scholarly journals Supplementation of vitamin C promotes early germ cell specification from human embryonic stem cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Zili Li ◽  
Fang Fang ◽  
Qian Zhao ◽  
Honggang Li ◽  
Chengliang Xiong
Keyword(s):  
Flow Cytometry ◽  
Vitamin C ◽  
Germ Cell ◽  
Embryonic Stem ◽  
Qrt Pcr ◽  
Differentiation Protocol ◽  
Germ Cell Specification ◽  
Tet Enzymes ◽  
In Vitro Induction

Abstract Background As the precursors of sperm and eggs, human primordial germ cells (hPGCs) emerge as early as weeks 2 to 3 of post-implantation development. Recently, robust hPGC induction models have been established in vitro with different protocols, but global 5mC/5hmC epigenetic reprogramming is not initiated in vitro. Previous studies found that vitamin C can enhance Tet (ten-eleven translocation) enzyme expression and improve 5hmC level in cells. But the effect of vitamin C supplementation on hPGC in vitro induction is still unknown. Methods We generated a gene-edited human embryonic stem cell (hESC) line carrying a BLIMP1-mkate2 reporter by CRISPR/Cas9 technology and used flow cytometry to optimize the PGC differentiation protocol; meanwhile, the expression of PGC genes (BLIMP1, TFAP2C, SOX17, OCT4) was evaluated by qRT-PCR. When different concentrations of vitamin C were added to the induction medium, the percentage of hPGCLCs (hPGC-like cells) was analyzed by flow cytometry; dot blot and ELISA were used to detect the levels of 5hmC and 5mC. The expression of TET enzymes was also evaluated by qRT-PCR. Results We optimized the PGC differentiation protocol with the BLIMP1-mkate reporter hESCs, and the efficiency of PGC induction in vitro can be improved to 30~40%. When 50 μg/mL vitamin C was added, the derived hPGCLCs not only upregulated the expression of key genes involved in human early germ cell development such as NANOS3, TFAP2C, BLIMP1, and SOX17, but also increased the levels of 5hmC and TET enzymes. Conclusions Taken together, supplementation of vitamin C can promote the in vitro induction of hPGCLCs from hESCs, which might be related to vitamin C-mediated epigenetic regulations during the differentiation process.

scholarly journals Oxygen tension modulates the mitochondrial genetic bottleneck and influences the segregation of a heteroplasmic mtDNA variant in vitro

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mikael G. Pezet ◽  
Aurora Gomez-Duran ◽  
Florian Klimm ◽  
Juvid Aryaman ◽  
Stephen Burr ◽  
...  
Keyword(s):  
Germ Cell ◽  
Oxygen Tension ◽  
Germ Line ◽  
Embryonic Stem ◽  
Mitochondrial Diseases ◽  
Genetic Bottleneck ◽  
Cellular Mechanisms ◽  
Germ Cell Specification ◽  
Vitro Model

AbstractMost humans carry a mixed population of mitochondrial DNA (mtDNA heteroplasmy) affecting ~1–2% of molecules, but rapid percentage shifts occur over one generation leading to severe mitochondrial diseases. A decrease in the amount of mtDNA within the developing female germ line appears to play a role, but other sub-cellular mechanisms have been implicated. Establishing an in vitro model of early mammalian germ cell development from embryonic stem cells, here we show that the reduction of mtDNA content is modulated by oxygen and reaches a nadir immediately before germ cell specification. The observed genetic bottleneck was accompanied by a decrease in mtDNA replicating foci and the segregation of heteroplasmy, which were both abolished at higher oxygen levels. Thus, differences in oxygen tension occurring during early development likely modulate the amount of mtDNA, facilitating mtDNA segregation and contributing to tissue-specific mutation loads.

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 Loss of Resf1 reduces the efficiency of embryonic stem cell self-renewal and germline entry

2021 ◽  
Vol 4 (12) ◽  
pp. e202101190
Author(s):  
Matúš Vojtek ◽  
Ian Chambers
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Cell Specification ◽  
Self Renewal ◽  
Downstream Target ◽  
Germ Cell Specification ◽  
Positive Effect

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 germline specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESC 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 the 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.

247. BMP signalling in the induction of germline precursors from mouse embryonic stem cells in vitro

2008 ◽  
Vol 20 (9) ◽  
pp. 47
Author(s):  
V. L. Dias ◽  
J. C. Young ◽  
K. L. Loveland
Keyword(s):  
Germ Cell ◽  
Embryoid Body ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Critical Window ◽  
Cell Markers ◽  
Germ Cell Specification ◽  
Bmp Signalling ◽  
Transient Elevation

BMP signalling is critical for germline lineage establishment during mouse embryogenesis. To assess its importance in the induction of germline precursors in vitro, a mouse embryonic stem (ES) cell line harbouring an Oct4 promoter-EGFP reporter construct was used to derive embryoid body (EB) aggregates cultured in the absence or presence of combinations of BMP2, BMP4 and BMP8b for 3 to 10 days. At both day 5 and 10 of culture, clearly defined clusters of Oct4-EGFP were observed in EBs cultured with BMPs, while these clusters were minimal to absent in untreated EBs. Quantitative mRNA analysis of day 3 to day 10 EBs revealed a significant elevation in the expression of genes associated with primordial germ cell specification in EBs grown in the presence of BMPs. Moreover, a transient elevation of early germ cell markers Blimp1, Fragilis and Stella was detected in day 3–4 EBs cultured with BMPs, followed their decline by day 5. In contrast, levels of the pluripotency markers, Oct3/4 and Nanog, and the later germ cell markers, Dazl and Vasa, progressively increased from day 3 to day 5. Levels of TGFβ superfamily signalling components ALK2, Smad1 and Smad5 remained relatively constant during this period. Wholemount immunofluorescence of day 5 Oct4-EGFP EBs exposed to BMP4 demonstrated co-localisation of primordial germ cell markers Oct3/4, Stella, and the cell surface antigen SSEA-1 with EGFP+ clusters. These results demonstrate that signalling by BMP2 and 4 enhances germ cell marker expression within EBs and identifies the day 3 to 5 timeframe in EB differentiation as a critical window when putative germ cells are first specified in vitro.

In vitro induction of apoptosis, necrosis and genotoxicity by cosmetic preservatives: application of flow cytometry as a complementary analysis by NRU

2011 ◽  
Vol 34 (2) ◽  
pp. 176-182 ◽  
Author(s):  
C. M. de Carvalho ◽  
P. F. C. Menezes ◽  
G. C. Letenski ◽  
C. E. O. Praes ◽  
I. H. S. Feferman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Induction Of Apoptosis ◽  
In Vitro Induction ◽  
Complementary Analysis

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.

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