Fibroblast-Adipocyte Lineage Cell Interactions Result in Differential Production of Extracellular Matrix Proteins
Academic Background
Scar formation is a common issue following trauma, burns, and other complications, significantly impacting the quality of life for millions of people worldwide. Fibroblasts play a central role in pathological scar formation, making them a common target for developing new therapies to promote healing and reduce scarring. Recent studies have shown that adipocyte lineage cells also play an important role in the wound healing process. Clinical reports indicate that placing autologous adipose micrografts at the surgical site can improve the appearance and pliability of existing scars. However, the specific cell types within fat grafts and their mechanisms in wound healing remain unclear. Therefore, this study aims to explore the interactions between adipocyte lineage cells and fibroblasts and how these interactions affect the production of extracellular matrix (ECM) proteins.
Source of the Paper
The study was conducted by Edward A. Sander, Mariam Y. El-Hattab, Kathryn R. Jacobson, Aloysius J. Klingelhutz, James A. Ankrum, and Sarah Calve. The research team is affiliated with the University of Iowa, University of Colorado Boulder, and University of Iowa Carver College of Medicine. The paper was published online on October 29, 2024, in the journal Cellular and Molecular Bioengineering.
Research Process
1. Cell Culture and Spheroid Formation
The study first utilized TERT-immortalized normal human dermal fibroblasts and adipose-derived stem cells (ADSCs) for culture. Fibroblasts and ADSCs were formed into spheroids using the droplet method and hanging-drop method, respectively. ADSC spheroids were further differentiated into pre-adipocytes and mature adipocytes.
2. Fibrin Gel Formation and Spheroid Co-Culture
The study used bovine fibrinogen and thrombin to prepare fibrin gels, and fibroblast, pre-adipocyte, and adipocyte spheroids were paired for co-culture. The co-culture system observed cell migration and collagen deposition through time-lapse imaging.
3. Fluorescent Labeling and Microscopy Imaging
Cells were labeled with Alexa Fluor 647-conjugated wheat germ agglutinin (WGA) or CellTracker Red before co-culture. Collagen deposition was visualized using CNA35-labeled fluorescent protein. The co-culture system was continuously imaged for 7 days using a Nikon Ti-E2 A1 confocal microscope.
4. Proteomics Analysis
The study used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze ECM proteins produced by different spheroid combinations. Protein data were analyzed using MaxQuant software and classified according to Gene Ontology and the Matrisome Project.
Key Findings
1. Observation of Collagen Deposition
The study found that fibroblast spheroids migrated and deposited collagen on fibrin gels, with collagen deposition primarily concentrated in the aligned fiber regions between the spheroids. When adipocyte spheroids were co-cultured with fibroblasts, collagen deposition was more pronounced around the adipocyte spheroids.
2. Differential Production of ECM Proteins
LC-MS/MS analysis revealed that the composition of ECM proteins produced by different spheroid combinations varied significantly. The co-culture of fibroblasts with pre-adipocytes (FP) produced the most collagen, followed by the co-culture of adipocytes with adipocytes (AA). The co-culture of fibroblasts with fibroblasts (FF) produced the least collagen.
3. Differences in Basement Membrane Proteins
Adipocyte spheroids (AA) produced the most diverse types of basement membrane proteins, while co-culturing adipocytes with fibroblasts (FA) or pre-adipocytes (PA) reduced the expression of certain basement membrane proteins.
Conclusion
The study demonstrates that interactions between adipocyte lineage cells and fibroblasts significantly influence the production and composition of ECM proteins. Specifically, co-culturing pre-adipocytes with fibroblasts significantly increased collagen production, while co-culturing adipocytes with fibroblasts suppressed the expression of certain basement membrane proteins. These findings provide new insights for developing adipose tissue-based therapeutic strategies, potentially improving wound healing and reducing scar formation.
Research Highlights
- Key Finding: Interactions between adipocyte lineage cells and fibroblasts significantly influence ECM protein production, particularly the significant increase in collagen deposition when pre-adipocytes are co-cultured with fibroblasts.
- Methodological Innovation: The study utilized a 3D spheroid co-culture system and time-lapse imaging, combined with LC-MS/MS analysis, to comprehensively reveal the impact of cell interactions on ECM protein composition.
- Application Value: The research findings provide a scientific basis for developing adipose tissue-based therapeutic strategies, potentially improving wound healing and reducing scar formation.
Other Valuable Information
The study also found that adipocyte spheroids exhibited higher stability on fibrin gels with less cell migration, which may be related to the basement membrane proteins produced by adipocyte spheroids. Additionally, the study revealed the potential roles of different collagen types (e.g., types I, III, and VI collagen) in wound healing.
Through systematic experimental design and advanced analytical techniques, this study thoroughly explored the interactions between adipocyte lineage cells and fibroblasts and their impact on ECM protein production, providing important scientific evidence for future clinical applications.