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Use of alginate hydrogel to improve long-term 3D culture of spermatogonial stem cells: stemness gene expression and structural features

Published online by Cambridge University Press:  13 October 2021

Masoud Hemadi ,
Vahideh Assadollahi ,
Ghasem Saki ,
Afshin Pirnia ,
Masoud Alasvand Open the ORCID record for Masoud Alasvand [Opens in a new window] ,
Abolfazl Zendehdel  and
Mohammadreza Gholami Open the ORCID record for Mohammadreza Gholami [Opens in a new window]
Masoud Hemadi
Affiliation:
Cellular and Molecular Research Center, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Vahideh Assadollahi
Affiliation:
Cancer and Immunology Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
Ghasem Saki
Affiliation:
Physiology Research Center, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Afshin Pirnia
Affiliation:
Medical Technology Research Center, Research Institute of Health Technology, Kermanshah University of Medical Sciences, Kermanshah, Iran
Masoud Alasvand
Affiliation:
Cancer and Immunology Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
Abolfazl Zendehdel
Affiliation:
Geriatric Department, Ziayian Hospital, Tehran University of Medical Sciences, Tehran, Iran
Mohammadreza Gholami*
Affiliation:
Medical Technology Research Center, Research Institute of Health Technology, Kermanshah University of Medical Sciences, Kermanshah, Iran
*
Author for correspondence: Mohammadreza Gholami. Medical Technology Research Center, Institute of Health Technology, Kermanshah University of Medical Sciences, Kermanshah Zip code: 67148-69914, Iran. Tel:/Fax: +98 8334276477. E-mail: [email protected]
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Summary

The quality and quantity of a spermatogonial stem-cell (SSC) culture can be measured in less time using a 3D culture in a scaffold. The present study investigated stemness gene expression and the morphological and structural characterization of SSCs encapsulated in alginate. SSCs were harvested from BALB/c neonatal mice testes through two-step mechanical and enzymatic digestion. The spermatogonial populations were separated using magnetic-activated cell sorting (MACS) using an anti-Thy1 antibody and c-Kit. The SSCs then were encapsulated in alginate hydrogel. After 2 months of SSC culturing, the alginate microbeads were extracted and stained to evaluate their histological properties. Real-time polymerase chain reaction (PCR) was performed to determine the stemness gene expression. Scanning electron microscopy (SEM) was performed to evaluate the SSC morphology, density and scaffold structure. The results showed that encapsulated SSCs had decreased expression of Oct4, Sox2 and Nanos2 genes, but the expression of Nanog, Bcl6b and Plzf genes was not significantly altered. Histological examination showed that SSCs with pale nuclei and numerous nucleolus formed colonies. SEM evaluation revealed that the alginate scaffold structure preserved the SSC morphology and density for more than 60 days. Cultivation of SSCs on alginate hydrogel can affect Oct4, Sox2 and Nanos2 expression.

Keywords

Alginate hydrogelCell culture techniqueEncapsulatedMouseSpermatogonial stem cellsStemness genes
Type
Research Article
Information
Zygote , Volume 30 , Issue 3 , June 2022 , pp. 312 - 318
Copyright
© The Author(s), 2021. Published by Cambridge University Press

