Poster L4 - Substrate Stiffness Influences in vitro Adipogenesis of Adipose-Derived Stem Cells

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Diego Valdes Cavazos

Undergraduate Research Assistant
University of Texas at San Antonio
San Antonio, Texas, United States

Co-Author(s)

LM

Linda Mota

Research Scientist
The University of Texas at San Antonio, United States

Primary Investigator(s)

EB

Eric Brey

Professor and Dean
The University of Texas at San Antonio, United States

Co-Author(s)

MP

Maria Prado-Gutierrez

Post-Doctorate
The University of Texas at San Antonio, United States

Introduction: Obesity is a major health concern linked to numerous metabolic disorders and chronic diseases. The behavior of adipocytes, the cells responsible for storing fat, is influenced by the extracellular niche, particularly the stiffness of the extracellular matrix (ECM). The ECM is a three-dimensional, multicellular network composed of proteins such as collagen that characterizes cells' fundamental properties and behavior. In addition to composition, the mechanical properties of the ECM play a role in stimulating stem cell differentiation and hold a role in certain disease progressions. In obesity, research has shown that ECM stiffness increases dramatically, however, there is minimal information on how this change affects adipogenesis, the development of adipose tissue. Consequently, this study works on optimizing the preparation of polyacrylamide (PA) gels to create an in vitro model of controlled stiffness to understand its effects on adipose-derived stem cell (ADSC) differentiation and maturation.

Materials and

Methods: To start, PA hydrogels were created following concentrations from a previous publication consisting of a mixture of acrylamide and bis-acrylamide, adjusting the bis-acrylamide amount to manipulate the stiffness. To confirm our desired modulus, PA hydrogels were characterized through a compression test using a Mach-1TM Mechanical Testing System. For cell culture, the gels were then coated with 0.5 mg/mL Type I Collagen. Mouse ADSCs were isolated from visceral and subcutaneous mouse tissues and seeded onto the PA gels. The cells were differentiated into brown adipocytes through induction and maturation media cocktails on PA hydrogels for four and ten days, respectively. Once differentiated, the adipocytes are either fixed or harvested with the PA hydrogels to be stained with DAPI and Bodipy for confocal imaging and to perform quantitative PCR.

Results, Conclusions, and Discussions: Gels were generated with a Young’s modulus within the range of different adipose tissue, with 3.58 kPa, close to the native stiffness of brown adipose tissue, up to 7.29 kPa, the stiffness of white adipose tissue. Brightfield microscopic images showed that ADSCs were able to attach and differentiate on top of the coated gels at both stiffness levels. Confocal images visibly suggest that a stiffer hydrogel of 7 kPA leads to larger lipid vacuoles and is corroborated by a higher expression of PPAR-γ, a transcription factor associated with lipid production. Additionally, adiponectin was significantly higher expressed in differentiating adipose stem cells at 7 kPa, suggesting a stiffer matrix could enhance the differentiation of ADSCs. These findings help us understand the role stiffness plays in cellular behavior in both healthy and diseased states and hold potential implications for developing therapeutic strategies for conditions such as obesity and diabetes, where tissue stiffness plays a significant role in disease progression. Further experimentation will focus on repeating the experiments to statistically confirm our findings and understand how the adipogenic pathways are affected.

Acknowledgements (Optional):