Development of a Human Adipose-Derived Stem Cell Model for Characterization of Chemical Modulation of Adipogenesis

Abstract

Abstract

Environmental influence is a significant factor governing the relationship over an individual's genetic predisposition toward obesity. A largely unexplored avenue is the notion of obesogens: environmental endocrine disrupting chemicals (EDCs) that may alter the propensity toward obesity, particularly during prenatal and early-life exposure. In an effort to establish a human-based platform for concentration–response assessments of novel candidate EDCs, human adipose-derived stem cells (ASCs) were selected as a biologically relevant primary cell model. A quantitative fluorescence-based readout of neutral lipid accumulation was used as a phenotypic endpoint to define a priming cocktail that could initiate lineage progression, but maintain an appreciable assay window (Z′-factor=0.85) with consistency across multiple human donor ASC populations. Concentration–response analysis of the uncoupled cocktail components demonstrates the feasibility of testing compounds with suspected modes of action for the myriad signaling pathways implicated in adipogenesis. The organotin tributyltin (TBT) was used as a reference compound for assay validation and found to enhance the effects of adipogenesis in the presence of priming cocktail. In addition, expression of key adipogenic genes WNT2, CEBPA, PPARG, FABP4, and PLIN1 were significantly modulated by TBT. Lactate dehydrogenase (LDH) and half-maximal toxicity concentration (TC₅₀) endpoint measurements were multiplexed with the protocol to quantitatively address chemical-mediated cytotoxicity. Application of an organotin training set on the platform demonstrated robust concentration–response curves with lowest observed effect levels of lipid accumulation well below respective LDH and TC₅₀ measurements. Finally, PPARG siRNA-mediated loss of function was used to distinguish the independent contributions of organotin-mediated lipid accumulation from transcriptional reprogramming of adipogenesis. The findings demonstrate an in vitro platform representative of human biology that is both scalable and quantitative for concentration–response characterization of novel EDCs that may alter the developmental dynamics of human adipogenesis.