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References

Afsartala, Z, Rezvanfar, MA, Hodjat, M, Tanha, S, Assadollahi, V, Bijangi, K, Abdollahi, M and Ghasemzadeh-Hasankolaei, M (2016). Amniotic membrane mesenchymal stem cells can differentiate into germ cells in vitro . In Vitro Cell Dev Biol Anim 52, 1060–71.CrossRefGoogle ScholarPubMed
Aponte, PM, Van Bragt, MP, De Rooij, DG and Van Pelt, AM (2005). Spermatogonial stem cells: characteristics and experimental possibilities. APMIS 113(11–12), 727–42.CrossRefGoogle ScholarPubMed
Assadollahi, V, Fathi, F, Abdi, M, Khadem Erfan, MB, Soleimani, F and Banafshi, O (2019a). Increasing maternal age of blastocyst affects on efficient derivation and behavior of mouse embryonic stem cells. J Cell Biochem 120, 3716–26.CrossRefGoogle ScholarPubMed
Assadollahi, V, Hassanzadeh, K, Abdi, M, Alasvand, M, Nasseri, S and Fathi, F (2019b). Effect of embryo cryopreservation on derivation efficiency, pluripotency, and differentiation capacity of mouse embryonic stem cells. J Cell Physiol 234, 21962–72.CrossRefGoogle ScholarPubMed
Azevedo, MA, Bourbon, AI, Vicente, AA and Cerqueira, MA (2014). Alginate/chitosan nanoparticles for encapsulation and controlled release of vitamin B2. Int J Biol Macromol 71, 141–6.CrossRefGoogle ScholarPubMed
Barralet, JE, Wang, L, Lawson, M, Triffitt, JT, Cooper, PR and Shelton, RM (2005). Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels. J Mater Sci Mater Med 16, 515–9.CrossRefGoogle ScholarPubMed
Bidarra, SJ and Barrias, CC (2019). 3D culture of mesenchymal stem cells in alginate hydrogels. Methods Mol Biol 2002, 165–80.CrossRefGoogle ScholarPubMed
Brinster, RL (2007). Male germline stem cells: from mice to men. Science 316(5823), 404–5.CrossRefGoogle Scholar
Buaas, FW, Kirsh, AL, Sharma, M, McLean, DJ, Morris, JL, Griswold, MD, de Rooij, DG and Braun, RE (2004). Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 36, 647–52.CrossRefGoogle ScholarPubMed
Caires, K, Broady, J and McLean, D (2010). Maintaining the male germline: regulation of spermatogonial stem cells. J Endocrinol 205, 133–45.CrossRefGoogle ScholarPubMed
de Oliveira, TV Tesser, RB Nunes, N Stumpp, T (2019).Dynamics of Sox2 expression during rat germ cell development and its relationship with emergence of spermatogonia. bioRciv doi: https://doi.org/10.1101/558015 [preprint]CrossRefGoogle Scholar
Gholami, M, Hemadi, M, Saki, G, Zendedel, A, Khodadadi, A and Mohammadi-Asl, J (2013a). Does prepubertal testicular tissue vitrification influence spermatogonial stem cells (SSCs) viability? J Assist Reprod Genet 30, 1271–7.CrossRefGoogle ScholarPubMed
Gholami, M, Saki, G, Hemadi, M, Khodadadi, A and Mohamma-di-Asl, J (2013b). Effect of melatonin on the expression of apoptotic genes in vitrified-thawed spermatogonia stem cells Type A of 6-day-old mice. Iran J Basic Med Sci 16, 906–9.Google ScholarPubMed
Gholami, M, Saki, G, Hemadi, M, Khodadadi, A and Mohammadi-Asl, J (2014). Melatonin improves spermatogonial stem cells transplantation efficiency in azoospermic mice. Iran J Basic Med Sci 17, 93–9.Google ScholarPubMed
Giudice, MG, De Michele, F, Poels, J, Vermeulen, M and Wyns, C (2017). Update on fertility restoration from prepubertal spermatogonial stem cells: how far are we from clinical practice? Stem Cell Res 21, 171–7.CrossRefGoogle ScholarPubMed
Hai, Y, Hou, J, Liu, Y, Liu, Y, Yang, H, Li, Z and He, Z (2014). The roles and regulation of Sertoli cells in fate determinations of spermatogonial stem cells and spermatogenesis. Semin Cell Dev Biol 29, 66–75.CrossRefGoogle ScholarPubMed
Hofmann, MC (2008). Gdnf signaling pathways within the mammalian spermatogonial stem cell niche. Mol Cell Endocrinol 288(1–2), 95–103.CrossRefGoogle ScholarPubMed
