Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture

OBJECTIVE: The properties of self-renewal and division in spermatogonial stem cells (SSCs) support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and selfrenewal of germline stem cells (...

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Autores principales: Azizi, Hossein, Skutella, Thomas, Shahverdi, Abdolhossein
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Royan Institute 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412782/
https://www.ncbi.nlm.nih.gov/pubmed/28670516
http://dx.doi.org/10.22074/cellj.2016.4184
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author Azizi, Hossein
Skutella, Thomas
Shahverdi, Abdolhossein
author_facet Azizi, Hossein
Skutella, Thomas
Shahverdi, Abdolhossein
author_sort Azizi, Hossein
collection PubMed
description OBJECTIVE: The properties of self-renewal and division in spermatogonial stem cells (SSCs) support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and selfrenewal of germline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system. MATERIALS AND METHODS: In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco’s modified Eagle’s medium (DMEM) medium (CTRL group), 10% fetal bovine serum (FBS)+DMEM (10% group), and growth factor (G group) that contained 2% FBS, glial cell-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry against the germ cell markers α6, β1, c-Kit, Thy-1, c-Ret, Plzf, and Oct4. The statistical significance between mean values in different groups was determined by one-way analysis of variance (ANOVA). RESULTS: We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression (P<0.05) of germ cell markers in the G and 10% groups versus the testis cells (T). Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermato- gonial stem-like colonies were partially positive. CONCLUSION: A non-adherent culture system could provide a favorable method for in vitro short-term culture of spermatogonial stem-like cell colonies.
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spelling pubmed-54127822017-07-01 Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture Azizi, Hossein Skutella, Thomas Shahverdi, Abdolhossein Cell J Original Article OBJECTIVE: The properties of self-renewal and division in spermatogonial stem cells (SSCs) support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and selfrenewal of germline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system. MATERIALS AND METHODS: In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco’s modified Eagle’s medium (DMEM) medium (CTRL group), 10% fetal bovine serum (FBS)+DMEM (10% group), and growth factor (G group) that contained 2% FBS, glial cell-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry against the germ cell markers α6, β1, c-Kit, Thy-1, c-Ret, Plzf, and Oct4. The statistical significance between mean values in different groups was determined by one-way analysis of variance (ANOVA). RESULTS: We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression (P<0.05) of germ cell markers in the G and 10% groups versus the testis cells (T). Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermato- gonial stem-like colonies were partially positive. CONCLUSION: A non-adherent culture system could provide a favorable method for in vitro short-term culture of spermatogonial stem-like cell colonies. Royan Institute 2017 2017-02-22 /pmc/articles/PMC5412782/ /pubmed/28670516 http://dx.doi.org/10.22074/cellj.2016.4184 Text en Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited http://creativecommons.org/licenses/by/2.5/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Azizi, Hossein
Skutella, Thomas
Shahverdi, Abdolhossein
Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture
title Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture
title_full Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture
title_fullStr Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture
title_full_unstemmed Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture
title_short Generation of Mouse Spermatogonial Stem-Cell-Colonies in A Non-Adherent Culture
title_sort generation of mouse spermatogonial stem-cell-colonies in a non-adherent culture
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412782/
https://www.ncbi.nlm.nih.gov/pubmed/28670516
http://dx.doi.org/10.22074/cellj.2016.4184
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