Influence of extracellular matrix on bovine mammary gland progenitor cell growth and differentiation

Margo S. Holland Department of Comparative Medicine, University of Tennessee, Knoxville, TN 37996-4543

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Judith A. Stasko USDA, National Animal Disease Center, Ames, IA 50010

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Robert E. Holland Department of Large Animal Clinical Sciences, University of Tennessee, Knoxville, TN 37996-4543

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Abstract

Objective—To examine the impact of simple versus complex extracellular matrices (ECMs) on morphologic development and differentiation of bovine mammary gland progenitor cells (BMGPCs).

Sample Population—Cultures of BMGPCs.

Procedures—BMGPCs were grown on the following extracellular matrices: collagen I, collagen IV, laminin, and a commercially available gelatinous protein mixture. Cells were examined with light microscopy and transmission electron microscopy.

Results—Formation of organoids and production of the gap junction protein, connexin 43, were the criteria for BMGPC differentiation. The BMGPCs formed a 2-dimensional monolayer when grown on plastic, laminin, collagen I, or collagen IV. These cells did not have a network of cells forming epithelial organoids resembling a honeycomb. However, they did produce gap junction proteins. When BMGPCs were cultured on the commercially available gelatinous protein mixture, 3-dimensional epithelial organoids resembling a honeycomb formed and connexin 43 was produced. The thickness of the commercially available gelatinous protein mixture also regulated cell shape reorganization. Cell density affected the formation organoid networks and the rate at which monolayers reached confluency.

Conclusions and Clinical Relevance—When plated on a commercially available gelatinous protein mixture, the BMGPC culture system allowed us to simulate, in vitro, the interaction between epithelial cells in varying stages of differentiation and the microenvironment. Thus, a heterogenous ECM, such as the commercially available gelatinous protein mixture, is more physiologically relevant in providing a microenvironment for BMGPC lineage pathway differentiation to mimic an in vivo environment. In contrast, BMGPCs grown on homogenous ECM, although able to produce connexin 43, are unable to form organoids.

Abstract

Objective—To examine the impact of simple versus complex extracellular matrices (ECMs) on morphologic development and differentiation of bovine mammary gland progenitor cells (BMGPCs).

Sample Population—Cultures of BMGPCs.

Procedures—BMGPCs were grown on the following extracellular matrices: collagen I, collagen IV, laminin, and a commercially available gelatinous protein mixture. Cells were examined with light microscopy and transmission electron microscopy.

Results—Formation of organoids and production of the gap junction protein, connexin 43, were the criteria for BMGPC differentiation. The BMGPCs formed a 2-dimensional monolayer when grown on plastic, laminin, collagen I, or collagen IV. These cells did not have a network of cells forming epithelial organoids resembling a honeycomb. However, they did produce gap junction proteins. When BMGPCs were cultured on the commercially available gelatinous protein mixture, 3-dimensional epithelial organoids resembling a honeycomb formed and connexin 43 was produced. The thickness of the commercially available gelatinous protein mixture also regulated cell shape reorganization. Cell density affected the formation organoid networks and the rate at which monolayers reached confluency.

Conclusions and Clinical Relevance—When plated on a commercially available gelatinous protein mixture, the BMGPC culture system allowed us to simulate, in vitro, the interaction between epithelial cells in varying stages of differentiation and the microenvironment. Thus, a heterogenous ECM, such as the commercially available gelatinous protein mixture, is more physiologically relevant in providing a microenvironment for BMGPC lineage pathway differentiation to mimic an in vivo environment. In contrast, BMGPCs grown on homogenous ECM, although able to produce connexin 43, are unable to form organoids.

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