title: Isolation of human male germ-line stem cells using enzymatic digestion and magnetic-activated cell sorting.
abstract: Mammalian spermatogenesis is a process whereby male germ-line stem cells (spermatogonial stem cells) divide and differentiate into sperm. Although a great deal of progress has been made in the isolation and characterization of spermatogonial stem cells (SSCs) in rodents, little is known about human SSCs. We have recently isolated human G protein-coupled receptor 125 (GPR125)-positive spermatogonia and GDNF family receptor alpha 1 (GFRA1)-positive spermatogonia using a 2-step enzymatic digestion and magnetic-activated cell sorting (MACS) from adult human testes. Cell purities of isolated human GPR125- and GFRA1-positive spermatogonia after MACS are greater than 95%, and cell viability is over 96%. The isolated GPR125- and GFRA1-positive spermatogonia coexpress GPR125, integrin, alpha 6 (ITGA6), THY1 (also known as CD90), GFRA1, and ubiquitin carboxyl-terminal esterase L1 (UCHL1), markers for rodent or pig SSCs/progenitors, suggesting that GPR125- and GFRA1-positive spermatogonia are phenotypically the SSCs in human testis. Human GPR125-positive spermatogonia can be cultured for 2 weeks with a remarkable increase in cell number. Immunocytochemistry further reveals that GPR125-positive spermatogonia can be maintained in an undifferentiated state in vitro. Collectively, the methods using enzymatic digestion and MACS can efficiently isolate and purify SSCs from adult human testis with consistent and high quality. The ability of isolating and characterizing human SSCs could provide a population of stem cells with high purity for mechanistic studies on human SSC self-renewal and differentiation as well as potential applications of human SSCs in regenerative medicine.
authors.affiliation: Clinical Stem Cell Research Center, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. zh27@georgetown.edu
authors.forename: Zuping
authors.initials: Z
authors.lastname: He
authors.forename: Maria
authors.initials: M
authors.lastname: Kokkinaki
authors.forename: Jiji
authors.initials: J
authors.lastname: Jiang
authors.forename: Wenxian
authors.initials: W
authors.lastname: Zeng
authors.forename: Ina
authors.initials: I
authors.lastname: Dobrinski
authors.forename: Martin
authors.initials: M
authors.lastname: Dym
mesh_terms: D000328:Adult; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013737:Testis / Q000166:cytology; D014019:Tissue Donors
chemical_list: C548160:ADGRA3 protein, human; C494987:GFRA1 protein, human; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D043562:Receptors, G-Protein-Coupled