Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells

Abstract

Objective Determine the molecular characteristics of human spermatogonia and optimize methods to enrich spermatogonial stem cells (SSCs). Design Laboratory study using human tissues Setting Research institute Patient(s)/Animal(s) Normal adult human testicular tissue. Interventions Human testicular tissue was fixed or digested with enzymes to produce a cell suspension. Human testis cells were fractionated by FACS and MACS. Main Outcome Measure(s) Immunostaining for selected markers, human-to-nude mouse xenotransplantation assay. Results Immunohistochemistry co-staining revealed the relative expression patterns of SALL4, UTF1, ZBTB16, UCHL1 and ENO2 in human undifferentiated spermatogonia as well as the extent of overlap with the differentiation marker, KIT. Whole mount analyses revealed that human undifferentiated spermatogonia (UCHL1+) were typically arranged in clones of 1–4 cells while differentiated spermatogonia (KIT+) were typically arranged in clones of 8 or more cells. The ratio of undifferentiated to differentiated spermatogonia is greater in humans than in rodents. SSC colonizing activity was enriched in the THY1dim and ITGA6+ fractions of human testes sorted by FACS. ITGA6 was effective for sorting human SSCs by MACS; THY1 and EPCAM were not. Conclusions Human spermatogonial differentiation correlates with increased clone size and onset of KIT expression, similar to rodents. The undifferentiated to differentiated developmental dynamics in human spermatogonia is different than rodents. THY1, ITGA6 and EPCAM can be used to enrich human SSC colonizing activity by FACS, but only ITGA6 is amenable to high throughput sorting by MACS.

Quantitative analysis of male germline stem cell differentiation reveals a role for the p53-mTORC1 pathway in spermatogonial maintenance

“…EpCAM was not used as a pan-spermatogonia marker as previously reported, 29 as recent studies as well as our own investigation suggest that EpCAM level is low in undifferentiated spermatogonia but increases along with early spermatogonial differentiation (data not shown). 30 Within the c-Kit negative a6-integrin high compartment that has been shown to be composed mostly of undifferentiated spermatogonia, we were able to further identify a GFP-negative population (population 1 or P1) and a GFP-positive population (population 2 or P2) (Fig. 1A).…”

Widespread transcriptional scanning in the testis modulates gene evolution rates

“…Testicular single-cell suspension was prepared by adapting existing protocols 54 . Specifically, samples from TESE surgery was washed 15 once with PBS and resuspended in 5mL PBS.…”

Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine

“…SSCs from humans also express GPR125 [49] , besides GFRα1 [50] . Another human SSC marker that adds to the known panel is ITGA6 [51] . Recently, a new SSC gene product has been reported: PAX7, that might represent a definite common SSC marker for several species including mice, cats, dogs, pigs, bulls, monkeys and humans [52] .…”