This study aimed to provide a molecular signature for enriched adult human stem/progenitor spermatogonia during short-term (<2 weeks) and long-term culture (up to more than 14 months) in comparison to human testicular fibroblasts and human embryonic stem cells. Human spermatogonia were isolated by CD49f magnetic activated cell sorting and collagen(-)/laminin(+) matrix binding from primary testis cultures obtained from ten adult men. For transcriptomic analysis, single spermatogonia-like cells were collected based on their morphology and dimensions using a micromanipulation system from the enriched germ cell cultures. Immunocytochemical, RT-PCR and microarray analyses revealed that the analyzed populations of cells were distinct at the molecular level. The germ- and pluripotency-associated genes and genes of differentiation/spermatogenesis pathway were highly expressed in enriched short-term cultured spermatogonia. After long-term culture, a proportion of cells retained and aggravated the "spermatogonial" gene expression profile with the expression of germ and pluripotency-associated genes, while in the majority of long-term cultured cells this molecular profile, typical for the differentiation pathway, was reduced and more genes related to the extracellular matrix production and attachment were expressed. The approach we provide here to study the molecular status of in vitro cultured spermatogonia may be important to optimize the culture conditions and to evaluate the germ cell plasticity in the future.

Department of Urology University Clinic Tübingen Hoppe Seyler Straße 3 72076 Tübingen Germany

Institute for Anatomy and Cell Biology Medical Faculty University of Heidelberg Im Neuenheimer Feld 307 69120 Heidelberg Germany

Institute for Anatomy and Cell Biology Medical Faculty University of Heidelberg Im Neuenheimer Feld 307 69120 Heidelberg Germany ; Amol University of Special Modern Technologies Special Modern Technologies P O Box 46168 49767 Amol Iran ; Department of Stem Cells and Developmental Biology Royan Institute P O Box 19395 4644 Tehran Iran

Institute of Anatomy University of Tübingen Österbergstraße 3 72074 Tübingen Germany

Institute of Anthropology and Human Genetics Microarray Facility University Clinic Calwerstraße 7 72076 Tübingen Germany

TATAA Biocenter AB Odinsgatan 28 41103 Göteborg Sweden and Institute of Biotechnology at the Czech Academy of Sciences Vídenská 1083 14220 Prague 4 Czech Republic

Zobrazit více v PubMed

Clermont Y. Renewal of spermatogonia in man. American Journal of Anatomy. 1966;118(2):509–524. PubMed

Gillan L, Matei D, Fishman DA, Gerbin CS, Karlan BY, Chang DD. Periostin secreted by epithelial ovarian carcinoma is a ligand for αVβ3 and αVβ5 integrins and promotes cell motility. Cancer Research. 2002;62(18):5358–5364. PubMed

Sadri-Ardekani H, Akhondi MA, van der Veen F, Repping S, Van Pelt AMM. In vitro propagation of human prepubertal spermatogonial stem cells. Journal of the American Medical Association. 2011;305(23):2416–2418. PubMed

Lee J, Shinohara T. Epigenetic modifications and self-renewal regulation of mouse germline stem cells. Cell Research. 2011;21(8):1164–1171. PubMed PMC

Conrad S, Renninger M, Hennenlotter J, et al. Generation of pluripotent stem cells from adult human testis. Nature. 2008;456(7220):344–349. PubMed

Sadri-Ardekani H, Mizrak SC, van Daalen SKM, et al. Propagation of human spermatogonial stem cells in vitro. Journal of the American Medical Association. 2009;302(19):2127–2134. PubMed

Chen B, Wang YB, Zhang ZL, et al. Xeno-free culture of human spermatogonial stem cells supported by human embryonic stem cell-derived fibroblast-like cells. Asian Journal of Andrology. 2009;11(5):557–565. PubMed PMC

He Z, Kokkinaki M, Jiang J, Dobrinski I, Dym MI. Isolation, characterization, and culture of human spermatogonia. Biology of Reproduction. 2010;82(2):363–372. PubMed PMC

Lim JJ, Sung S-Y, Kim HJ, et al. Long-term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non-obstructive azoospermia under exogenous feeder-free culture conditions. Cell Proliferation. 2010;43(4):405–417. PubMed PMC

Izadyar F, Wong J, Maki C, et al. Identification and characterization of repopulating spermatogonial stem cells from the adult human testis. Human Reproduction. 2011;26(6):1296–1306. PubMed

