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

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.