skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: The impact of extracellular matrix proteins on bovine fibro‐adipogenic progenitor cell adhesion, proliferation, and differentiation in vitro
Abstract Fibro‐adipogenic progenitor cells (FAPs) are mesenchymal stem cells that produce extracellular matrix (ECM) and intramuscular adipocytes in skeletal muscle. While FAPs have demonstrated responsiveness to their physical environment, there is limited knowledge of how the ECM substrate of FAPs impacts their differentiation, particularly in livestock animals. We hypothesized that the ECM substrate FAPs are cultured on will differentially impact their adherence, proliferation, and differentiation. Through an initial screen of 9 ECM proteins and their combinations, significant variation of bovine FAP attachment and differentiation across coatings was observed. The ECM substrates fibronectin, collagen 6, vitronectin, and a combination of fibronectin and collagen 6 were selected for further testing. Notably, fibronectin increased cell proliferation and attachment rates, without impairing FAP adipogenic or fibrogenic differentiation compared to the other coatings. Benefits of fibronectin were maintained at lower concentrations and when combined with less favorable coatings such as collagen 6. When assessed for their adipogenic potential on each coating at different substrate stiffnesses, lipid accumulation decreased with increasing substrate stiffness, while cell attachment increased on stiffer substrates. Overall, these results demonstrate the high responsiveness of FAPs to their ECM substrate, along with highlighting fibronectin as a preferred substrate for in vitro experiments with bovine FAPs.  more » « less
Award ID(s):
2320899
PAR ID:
10586961
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
Physiological Reports
Volume:
13
Issue:
9
ISSN:
2051-817X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Jian Yang (Ed.)
    Dynamic regulation of cell-extracellular matrix (ECM)-material interactions is crucial for various biomedical applications. In this study, a light-activated molecular switch for the modulation of cell attachment/detachment behaviors was established on monolayer graphene (Gr)/n-type Silicon substrates (Gr/Si). Initiated by light illumination at the Gr/Si interface, pre-adsorbed proteins (bovine serum albumin, ECM proteins collagen-1, and fibronectin) underwent protonation to achieve negative charge transfer to Gr films (n-doping) through π-π interactions. This n-doping process stimulated the conformational switches of ECM proteins. The structural alterations in these ECM interactors significantly reduced the specificity of the cell surface receptor-ligand interaction (e.g., integrin recognition), leading to dynamic regulation of cell adhesion and eventual cell detachment. RNA-sequencing results revealed that the detached bone marrow mesenchymal stromal cell sheets from the Gr/Si system manifested regulated immunoregulatory properties and enhanced osteogenic differentiation, implying their potential application in bone tissue regeneration. This work not only provides a fast and feasible method for controllable cells/cell sheets harvesting but also gives new insights into the understanding of cell-ECM-material communications. 
    more » « less
  2. The extracellular matrix (ECM) regulates carcinogenesis by interacting with cancer cells via cell surface receptors. Discoidin Domain Receptor 2 (DDR2) is a collagen‐activated receptor implicated in cell survival, growth, and differentiation. Dysregulated DDR2 expression has been identified in various cancer types, making it as a promising therapeutic target. Additionally, cancer cells exhibit mechanosensing abilities, detecting changes in ECM stiffness, which is particularly important for carcinogenesis given the observed ECM stiffening in numerous cancer types. Despite these, whether collagen‐activated DDR2 signaling and ECM stiffness‐induced mechanosensing exert similar effects on cancer cell behavior and whether they operate through analogous mechanisms remain elusive. To address these questions, we performed bulk RNA sequencing (RNA‐seq) on human SH‐SY5Y neuroblastoma cells cultured on collagen‐coated substrates. Our results show that DDR2 downregulation induces significant changes in the cell transcriptome, with changes in expression of 15% of the genome, specifically affecting the genes associated with cell division and differentiation. We validated the RNA‐seq results by showing that DDR2 knockdown redirects the cell fate from proliferation to senescence. Like DDR2 knockdown, increasing substrate stiffness diminishes cell proliferation. Surprisingly, RNA‐seq indicates that substrate stiffness has no detectable effect on the transcriptome. Furthermore, DDR2 knockdown influences cellular responses to substrate stiffness changes, highlighting a crosstalk between these two ECM‐induced signaling pathways. Based on our results, we propose that the ECM could activate DDR2 signaling and mechanosensing in cancer cells to orchestrate their cell fate through distinct mechanisms, with or without involving gene expression, thus providing novel mechanistic insights into cancer progression. 
    more » « less
  3. Abstract Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, a novel hydrophobic hydrogel system is presented via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non‐protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, it is shown that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, it is revealed that stiffness‐mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. The material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function. 
    more » « less
  4. Abstract Cells are known to continuously remodel their local extracellular matrix (ECM) and in a reciprocal way, they can also respond to mechanical and biochemical properties of their fibrous environment. In this study, we measured how stiffness around dermal fibroblasts (DFs) and human fibrosarcoma HT1080 cells differs with concentration of rat tail type 1 collagen (T1C) and type of ECM. Peri-cellular stiffness was probed in four directions using multi-axes optical tweezers active microrheology (AMR). First, we found that neither cell type significantly altered local stiffness landscape at different concentrations of T1C. Next, rat tail T1C, bovine skin T1C and fibrin cell-free hydrogels were polymerized at concentrations formulated to match median stiffness value. Each of these hydrogels exhibited distinct fiber architecture. Stiffness landscape and fibronectin secretion, but not nuclear/cytoplasmic YAP ratio differed with ECM type. Further, cell response to Y27632 or BB94 treatments, inhibiting cell contractility and activity of matrix metalloproteinases, respectively, was also dependent on ECM type. Given differential effect of tested ECMs on peri-cellular stiffness landscape, treatment effect and cell properties, this study underscores the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction. 
    more » « less
  5. Cells are known to continuously remodel their local extracellular matrix (ECM) and in a reciprocal way, they can also respond to mechanical and biochemical properties of their fibrous environment. In this study, we measured how stiffness around dermal fibroblasts (DFs) and human fibrosarcoma HT1080 cells differs with concentration of rat tail type 1 collagen (T1C) and type of ECM. Peri-cellular stiffness was probed in four directions using multi-axes optical tweezers active microrheology (AMR). First, we found that neither cell type significantly altered local stiffness landscape at different concentrations of T1C. Next, rat tail T1C, bovine skin T1C and fibrin cell-free hydrogels were polymerized at concentrations formulated to match median stiffness value. Each of these hydrogels exhibited distinct fiber architecture. Stiffness landscape and fibronectin secretion, but not nuclear/cytoplasmic YAP ratio differed with ECM type. Further, cell response to Y27632 or BB94 treatments, inhibiting cell contractility and activity of matrix metalloproteinases, respectively, was also dependent on ECM type. Given differential effect of tested ECMs on peri-cellular stiffness landscape, treatment effect and cell properties, this study underscores the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction. 
    more » « less