Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells

Abstract

The milieu of testicular germline stem cells (mGSCs) is characterized as low oxygen (O 2 ) environment, whereas, there in-vitro expansion is typically performed under normoxia (20-21% O 2 ). Here, we evaluated and compared the culture and multilineage differentiation characteristics of enriched (through differential platting and percoll density centrifugation) caprine mGSCs (cmGSCs) under hypoxic (5% O 2 ) and normoxic (21% O 2 ) culture conditions. For this, in addition to growth characteristics and populationdoubling time (PDT); viability, proliferation, senescence, and expression of key-markers of adhesion (βintegrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated and compared under normoxia and hypoxia. Moreover, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) was assessed. The survival, viability and proliferation were signi cantly promoted and PDT was reduced (p < 0.05), thus yielding a higher number of viable cells with larger colonies under hypoxia. Furthermore, expression of stemness and adhesion markers was distinctly increased under lowered O 2 condition. Conversely, the presence of differentiated regions and expression of differentiation speci c key genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were signi cantly (p < 0.05) reduced under hypoxic conditions. These data demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics, and stemness but not the multilineage differentiation potential of cmGSCs as compared with normoxia. These data are important for the development of robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

Different effects of sugars and methods to preserve post-thaw functional properties of cryopreserved caprine spermatogonial stem cells

“…Spermatogonial stem cells (SSCs) are the male germline stem cells of the seminiferous tubules in the testis and are the creator cells of spermatogenesis [Kanatsu-Shinohara et al, 2008]. The culture of SSCs is required for their expansion, differentiation [Singh et al, 2021], and manipulation. However, subsequent subcultures over time resulted in a gradual decline in proliferation rate, differentiation into other cell types, and higher senescence and apoptosis of putative SSCs [Zhu et al, 2013].…”