Improved Protocol to Study Osteoblast and Adipocyte Differentiation Balance

Adipogenesis-osteoblastogenesis balance-rupture is relevant in multiple diseases. Current human mesenchymal stem cells (hMSCs) in vitro differentiation models are expensive, and are hardly reproducible. Their scarcity and variability make an affordable and reliable method to study adipocyte-osteoblast-equilibrium difficult. Moreover, media composition has been inconstant throughout the literature. Our aims were to compare improved differentiation lab-made media with consensus/commercial media, and to identify a cell-line to simultaneously evaluate both MSCs differentiations. Lab-made media were compared with consensus and commercial media in C3H10T1/2 and hMSC, respectively. Lab-made media were tested on aged women primary pre-osteoblast-like cells. To determine the optimum cell line, C3H10T1/2 and hMSC-TERT cells were differentiated to both cell fates. Differentiation processes were evaluated by adipocytic and osteoblastic gene-markers expression and staining. Lab-made media significantly increased consensus medium induction and overcame commercial media in hMSCs differentiation to adipocytes and osteoblasts. Pre-osteoblast-like cells only properly differentiate to adipocyte. Lab-made media promoted adipocyte gene-markers expression in C3H10T1/2 and hMSC-TERT, and osteoblast gene-markers in C3H10T1/2. Oil Red O and Alizarin Red staining supported these findings. Optimized lab-made media were better at differentiating MSCs compared to consensus/commercial media, and evidenced the adipogenic commitment of pre-osteoblast-like cells from aged-women. C3H10T1/2 is an optimum MSC line by which to study adipocyte-osteoblast differentiation balance.

Automated summary

The balance between adipogenesis and osteoblastogenesis is relevant in multiple diseases. Current in vitro differentiation models for human mesenchymal stem cells (hMSCs) are expensive and hardly reproducible, making it difficult to study the equilibrium between adipocytes and osteoblasts in an affordable and reliable manner. In this study, lab-made media were compared with consensus and commercial media, and a cell-line (C3H10T1/2) was identified to simultaneously evaluate both MSCs differentiations. Lab-made media significantly increased the differentiation of hMSCs into adipocytes and osteoblasts. Additionally, the lab-made media promoted adipocyte gene-markers expression in C3H10T1/2 and hMSC-TERT and osteoblast gene-markers in C3H10T1/2. The optimized lab-made media proved to be better at differentiating MSCs compared to consensus/commercial media and demonstrated the adipogenic commitment of pre-osteoblast-like cells from aged-women. C3H10T1/2 is an ideal MSC line for studying the balance between adipocytes and osteoblasts.