title: Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (Felis catus).
abstract: 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.
authors.affiliation: Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Narong
authors.initials: N
authors.lastname: Tiptanavattana
authors.forename: Mongkol
authors.initials: M
authors.lastname: Techakumphu
authors.forename: Theerawat
authors.initials: T
authors.lastname: Tharasanit
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology / Q000502:physiology*; D000818:Animals; D015415:Biomarkers; D002415:Cats / Q000502:physiology*; D018929:Cell Culture Techniques; D002470:Cell Survival; D008297:Male; D012741:Sexual Maturation / Q000502:physiology*; D013093:Spermatogonia / Q000166:cytology / Q000502:physiology*; D013737:Testis / Q000166:cytology* / Q000502:physiology
chemical_list: D015415:Biomarkers