Bita Nickkholgh1, S Canan Mizrak1, Saskia K M van Daalen1, Cindy M Korver1, Hooman Sadri-Ardekani1, Sjoerd Repping1, Ans M M van Pelt2. 1. Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. 2. 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.
Abstract
OBJECTIVE: To determine the genetic and epigenetic stability of human spermatogonial stem cells (SSCs) during long-term culture. DESIGN: Experimental basic science study. SETTING: Reproductive biology laboratory. PATIENT(S): Cryopreserved human testicular tissue from two prostate cancer patients with normal spermatogenesis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): 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(S): 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. CONCLUSION(S): 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.
OBJECTIVE: To determine the genetic and epigenetic stability of human spermatogonial stem cells (SSCs) during long-term culture. DESIGN: Experimental basic science study. SETTING: Reproductive biology laboratory. PATIENT(S): Cryopreserved human testicular tissue from two prostate cancerpatients with normal spermatogenesis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): 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(S): 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. CONCLUSION(S): 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: Callista L Mulder; Yi Zheng; Sabrina Z Jan; Robert B Struijk; Sjoerd Repping; Geert Hamer; Ans M M van Pelt Journal: Hum Reprod Update Date: 2016-05-30 Impact factor: 15.610
Authors: Callista L Mulder; Lisa A E Catsburg; Yi Zheng; Cindy M de Winter-Korver; Saskia K M van Daalen; Madelon van Wely; Steven Pals; Sjoerd Repping; Ans M M van Pelt Journal: Hum Reprod Date: 2018-01-01 Impact factor: 6.918
Authors: Federico Del Vento; Maxime Vermeulen; Francesca de Michele; Maria Grazia Giudice; Jonathan Poels; Anne des Rieux; Christine Wyns Journal: Int J Mol Sci Date: 2018-01-18 Impact factor: 5.923