Enhancing adipogenic differentiation efficiency and gene expression in yak stromal vascular cells through 3D spheroid...

Intramuscular fat deposition is a key determinant of meat quality in livestock species; however, yak (Bos grunniens) stromal vascular cells (SVCs) exhibit relatively low adipogenic differentiation efficiency under conventional in vitro conditions. In this study, we established a three-dimensional (3D) spheroid culture system to improve adipogenic differentiation of yak SVCs and compared it with traditional two-dimensional (2D) monolayer culture. Yak SVCs were isolated from longissimus dorsi muscle and characterized prior to differentiation. Cells were subjected to adipogenic induction under both 2D and 3D conditions. Adipogenic differentiation was evaluated by morphological observation, Oil Red O staining and quantification, and quantitative real-time PCR (RT-qPCR) analysis of adipogenic marker genes. Additionally, Western blot analysis of key adipogenic proteins was performed specifically on yak SVCs. To validate the induction system, murine C3H10T1/2 cells were included as a parallel control group and evaluated using the aforementioned non-protein assays. The results showed that 3D culture significantly enhanced lipid accumulation and increased the expression of adipogenic marker genes compared with 2D culture in yak SVCs. Consistently, protein levels of PPAR γ were significantly elevated in the 3D group of yak SVCs, while FABP4 showed an upward trend. Furthermore, the C3H10T1/2 control group exhibited successful adipogenic differentiation with similarly enhanced lipid accumulation and gene expression under 3D conditions, confirming the reliability of our system. In conclusion, 3D spheroid culture improves adipogenic differentiation of yak SVCs and provides a useful in vitro model for studying adipose development in yak. This system may facilitate future studies on the regulation of intramuscular fat deposition in livestock species.