Investigation of Tumorigenicity ability of Immature Male Mouse Spermatogonial Stem Cells after In Vitro Cultivation and Inoculation in Athymic Animals

Authors
Z. Mazaheri 1
M. Haddadi 2
Z. Mazaheri 1
S. Amanpour 2
S. Muhammadnejad 2
A. Muhammadnejad 2
M. Movahedin 3
1 Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2 Cancer Research Center, Cancer Institute of Iran, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Anatomical sciences, Medical Sciences Faculty, Tarbiat Modares University, Tehran, Iran
Abstract
Objective: It is hypothesized that stem cells have the capability to form tumors after transplantation. Spermatogonial stem cells have proliferation potency and colonization ability related to express pluripotency genes such as c-Myc. The primary aim of this study is to investigate tumorigenicity ability of these cells after in vitro cultivation and inoculation in athymic animals. Methods: Spermatogonial stem cells from 3-5 day-old neonatal mice testes (NMRI) were cultured following two-step enzymatic digestion. After one month of culturing the spermatogonial stem cells, the obtained colonies were identified by Oct4 and PLZF markers. Expressions of Nanog, Oct4 and c-Myc pluripotency genes were subsequently studied. We subcutaneously inoculated 5 x 106 cells into athymic mice and assessed tumor formation after 8 weeks. Mouse embryonic stem cells (CCE line) were used as the positive control. Generated tumors were measured by a caliper. Results: The colonies expressed Oct4 and PLZF proteins. Ratio of pluripotency gene expressions in these cells compared to embryonic stem cells significantly decreased (P≤0.05). Mouse embryonic stem cells formed tumors however the spermatogonial colonies did not form any tumors. Conclusion: Mouse spermatogonial stem cells in comparison with embryonic stem cells are not capable of forming tumors in vivo. We have observed that the tumorigenic ability of these cells decreased significantly with down regulation of pluripotency gene expressions, particularly c-Myc. However, this study should be reassessed by using human tissue samples.

