Volume 11, Issue 7 (10-2013)                   IJRM 2013, 11(7): 551-0 | Back to browse issues page

XML Persian Abstract Print


Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Akhondi M M, Mohazzab A, Jeddi-Tehrani M, Sadeghi M R, Eidi A, Khodadadi A et al . Propagation of human germ stem cells in long-term culture. IJRM 2013; 11 (7) :551-0
URL: http://ijrm.ir/article-1-444-en.html
1- Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
2- Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
3- Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
4- Research and Preparation Center, Iranian Tissue Bank, Tehran University of Medical Science, Tehran, Iran
5- Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran , saba.piravar@gmail.com
Abstract:   (3202 Views)
Background: Spermatogonial stem cells (SSCs), a subset of undifferentiated type A spermatogonia, are the foundation of complex process of spermatogenesis and could be propagated in vitro culture conditions for long time for germ cell transplantation and fertility preservation.
Objective: The aim of this study was in vitro propagation of human spermatogonial stem cells (SSCs) and improvement of presence of human Germ Stem Cells (hGSCs) were assessed by specific markers POU domain, class 5, transcription factor 1 (POU5F1), also known as Octamer-binding transcription factor 4 (Oct-4) and PLZF (Promyelocytic leukaemia zinc finger protein).
Materials and Methods: Human testicular cells were isolated by enzymatic digestion (Collagenase IV and Trypsin). Germ cells were cultured in Stem-Pro 34 media supplemented by growth factors such as glial cell line-derived neurotrophic factor, basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor to support self-renewal divisions. Germline stem cell clusters were passaged and expanded every week. Immunofluorecent study was accomplished by Anti-Oct4 antibody through the culture. The spermatogonial stem cells genes expression, PLZF, was studied in testis tissue and germ stem cells entire the culture.
Results: hGSCs clusters from a brain dead patient developed in testicular cell culture and then cultured and propagated up to 6 weeks. During the culture Oct4 were a specific marker for identification of hGSCs in testis tissue. Expression of PLZF was applied on RNA level in germ stem cells.
Conclusion: hGSCs indicated by SSCs specific marker can be cultured and propagated for long-term in vitro conditions.
Full-Text [PDF 580 kb]   (557 Downloads) |   |   Full-Text (HTML)  (274 Views)  
Type of Study: Original Article |

References
1. Clermont Y. Renewal of spermatogonia in man. Am J Anat 1966; 118: 509-524. [DOI:10.1002/aja.1001180211]
2. Wu X, Goodyear SM, Tobias JW, Avarbock MR, Brinster RL. Spermatogonial Stem Cell Self-Renewal Requires ETV5-Mediated Downstream Activation of Brachyury in Mice. Biol Reprod 2011; 85:1114-1123. [DOI:10.1095/biolreprod.111.091793]
3. Hofmann MC, Braydich-Stolle L, Dym M. Isolation of male germ-line stem cells; influence of GDNF. Dev Biol 2005; 279: 114-124. [DOI:10.1016/j.ydbio.2004.12.006]
4. Kubota H, Avarbock MR, Brinster RL. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 2004; 101: 16489-16494. [DOI:10.1073/pnas.0407063101]
5. Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Ogura A, et al. Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions. Biol Reprod 2005; 72: 985-991. [DOI:10.1095/biolreprod.104.036400]
6. Hermann BP, Sukhwani M, Lin CC, Sheng Y, Tomko J, Rodriguez M, et al. Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques. Stem Cells 2007; 25: 2330-2338. [DOI:10.1634/stemcells.2007-0143]
7. Clermont Y. The cycle of the seminiferous epithelium in man. Am J Anat 1963; 112: 35-51. [DOI:10.1002/aja.1001120103]
8. Clermont Y. Spermatogenesis in man. A study of the spermatogonial population. Fertil Steril 1966; 17: 705-721. [DOI:10.1016/S0015-0282(16)36120-9]
9. Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 1972; 52: 198-236. [DOI:10.1152/physrev.1972.52.1.198]
10. Dym M, Kokkinaki M, He Z. Spermatogonial stem cells: mouse and human comparisons. Birth Defects Res C Embryo Today 2009; 87: 27-34. [DOI:10.1002/bdrc.20141]
11. Kanatsu-Shinohara M, Ogonuki N, Morimoto H, Ogura A, Shinohara T. Serum- and feeder-free culture of mouse germline stem cells. Biol Reprod 2011; 84: 97-105. [DOI:10.1095/biolreprod.110.086462]
12. Hermann BP, Sukhwani M, Simorangkir DR, Chu T, Plant TM, Orwig KE. Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques. Hum Reprod 2009; 24: 1704-1716. [DOI:10.1093/humrep/dep073]
13. Seandel M, James D, Shmelkov SV, Falciatori I, Kim J, Chavala S, et al. Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature 2007; 449: 346-350. [DOI:10.1038/nature06129]
14. He Z, Kokkinaki M, Jiang J, Dobrinski I, Dym M. Isolation, characterization, and culture of human spermatogonia. Biol Reprod 2010; 82: 363-372. [DOI:10.1095/biolreprod.109.078550]
15. Meng X, Lindahl M, Hyvönen ME, Parvinen M, de Rooij DG, Hess MW, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000; 287: 1489-1493. [DOI:10.1126/science.287.5457.1489]
16. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci USA 1994; 91: 11303-11307. [DOI:10.1073/pnas.91.24.11303]
17. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA 1994; 91: 11298-11302. [DOI:10.1073/pnas.91.24.11298]
18. Dann CT, Alvarado AL, Molyneux LA, Denard BS, Garbers DL, Porteus MH. Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation. Stem Cells 2008; 26: 2928-2937. [DOI:10.1634/stemcells.2008-0134]
19. Sadri-Ardekani H, Mizrak S, van Daalen S, Korver C, Roepers-Gajadien H, Koruji M, et al. Propagation of Human Spermatogonial Stem Cells In Vitro. JAMA 2009; 302: 2127-2134. [DOI:10.1001/jama.2009.1689]
20. Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med 1996; 2: 693-696. [DOI:10.1038/nm0696-693]
21. Mizrak SC, Chikhovskaya JV, Sadri-Ardekani H, van Daalen S, Korver CM, Hovingh SE, et al. Embryonic stem cell-like cells derived from adult human testis. Hum Reprod 2010; 25: 158-167. [DOI:10.1093/humrep/dep354]
22. 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. [DOI:10.1242/dev.02004]
23. Ebata KT, Yeh JR, Zhang X, Nagano MC. The application of biomarkers of spermatogonial stem cells for restoring male fertility. Dis Markers 2008; 24: 267-276. [DOI:10.1155/2008/536020]
24. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, et al. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 2004; 36: 653-659. [DOI:10.1038/ng1367]

Send email to the article author


Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Designed & Developed by : Yektaweb