Development and Validation of a Standardized Adipogenesis Assay Using Human Bone Marrow Mesenchymal Stem Cells

Obesity is a global health concern linked to various diseases. Recently, chemicals known as "obesogens" have been identified as significant contributors to obesity, which highlighting the need for reliable methods to screen chemicals for their obesity-promoting potential. This study aimed to develop a standardized adipogenesis assay using human bone marrow-derived mesenchymal stem cells (hBM-MSCs) to evaluate the adipogenic potential of chemicals. Lipid accumulation in hBM-MSCs was measured after chemical exposure. The assay conditions of cell confluency and detection methods were optimized. Results showed that initiating differentiation at complete confluency was crucial for reliable outcomes. Reliable data was ensured by detecting at least nine points per well, and fluorescence signals remained stable for up to ten days post-staining. Using the established method, we evaluated the effects of six selected chemicals on adipogenesis. Testing adipogenic responses of hBM-MSCs from different donors identified specific cell lots suitable for studies. Comparisons with the 3T3-L1 adipogenesis assay validated the hBM-MSCs model. Additionally, we immortalized an hBM-MSC cell line which displayed similar gene expression profiles to primary cells showed greater sensitivity to adipogenesis-inducing chemicals. These findings confirm the robustness and reliability of the hBM-MSC adipogenesis assay for studying the adipogenesis effects of chemicals. Overall design: Primary human mesenchymal stem cells were immortalized using Lenti-hTERT method and RNA-seqencing was performed to examine transcriptomal similiarity between original cells verse immortalized cells after 14-day adipogenesis