Simplified isolation and enrichment of spermatogonial stem-like cells frompubertal domestic cats (Felis catus)

[摘要] References(37)Cited-By(1)The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertaldonors is currently poor primarily, because of contamination with other testicular cells.This study aimed to purify SSC-like cells using different extracellular matrixes and adiscontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histologyand SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymaticdigestion, 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 (experiment2). Experiment 3 was performed to test whether or not the additional step of Percollgradient density centrifugation could really improve purification of SSC-like cells.Testicular histology revealed complete spermatogenesis with laminin expression essentiallyat the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4in the spermatogonia. The relative percentage of SSC-like cells, as determined by cellsexpressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highestSSC-like cell purity was obtained with the 15-min laminin-coated dish compared with otherincubation times and gelatin treatment (P<0.05). Percoll treatmentprior 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 typicalSSC-like cell morphology and also expressed POU5F1, RETand ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradientdensity centrifugation and laminin plating highly enriched the SSC-like cellspopulation.

[学科分类] 兽医学

[关键词] extracellular matrix;feline;purity;spermatogonial stem cell