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Isolation and differentiation of bovine mammary gland progenitor cell populations

Margo S. HollandDepartment of Veterinary Pathology, Michigan State University, East Lansing, MI 48824.
Present address is Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50010.

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Mei-Hui TaiCollege of Veterinary Medicine, and the Department of Pediatrics, College of Human Medicine, Michigan State University, East Lansing, MI 48824.

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James E. TroskoCollege of Veterinary Medicine, and the Department of Pediatrics, College of Human Medicine, Michigan State University, East Lansing, MI 48824.

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Lorri D. GriffinDepartment of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Judith A. StaskoUSDA-National Animal Disease Center, 2300 Dayton Ave, Ames, IA 50010.

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Norman C. ChevilleDepartment of Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Robert E. HollandDepartment of Large Animal Clinical Sciences, Michigan State University, East Lansing, MI 48824.
Present address is Veterinary Diagnostics and Production Animal Medicine, Iowa State University, Ames, IA 50011.

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Abstract

Objective—To isolate bovine mammary gland cells with stem cell characteristics.

Sample Population—Monolayers of bovine mammary gland cells.

Procedure—Mammary gland cell populations were separated by use of selected media supplements. Phenotypic characteristics were examined via light and transmission electron microscopy. Cellular expression of casein and connexin 43 was identified immunohistochemically. A scrape-loading and dye transfer assay was used to examine the mammary gland cell populations for homogenous gap junctional intercellular communication (GJIC).

Results—Subpopulations of mammary gland cells grown in vitro are classified on the basis of their distinct morphologic features and ability to communicate via gap junctions. Ultrastructurally, 2 morphologically distinct cell types were classified as type I and II cells. Type I cells were small light undiffertiated cells and large light undifferentiated cells that were deficient in functional gap junctions (as is characteristic of stem cells). Type II cells included large light differentiated cells and terminally differentiated cells; GJIC was functional in type II cells. Type II cells had cytoplasmic expression of connexin 43, whereas, type I cells did not. All cells expressed casein.

Conclusions and Clinical Relevance—Subpopulations of bovine mammary gland cells with stem cell characteristics were identified. Phenotypic differences are observed among type I bovine mammary gland cells with stem cell characteristics. Gap junctional intercellular communication may be necessary for the differentiation of stem cells. Characterization of bovine mammary gland stem cells and their progeny may provide a new tool with which to study mammary gland health. (Am J Vet Res 2003;63:396–403)

Abstract

Objective—To isolate bovine mammary gland cells with stem cell characteristics.

Sample Population—Monolayers of bovine mammary gland cells.

Procedure—Mammary gland cell populations were separated by use of selected media supplements. Phenotypic characteristics were examined via light and transmission electron microscopy. Cellular expression of casein and connexin 43 was identified immunohistochemically. A scrape-loading and dye transfer assay was used to examine the mammary gland cell populations for homogenous gap junctional intercellular communication (GJIC).

Results—Subpopulations of mammary gland cells grown in vitro are classified on the basis of their distinct morphologic features and ability to communicate via gap junctions. Ultrastructurally, 2 morphologically distinct cell types were classified as type I and II cells. Type I cells were small light undiffertiated cells and large light undifferentiated cells that were deficient in functional gap junctions (as is characteristic of stem cells). Type II cells included large light differentiated cells and terminally differentiated cells; GJIC was functional in type II cells. Type II cells had cytoplasmic expression of connexin 43, whereas, type I cells did not. All cells expressed casein.

Conclusions and Clinical Relevance—Subpopulations of bovine mammary gland cells with stem cell characteristics were identified. Phenotypic differences are observed among type I bovine mammary gland cells with stem cell characteristics. Gap junctional intercellular communication may be necessary for the differentiation of stem cells. Characterization of bovine mammary gland stem cells and their progeny may provide a new tool with which to study mammary gland health. (Am J Vet Res 2003;63:396–403)