Huleihel, M, Nourashrafeddin, S and Plant, TM (2015). Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta). Asian J Androl 17, 972–80.CrossRefGoogle Scholar
Ishii, K, Kanatsu-Shinohara, M, Toyokuni, S and Shinohara, T (2012). FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b through MAP2K1 activation. Development 139, 1734–43.CrossRefGoogle ScholarPubMed
Jalayeri, M, Pirnia, A, Najafabad, EP, Varzi, AM and Gholami, M (2017). Evaluation of alginate hydrogel cytotoxicity on three-dimensional culture of type A spermatogonial stem cells. Int J Biol Macromol 95, 888–94.CrossRefGoogle ScholarPubMed
Kanatsu-Shinohara, M and Shinohara, T (2013). Spermatogonial stem cell self-renewal and development. Annu Rev Cell Dev Biol 29, 163–87.CrossRefGoogle ScholarPubMed
Kanatsu-Shinohara, M, Lee, J, Inoue, K, Ogonuki, N, Miki, H, Toyokuni, S, Ikawa, M, Nakamura, T, Ogura, A and Shinohara, T (2008). Pluripotency of a single spermatogonial stem cell in mice. Biol Reprod 78, 681–7.CrossRefGoogle ScholarPubMed
Ketkar, A and Reddy, KVR (2012). Effect of Oct-4 silencing on proliferation and differentiation of mouse undifferentiated Type A spermatogonial cells. J Cell Sci Ther 3, 131.Google Scholar
Khajavi, N, Akbari, M, Abdolsamadi, HR, Abolhassani, F, Dehpour, AR, Koruji, M and Habibi Roudkenar, M (2014). Role of somatic testicular cells during mouse spermatogenesis in three-dimensional collagen gel culture system. Cell J 16, 79–90.Google ScholarPubMed
Kubota, H, Avarbock, MR and Brinster, RL (2004). Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA 101, 16489–94.CrossRefGoogle ScholarPubMed
Lewicki, J, Bergman, J, Kerins, C and Hermanson, O (2019). Optimization of 3D bioprinting of human neuroblastoma cells using sodium alginate hydrogel. Bioprinting 16, e00053.CrossRefGoogle Scholar
Lim, F and Sun, AM (1980). Microencapsulated islets as bioartificial endocrine pancreas. Science 210(4472), 908–10.CrossRefGoogle ScholarPubMed
Mahmoud, H (2012). Concise review: spermatogenesis in an artificial three-dimensional system. Stem Cell 30, 2355–60.CrossRefGoogle Scholar
McLean, DJ, Friel, PJ, Johnston, DS and Griswold, MD (2003). Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice. Biol Reprod 69, 2085–91.CrossRefGoogle ScholarPubMed
Meng, X, Lindahl, M, Hyvönen, ME, Parvinen, M, de Rooij, DG, Hess, MW, Raatikainen-Ahokas, A, Sainio, K, Rauvala, H, Lakso, M, Pichel, JG, Westphal, H, Saarma, M and Sariola, H (2000). Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287(5457), 1489–93.CrossRefGoogle ScholarPubMed
Mohammadzadeh, E, Mirzapour, T, Nowroozi, MR, Nazarian, H, Piryaei, A, Alipour, F, Modarres Mousavi, SM and Ghaffari Novin, M (2019). Differentiation of spermatogonial stem cells by soft agar three-dimensional culture system. Artif Cells Nanomed Biotechnol 47, 1772–81.CrossRefGoogle ScholarPubMed
Nagano, M, Ryu, BY, Brinster, CJ, Avarbock, MR and Brinster, RL (2003). Maintenance of mouse male germ line stem cells in vitro . Biol Reprod 68, 2207–14.CrossRefGoogle ScholarPubMed
O’Shea, GM and Sun, AM (1986). Encapsulation of rat islets of Langerhans prolongs xenograft survival in diabetic mice. Diabetes 35, 943–6.CrossRefGoogle ScholarPubMed
Oatley, JM, Avarbock, MR, Telaranta, AI, Fearon, DT and Brinster, RL (2006). Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc Natl Acad Sci USA 103, 9524–9.CrossRefGoogle ScholarPubMed
Oatley, JM, Avarbock, MR and Brinster, RL (2007). Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. J Biol Chem 282, 25842–51.CrossRefGoogle ScholarPubMed
Oatley, JM, Kaucher, AV, Avarbock, MR and Brinster, RL (2010). Regulation of mouse spermatogonial stem cell differentiation by STAT3 signaling. Biol Reprod 83, 427–33.CrossRefGoogle ScholarPubMed
