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.

Keywords: bovine; extracellular matrix; fibronectin; fibro‐adipogenic progenitor; mesenchymal stem cell.

FIGURE 1

Bovine FAP isolation and verification. (a) Schematic of primary cell isolation protocol and FAP sorting using MACS. (b) Composite and single channel image of PDGFRα signal on animal 1 bovine FAPs. (c) Perilipin expression in FAPs after 10 days of adipogenic differentiation. (d) αSMA expression in FAPs after 10 days of fibrogenic differentiation. All cells plated on fibronectin (b–d). Scale bars are 50 μm (b) and 200 μm (c, d).

FIGURE 2

ECM Array Chip. (a) Advanced Biomatrix ECM Select Array Kit Ulta‐36 legend and coating replicate layout. (b) Representative immunofluorescent images showing Perilipin and αSMA signal after 5 days of differentiation in BoFat media. Chip cultured using P4 Animal 2 FAPs. Scale bars are 200 μm.

FIGURE 3

Quantification of FAP performance on the ECM Array Chip depicted in Figure 2. X‐axis values represent each coating treatment depicted in Figure 2a. (a) Quantification of total Hoechst+ cells. (b) Quantification of total Actin+ area. (c) Perilipin+ area as a percentage of the total Actin+ area. (d) αSMA+ area as a percentage of total Actin+ area. Coatings used in following select assays: B3 = Fibronectin + Collagen6, E1 = Collagen6, F1 = Fibronectin, H1 = Vitronectin. n = 9 replicates. Chip cultured using animal 2 FAPs.

FIGURE 4

FAP attachment and proliferation on select substrates. (a) FAPs on fibronectin at 1, 3, and 6 h timepoints. (b) Quantification of total Hoechst+ FAPs for each coating at 1, 3, and 6 h. (c) Quantification of Actin+ area (μm2)/Hoechst+ FAPs for each coating at 1, 3, and 6 h. (d) FAPs after 24 h. treatment with EdU from Animal 2. (e) Quantification of total Hoechst+ cells 24 h. post‐seeding. (f) Quantification of EdU+/Hoechst+ FAPs 24 h post‐seeding and EdU treatment. N = 3 cattle. Scale bars are 200 μm.

FIGURE 5

FAP differentiation across different substrates. (a) Immunofluorescent images of FAPs after 10 days of differentiation in the indicated media. (b) Quantification of total Actin+ area between coatings and media conditions. (c) Quantification of percent perilipin+/Actin+ area across media and coating conditions. (d) Quantification of percent αSMA+/Actin+ area between media and coating conditions. N = 3 cattle and n = 3 medias. Scale bars are 200 μm.

FIGURE 6

FAP differentiation on each coating at different stiffnesses. (a) Immunofluorescent images of FAPs after 10 days of differentiation on the indicated coating at 2, 8, or 25 kPa. (b) Quantification of percent Perilipin+/Actin+ area across coating types and stiffnesses. (c) Quantification of percent αSMA +/Actin+ area across coating types and stiffnesses. (d) Quantification of number of total Hoechst+ nuclei for each coating type and stiffness. (e) Quantification of Actin+ area for each coating type and stiffness. N = 3 cattle and n = 4 stiffnesses. Scale bars are 200 μm.