Mirzapour T, Movahedin M, Tengku Ibrahim TA, et al. Effects of basic fibroblast growth factor and leukaemia inhibitory factor on proliferation and short-term culture of human spermatogonial stem cells. Andrologia. 2012;44(supplement 1):41–55. PubMed

Golestaneh N, Kokkinaki M, Pant D, et al. Pluripotent stem cells derived from adult human testes. Stem Cells and Development. 2009;18(8):1115–1126. PubMed PMC

Kossack N, Meneses J, Shefi S, et al. Isolation and characterization of pluripotent human spermatogonial stem cell-derived cells. Stem Cells. 2009;27(1):138–149. PubMed PMC

Mizrak SC, Chikhovskaya JV, Sadri-Ardekani H, et al. Embryonic stem cell-like cells derived from adult human testis. Human Reproduction. 2010;25(1):158–167. PubMed

Chikhovskaya JV, Jonker MJ, Meissner A, Breit TM, Repping S, van Pelt AMM. Human testis-derived embryonic stem cell-like cells are not pluripotent, but possess potential of mesenchymal progenitors. Human Reproduction. 2012;27(1):210–221. PubMed

Gonzalez R, Griparic L, Vargas V, et al. A putative mesenchymal stem cells population isolated from adult human testes. Biochemical and Biophysical Research Communications. 2009;385(4):570–575. PubMed

Stimpfel M, Skutella T, Kubista M, Malicev E, Conrad S, Virant-Klun I. Potential stemness of frozen-thawed testicular biopsies without sperm in infertile men included into the in vitro fertilization programme. Journal of Biomedicine and Biotechnology. 2012;2012:15 pages.291038 PubMed PMC

Lim JJ, Kim HJ, Kim KS, Hong JY, Lee DR. In vitro culture-induced pluripotency of human spermatogonial stem cells. BioMed Research International. 2013;2013:9 pages.143028 PubMed PMC

Heer R, Hepburn AC, Williamson SC, et al. Renal differentiation from adult spermatogonial stem cells. Renal failure. 2013;35(10):1387–1391. PubMed

Zhang Z, Liu J, Liu Y, et al. Generation, characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells. Journal of Cellular Physiology. 2013;228(2):298–305. PubMed

Kossack N, Terwort N, Wistuba J, et al. A combined approach facilitates the reliable detection of human spermatogonia in vitro. Human Reproduction. 2013;28(11):3012–3025. PubMed

Ko K, Arauzo-Bravo MJ, Tapia N, et al. Human adult germline stem cells in question. Nature. 2010;465(7301):E1–E3. PubMed

Tichopad A, Kitchen R, Riedmaier I, Becker C, Ståhlberg A, Kubista M. Design and optimization of reverse-transcription quantitative PCR experiments. Clinical Chemistry. 2009;55(10):1816–1823. PubMed

Castrillon DH, Quade BJ, Wang TY, Quigley C, Crum CP. The human VASA gene is specifically expressed in the germ cell lineage. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(17):9585–9590. PubMed PMC

Menke DB, Mutter GL, Page DC. Expression of DAZ, an Azoospermia factor candidate, in human spermatogonia. American Journal of Human Genetics. 1997;60(1):237–241. PubMed PMC

Szczerba A, Jankowska A, Andrusiewicz M, Karczewski M, Turkiewicz W, Warchoł JB. Distribution of the DAZ gene transcripts in human testis. Folia Histochemica et Cytobiologica. 2004;42(2):119–121. PubMed

Wang F, Zhang Q, Cao J, Huang Q, Zhu X. The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules. Experimental Cell Research. 2008;314(1):213–226. PubMed

Reid A, Gould A, Brand N, et al. Leukemia translocation gene, PLZF, is expressed with a speckled nuclear pattern in early hematopoietic progenitors. Blood. 1995;86(12):4544–4552. PubMed

Buaas FW, Kirsh AL, Sharma M, et al. Plzf is required in adult male germ cells for stem cell self-renewal. Nature Genetics. 2004;36(6):647–652. PubMed

Tureci O, Sahin U, Koslowski M, et al. A novel tumour associated leucine zipper protein targeting to sites of gene transcription and splicing. Oncogene. 2002;21(24):3879–3888. PubMed

Mendoza-Lujambio I, Burfeind P, Dixkens C, et al. The Hook1 gene is non-functional in the abnormal spermatozoon head shape (azh) mutant mouse. Human Molecular Genetics. 2002;11(14):1647–1658. PubMed

