Human Differentiated Adipocytes as Surrogate Mature Adipocytes for Adipocyte-Derived Extracellular Vesicle Analysis

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  Figure 1. Workflow for the isolation, differentiation, and characterization of human preadipocytes, differentiated adipocytes, and their EVs. (A) Visceral adipose tissue obtained from bariatric patients was enzymatically digested using Collagenase A at 37 °C for 30–40 min, followed by filtration through 300 µm and 100 µm cell strainers. Subsequent centrifugation (500× g, RT, 10 min) yielded three distinct fractions: stromal vascular fraction (SVF), SVF supernatant (SVF-SN), and mature adipocytes (mAdipos). The SVF-derived preadipocytes (preAdipos) were cultured and induced to differentiate into differentiated mature adipocytes (difAdipos) over 28 days. Conditioned media from adipocyte cultures were collected and processed using tangential flow filtration (TFF) for EV isolation. (B) Characterization of preAdipos, difAdipos, and mAdipos included functional assays assessing lipolysis, morphological analysis via brightfield microscopy, and gene expression analysis using quantitative PCR. (C) Isolated EVs from preAdipos (preAdipo-EVs) and difAdipos (difAdipo-EVs) underwent extensive characterization, including immunoblot analysis of EV and adipocyte markers (CD9, CD81, CD63, and ADIPOQ), size…

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  Figure 2. Human preadipocytes undergo differentiation and acquire a mature adipocyte phenotype. (A) Differentiation timeline: At Day 1, cells reached ~80% confluency in growth medium (GM). Differentiation was initiated on Day 0 following a 24 h incubation in GM by replacing the medium with adipogenic induction medium 1 (AIM1). The transition to adipogenic induction medium 2 (AIM2) was performed to facilitate further maturation, leading to the acquisition of a differentiated mature adipocyte (difAdipo) phenotype by Day 28. (B) Morphological changes during adipogenesis: Preadipocytes were sourced from three independent bariatric surgical patients. Morphological changes were documented every seven days for each patient. (a) Representative brightfield microscopy images illustrate the progressive morphological changes occurring during preadipocyte differentiation into difAdipos. Lipid droplet accumulation becomes progressively evident throughout the differentiation process. (b) The morphology of difAdipos at Day 28 closely resembles that of primary mature adipocytes (mAdipos), which were directly isolated from patient-derived visceral adipose tissue and maintained in culture for 24 h. Images…

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  Figure 3. Transcript profile in preadipocytes, differentiated adipocytes, and mature adipocytes. (A) Relative mRNA expression levels of preadipocyte markers (OCT4, PREF1, GATA3) and (B) mature adipocyte markers (ADIPOQ, PLIN1, PPARG) were measured at Day 0 (D0), D7, D14, D21, D28, and in terminally differentiated mAdipos. OCT4 expression peaked at D7 and decreased thereafter, indicating early differentiation. PREF-1 expression increased to a peak at D7 and decreased towards D28, consistent with its inhibitory role in adipogenesis. GATA3 expression peaked at D7 and D14 and remained elevated in mAdipos, indicating its involvement in early differentiation. Adipocyte markers ADIPOQ, PLIN1, and PPARG exhibited a progressive increase, peaking at D28 and in mAdipos, reflecting mature adipocyte function (n = 3 independent biological replicates). The statistical analysis was performed using ordinary one-way ANOVA with Bartlett’s test. OCT4, octamer-binding transcription factor 4; PREF-1, preadipocyte factor 1; GATA3, GATA-binding protein 3; ADIPOQ, adiponectin; PLIN, perilipin; PPARG, peroxisome proliferator-activated receptor gamma. A value of p < 0.05 was deemed statistically significant (* p <…

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  Figure 4. Differentiated adipocytes (difAdipos) exhibit lipolytic function comparable to mature adipocytes (mAdipos). Lipolytic activity was assessed in preAdipos, difAdipos, and mAdipos by stimulating cells with isoproterenol and measuring glycerol release. D0 difAdipos reflected undifferentiated adipocytes and exhibited low-level lipolytic activity similar to HEK293T cells (negative control). D28 difAdipos exhibited lipolytic activity comparable to mAdipos and glycerol (positive control), validating the differentiation protocol and confirming that difAdipos functionally resemble mature adipocytes in their lipolytic activity (n = 3 independent biological replicates). Statistical analysis was performed using one-way ANOVA with Sidak’s multiple comparison test. A value of p < 0.05 was deemed statistically significant (**** p < 0.0001); ns, not significant.

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  Figure 5. Multiparametric characterization of matched adipose tissue-derived EVs from the same bariatric patients. (A) MRPS analysis of EVs from preAdipos, difAdipos, and mAdipos from the same individuals (n = 3 independent biological replicates). (B) TEM imaging showed a polydispersed population of particles, including small EVs with a characteristic ‘cup-shaped’ morphology. Images are representative of EVs from preAdipo, difAdipo, and mAdipo groups, which exhibited similar ultrastructural features. Yellow arrows indicate individual EVs. (C) Immunoblot analysis of EVs from difAdipos reveals heterogeneous expression of typical EV (CD9, CD81, CD63, TSG101, and Alix), non-EV (CANX), and adipose-specific (ADIPOQ) markers. Full-size uncropped blot images for panel (C) are provided in Supplementary Figure S1. MRPS, microfluidic resistive pulse sensing; TEM, transmission electron microscopy.

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  Figure 6. Single EV multiparametric characterization comparing preAdipo-EVs (Day 2) and difAdipo-EVs (Day 28). (A) Representative TIRFM images illustrating difAdio-EVs were captured by anti-CD9 and anti-CD63 antibodies and immobilized on a biochip surface. Detection antibodies against CD63, OCT4, and APMAP were used to identify EV surface proteins. A fluorescence-labeled molecular beacon detected the mRNA ADIPOQ within those EVs. The merged image reveals difAdipo-EVs defined by colocalization of OCT4 and mRNA ADIPOQ, CD63 and OCT4, CD63 and mRNA ADIPOQ, and CD63 and APMAP; scale 10 µm. (B) The relative fluorescence intensity of EVs collected at Day 2 and Day 28 was analyzed for the tetraspanin marker CD63, the undifferentiated cell marker OCT4, the adipocyte plasma membrane-associated protein APMAP, and the adipose tissue gene marker mRNA ADIPOQ. OCT4, octamer-binding transcription factor 4; ADIPOQ, adiponectin; APMAP, astrocyte membrane-associated protein (n = 3 independent biological replicates). Statistical analysis was performed using Student’s t-test. A value of p < 0.05 was deemed statistically significant (* p < 0.05; *** p < 0.001); ns, not significant.

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  Figure 7. Uptake of difAdipo- and mAdipo-EVs by HMC3 microglia and THP-1 macrophages. HMC3 microglia and PMA-differentiated THP-1 macrophages were incubated for 24 h at 37 °C with PBS (control), MemGlow™ 488-labeled difAdipo-EVs or MemGlow™ 488-labeled mAdipo-EVs (1 × 109 EVs/well). EVs appear in green; nuclei were counterstained with Hoechst 33342 (NucBlue™, Invitrogen; blue). Representative fields from three independent biological replicates (n = 3). Scale bars, 50 μm.