Comparison of different in vitro differentiation conditions for murine female germline stem cells

Background/Aims: The isolation and establishment of female germline stem cells (FGSCs) is controversial because of questions regarding the reliability and stability of the isolation method using antibody targeting mouse vasa homologue (MVH), and the molecular mechanism of FGSCs self-renewal remains unclear. Thus, there needs to be a simple and reliable method for sorting FGSCs to study them. Methods: We applied the differential adhesion method to enrich FGSCs (DA-FGSCs) from mouse ovaries. Through four rounds of purification and 7-9 subsequent passages, DA-FGSC lines were established. In addition, we assessed the role of the phosphoinositide-3 kinase (PI3K)-AKT pathway in regulating FGSC self-renewal. Results: The obtained DA-FGSCs spontaneously differentiated into oocyte-like cells in vitro and formed functional eggs in vivo that were fertilized and produced healthy offspring. AKT was rapidly phosphorylated when the proliferation rate of FGSCs increased after 10 passages, and the addition of a chemical PI3K inhibitor prevented FGSCs self-renewal. Furthermore, over-expression of AKT-induced proliferation and differentiation of FGSCs, c-Myc, Oct-4 and Gdf-9 levels were increased. Conclusions: The differential adhesion method provides a more feasible approach and is an easier procedure to establish FGSC lines than traditional methods. The AKT pathway plays an important role in regulation of the proliferation and maintenance of FGSCs. These findings could help promote stem cell studies and provide a better understanding of causes of ovarian infertility, thereby providing potential treatments for infertility.

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GAS5/miR-21 Axis as a Potential Target to Rescue ZCL-082-Induced Autophagy of Female Germline Stem Cells In Vitro

Molecular Therapy — Nucleic Acids ◽

10.1016/j.omtn.2019.06.012 ◽

2019 ◽

Vol 17 ◽

pp. 436-447 ◽

Cited By ~ 3

Author(s):

Bo Li ◽

Xiaopeng Hu ◽

Yanzhou Yang ◽

Mingyan Zhu ◽

Jiong Zhang ◽

...

Keyword(s):

Stem Cells ◽

Germline Stem Cells ◽

Potential Target ◽

Female Germline

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C89 Induces Autophagy of Female Germline Stem Cells via Inhibition of the PI3K-Akt Pathway In Vitro

Cells ◽

10.3390/cells8060606 ◽

2019 ◽

Vol 8 (6) ◽

pp. 606 ◽

Cited By ~ 3

Author(s):

Xinyue Li ◽

Xiaopeng Hu ◽

Geng G. Tian ◽

Ping Cheng ◽

Zezhong Li ◽

...

Keyword(s):

Stem Cells ◽

Cell Counting ◽

Akt Pathway ◽

Germline Stem Cell ◽

Germline Stem Cells ◽

Sequencing Data ◽

Sequestosome 1 ◽

Small Molecule Compound ◽

Female Germline

Postnatal female germline stem cells (FGSCs) are a type of germline stem cell with self-renewal ability and the capacity of differentiation toward oocyte. The proliferation, differentiation, and apoptosis of FGSCs have been researched in recent years, but autophagy in FGSCs has not been explored. This study investigated the effects of the small-molecule compound 89 (C89) on FGSCs and the underlying molecular mechanism in vitro. Cytometry, Cell Counting Kit-8 (CCK8), and 5-ethynyl-2’-deoxyuridine (EdU) assay showed that the number, viability, and proliferation of FGSCs were significantly reduced in C89-treated groups (0.5, 1, and 2 µM) compared with controls. C89 had no impact on FGSC apoptosis or differentiation. However, C89 treatment induced the expression of light chain 3 beta II (LC3BII) and reduced the expression of sequestosome-1 (SQSTM1) in FGSCs, indicating that C89 induced FGSC autophagy. To investigate the mechanism of C89-induced FGSC autophagy, RNA-seq technology was used to compare the transcriptome differences between C89-treated FGSCs and controls. Bioinformatics analysis of the sequencing data indicated a potential involvement of the phosphatidylinositol 3 kinase and kinase Akt (PI3K-Akt) pathway in the effects of C89′s induction of autophagy in FGSCs. Western blot confirmed that levels of p-PI3K and p-Akt were significantly reduced in the C89- or LY294002 (PI3K inhibitor)-treated groups compared with controls. Moreover, we found cooperative functions of C89 and LY294002 in inducing FGSC autophagy through suppressing the PI3K-Akt pathway. Taken together, this research demonstrates that C89 can reduce the number, viability, and proliferation of FGSCs by inducing autophagy. Furthermore, C89 induced FGSC autophagy by inhibiting the activity of PI3K and Akt. The PI3K-Akt pathway may be a target to regulate FGSC proliferation and death.

