Long-Term Proliferation in Culture and Germline Transmission of Mouse Male Germline Stem Cells1

Kanatsu-Shinohara, Mito; Ogonuki, Narumi; Inoue, Kimiko; Miki, Hiromi; Ogura, Atsuo; Toyokuni, Shinya; Shinohara, Takashi

doi:10.1095/biolreprod.103.017012

Cited by 922 publications

(1,052 citation statements)

References 33 publications

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“…GS cells were also established from WBB6F1-Sl/Sl d mice, at 6-weeks-old. For the derivation of GS cells 8 , testis cells were dissociated enzymatically with 2 mg ml − 1 of collagenase digestion (Type IV) for 15 min, followed by 1 mM EDTA and 0.25% trypsin for 10 min at 37 °C (ref. 38).…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, the in vitro proliferation of murine SSCs has become possible by adding glial cell line-derived neurotrophic factor to the SSCs culture media 8,9 . The cultured SSCs, termed germline stem (GS) cells or SSC lines, proliferated in a self-renewing manner for up to or beyond 2 years, while remaining stable genetically, epigenetically and karyotypically 10 .…”

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confidence: 99%

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In vitro production of fertile sperm from murine spermatogonial stem cell lines

et al. 2011

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spermatogonial stem cells (ssCs) are the only stem cells in the body that transmit genetic information to the next generation. The long-term propagation of rodent ssCs is now possible in vitro, and their genetic modification is feasible. However, their differentiation into sperm is possible only under in vivo conditions. Here we show a new in vitro system that can induce full spermatogenesis from ssC lines or any isolated ssCs. The method depends on an organ culture system onto which ssCs are transplanted. The settled ssCs form colonies and differentiate up into sperm. The resultant haploid cells are fertile, and give rise to healthy offspring through micro-insemination. In addition, the system can induce spermatogenesis from ssCs that show spermatogenic failure due to a micro-environmental defect in their original testes. Thus, an in vitro system is established that can be used to correct or manipulate the micro-environmental conditions required for proper spermatogenesis from murine ssC lines.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

In vitro production of fertile sperm from murine spermatogonial stem cell lines

et al. 2011

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“…For the in vitro culture of stem cells including SSCs, feeder layer with supplements that promote cell growth, self-renewal and signal transduction are important elements. In the case of SSCs, commonly used feeder cells are STO and mouse embryonic fibroblast (MEF) (Kanatsu-Shinohara et al, 2003;Kubota et al, 2004;Ryu et al, 2005). However, the effect of STO and MEF on the transduction and survival rate of stem cells including SSCs has not been studied.…”

Section: Discussionmentioning

confidence: 99%

“…For most of these studies, the transgenic animals were produced by lentiviral infection of highly purified SSCs and transplanting them right into testes of the recipient animals. With the recent developments of the in vitro culture technique of rodent SSCs, the possibility of germ-line modification for cultured cells has been proposed (Kanatsu-Shinohara et al, 2003;Kubota et al, 2004). If genetic modification and culture techniques of SSC are used simultaneously with a drug selection method, improvements in purity and mass propagation of trans-duced cells are possible.…”

Section: Introductionmentioning

confidence: 99%

Efficient Enhancement of Lentiviral Transduction Efficiency in Murine Spermatogonial Stem Cells

Kim

et al. 2012

Molecules and Cells

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Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout postnatal life in male and have the ability to transmit genetic information to the subsequent generation. In this study, we have optimized the transduction efficiency of SSCs using a lentiviral vector by considering different multiplicity of infection (MOI), duration of infection, presence or absence of feeder layer and polycationic agents. We tested MOI of 5, 10 or 20 and infection duration of 6, 9 or 12 h respectively. After infection, cells were cultured for 1 week and as a result, the number of transduced SSCs increased significantly for MOI of 5 and 10 with 6 h of infection. When the same condition (MOI of 5 with 6 hours) was applied in presence or absence of STO feeder layer and infected SSCs were cultured for 3 weeks on the STO feeder layer, a significant increase in the number of transduced cells was observed for without the feeder layer during infection. We subsequently studied the effects of polycationic agents, polybrene and dioctadecylamidoglycyl spermine (DOGS), on the transduction efficiency. Compared with the polybrene treatment, the recovery rate of the transduced SSCs was significantly higher for the DOGS treatment. Therefore, our optimization study could contribute to the enhancement of germ-line modification of SSCs using lentiviral vectors and in generation of transgenic animals.

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“…Long-term culture of SSCs provides a theoretical basis for researches about SSCs. The established SSCs strains provided not only a novel tool to analyze spermatogenesis or stem cell selfrenewal but also opened up new areas for practical application (Kanatsu-Shinohara et al 2003). This concerned widely the supplement of growth factors for regulation of in vitro and in vivo cell growth and proliferation.…”

Section: Introductionmentioning

confidence: 99%

Effects of GDNF and LIF on mouse spermatogonial stem cells proliferation in vitro

et al. 2013

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Spermatogonial stem cells (SSCs) are the only type of cells that transmit genes to the subsequent generations. The proliferation, cultivation and identification of SSCs in vitro are critical to understanding of male infertility, genetic resources and conservation of endangered species. To investigate the effects of glial cell-derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) on the proliferation of mouse SSCs in vitro, supplement of GDNF and/or LIF were designed to culture SSCs. The testes of 6-8 d mouse were harvested and digested by two-step enzyme digestion method. The SSCs and Sertoli cells were separated by differential plating. Then the SSCs were identified by alkaline phosphatase staining, RT-PCR and indirect immunofluorescence cell analysis. The cellular proliferation capacity was measured by methyl thiazolyl tetrazolium assay. The results showed that addition of 20 and 40 ng/ml of GDNF could strongly promote growth of mouse SSCs (p \ 0.05). There was no significant difference between LIF treatment groups and the control group in promoting proliferation of the mouse SSCs (p[0.05). However, the combination of 20 ng/ml GDNF and 1,000 U/ml LIF could significantly enhance the invitro proliferation of mouse SSCs (p \ 0.05), and the OD 490 value was 0.696 at day 5 of culture when the density of SSCs was 5-10 9 10 4 cells/ml.

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Long-Term Proliferation in Culture and Germline Transmission of Mouse Male Germline Stem Cells1

Cited by 922 publications

References 33 publications

In vitro production of fertile sperm from murine spermatogonial stem cell lines

In vitro production of fertile sperm from murine spermatogonial stem cell lines

Efficient Enhancement of Lentiviral Transduction Efficiency in Murine Spermatogonial Stem Cells

Effects of GDNF and LIF on mouse spermatogonial stem cells proliferation in vitro

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