Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

Hou, Jingmei; Niu, Minghui; Liu, Linhong; Zhu, Zijue; Wang, Xiaobo; Sun, Min; Yuan, Quan; Yang, Shi; Zeng, Wenxian; Liu, Yang; Li, Zheng; He, Zuping

doi:10.1038/srep16922

Cited by 46 publications

(61 citation statements)

References 48 publications

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“…Chromosomal karyotype analysis of the exponentially growing immortalized human Sertoli cells at passage 15 (P15) was conducted pursuant to the procedure described previously [12]. In brief, cells were inhibited with 5 μg/ml colchicine for 3 hours, followed by hypotonic treatment with 0.075 M KCl solution, fixed with 3:1 methanol-glacial acetic acid, and dropped onto chilled slides.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, it is necessary to establish a human Sertoli cell line to obtain adequate human Sertoli cells for basic research to uncover the mechanisms underlying human spermatogenesis as well as for their applications in both reproductive and regenerative medicine. Currently, several germ cell lines in human or rodents and Sertoli cell lines in rodents have been established with either simian virus large T antigen (SV40-LTAg) or telomerase (TERT) [12–15]. However, human Sertoli cell line has not yet been available.…”

Section: Introductionmentioning

confidence: 99%

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Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

et al. 2017

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Sertoli cells are required for normal spermatogenesis and they can be reprogrammed to other types of functional cells. However, the number of primary Sertoli cells is rare and human Sertoli cell line is unavailable. In this study, we have for the first time reported a stable human Sertoli cell line, namely hS1 cells, by overexpression of human telomerase. The hS1 cells expressed a number of hallmarks for human Sertoli cells, including SOX9, WT1, GDNF, SCF, BMP4, BMP6, GATA4, and VIM, and they were negative for 3β-HSD, SMA, and VASA. Higher levels of AR and FSHR were observed in hS1 cells compared to primary human Sertoli cells. Microarray analysis showed that 70.4% of global gene profiles of hS1 cells were similar to primary human Sertoli cells. Proliferation assay demonstrated that hS1 cells proliferated rapidly and they could be passaged for more than 30 times in 6 months. Neither Y chromosome microdeletion nor tumorgenesis was detected in this cell line and 90% normal karyotypes existed in hS1 cells. Collectively, we have established the first human Sertoli cell line with phenotype of primary human Sertoli cells, an unlimited proliferation potential and high safety, which could offer sufficient human Sertoli cells for basic research as well as reproductive and regenerative medicine.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

et al. 2017

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“…Production of functional germ cells is essential for the damental characteristic of germline stem cells (Pech et al 2015). In contrast, SV40 large T antigen was an efficient factor for animal cell immortalization (hofmann et al 2005;Hou et al 2015). In this study, bovine mGSCs were immortalized by SV40 large T antigen, and the cells fulfilled the criteria of mGSCs and maintained the stemness markers and self-renewal and meiosis potential.…”

Section: Introductionmentioning

confidence: 74%

“…Previous studies have mainly focused on the biological features of mouse or human spermatogonial stem cells. At present, the immortalized mouse and human germline cells have been established (Hofmann et al 2005;hou et al 2015;Wang H et al 2016). however, no bovine mGSC line has been reported, and study on livestock is relatively rare.…”

Section: Discussionmentioning

confidence: 99%

Establishment and characterization of immortalized bovine male germline stem cell line

Lei

Pan

et al. 2017

Journal of Integrative Agriculture

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Male germline stem cells (mGSCs) are unique adult germ cells with self-renewal potential and spermatogenesis function in the testis. However, further studies are needed to establish a long-term cultural system of mGSCs in vitro, especially for large animals such as bovine mGSCs. In this study, we first established a stable immortalized bovine male germline stem cell line by transducing Simian virus 40 (SV40) large T antigen. The proliferation of these cells was improved significantly. These cells could express spermatogonial stem cell (SSC)-specific markers, such as PLZF, PGP9.5, VASA, LIN28A, and CD49F, both in the mRNA and protein levels. Additionally, these cells could be differentiated into three germ layer cells to enter meiosis, form colonies, and proliferate in the seminiferous tubules of busulfan-induced infertile mice. The immortalized bovine mGSCs maintain the criteria of mGSCs.

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“…The large T antigen forms complexes with tumor suppressors, such as pRB-1 and p53, and induces DNA synthesis in both dividing and quiescent cells, leading to extension of the lifespan of the cells [26]. This approach has been used for transformation of various primary cell types [27][28][29]. One of the major limitations of permanent immortal cell lines is their proliferative status, which prevents the development of the full physiological phenotype [30].…”

Section: Discussionmentioning

confidence: 99%

Establishment of A Reversibly Inducible Porcine Granulosa Cell Line

Bai

Zhu

Mao

et al. 2020

Cells

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Granulosa cells (GCs) are the key components of ovarian follicles for regulating oocyte maturation. Previous established GC lines have allowed prolonged proliferation, but lost some physiological features owing to long-term immortalization. This study was to establish an induced immortal porcine GC line with reversible proliferation status by the tetracycline inducible (Tet-on) 3G system. Our conditional immortal porcine GCs (CIPGCs) line steadily propagated for at least six months and displayed primary GC morphology when cultured in the presence of 50 ng/mL doxycycline [Dox (+)]. Upon Dox withdrawal [Dox (–)], Large T-antigen expression, reflected by mCherry fluorescence, gradually became undetectable within 48 h, accompanied by less proliferation and size increase. The levels of estradiol and progesterone, and the expression of genes associated with steroid production, such as CYP11A1 (cytochrome P450 family 11), 3β-HSD (3β-hydroxysteroid dehydrogenase), StAR (steroidogenic acute regulatory protein), and CYP19A1 (cytochrome P450 family 19 subfamily a member 1), were all significantly higher in the Dox (–) group than Dox (+) group. The CIPGCs could switch into a proliferative state upon Dox induction. Interestingly, the expression of StAR and CYP19A1 in the CIPGCs (–Dox) was significantly increased by adding porcine follicular fluid (PFF) to mimic an ovary follicle environment. Moreover, PFF priming the CIPGCs in Dox (–) group resulted in similar estradiol production as that of primary GC, and enabled this cell line to respond to gonadotrophins in estradiol production. Collectively, we have established an inducible immortal porcine GC line, which offers a unique and valuable model for future research on the regulation of ovarian functions.

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Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

Cited by 46 publications

References 48 publications

Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

Establishment and characterization of immortalized bovine male germline stem cell line

Establishment of A Reversibly Inducible Porcine Granulosa Cell Line

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