Advertisement

Genetic and epigenetic stability of human spermatogonial stem cells during long-term culture

Published:September 25, 2014DOI:https://doi.org/10.1016/j.fertnstert.2014.08.022

      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.

      Key Words

      To read this article in full you will need to make a payment

      References

        • Pulte D.
        • Gondos A.
        • Brenner H.
        Trends in 5- and 10-year survival after diagnosis with childhood hematologic malignancies in the United States, 1990–2004.
        J Natl Cancer Inst. 2008; 100: 1301-1309
        • Pui C.H.
        • Carroll W.L.
        • Meshinchi S.
        • Arceci R.J.
        Biology, risk stratification, and therapy of pediatric acute leukemias: an update.
        J Clin Oncol. 2011; 29: 551-565
        • Wallace W.H.
        • Anderson R.A.
        • Irvine D.S.
        Fertility preservation for young patients with cancer: who is at risk and what can be offered?.
        Lancet Oncol. 2005; 6: 209-218
        • Bleyer W.A.
        The impact of childhood cancer on the United States and the world.
        CA Cancer J Clin. 1990; 40: 355-367
        • Blatt J.
        Pregnancy outcome in long-term survivors of childhood cancer.
        Med Pediatr Oncol. 1999; 33: 29-33
        • Balcerek M.
        • Reinmuth S.
        • Hohmann C.
        • Keil T.
        • Borgmann-Staudt A.
        Suspected infertility after treatment for leukemia and solid tumors in childhood and adolescence.
        Dtsch Arztebl Int. 2012; 109: 126-131
        • Tromp K.
        • Claessens J.J.
        • Knijnenburg S.L.
        • van der Pal H.J.
        • van Leeuwen F.E.
        • Caron H.N.
        • et al.
        Reproductive status in adult male long-term survivors of childhood cancer.
        Hum Reprod. 2011; 26: 1775-1783
        • Struijk R.B.
        • Mulder C.L.
        • van der Veen F.
        • van Pelt A.M.
        • Repping S.
        Restoring fertility in sterile childhood cancer survivors by autotransplanting spermatogonial stem cells: are we there yet?.
        Biomed Res Int. 2013; 2013: 903142
        • Sadri-Ardekani H.
        • Mizrak S.C.
        • van Daalen S.K.
        • Korver C.M.
        • Roepers-Gajadien H.L.
        • Koruji M.
        • et al.
        Propagation of human spermatogonial stem cells in vitro.
        J Am Med Assoc. 2009; 302: 2127-2134
        • Sadri-Ardekani H.
        • Akhondi M.A.
        • van der Veen F.
        • Repping S.
        • van Pelt A.M.
        In vitro propagation of human prepubertal spermatogonial stem cells.
        J Am Med Assoc. 2011; 305: 2416-2418
        • Grafodatskaya D.
        • Choufani S.
        • Ferreira J.C.
        • Butcher D.T.
        • Lou Y.
        • Zhao C.
        • et al.
        EBV transformation and cell culturing destabilizes DNA methylation in human lymphoblastoid cell lines.
        Genomics. 2010; 95: 73-83
        • Cairns J.
        Somatic stem cells and the kinetics of mutagenesis and carcinogenesis.
        Proc Natl Acad Sci U S A. 2002; 99: 10567-10570
        • Frost J.
        • Monk D.
        • Moschidou D.
        • Guillot P.V.
        • Stanier P.
        • Minger S.L.
        • et al.
        The effects of culture on genomic imprinting profiles in human embryonic and fetal mesenchymal stem cells.
        Epigenetics. 2011; 6: 52-62
        • Kanatsu-Shinohara M.
        • Ogonuki N.
        • Iwano T.
        • Lee J.
        • Kazuki Y.
        • Inoue K.
        • et al.
        Genetic and epigenetic properties of mouse male germline stem cells during long-term culture.
        Development. 2005; 132: 4155-4163
        • Schmidt J.A.
        • Abramowitz L.K.
        • Kubota H.
        • Wu X.
        • Niu Z.
        • Avarbock M.R.
        • et al.
        In vivo and in vitro aging is detrimental to mouse spermatogonial stem cell function.
        Biol Reprod. 2010; 84: 698-706
        • Garner J.L.
        • Niles K.M.
        • McGraw S.
        • Yeh J.R.
        • Cushnie D.W.
        • Hermo L.
        • et al.
        Stability of DNA methylation patterns in mouse spermatogonia under conditions of MTHFR deficiency and methionine supplementation.
        Biol Reprod. 2013; 89: 125
        • Nickkholgh B.
        • Mizrak S.C.
        • Korver C.M.
        • van Daalen S.K.
        • Meissner A.
        • Repping S.
        • et al.
        Enrichment of spermatogonial stem cells from long-term cultured human testicular cells.
        Fertil Steril. 2014; 102: 558-565.e5
