mouse or human EBs previously reported. Although detailed properties such as the functions of these Vasa-positive cells have not been confirmed, these results demonstrate that cyES cells obtained in the current study might contribute to putative germ cells in vitro by differentiating to EBs. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

scholarly journals ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Nataliya Nady ◽  
Ankit Gupta ◽  
Ziyang Ma ◽  
Tomek Swigut ◽  
Akiko Koide ◽  
...  
Keyword(s):  
Stem Cell ◽  
Germ Cell ◽  
Epigenetic Modification ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
Embryonic Stem ◽  
Stem Cell Maintenance ◽  
Family Protein

Prdm14 is a sequence-specific transcriptional regulator of embryonic stem cell (ESC) pluripotency and primordial germ cell (PGC) formation. It exerts its function, at least in part, through repressing genes associated with epigenetic modification and cell differentiation. Here, we show that this repressive function is mediated through an ETO-family co-repressor Mtgr1, which tightly binds to the pre-SET/SET domains of Prdm14 and co-occupies its genomic targets in mouse ESCs. We generated two monobodies, synthetic binding proteins, targeting the Prdm14 SET domain and demonstrate their utility, respectively, in facilitating crystallization and structure determination of the Prdm14-Mtgr1 complex, or as genetically encoded inhibitor of the Prdm14-Mtgr1 interaction. Structure-guided point mutants and the monobody abrogated the Prdm14-Mtgr1 association and disrupted Prdm14's function in mESC gene expression and PGC formation in vitro. Altogether, our work uncovers the molecular mechanism underlying Prdm14-mediated repression and provides renewable reagents for studying and controlling Prdm14 functions.

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.

scholarly journals Generation of multipotent cell lines from a distinct population of male germ line stem cells

Reproduction ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 771-784 ◽  
Author(s):  
Fariborz Izadyar ◽  
Francis Pau ◽  
Joel Marh ◽  
Natalia Slepko ◽  
Tracy Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Complex Mixture ◽  
Neural Cells ◽  
Differentiation Potential

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

scholarly journals In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

PLoS ONE ◽  
2009 ◽  
Vol 4 (4) ◽  
pp. e5338 ◽  
Author(s):  
Kaori Yamauchi ◽  
Kouichi Hasegawa ◽  
Shinichiro Chuma ◽  
Norio Nakatsuji ◽  
Hirofumi Suemori
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Embryonic Stem Cells ◽  
Germ Cell ◽  
Cynomolgus Monkey ◽  
Embryonic Stem ◽  
Germ Cell Differentiation

scholarly journals Resf1 supports embryonic stem cell self-renewal and effective germline entry

2021 ◽  
Author(s):  
Matus Vojtek ◽  
Ian Chambers
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Line ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Cell Specification ◽  
Self Renewal ◽  
Downstream Target ◽  
Germ Cell Specification

Retroelement silencing factor 1 (Resf1) interacts with the key regulators of mouse embryonic stem cells (ESCs) Oct4 and Nanog, and its absence results in sterility of mice. However, the function of Resf1 in ESCs and germ line specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESCs self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification.

scholarly journals Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3099
Author(s):  
Aline Fernanda de Souza ◽  
Fabiana Fernandes Bressan ◽  
Naira Caroline Godoy Pieri ◽  
Ramon Cesar Botigelli ◽  
Tamas Revay ◽  
...  
Keyword(s):  
Germ Cell ◽  
Turner Syndrome ◽  
Genetic Disorder ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
In Vitro Models ◽  
Germline Development ◽  
Germ Cell Specification

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.

P53 and H3K4me2 activate N6‐methylated LncPGCAT‐1 to regulate primordial germ cell formation via MAPK signaling

2020 ◽  
Vol 235 (12) ◽  
pp. 9895-9909 ◽  
Author(s):  
Qisheng Zuo ◽  
Jing Jin ◽  
Kai Jin ◽  
Jing Zhou ◽  
Changhua Sun ◽  
...  
Keyword(s):  
Germ Cell ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
Mapk Signaling ◽  
Germ Cell Formation

scholarly journals Dazl is a critical player for primordial germ cell formation in medaka

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mingyou Li ◽  
Feng Zhu ◽  
Zhendong Li ◽  
Ni Hong ◽  
Yunhan Hong
Keyword(s):  
Germ Cell ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
Germ Cell Formation

scholarly journals Long Noncoding RNA LncPGCR Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 292
Author(s):  
Jingyi Jiang ◽  
Chen Chen ◽  
Shaoze Cheng ◽  
Xia Yuan ◽  
Jing Jin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Noncoding Rna ◽  
Marker Gene ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
Embryonic Stem ◽  
Positive Role ◽  
Dependent Inhibition

Although lncRNAs have been identified as playing critical roles in the development of germ cells, their potential involvement in the development of PGCs in chickens remains poorly understood. Differentially expressed lncRNAs (DELs) from previous RNA-seq of embryonic stem cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) were analyzed by K-means clustering, from which a key candidate, lncRNA (lncRNA PGC regulator, LncPGCR) was obtained. We confirmed that LncPGCR plays a positive role in the development of PGCs by increasing the expression of the PGC marker gene (Cvh and C-kit), while downregulating the pluripotency-associated gene (Nanog) in vitro and in vivo. The activation and expression of LncPGCR are regulated by histone acetylation, and transcription factor TCF7L2. Mechanistically, a rescue assay was performed to further confirm that LncPGCR contributed to the development of PGCs by regulating the gga-miR-6577-5p/Btrc signaling pathway. Adsorption of gga-miR-6577-5p activated the WNT signaling cascade by relieving the gga-miR-6577-5p-dependent inhibition of Btrc expression. Taken together, our study discovered the growth-expedited role of LncPGCR in PGCs development, showing the potential LncPGCR/miR-6577-5p/Btrc pathway. The results and findings provide a novel insight into the development of PGCs.

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