Tissue Eng Regen Med.  2017 Oct;14(5):557-566. 10.1007/s13770-017-0058-x.

Enrichment and In Vitro Culture of Spermatogonial Stem Cells from Pre-Pubertal Monkey Testes

• Kim YH
• Kang HG
• Kim BJ
• Jung SE
• Karmakar P
• Kim SM
• Hwang S
• Ryu BY

• KMID: 2394989
• DOI: http://doi.org/10.1007/s13770-017-0058-x

Abstract

Spermatogonial stem cells (SSCs) are essential for spermatogenesis throughout the lifespan of the male. However, the rarity of SSCs has raised the need for an efficient selection method, but little is known about culture conditions that stimulate monkey SSC proliferation in vitro. In this study, we report the development of effective enrichment techniques and in vitro culturing of germ cells from pre-pubertal monkey testes. Testis cells were analyzed by fluorescence-activated cell sorting techniques and were transplanted into the testes of nude mice to characterize SSCs. Thy-1-positive cells showed a higher number of colonies than the unselected control after xenotransplantation. Extensive colonization of monkey cells in the mouse testes indicated the presence of highly enriched populations of SSCs in the Thy-1-positive sorted cells. Furthermore, monkey testis cells were enriched by differential plating using extracellular matrix, laminin, and gelatin, and then cultured under various conditions. Isolation of monkey testicular germ cells by differential plating increased germ cell purity by 2.7-fold, following the combinational isolation method using gelatin and laminin. These enriched germ cells actively proliferated under culture conditions involving StemPro medium supplemented with bFGF, GDNF, LIF, and EGF at 37 ℃. These results suggest that the enrichment and in vitro culture method proposed in the present study for harvesting a large number of functionally active monkey SSCs can be applied as the basis for efficient in vitro expansion of human SSCs.

Keyword

Monkey spermatogonial stem cells; Enrichment; in vitro culture

MeSH Terms

Animals
Colon
Epidermal Growth Factor
Extracellular Matrix
Flow Cytometry
Gelatin
Germ Cells
Glial Cell Line-Derived Neurotrophic Factor
Haplorhini*
Humans
In Vitro Techniques*
Laminin
Male
Methods
Mice
Mice, Nude
Spermatogenesis
Stem Cells*
Testis*
Transplantation, Heterologous
Epidermal Growth Factor
Gelatin
Glial Cell Line-Derived Neurotrophic Factor
Laminin