Human differentiated adipocytes can serve as surrogate mature adipocytes for adipocyte-derived extracellular vesicle analysis

Abstract Obesity is a growing global health concern, contributing to diseases such as cancer, autoimmune disorders, and neurodegenerative conditions. Adipose tissue dysfunction, characterized by abnormal adipokine secretion and chronic inflammation, plays a key role in these conditions. Adipose-derived extracellular vesicles (ADEVs) have emerged as critical mediators in obesity-related diseases. However, the study of mature adipocyte-derived EVs (mAdipo-EVs) is limited due to the short lifespan of mature adipocytes in culture, low EV yields, and the low abundance of these EV subpopulations in the circulation. Additionally, most studies rely on rodent models, which have differences in adipose tissue biology compared to humans. To overcome these challenges, we developed a standardized approach for differentiating human preadipocytes (preAdipos) into mature differentiated adipocytes (difAdipos), which produce high-yield, human adipocyte EVs (Adipo-EVs). Using visceral adipose tissue from bariatric surgical patients, we isolated the stromal vascular fraction (SVF) and differentiated preAdipos into difAdipos. Brightfield microscopy revealed that difAdipos exhibited morphological characteristics comparable to mature adipocytes (mAdipos) directly isolated from visceral adipose tissue, confirming their structural similarity. Additionally, qPCR analysis demonstrated decreased preadipocyte markers and increased mature adipocyte markers, further validating successful differentiation. Functionally, difAdipos exhibited lipolytic activity comparable to mAdipos, supporting their functional resemblance to native adipocytes. We then isolated preAdipo-EVs and difAdipo-EVs using tangential flow filtration and characterized them using bulk and single EV analysis. DifAdipo-EVs displayed classical EV and adipocyte-specific markers, with significant differences in biomarker expression compared to preAdipo-EVs. These findings demonstrate that difAdipos serve as a reliable surrogate for mature adipocytes, offering a consistent and scalable source of adipocyte-derived EVs for studying obesity and its associated disorders. Competing Interest Statement The authors have declared no competing interest. Footnotes We have updated the manuscript by including full-size, uncropped Western blot images as Supplementary Figure 1, corresponding to the cropped blots shown in Figure 5C. These blots show the expression of canonical EV markers (CD9 and CD81), a negative control for cellular contamination (Calnexin/CANX), and an adipocyte-specific marker (Adiponectin/ADIPOQ) in EVs isolated from differentiated adipocytes (difAdipos) derived from two bariatric patients (EV1 Patient 1, EV2 Patient 2). EVs from HEK293 cells were included as a positive control to confirm antibody specificity, showing robust expression of CD9 and CD81 and no Calnexin signal. Each panel (AD) of Supplementary Figure 1 corresponds to a specific target protein and includes molecular weight standards and all sample lanes for full transparency. Additionally, Figure 5B has been updated with improved TEM images that more clearly depict the cup-shaped morphology of EVs. Finally, the author sequence has been revised to accurately reflect individual contributions. These updates enhance the clarity, rigor, and reproducibility of the data presented in the manuscript.