A simple method for isolation culture and in vit

Authors: Madjid MomeniMoghaddam Maryam M Matin Sohrab Boozarpour Sajjad Sisakhtnezhad Hossein Kazemi Mehrjerdi Moein Farshchian Mahtab Dastpak Ahmad Reza Bahrami

Publish Date: 2013/11/21

Volume: 50, Issue: 2, Pages: 155-161

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Abstract

Spermatogonial stem cells SSCs are expected to participate in male infertility therapy endangered species preservation and transgenic animal technology by their unique unipotency to differentiate into spermatozoa The main challenges however remain to be addressed including the appropriate conditions to reach good number of these cells and how to derive culture and maintain them in vitro In the present study the testicular tissues were isolated from 1dold male chickens to establish primary cell cultures This culture led to development of distinguished colonies which were further characterized by alkaline phosphatase AP activity assay and gene expression analysis They were shown to be positive for AP activity and expressed two main transcription factors of OCT4 and STRA8 as indicated by reverse transcriptionpolymerase chain reaction These were indications of carrying characteristics of SSCs by these colonies The cultures were also exposed to different concentrations of glial cell linederived neurotrophic factor GDNF basic fibroblast growth factor bFGF and leukemia inhibitory factor LIF growth factors to seek optimum colonyforming conditions Colonyforming activity assay indicated that they were able to propagate in vitro with an increased selfrenewal property when cultured in the presence of 15 ng/mL of GDNF 20 ng/mL of bFGF and 15 ng/mL of LIF The present work provides an easy and practical method for isolation culture and in vitro maintenance of chicken spermatogonial stem cells and introduces appropriate cell culture conditions to improve and maintain their selfrenewal property based on supplying the necessary growth factorsThis study was supported by grants from Iranian Council of Stem cell Technology ICST grant number 100313 and Ferdowsi University of Mashhad 20683 which are greatly appreciated and was performed at the Institute of Biotechnology Authors are grateful to Morghak Company of Mashhad for very generous supplying of the chickens We are also thankful to Manouchehr TeymouriShaban Aliakbar HaddadMashadrizeh and Hojjat NaderiMeshkin for their great assistance

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