Keywords

[1]     Hou J, Yang S, Yang H, Liu Y, Liu Y, Hai Y, Chen Z, Guo Y, Gong Y, Gao WQ, Li Z, He Z. Generation of male differentiated germ cells from various types of stem cells. Reproduction 2014; 147(6): R179-R188.
[2]     Jain M, Halder A.Sertoli cell only syndrome: Status of sertoli cell maturation and function. Indian J Endocrinol Metab 2012; 16(Suppl 2): S512-3.
[3]     Marques-Mari AI, Lacham-Kaplan O, Medrano JV, Pellicer A, Simón C. Differentiation of germ cells and gametes from stem cells. Hum Reprod Update 2009; 15(3): 379-90.
[4]     Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126(4): 663-76.
[5]     Gardner LB, Lee LA, Dang CV. c-Myc protooncogene. In: Bertino J, editor. Encyclopedia of cancer. Vol 1. 2nd ed. San Diego (CA): Academic Press, 2002; P: 555–61.
[6]     Toyooka Y, Tsunekawa N, Akasu R, Noce T. Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A 2003; 100(20): 11457-62.
[7]     Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004; 427(6970): 148-54.
[8]     Hübner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF 3rd, Boiani M, Schöler HR. Derivation of oocytes from mouse embryonic stem cells. Science 2003; 300(5623): 1251-6.
[9]     Koruji M, Movahedin M, Mowla SJ, Gourabi H, Arfaee AJ. Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions. In Vitro Cell Dev Biol Anim 2009; 45(5-6): 281-9.
[10] Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994; 91(24): 11298-302.
[11] Tokuda M, Kadokawa Y, Kurahashi H, Marunouchi T. CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes. Biol Reprod 2007; 76(1): 130-41.
[12] Hofmann MC, Braydich-Stolle L, Dym M. Isolation of male germ-line stem cells; influence of GDNF. Dev Biol 2005; 279(1): 114-24.
[13] Makoolati Z, Movahedin M, Forouzandeh-Moghadam M. Effects of different doses of bone morphogenetic protein 4 on viability and proliferation rates of mouse embryonic stem cells. Yakhteh 2009; 11(1): 29-34.
[14] Mazaheri Z, Movahedin M, Rahbarizadeh F, Amanpour S. Generation of In-vitro Spermatogonial Stem Cells following Genetic Manipulation of Primordial Germ-like Cells. Avicenna J Med Biotechnol 2012; 4(2): 55-63.
[15] Rous P. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J Exp Med 1911; 13(4): 397-411.
[16] Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448(7151): 313-7.
[17] Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008; 26(1): 101-6.
[18] Alitalo K, Bishop JM, Smith DH, Chen EY, Colby WW, Levinson AD. Nucleotide sequence to the v-myc oncogene of avian retrovirus MC29. Proc Natl Acad Sci U S A 1983; 80(1): 100-4.
[1]     Hou J, Yang S, Yang H, Liu Y, Liu Y, Hai Y, Chen Z, Guo Y, Gong Y, Gao WQ, Li Z, He Z. Generation of male differentiated germ cells from various types of stem cells. Reproduction 2014; 147(6): R179-R188.
[2]     Jain M, Halder A.Sertoli cell only syndrome: Status of sertoli cell maturation and function. Indian J Endocrinol Metab 2012; 16(Suppl 2): S512-3.
[3]     Marques-Mari AI, Lacham-Kaplan O, Medrano JV, Pellicer A, Simón C. Differentiation of germ cells and gametes from stem cells. Hum Reprod Update 2009; 15(3): 379-90.
[4]     Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126(4): 663-76.
[5]     Gardner LB, Lee LA, Dang CV. c-Myc protooncogene. In: Bertino J, editor. Encyclopedia of cancer. Vol 1. 2nd ed. San Diego (CA): Academic Press, 2002; P: 555–61.
[6]     Toyooka Y, Tsunekawa N, Akasu R, Noce T. Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A 2003; 100(20): 11457-62.
[7]     Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004; 427(6970): 148-54.
[8]     Hübner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF 3rd, Boiani M, Schöler HR. Derivation of oocytes from mouse embryonic stem cells. Science 2003; 300(5623): 1251-6.
[9]     Koruji M, Movahedin M, Mowla SJ, Gourabi H, Arfaee AJ. Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions. In Vitro Cell Dev Biol Anim 2009; 45(5-6): 281-9.
[10] Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994; 91(24): 11298-302.
[11] Tokuda M, Kadokawa Y, Kurahashi H, Marunouchi T. CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes. Biol Reprod 2007; 76(1): 130-41.
[12] Hofmann MC, Braydich-Stolle L, Dym M. Isolation of male germ-line stem cells; influence of GDNF. Dev Biol 2005; 279(1): 114-24.
[13] Makoolati Z, Movahedin M, Forouzandeh-Moghadam M. Effects of different doses of bone morphogenetic protein 4 on viability and proliferation rates of mouse embryonic stem cells. Yakhteh 2009; 11(1): 29-34.
[14] Mazaheri Z, Movahedin M, Rahbarizadeh F, Amanpour S. Generation of In-vitro Spermatogonial Stem Cells following Genetic Manipulation of Primordial Germ-like Cells. Avicenna J Med Biotechnol 2012; 4(2): 55-63.
[15] Rous P. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J Exp Med 1911; 13(4): 397-411.
[16] Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448(7151): 313-7.
[17] Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008; 26(1): 101-6.
[18] Alitalo K, Bishop JM, Smith DH, Chen EY, Colby WW, Levinson AD. Nucleotide sequence to the v-myc oncogene of avian retrovirus MC29. Proc Natl Acad Sci U S A 1983; 80(1): 100-4.
Pathobiology Reserach
Volume 17, Issue 1
April 2014
Pages 17-27