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

ABSTRACT: To determine the molecular characteristics of human spermatogonia and optimize methods to enrich spermatogonial stem cells (SSCs).Laboratory study using human tissues.Research institute.Healthy adult human testicular tissue.Human testicular tissue was fixed or digested with enzymes to produce a cell suspension. Human testis cells were fractionated by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS).Immunostaining for selected markers, human-to-nude mouse xenotransplantation assay.Immunohistochemistry costaining 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 one to four cells whereas differentiated spermatogonia (KIT+) were typically arranged in clones of eight or more cells. The ratio of undifferentiated-to-differentiated spermatogonia is greater in humans than in rodents. The 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.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.

SUBMITTER: Valli H

PROVIDER: S-EPMC4128386 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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

Valli Hanna H Sukhwani Meena M Dovey Serena L SL Peters Karen A KA Donohue Julia J Castro Carlos A CA Chu Tianjiao T Marshall Gary R GR Orwig Kyle E KE

Fertility and sterility 20140602 2

<h4>Objective</h4>To determine the molecular characteristics of human spermatogonia and optimize methods to enrich spermatogonial stem cells (SSCs).<h4>Design</h4>Laboratory study using human tissues.<h4>Setting</h4>Research institute.<h4>Patient(s)</h4>Healthy adult human testicular tissue.<h4>Intervention(s)</h4>Human testicular tissue was fixed or digested with enzymes to produce a cell suspension. Human testis cells were fractionated by fluorescence-activated cell sorting (FACS) and magnetic ...[more]

PMID: 24890267

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Project description:BackgroundDue to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions.Methodology/principal findingsHESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions.Conclusions/significanceThe application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.

Dataset Information

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

Publications

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

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