        • Fiorentino F.
        • Caiazzo F.
        • Napolitano S.
        • Spizzichino L.
        • Bono S.
        • Sessa M.
        • et al.
        Introducing array comparative genomic hybridization into routine prenatal diagnosis practice: a prospective study on over 1,000 consecutive clinical cases.
        Prenat Diagn. 2011; 31: 1270-1282
        • Valli H.
        • Sukhwani M.
        • Dovey S.L.
        • Peters K.A.
        • Donohue J.
        • Castro C.A.
        • et al.
        Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells.
        Fertil Steril. 2014; 102: 566-580.e7
        • Fragouli E.
        • Alfarawati S.
        • Daphnis D.D.
        • Goodall N.N.
        • Mania A.
        • Griffiths T.
        • et al.
        Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: scientific data and technical evaluation.
        Hum Reprod. 2011; 26: 480-490
        • Hayashi K.
        • Ohta H.
        • Kurimoto K.
        • Aramaki S.
        • Saitou M.
        Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
        Cell. 2011; 146: 519-532
        • Oakes C.C.
        • la Salle S.
        • Smiraglia D.J.
        • Robaire B.
        • Trasler J.M.
        Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells.
        Dev Biol. 2007; 307: 368-379
        • Marques C.J.
        • Joao Pinho M.
        • Carvalho F.
        • Bieche I.
        • Barros A.
        • Sousa M.
        DNA methylation imprinting marks and DNA methyltransferase expression in human spermatogenic cell stages.
        Epigenetics. 2011; 6: 1354-1361
        • Feng S.
        • Jacobsen S.E.
        • Reik W.
        Epigenetic reprogramming in plant and animal development.
        Science. 2010; 330: 622-627
        • Harper J.C.
        • Geraedts J.
        • Borry P.
        • Cornel M.C.
        • Dondorp W.
        • Gianaroli L.
        • et al.
        European Society of Human Genetics, European Society of Human Reproduction and Embryology. Current issues in medically assisted reproduction and genetics in Europe: research, clinical practice, ethics, legal issues and policy.
        Eur J Hum Genet. 2013; 21: S1-S21
        • Conrad S.
        • Renninger M.
        • Hennenlotter J.
        • Wiesner T.
        • Just L.
        • Bonin M.
        • et al.
        Generation of pluripotent stem cells from adult human testis.
        Nature. 2008; 456: 344-349
        • Mizrak S.C.
        • Chikhovskaya J.V.
        • Sadri-Ardekani H.
        • van Daalen S.
        • Korver C.M.
        • Hovingh S.E.
        • et al.
        Embryonic stem cell-like cells derived from adult human testis.
        Hum Reprod. 2010; 25: 158-167
        • Kerjean A.
        • Dupont J.M.
        • Vasseur C.
        • le Tessier D.
        • Cuisset L.
        • Paldi A.
        • et al.
        Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis.
        Hum Mol Genet. 2000; 9: 2183-2187
        • Minor A.
        • Chow V.
        • Ma S.
        Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal.
        Reproduction. 2011; 141: 749-757
        • Ouko L.A.
        • Shantikumar K.
        • Knezovich J.
        • Haycock P.
        • Schnugh D.J.
        • Ramsay M.
        Effect of alcohol consumption on CpG methylation in the differentially methylated regions of H19 and IG-DMR in male gametes: implications for fetal alcohol spectrum disorders.
        Alcohol Clin Exp Res. 2009; 33: 1615-1627
        • Stouder C.
        • Paoloni-Giacobino A.
        Transgenerational effects of the endocrine disruptor vinclozolin on the methylation pattern of imprinted genes in the mouse sperm.
        Reproduction. 2010; 139: 373-379
        • Sato A.
        • Otsu E.
        • Negishi H.
        • Utsunomiya T.
        • Arima T.
        Aberrant DNA methylation of imprinted loci in superovulated oocytes.
        Hum Reprod. 2007; 22: 26-35
        • Kawakami T.
        • Chano T.
        • Minami K.
        • Okabe H.
        • Okada Y.
        • Okamoto K.
        Imprinted DLK1 is a putative tumor suppressor gene and inactivated by epimutation at the region upstream of GTL2 in human renal cell carcinoma.
        Hum Mol Genet. 2006; 15: 821-830
        • Nelissen E.C.
        • Dumoulin J.C.
        • Daunay A.
        • Evers J.L.
        • Tost J.
        • van Montfoort A.P.
        Placentas from pregnancies conceived by IVF/ICSI have a reduced DNA methylation level at the H19 and MEST differentially methylated regions.
        Hum Reprod. 2013; 28: 1117-1126