Long‐term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non‐obstructive azoospermia under exogenous feeder‐free culture conditions

Lim, Jinho; Sung, Suye; Kim, H. J.; Song, Seung-Hun; Hong, Jun; Yoon, Tae Ki; Kim, J. K.; Kim, K.-S.; Lee, D. R.

doi:10.1111/j.1365-2184.2010.00691.x

Cited by 103 publications

(95 citation statements)

References 36 publications

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“…To reduce the interference of somatic cells, the subculturing MGSCs are maintained in media supplemented with growth factors. Glial cell linederived neurotrophic factor (GDNF) has far been demonstrated to play vital roles in SSC self-renewal and long-term cultures of SSCs across species [2,9,16,17,19,21,[25][26][27]32]. However, we observed that GDNF showed little positive effects on porcine MGSC colony formation (data not shown), which is in line with the previous report by Kuijk et al [20].…”

Section: Discussionsupporting

confidence: 76%

In vitro propagation of male germline stem cells from piglets

Zheng

Tian

Zhang

et al. 2013

J Assist Reprod Genet

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Purpose To study the effects of serum and growth factors on propagation of porcine male germline stem cells (MGSCs) in vitro and develop a culture system for these stem cells. Methods Fresh testicular cells from neonatal piglets were obtained by mechanical dissociation and collagenase-trypsin digestion. After differential plating, non-adhering cells were cultured in media supplemented with different concentrations of serum (0, 1 %, 2 %, 5 %, 10 %). After 10 days of primary culture, the cells were maintained in media supplemented with different concentrations of growth factors (basic fibroblast growth factor and epidermal growth factor at 1, 5, 10 ng/ml). The number of MGSC-derived colonies with different sizes was determined in each treatment to assess the effects of serum concentrations and growth factors. Results The number of MGSC-derived colonies was significantly higher in the presence of 1 % rather than 10 % fetal bovine serum (FBS). Basic fibroblast growth factor (bFGF) at 1, 5 ng/ml and epidermal growth factor (EGF) at 5, 10-ng/ml significantly promoted colony formation. Immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and xenotransplantation assays demonstrated the presence of functional stem cells in cultured cell population.Conclusions In vitro propagation of porcine MGSCs could be maintained in the presence of 1 % FBS and supplementation of growth factors for 1 month.

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Section: Discussionsupporting

confidence: 76%

In vitro propagation of male germline stem cells from piglets

Zheng

Tian

Zhang

et al. 2013

J Assist Reprod Genet

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“…Although mGSCs have been differentiated into spermatogenic cells when they were co-cultured in vitro with feeder layer cells, treated with RA or transplantated in vivo (Feng et al 2002;Kanatsu-Shinohara et al 2003), few reports have shown that murine SSCs are induced in vitro to form haploid male germ cells using RA. Sycp3 and TH2B, specific markers of haploid male germ cells (Lim et al 2010;Li et al 2011), are expressed by haploid male germ cells, as seen in our result. In addition, the result from cell analysis by indirect immunofluorescence demonstrated that haploid male germ cells largely expressed protamine 1 (Fig.…”

Section: Discussionsupporting

confidence: 89%

Differentiation of spermatogonial stem cell-like cells from murine testicular tissue into haploid male germ cells in vitro

et al. 2013

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In vitro differentiation of spermatogonial stem cells (SSCs) promotes the understanding of the mechanism of spermatogenesis. The purpose of this study was to isolate spermatogonial stem cell-like cells from murine testicular tissue, which then were induced into haploid germ cells by retinoic acid (RA). The spermatogonial stem cell-like cells were purified and enriched by a two-step plating method based on different adherence velocities of SSCs and somatic cells. Cell colonies were present after culture in M1-medium for 3 days. Through alkaline phosphatase, RT-PCR and indirect immunofluorescence cell analysis, cell colonies were shown to be SSCs. Subsequently, cell colonies of SSCs were cultured in M2-medium containing RA for 2 days. Then the cell colonies of SSCs were again cultured in M1-medium for 6-8 days, RT-PCR and indirect immunofluorescence cell analysis were chosen to detect haploid male germ cells. It could be demonstrated that 10 -7 mol l -1 of RA effectively induced the SSCs into haploid male germ cells in vitro.

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“…As for OA patient, all genes were expressed; SCP3, H2B and TP1, which represent advanced stages of spermatogenesis (meiotic spermatocytes and spermatid respectively). All the gene markers for meiotic spermatogenesis were proven by previous study [16]. Our study has shown positive result with the present of SSC colonies in OA patient with HESC and BFGF media for 49 days of culture.…”

Section: Gene Expression Analysissupporting

confidence: 74%

Identification of Spermatogonial Stem Cell-Like Cell Differentiation In Vitro from Azoospermia Patient Using Modified Human Embryonic Stem Cell (HESC) Media

YAW¹,

MI²

2015

Andrology (Los Angel)

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Background: Spermatogenesis is a process where proliferation and differentiation of spermatogonial stem cell (SSC) into haploid sperm cells in the testis. Azoospermia patients have impaired spermatogenesis where they have difficulty to get normal sperm. The purpose of this study is to proliferate and differentiate SSC-like cell in in vitro culture from azoospermia patient samples and to determine the pattern of gene expressions involved. Methods and Results:Testicular biopsy tissue obtained from obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) patients were dissociated and cultured in different human embryonic stem cell (HESC) media added with specific growth factors and reproductive hormones according to the day of culture using 24 well plate dishes. SSC-like cell colonies were formed after 5 weeks of culture. Gene expression analyses were done on day 49 and day 90 using quantitative reverse transcription polymerase chain reaction (q-RT PCR). OCT4 was detected in NOA patient and ITGB1 was identified in OA patient for 49 days of culture. Gene expression for all stages of spermatogenic cells in day 90 were mostly detected in OA patient; ITGB1, SCP3, H2B and TNP1. However, gene TNP1 was only expressed in NOA patient shown spermatid formation stage. Conclusions:Our result shown that there is a potential to develop sperm in vitro culture from testis biopsy of azoospermic patients for further clinical application.

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Long‐term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non‐obstructive azoospermia under exogenous feeder‐free culture conditions

Abstract: These finding may support the idea that in vitro propagation of SSCs could be a useful tool for infertility treatment and study of reproductive biology.

Cited by 103 publications

References 36 publications

In vitro propagation of male germline stem cells from piglets

In vitro propagation of male germline stem cells from piglets

Differentiation of spermatogonial stem cell-like cells from murine testicular tissue into haploid male germ cells in vitro

Identification of Spermatogonial Stem Cell-Like Cell Differentiation In Vitro from Azoospermia Patient Using Modified Human Embryonic Stem Cell (HESC) Media

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