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C28-induced autophagy of female germline stem cells in vitro and its potential mechanisms

10.21203/rs.2.18819/v1 ◽

2019 ◽

Author(s):

Rong Hu ◽

Xiuying Pei ◽

Huchen Zhou ◽

Ji Wu ◽

Ping Chen ◽

...

Keyword(s):

Stem Cells ◽

Er Stress ◽

Cellular Response ◽

Germline Stem Cells ◽

Rna Seq ◽

Sequestosome 1 ◽

Expression Of Genes ◽

Female Germline ◽

Molecular Compounds

Abstract BackgroundThere are few studies indicating that small molecular compounds affect the proliferation, differentiation, apoptosis, and autophagy of female germline stem cells (FGSCs). However, the epigenetic regulatory mechanism of small molecular compounds that induce autophagy in FGSCs remains unknown.ResultsIn this study, we found that C28 reduced the viability and proliferation of FGSCs, respectively. Additionally, western blotting showed that the expression of autophagy marker light chain 3 beta II (LC3B-II) was significantly increased and expression of sequestosome-1 (SQSTM1) was significantly reduced in C28-treated groups. Immunofluorescence showed that, in C28-treated groups, the number of LC3B-II-positive puncta was increased significantly. These results indicated that C28 induced autophagy of FGSCs in vitro. ChIP-seq data showed that autophagy-related biological processes such as regulation of mitochondrial membrane potential, Golgi vesicle transport, and cellular response to reactive oxygen species were enriched. In addition, RNA-Seq showed that the expression of genes (Trib3, DDIT3, and ATF4) related to endoplasmic reticulum (ER) stress was enhanced by C28.ConclusionC28 could induce FGSC autophagy in vitro leading to a decrease in the number of FGSCs. H3K27ac and ER stress might play roles in C28-induced autophagy of FGSCs in vitro.

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Offspring production of haploid spermatid-like cells derived from mouse female germline stem cells with chromatin condensation

Cell & Bioscience ◽

10.1186/s13578-021-00697-z ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Xiaopeng Hu ◽

Hu Wang ◽

Geng. G. Tian ◽

Changliang Hou ◽

Bo Xu ◽

...

Keyword(s):

Stem Cells ◽

Retinoic Acid ◽

Y Chromosome ◽

Germ Cells ◽

Chromatin Condensation ◽

Germline Stem Cells ◽

Chromosome Conformation ◽

Female Germline

Abstract Background During male meiosis, the Y chromosome can form perfect pairing with the X chromosome. However, it is unclear whether mammalian Female germline stem cells (FGSCs) without a Y chromosome can transdifferentiate into functional haploid spermatid-like cells (SLCs). Results We found that spermatogenesis was restarted by transplanting FGSCs into Kitw/wv mutant testes. Complete meiosis and formation of SLCs was induced in vitro by testicular cells of Kitw/wv mutant mice, cytokines and retinoic acid. Healthy offspring were produced by sperm and SLCs derived from the in vivo and in vitro transdifferentiation of FGSCs, respectively. Furthermore, high-throughput chromosome conformation capture sequencing(Hi-C-seq) and “bivalent” (H3K4me3-H3K27me3) micro chromatin immunoprecipitation sequencing (μChIP-seq) experiments showed that stimulated by retinoic acid gene 8 (STRA8)/protamine 1 (PRM1)-positive transdifferentiated germ cells (tGCs) and male germ cells (mGCs) display similar chromatin dynamics and chromatin condensation during in vitro spermatogenesis. Conclusion This study demonstrates that sperm can be produced from FGSCs without a Y chromosome. This suggests a strategy for dairy cattle breeding to produce only female offspring with a high-quality genetic background.

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ID3019 Female germline stem Cells: A source for application in reproductive and regenerative medicine

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.240 ◽

2017 ◽

Vol 4 (S) ◽

pp. 31

Author(s):

Thuy Hong Bui

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Ovarian Function ◽

Primordial Germ Cells ◽

Postnatal Life ◽

Proliferative Potential ◽

Germline Stem Cells ◽

Mammalian Female ◽

Female Germline

Studies suggest a renewable source of eggs and stir more controversy, especially about the origin of female germline stem cells (FGSCs). It should be elucidated whether or not neo-oogenesis continues in the ovaries of mammalian female during postnatal life. Therefore, the establishment of FGSCs is very important for many applications. Here, using adult pig ovary, we isolate, identify, characterize FGSCs to elucidate their origin, then examined the proliferation, growth and differentiation of them. These cells were heterogeneous, depending on both of c-kit expression and cell size, and also express stem cell and germ cell markers. Importantly, we show clearly that the cells with the characteristics of early primordial germ cells are present in the adult pig ovary. Once FGSCs were established, they could be expanded in vitro for months without loss of the identifying markers and proliferative potential. Under appropriate conditions, the FGSCs differentiated into primordial oocyte-like cells and grow close to full-sized oocytes. These may assist in therapeutic strategies in human with their potential to make new oocytes and support ovarian function and fertility. Our results support the theory that the ovary contains a small number of undifferentiated cells with stem cell characteristics. These might remain in the postnatal and adult ovary and under certain conditions could resume mitosis, enter meiosis and give rise to oocytes. Given the existence of these FGSCs in mammalian ovaries and the depletion in ovarian reserve during female reproductive aging, one can hypothesize that such “neo-oogenesis” was present in ancestral forms, is still present in insects, some fish and mollusks, but has been lost in land vertebrates through evolution. FGSCs cannot proliferate in the ovary normally because of inhibitory factors, but under appropriate conditions, they can undergo proliferation and differentiation, and provide a potential mechanism for the self-renewal of germline stem cells.

