Z4 - 3D Human Heterotypic Spheroids for Exploring Fibroblast Impact On Preadipocyte Differentiation into Adipocytes

Introduction: : Among the many cell types present in adipose tissues, preadipocytes and fibroblasts, in particular, are thought to play an instrumental role in coordinating changes to the ECM that accommodate dramatic shifts in adipose tissue size[1]. These cells remodel the ECM to mechanically support adipocytes as they expand or shrink as a consequence of storing or releasing lipids[2]. This process becomes impaired in unhealthy adipose tissues and is associated with accumulation of ECM proteins and matrix stiffening (i.e., fibrosis). Furthermore, adipocyte hyperplasia (i.e., formation of new adipocytes via preadipocyte differentiation into adipocytes) is an ECM guided process associated with metabolically healthy adipose tissue that also becomes impaired in fibrotic tissues[3]. The direct impact that fibroblasts have on the capacity of preadipocytes to differentiate into adipocytes, however, remains unclear. We hypothesize that mixing fibroblasts with preadipocytes into heterotypic spheroids prior to induction with differentiation medium will significantly reduce adipocyte differentiation capacity and spheroid size due to shifts in ECM composition and mechanical environment. To test this hypothesis, different ratios of human fibroblasts and preadipocytes were formed into spheroids, cultured in differentiation media, and analyzed for changes in spheroid size, adipogenic capacity, lipid accumulation, mechanical environment, adipokine secretion profile, and ECM composition for comparison with previous work[4].

Materials and

Methods: : Immortalized human preadipocytes (NPADs) and human dermal fibroblasts (HDFs), prelabeled with CellTracker Red, were mixed into cell suspensions to produce NPAD:HDF ratios of 100:0, 75:25, 50:50, 25:75, and 0:100. Suspensions were then added to sterile agarose micromolds to form 35 spheroids containing 5,000 total cells and cultured in preadipocyte differentiation media (PDM-2, Lonza) consistent with our earlier work[5]. Changes in spheroid size were quantified from images acquired over 24 days of culture. Lipid droplets were visualized by fixing spheroids in 10% NBF and labeling with BODIPY 493/503 (TargetMol). On day 24, an additional set of spheroids were fixed in 10% NBF, embedded in HistoGel (Epredia), sectioned into 8 μm thick slices, and either stained with H&E or labeled with immunofluorescent antibodies to visualize the distribution of ECM and adhesion proteins. Separately, HDFs and NPADs were characterized using flow cytometry to determine if common MSC markers indicative of their capacity to differentiate into adipocytes were expressed.

Results, Conclusions, and Discussions:: All spheroids initially decreased approximately 75% in size over the first 3 days of culture (Fig. 1C). Thereafter, spheroids that contained NPADs expanded in size in proportion to the NPAD:HDF ratio. Confocal imaging of the spheroids (Fig. 1 D-H) revealed lipid droplet formation in all spheroids, including the 0:100 NPAD:HDF spheroid, indicating that human dermal fibroblasts are capable of producing lipid droplets when exposed to adipocyte differentiation media (Fig. 1H). The number of lipid droplets in the HDF-only spheroids appeared to be substantially greater than the droplets observed in the NPAD-containing spheroids. Lipid content of HDF spheroids, assessed by normalizing the lipid positive area to spheroid total nucleus area, was significantly higher than other spheroid ratios (Fig. 1I), and increased with the proportion of fibroblasts present. To better understand why lipids formed in HDFs, we used flow cytometry to determine if mesenchymal stem cell (MSC) markers were present on the HDFs and found that about 70% of them were positive for CD73, CD105, and CD90, and negative for endothelial and immune cell markers, indicating an MSC phenotype[6]. Given that the widely used 3T3-L1 mouse cell line is derived from spontaneously transformed dermal fibroblasts[7], reports of a specialized adipocyte depot located in the dermis that contributes to wound healing[8], and other examples of fibroblast/myofibroblast accumulating lipids[9], it is not entirely surprising to observe this behavior in HDF spheroids. Current efforts are underway to characterize the mechanical environment, ECM, and secretion profiles produced by these ratios, and to evaluate the robustness of HDF lipid accumulation in fibroblasts from other sources. In conclusion, at least with respect to de novo ECM production and lipid accumulation, these findings refute our hypothesis.

Acknowledgements and/or References (Optional):: [1] C. M. Gliniak, L. Pedersen, and P. E. Scherer, ‘Adipose tissue fibrosis: the unwanted houseguest invited by obesity’, 2023. doi: 10.1530/JOE-23-0180.
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