Expression of neural markers on bone marrow–derived canine mesenchymal stem cells

Hiroaki Kamishina Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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Jie Deng Department of Anatomy and Cell Biology, Program in Stem Cell Biology and Regenerative Medicine, College of Medicine, University of Florida, Gainesville, FL 32610.

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 PhD
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Takashi Oji Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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Jennifer A. Cheeseman Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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Roger M. Clemmons Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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Abstract

Objective—To evaluate cell surface markers of bone marrow–derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells.

Sample Population—Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs.

Procedures—Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (β III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation.

Results—Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed β III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in β III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions.

Conclusions and Clinical Relevance—Canine bone marrow–derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.

Abstract

Objective—To evaluate cell surface markers of bone marrow–derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells.

Sample Population—Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs.

Procedures—Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (β III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation.

Results—Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed β III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in β III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions.

Conclusions and Clinical Relevance—Canine bone marrow–derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.

Contributor Notes

Dr. Deng's present address is Center for Human Genetics, Duke University Medical Center, Durham, NC 27710.

Supported by the University of Florida College of Veterinary Medicine Consolidated Faculty Research Development Award Grant.

Presented in part at the Experimental Biology 2006 meeting, San Francisco, April 2006.

The authors thank Neal Benson and Bhavna Bhardwaj for assistance with flow cyotometric analysis.

Address correspondence to Dr. Kamishina.
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