Bowles J, Teasdale RP, James K, Koopman P. Dppa3 is a marker of pluripotency and has a human homologue that is expressed in germ cell tumours. Cytogenetic and Genome Research. 2003;101(3-4):261–265. PubMed

West JA, Viswanathan SR, Yabuuchi A, et al. A role for Lin28 in primordial germ-cell development and germ-cell malignancy. Nature. 2009;460(7257):909–913. PubMed PMC

Zhang N, Yeh HJ, Zhong R, Li YS, Deuel TF. A dominant-negative pleiotrophin mutant introduced by homologous recombination leads to germ-cell apoptosis in male mice. Proceedings of the National Academy of Sciences of the United States of America. 1999;96(12):6734–6738. PubMed PMC

van Gurp RJHLM, Oosterhuis JW, Kalscheuer V, Mariman ECM, Looijenga LHJ. Biallelic expression of the H19 and IGF2 genes in human testicular germ cell tumors. Journal of the National Cancer Institute. 1994;86(14):1070–1075. PubMed

Bafico A, Gazit A, Pramila T, Finch PW, Yaniv A, Aaronson SA. Interaction of Frizzled Related Protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling. Journal of Biological Chemistry. 1999;274(23):16180–16187. PubMed

Walsh J, Andrews PW. Expression of Wnt and Notch pathway genes in a pluripotent human embryonal carcinoma cell line and embryonic stem cell. Acta Pathologica, Microbiologica, et Immunologica Scandinavica. 2003;111(1):197–210. PubMed

Freemantle SJ, Kerley JS, Olsen SL, Gross RH, Spinella MJ. Developmentally-related candidate retinoic acid target genes regulated early during neuronal differentiation of human embryonal carcinoma. Oncogene. 2002;21(18):2880–2889. PubMed

Pezzolesi MG, Zbuk KM, Waite KA, Eng C. Comparative genomic and functional analyses reveal a novel cis-acting PTEN regulatory element as a highly conserved functional E-box motif deleted in Cowden syndrome. Human Molecular Genetics. 2007;16(9):1058–1071. PubMed

Wang CQF, Mruk DD, Lee WM, Cheng CY. Coxsackie and Adenovirus Receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli-Sertoli and Sertoli-germ cell interface. Experimental Cell Research. 2007;313(7):1373–1392. PubMed PMC

Mosevitsky MI, Snigirevskaya ES, Komissarchik YY. Immunoelectron microscopic study of BASP1 and MARCKS location in the early and late rat spermatids. Acta Histochemica. 2012;114(3):237–243. PubMed

Chang H, Matzuk MM. Smad5 is required for mouse primordial germ cell development. Mechanisms of Development. 2001;104(1-2):61–67. PubMed

Itman C, Loveland KL. SMAD expression in the testis: an insight into BMP regulation of spermatogenesis. Developmental Dynamics. 2008;237(1):97–111. PubMed

Pellegrini M, Grimaldi P, Rossi P, Geremia R, Dolci S. Developmental expression of BMP4∖ALK3∖SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation. Journal of Cell Science. 2003;116(16):3363–3372. PubMed

Wu J-W, Wang R-Y, Guo Q-S, Xu C. Expression of the retinoic acid-metabolizing enzymes RALDH2 and CYP26b1 during mouse postnatal testis development. Asian Journal of Andrology. 2008;10(4):569–576. PubMed

Wang S, Zheng H, Esaki Y, Kelly F, Yan W. Cullin3 is a KLHL10-interacting protein preferentially expressed during late spermiogenesis. Biology of Reproduction. 2006;74(1):102–108. PubMed

Vinci G, Brauner R, Tar A, et al. Mutations in the TSPYL1 gene associated with 46,XY disorder of sex development and male infertility. Fertility and Sterility. 2009;92(4):1347–1350. PubMed

Mizuno K, Tokumasu A, Nakamura A, et al. Genes associated with the formation of germ cells from embryonic stem cells in cultures containing different glucose concentrations. Molecular Reproduction and Development. 2006;73(4):437–445. PubMed

Sato T, Katagiri K, Gohbara A, et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature. 2011;471(7339):504–507. PubMed

Quackenbush J. Microarray data normalization and transformation. Nature Genetics. 2002;32:496–501. PubMed

Optimization of in vitro culture conditions of common carp germ cells for purpose of surrogate production

Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells