Abstract
Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.
*These authors contributed equally to this work
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sehgal L, Usmani A, Dalal SN, Majumdar SS (2014) Generation of transgenic mice by exploiting spermatogonial stem cells in vivo. Methods Mol Biol 1194:327–337. doi:10.1007/978-1-4939-1215-5_18
Wu Y, Zhou H, Fan X, Zhang Y, Zhang M, Wang Y, Xie Z, Bai M, Yin Q, Liang D, Tang W, Liao J, Zhou C, Liu W, Zhu P, Guo H, Pan H, Wu C, Shi H, Wu L, Tang F, Li J (2015) Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res 25(1):67–79. doi:10.1038/cr.2014.160
Sato T, Sakuma T, Yokonishi T, Katagiri K, Kamimura S, Ogonuki N, Ogura A, Yamamoto T, Ogawa T (2015) Genome editing in mouse spermatogonial stem cell lines using TALEN and double-nicking CRISPR/Cas9. Stem Cell Rep 5(1):75–82. doi:10.1016/j.stemcr.2015.05.011
Aponte PM (2015) Spermatogonial stem cells: current biotechnological advances in reproduction and regenerative medicine. World J Stem Cells 7(4):669–680. doi:10.4252/wjsc.v7.i4.669
Kanatsu-Shinohara M, Lee J, Inoue K, Ogonuki N, Miki H, Toyokuni S, Ikawa M, Nakamura T, Ogura A, Shinohara T (2008) Pluripotency of a single spermatogonial stem cell in mice. Biol Reprod 78(4):681–687. doi:10.1095/biolreprod.107.066068
Simon L, Ekman GC, Kostereva N, Zhang Z, Hess RA, Hofmann MC, Cooke PS (2009) Direct transdifferentiation of stem/progenitor spermatogonia into reproductive and nonreproductive tissues of all germ layers. Stem Cells 27(7):1666–1675. doi:10.1002/stem.93
Ning L, Goossens E, Geens M, Van Saen D, Van Riet I, He D, Tournaye H (2010) Mouse spermatogonial stem cells obtain morphologic and functional characteristics of hematopoietic cells in vivo. Hum Reprod 25(12):3101–3109. doi:10.1093/humrep/deq269
Kossack N, Meneses J, Shefi S, Nguyen HN, Chavez S, Nicholas C, Gromoll J, Turek PJ, Reijo-Pera RA (2009) Isolation and characterization of pluripotent human spermatogonial stem cell-derived cells. Stem Cells 27(1):138–149. doi:10.1634/stemcells.2008-0439
Dym M, He Z, Jiang J, Pant D, Kokkinaki M (2009) Spermatogonial stem cells: unlimited potential. Reprod Fertil Dev 21(1):15–21
Golestaneh N, Kokkinaki M, Pant D, Jiang J, DeStefano D, Fernandez-Bueno C, Rone JD, Haddad BR, Gallicano GI, Dym M (2009) Pluripotent stem cells derived from adult human testes. Stem Cells Dev 18(8):1115–1126. doi:10.1089/scd.2008.0347
Izadyar F, Wong J, Maki C, Pacchiarotti J, Ramos T, Howerton K, Yuen C, Greilach S, Zhao HH, Chow M, Chow YC, Rao J, Barritt J, Bar-Chama N, Copperman A (2011) Identification and characterization of repopulating spermatogonial stem cells from the adult human testis. Hum Reprod 26(6):1296–1306. doi:10.1093/humrep/der026
Hamra FK, Gatlin J, Chapman KM, Grellhesl DM, Garcia JV, Hammer RE, Garbers DL (2002) Production of transgenic rats by lentiviral transduction of male germ-line stem cells. Proc Natl Acad Sci U S A 99(23):14931–14936. doi:10.1073/pnas.222561399
Hofmann MC, Braydich-Stolle L, Dym M (2005) Isolation of male germ-line stem cells; influence of GDNF. Dev Biol 279(1):114–124. doi:10.1016/j.ydbio.2004.12.006
Izadyar F, Spierenberg GT, Creemers LB, den Ouden K, de Rooij DG (2002) Isolation and purification of type A spermatogonia from the bovine testis. Reproduction 124(1):85–94
Shafa M, Sjonnesen K, Yamashita A, Liu S, Michalak M, Kallos MS, Rancourt DE (2012) Expansion and long-term maintenance of induced pluripotent stem cells in stirred suspension bioreactors. J Tissue Eng Regen Med 6(6):462–472. doi:10.1002/term.450
Krawetz R, Taiani JT, Liu S, Meng G, Li X, Kallos MS, Rancourt DE (2010) Large-scale expansion of pluripotent human embryonic stem cells in stirred-suspension bioreactors. Tissue Eng Part C Methods 16(4):573–582. doi:10.1089/ten.TEC.2009.0228
Fluri DA, Tonge PD, Song H, Baptista RP, Shakiba N, Shukla S, Clarke G, Nagy A, Zandstra PW (2012) Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures. Nat Methods 9(5):509–516. doi:10.1038/nmeth.1939
Dores C, Rancourt D, Dobrinski I (2015) Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis. Andrology 3(3):590–597. doi:10.1111/andr.12031
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sakib, S., Dores, C., Rancourt, D., Dobrinski, I. (2016). Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment. In: Turksen, K. (eds) Bioreactors in Stem Cell Biology. Methods in Molecular Biology, vol 1502. Humana Press, New York, NY. https://doi.org/10.1007/7651_2016_334
Download citation
DOI: https://doi.org/10.1007/7651_2016_334
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6476-5
Online ISBN: 978-1-4939-6478-9
eBook Packages: Springer Protocols