Z7 - Cytokine-Induced Phenotypic Remodeling of Adipocytes Suggests a Reparative Role in Wound Healing

Introduction: : The use of adipose tissues and adipose-based products has emerged as a promising therapeutic strategy for enhancing wound healing and minimizing scar formation. However, the mechanisms underlying adipocyte-mediated tissue repair remain poorly understood. Given that adipocytes undergo morphological and functional reprogramming to support wound remodeling and lipolysis provides essential metabolic support during repair, many questions remain about how these transformed adipocytes (i.e., wound-associated adipocytes) interact with fibroblasts to facilitate wound remodeling. Before we can answer these questions, a method to generate wound-associated adipocytes for in vitro studies is needed. To accomplish this goal, we stimulated our 3D human adipocyte spheroid model with the wound-associated cytokines transforming growth factor beta-1 (TGF-β1), which induces adipocyte dedifferentiation and migration, and tumor necrosis factor alpha (TNF), which promotes lipolysis. This study uncovers the coordinated role. of both cytokines in regulating adipocyte behavior and provides new tools for studying the mechanisms of adipose-based wound therapies.

Materials and

Methods: : Immortalized human preadipocytes (NPAD) were cultured in PDM-2 differentiation medium (Lonza) for 10 days to generate mature adipocytes (Ad). To achieve high-throughput and reproducible 3D cultures, an agarose micromold containing 35 microwells was used to form uniform adipocyte spheroids containing 1,000 cells per spheroid (Fig. 1A). On day 10 of differentiation, spheroids were treated with TGF-β1 (5 ng/mL) and/or TNF (10 ng/mL) for 48 hours. Following cytokine treatment, spheroid size was analyzed by microscopy, lipid content assessed by BODIPY staining, and spheroids were dissociated for analysis of surface marker expression by flow cytometry.

Results, Conclusions, and Discussions:: We developed a standardized 3D spheroid culture platform to investigate cytokine-induced adipocyte conversion during wound repair. 48-hour treatment of both TGF-β1 and TNF significantly reduced adipocyte spheroid size, consistent with those seen in vivo (Figure 1B-C). TNF, but not TGFβ1, led to a marked ~50% decrease in lipid droplet content and size, which is consistent with lipolytic activation (Figure D-F). Flow cytometry analysis revealed that the median fluorescence intensity (MFI) of CD36⁺ adipocytes decreased 25%, while CD105⁺ cells increased 46% following combined TGFβ1 and TNFα stimulation (Figure G-H). This shows a shift away from mature adipocyte markers (i.e. CD36) and toward adipose progenitor markers (i.e. CD105).
These results are in agreement with reports that TNF promotes lipolysis and cooperates with TGF-β1 to induce phenotypic (i.e. size) and metabolic reprogramming (i.e. release of lipids) of adipocytes. The observed changes are consistent with a shift toward a wound-associated adipocyte state which has been reported in vivo to facilitate adipocyte-mediated wound remodeling. Critically, our study provides a reproducible in vitro model that allows researchers to dissect the role of wound-associated adipocytes on wound healing directly and through interaction with other cell types. Future work will investigate the downstream signaling pathways involved in this transition and evaluate functional outcomes such as cell migration or matrix remodeling through modulation of dermal fibroblasts. These findings may contribute to the development of adipose-based therapies aimed at enhancing tissue repair.

Acknowledgements and/or References (Optional):: Support for this work was provided by the National Institutes of Health, NIGMS R01GM145626 and NIEHS P42 ES013661. Adipose was supplied by the Tissue Procurement Core at the University of Iowa which is supported by an award from NIH (NCI award number P30CA086862) and the University of Iowa Carver College of Medicine.