title: Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells.
abstract: OBJECTIVE
  To determine the molecular characteristics of human spermatogonia and optimize methods to enrich spermatogonial stem cells (SSCs).
  
  
  METHODS
  Laboratory study using human tissues.
  
  
  METHODS
  Research institute.
  
  
  METHODS
  Healthy adult human testicular tissue.
  
  
  METHODS
  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).
  
  
  METHODS
  Immunostaining for selected markers, human-to-nude mouse xenotransplantation assay.
  
  
  RESULTS
  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.
  
  
  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.
authors.affiliation: Department of Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Magee-Womens Research Institute, Pittsburgh, Pennsylvania.
authors.forename: Hanna
authors.initials: H
authors.lastname: Valli
authors.affiliation: Magee-Womens Research Institute, Pittsburgh, Pennsylvania.
authors.forename: Meena
authors.initials: M
authors.lastname: Sukhwani
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Magee-Womens Research Institute, Pittsburgh, Pennsylvania.
authors.forename: Serena L
authors.initials: SL
authors.lastname: Dovey
authors.affiliation: Magee-Womens Research Institute, Pittsburgh, Pennsylvania.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Peters
authors.affiliation: Magee-Womens Research Institute, Pittsburgh, Pennsylvania.
authors.forename: Julia
authors.initials: J
authors.lastname: Donohue
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Magee-Womens Research Institute, Pittsburgh, Pennsylvania.
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Castro
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Magee-Womens Research Institute, Pittsburgh, Pennsylvania.
authors.forename: Tianjiao
authors.initials: T
authors.lastname: Chu
authors.affiliation: Department of Natural Sciences, Chatham University, Pittsburgh, Pennsylvania.
authors.forename: Gary R
authors.initials: GR
authors.lastname: Marshall
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Department of Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Magee-Womens Research Institute, Pittsburgh, Pennsylvania. Electronic address: orwigke@upmc.edu.
authors.forename: Kyle E
authors.initials: KE
authors.lastname: Orwig
mesh_terms: D053687:Adult Stem Cells / Q000378:metabolism* / Q000637:transplantation; D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D000071858:Epithelial Cell Adhesion Molecule; D005434:Flow Cytometry; D006801:Humans; D018189:Immunomagnetic Separation; D039503:Integrin alpha6 / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D013093:Spermatogonia / Q000378:metabolism* / Q000637:transplantation; D013737:Testis / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D018800:Thy-1 Antigens / Q000378:metabolism; D014183:Transplantation, Heterologous
chemical_list: D000951:Antigens, Neoplasm; D015415:Biomarkers; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; C588002:ITGA6 protein, human; D039503:Integrin alpha6; D018800:Thy-1 Antigens; D019009:Proto-Oncogene Proteins c-kit
keywords: FACS; MACS; Testis; human spermatogonia; spermatogonial stem cell; stem cell