Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (Felis catus)
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- TIPTANAVATTANA Narong
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
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- TECHAKUMPHU Mongkol
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
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- THARASANIT Theerawat
- Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
書誌事項
- タイトル別名
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- Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (<i>Felis catus</i>)
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説明
The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal donors is currently poor primarily, because of contamination with other testicular cells. This study aimed to purify SSC-like cells using different extracellular matrixes and a discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymatic digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish. The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment 2). Experiment 3 was performed to test whether or not the additional step of Percoll gradient density centrifugation could really improve purification of SSC-like cells. Testicular histology revealed complete spermatogenesis with laminin expression essentially at the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4 in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells expressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highest SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other incubation times and gelatin treatment (P<0.05). Percoll treatment prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33 ± 0.14%, P<0.001) and purity (83.82 ± 2.05% for SSEA-4 and 64.39 ± 1.51% for GFRα-1, P<0.05). These attached cells demonstrated a typical SSC-like cell morphology and also expressed POU5F1, RET and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient density centrifugation and laminin plating highly enriched the SSC-like cells population.
収録刊行物
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- The Journal of Veterinary Medical Science
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The Journal of Veterinary Medical Science 77 (11), 1347-1353, 2015
公益社団法人 日本獣医学会
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詳細情報 詳細情報について
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- CRID
- 1390282681406486912
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- NII論文ID
- 130005112084
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- NII書誌ID
- AA10796138
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- ISSN
- 13477439
- 09167250
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- NDL書誌ID
- 026964051
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- PubMed
- 26074411
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- NDL
- Crossref
- PubMed
- CiNii Articles
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- 抄録ライセンスフラグ
- 使用不可