Orwig, KE, Ryu, BY, Master, SR, Phillips, BT, Mack, M, Avarbock, MR, Chodosh, L and Brinster, RL (2008). Genes involved in post-transcriptional regulation are overrepresented in stem/progenitor spermatogonia of cryptorchid mouse testes. Stem Cell 26, 927–38.CrossRefGoogle ScholarPubMed
Patra, SK, Vemulawada, C, Soren, MM, Sundaray, JK, Panda, MK and Barman, HK (2018). Molecular characterization and expression patterns of Nanog gene validating its involvement in the embryonic development and maintenance of spermatogonial stem cells of farmed carp, Labeo rohita . J Anim Sci Biotechnol 9, 45.CrossRefGoogle ScholarPubMed
Pesce, M, Wang, X, Wolgemuth, DJ and Schöler, HR (1998). Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation. Mech Dev 71(1–2), 89–98.CrossRefGoogle ScholarPubMed
Phillips, BT, Gassei, K and Orwig, KE (2010). Spermatogonial stem cell regulation and spermatogenesis. Phil Trans Roy Soc Lond B 365(1546), 1663–78.CrossRefGoogle ScholarPubMed
Pirnia, A, Parivar, K, Hemadi, M, Yaghmaei, P and Gholami, M (2017). Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation. Andrologia 49, e12650.CrossRefGoogle ScholarPubMed
Pravdyuk, AI, Petrenko, YA, Fuller, BJ and Petrenko, AY (2013). Cryopreservation of alginate encapsulated mesenchymal stromal cells. Cryobiology 66, 215–22.CrossRefGoogle ScholarPubMed
Sarker, B, Rompf, J, Silva, R, Lang, N, Detsch, R, Kaschta, J, Fabry, B and Boccaccini, AR (2015). Alginate-based hydrogels with improved adhesive properties for cell encapsulation. Int J Biol Macromol 78, 72–8.CrossRefGoogle ScholarPubMed
Schmidt, JA, Avarbock, MR, Tobias, JW and Brinster, RL (2009). Identification of glial cell line-derived neurotrophic factor-regulated genes important for spermatogonial stem cell self-renewal in the rat. Biol Reprod 81, 56–66.CrossRefGoogle ScholarPubMed
Sharpe, R (1994) Regulation of spermatogenesis. In The Physiology of Reproduction (Knobil, E and Neill, JD eds). Raven Press: New York. pp. 1363–434.Google Scholar
Sofikitis, N, Pappas, E, Kawatani, A, Baltogiannis, D, Loutradis, D, Kanakas, N, Giannakis, D, Dimitriadis, F, Tsoukanelis, K, Georgiou, I, Makrydimas, G, Mio, Y, Tarlatzis, V, Melekos, M and Miyagawa, I (2005). Efforts to create an artificial testis: culture systems of male germ cells under biochemical conditions resembling the seminiferous tubular biochemical environment. Hum Reprod Update 11, 229–59.CrossRefGoogle ScholarPubMed
Song, HW and Wilkinson, MF (2014). Transcriptional control of spermatogonial maintenance and differentiation. Semin Cell Dev Biol 30, 14–26.CrossRefGoogle ScholarPubMed
Wang, N, Adams, G, Buttery, L, Falcone, FH and Stolnik, S (2009). Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells. J Biotechnol 144, 304–12.CrossRefGoogle ScholarPubMed
Wu, X, Oatley, JM, Oatley, MJ, Kaucher, AV, Avarbock, MR and Brinster, RL (2010). The POU domain transcription factor POU3F1 is an important intrinsic regulator of GDNF-induced survival and self-renewal of mouse spermatogonial stem cells. Biol Reprod 82, 1103–11.CrossRefGoogle ScholarPubMed
Yang, L, Wu, W and Qi, H (2013). Gene expression profiling revealed specific spermatogonial stem cell genes in mouse. Genesis 51, 83–96.CrossRefGoogle Scholar
Yeh, JR, Zhang, X and Nagano, MC (2007). Establishment of a short-term in vitro assay for mouse spermatogonial stem cells. Biol Reprod 77, 897–904.CrossRefGoogle ScholarPubMed
Ziloochi Kashani, M, Bagher, Z, Asgari, HR, Najafi, M, Koruji, M and Mehraein, F (2020). Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold. Syst Biol Reprod Med 66, 202–15.CrossRefGoogle ScholarPubMed