Development and characterization of a standardized adipogenesis assay for testing metabolism disrupting chemicals using human bone marrow derived mesenchymal stem cells

• Standardized hBM-MSCs assay to screen chemicals promoting adipogenesis. • Optimized cell density, fluorescence detection, and assay reproducibility. • Evaluated six chemicals’ effects on adipogenesis with high assay reliability. • Established an immortalized hBM-MSC line with results matching primary cells. • hBM-MSCs assay showed strong correlation with the 3T3-L1 adipogenesis model. Obesity is a global health concern linked to various diseases. Recently, chemicals known as "obesogens" have been identified as significant contributors to obesity, highlighting the need for reliable methods to screen chemicals for their obesity-promoting potential. This study aimed to develop a standardized adipogenesis assay using primary human bone marrow-derived multipotent mesenchymal stromal stem cells (hBM-MSCs) to evaluate the adipogenic potential of chemicals. Lipid accumulation in hBM-MSCs was measured after chemical exposure. Assay conditions including cell confluency, fluorescent staining of neutral lipids and fluorometric detection were optimized and standardized. Using the established method, we evaluated the effects of six selected chemicals on the adipogenic responses of hBM-MSCs from different donors, including an in-house generated immortalized hBM-MSC cell line. Not all donor-derived hBM-MSCs were shown to be suitable for the assay, but we observed comparable results among suitable hBM-MSCs and between primary hBM-MSCs and their immortalized derivative cell line. Comparisons with the well-established 3T3-L1 preadipocyte model revealed a strong correlation with the hBM-MSCs assay. We observed that experimental details such as cell density, fluorescent measurement and cell donors can greatly affect outcomes; this stresses the importance of a well described standard operating procedure (SOP). Our developed test method, including a detailed SOP, paves the way for a validated test methodology that is urgently needed and currently lacking for the identification of obesogenic endocrine disruptors.