title: Genetic and epigenetic stability of human spermatogonial stem cells during long-term culture.
abstract: OBJECTIVE
  To determine the genetic and epigenetic stability of human spermatogonial stem cells (SSCs) during long-term culture.
  
  
  METHODS
  Experimental basic science study.
  
  
  METHODS
  Reproductive biology laboratory.
  
  
  METHODS
  Cryopreserved human testicular tissue from two prostate cancer patients with normal spermatogenesis.
  
  
  METHODS
  None.
  
  
  METHODS
  Testicular cells before and 50 days after culturing were subjected to ITGA6 magnetic-activated cell sorting to enrich for SSCs. Individual spermatogonia were analyzed for aneuploidies with the use of single-cell 24-chromosome screening. Furthermore, the DNA methylation statuses of the paternally imprinted genes H19, H19-DMR (differentially methylated region), and MEG3 and the maternally imprinted genes KCNQ1OT1 and PEG3 were identified by means of bisulfite sequencing.
  
  
  RESULTS
  Aneuploidy screening showed euploidy with no chromosomal abnormalities in all cultured and most noncultured spermatogonia from both patients. The methylation assays demonstrated demethylation of the paternally imprinted genes H19, H19-DMR, and MEG3 of 11%-28%, 43%-68%, and 18%-26%, respectively, and increased methylation of the maternally imprinted genes PEG 3 and KCNQ1OT of 13%-50% and 30%-38%, respectively, during culture.
  
  
  CONCLUSIONS
  In the current culture system for human SSCs propagation, genomic stability is preserved, which is important for future clinical use. Whether the observed changes in methylation status have consequences on functionality of SSCs or health of offspring derived from transplanted SSCs requires further investigation.
authors.affiliation: Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Bita
authors.initials: B
authors.lastname: Nickkholgh
authors.affiliation: Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: S Canan
authors.initials: SC
authors.lastname: Mizrak
authors.affiliation: Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Saskia K M
authors.initials: SK
authors.lastname: van Daalen
authors.affiliation: Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Cindy M
authors.initials: CM
authors.lastname: Korver
authors.affiliation: Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Hooman
authors.initials: H
authors.lastname: Sadri-Ardekani
authors.affiliation: Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Sjoerd
authors.initials: S
authors.lastname: Repping
authors.affiliation: Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. Electronic address: a.m.vanpelt@amc.uva.nl.
authors.forename: Ans M M
authors.initials: AM
authors.lastname: van Pelt
mesh_terms: D053687:Adult Stem Cells; D000782:Aneuploidy; D002469:Cell Separation; D002478:Cells, Cultured; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D018392:Genomic Imprinting; D006801:Humans; D039503:Integrin alpha6 / Q000235:genetics; D008280:Magnetics; D008297:Male; D013093:Spermatogonia / Q000378:metabolism*
chemical_list: C588002:ITGA6 protein, human; D039503:Integrin alpha6
keywords: Human spermatogonial stem cell; aneuploidy screening; epigenetic imprinting; genomic stability; testicular culture