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ID: 1063 Isolation and characterization of female germline stem cell derived from porcine ovary

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.337 ◽

2017 ◽

Vol 4 (S) ◽

pp. 145

Author(s):

Nguyen Huy Hoang ◽

Nguyen Thi Bao Tran ◽

Nguyen Van Thuan ◽

Bui Hong Thuy

Keyword(s):

Stem Cells ◽

Stem Cell ◽

In Vitro Culture ◽

Germline Stem Cell ◽

Stem Cell Markers ◽

Significant Finding ◽

Germline Stem Cells ◽

Isolation And Characterization ◽

Female Germline

One of the most significant finding in stem cell area in the early 21st century is the founding of female germline stem cells (FGSCs). Establishment of FGSCs allowed new possibilities for the use of them in biotechnology and medicine. Hence, the purpose of this study was to establish, characterize the porcine female germline stem cells (pFGSCs) from porcine ovary. The result revealed the success in establishing pFGSCs from ovarian tissue. Most of the pFGSCs were round shape after in vitro culture, forming groups of cells that cluster around the ovarian cells colonies. Immunofluorescent analysis of pFGSCs showed that these cells expressed germ cell and stem cell markers such as: Vasa, Stella, c-kit and Oct4. After several weeks in in vitro culture, pFGSCs increased in number without the loss of proliferative potential. Our results suggested that pFGSCs isolated from adult mammalian ovary, under appropriate conditions, could undergo proliferation.

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C28 induced autophagy of female germline stem cells in vitro with changes of H3K27 acetylation and transcriptomics

Gene ◽

10.1016/j.gene.2020.145150 ◽

2021 ◽

Vol 766 ◽

pp. 145150

Author(s):

Ping Chen ◽

Xinyan Zhao ◽

Geng G. Tian ◽

Xiaoyan Yuan ◽

Xinyue Li ◽

...

Keyword(s):

Stem Cells ◽

Germline Stem Cells ◽

Female Germline

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Spermidine induces cytoprotective autophagy of female germline stem cells in vitro and ameliorates aging caused by oxidative stress through upregulated sequestosome-1/p62 expression

Cell & Bioscience ◽

10.1186/s13578-021-00614-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Xiaoyan Yuan ◽

Geng. G. Tian ◽

Xiuying Pei ◽

Xiaopeng Hu ◽

Ji Wu

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Protein Expression ◽

Cellular Senescence ◽

Critical Role ◽

Autophagic Flux ◽

Germline Stem Cells ◽

Sequestosome 1 ◽

Female Germline

Abstract Background Autophagy is required for oogenesis and plays a critical role in response to aging caused by oxidative stress. However, there have been no reports on regulation of cytoprotective autophagy in female germline stem cells (FGSCs) in response to aging caused by oxidative stress. Results We found that Spermidine (SPD) significantly increased protein expression of autophagy markers microtubule-associated protein 1 light chain 3 beta-II (MAP1LC3B-II/LC3B-II) and sequestosome-1/p62 (SQSTM1/p62), and evoked autophagic flux in FGSCs. Moreover, SPD increased the number and viability of FGSCs in vitro. Further, we found that SPD significantly reduced basal or hydrogen peroxide (H2O2)-induced up-regulated protein expression of the aging markers, cyclin dependent kinase inhibitor 2A (p16/CDKN2A) and tumor protein 53 (p53). After knockdown of p62 in FGSCs, p16 protein levels were significant higher compared with controls. However, protein p16 levels were not significantly changed in p62 knockdown FGSCs with SPD treatment compared with without SPD. Moreover, SPD significantly changed the expression of autophagy-related genes and pathways in FGSCs, as shown by bioinformatics analysis of RNA sequencing data. Additionally, SPD significantly inhibited AKT/mTOR phosphorylation. Conclusions SPD induces cytoprotective autophagy in FGSCs in vitro and ameliorates cellular senescence of FGSCs induced by H2O2. Furthermore, SPD can ameliorate cellular senescence of FGSCs through p62. SPD might induce autophagy in FGSCs via the PI3K/Akt pathway. Our findings could be helpful for delaying aging of female germ cells due to oxidative stress and preserving female fertility.

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