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Evaluation of the osteogenic and chondrogenic differentiation capacities of equine adipose tissue-derived mesenchymal stem cells

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  • 1 Center for Regenerative Medicine, University Hospital Tübingen, D-72076 Tübingen, Germany.
  • | 2 Departments of Orthopaedic Surgery, University Hospital Tübingen, D-72076 Tübingen, Germany.
  • | 3 Departments of Orthopaedic Surgery, University Hospital Tübingen, D-72076 Tübingen, Germany.
  • | 4 Institute for Anatomy Eberhard-Karls-University D-72076 Tübingen, Germany.
  • | 5 Internal Medicine II, University Hospital Tübingen, D-72076 Tübingen, Germany.
  • | 6 Center for Regenerative Medicine, University Hospital Tübingen, D-72076 Tübingen, Germany.
  • | 7 Horse Clinic Walliser, Nurtingerstr 200, D-73230 Kirchheim unter Teck, Germany.
  • | 8 Center for Regenerative Medicine, University Hospital Tübingen, D-72076 Tübingen, Germany.
  • | 9 Institute for Anatomy Eberhard-Karls-University D-72076 Tübingen.
  • | 10 Center for Regenerative Medicine, University Hospital Tübingen, D-72076 Tübingen, Germany.
  • | 11 Departments of Orthopaedic Surgery, University Hospital Tübingen, D-72076 Tübingen, Germany.

Abstract

Objective—To evaluate the proliferative behavior, telomere length, immunophenotype, and differentiation capacity of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs).

Animals—6 adult racing horses treated for articular Injury but otherwise healthy

Procedures—AT-MSCs were Isolated from horses and expanded In Dulbecco modified Eagle medium enriched with fetal bovine serum and antimicrobials. Expression of cell surface antigens and telomere length were Investigated via flow cytometry Differentiation of MSCs Into chondrocytes, osteoblasts, and adipocytes was Induced In vitro by specific stimuli and was evaluated by analyzing marker genes with quantitative reverse transcriptase PCR assays and immunocytochemical and cytologie evaluations.

Results—Equine MSCs could be cultured up to the fifth passage before signs of senescence, apoptosis, and detachment Indicated cellular exhaustion. However, the AT-MSCs from 2 of 6 horses survived to later passages with Increased doubling rates and telomere lengths. The cells had a typical phenotype, with expression of CD14, CD73, CD90, CD105, CD140b, and CD164 antigens and a lack of CD34 and CD45 antigens. The cells also had a strong potential to differentiate Into osteoblasts, as characterized by Intense von Kossa and alizarin red staining as well as high Induction of osteopontin. Chondrogenic differentiation was detected via Alelan blue staining and expression of aggrecan and type II collagen Adipogenesis was Induced in AT-MSCs by supplementation of differentiation media with rabbit serum.

Conclusions and Clinical Relevance—Equine AT-MSCs representa suitable cellular source for regenerative treatment of bone or cartilage defects, particularly when expanded In vitro for only a few passages. (Am J Vet Res 2010;71:1228-1236)

Abstract

Objective—To evaluate the proliferative behavior, telomere length, immunophenotype, and differentiation capacity of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs).

Animals—6 adult racing horses treated for articular Injury but otherwise healthy

Procedures—AT-MSCs were Isolated from horses and expanded In Dulbecco modified Eagle medium enriched with fetal bovine serum and antimicrobials. Expression of cell surface antigens and telomere length were Investigated via flow cytometry Differentiation of MSCs Into chondrocytes, osteoblasts, and adipocytes was Induced In vitro by specific stimuli and was evaluated by analyzing marker genes with quantitative reverse transcriptase PCR assays and immunocytochemical and cytologie evaluations.

Results—Equine MSCs could be cultured up to the fifth passage before signs of senescence, apoptosis, and detachment Indicated cellular exhaustion. However, the AT-MSCs from 2 of 6 horses survived to later passages with Increased doubling rates and telomere lengths. The cells had a typical phenotype, with expression of CD14, CD73, CD90, CD105, CD140b, and CD164 antigens and a lack of CD34 and CD45 antigens. The cells also had a strong potential to differentiate Into osteoblasts, as characterized by Intense von Kossa and alizarin red staining as well as high Induction of osteopontin. Chondrogenic differentiation was detected via Alelan blue staining and expression of aggrecan and type II collagen Adipogenesis was Induced in AT-MSCs by supplementation of differentiation media with rabbit serum.

Conclusions and Clinical Relevance—Equine AT-MSCs representa suitable cellular source for regenerative treatment of bone or cartilage defects, particularly when expanded In vitro for only a few passages. (Am J Vet Res 2010;71:1228-1236)

Contributor Notes

Supported in part by the Deutsche Forschungsgemenischaft and Federal Ministry for Education and Research and the Center for Regenerative Medicine of the Tübingen University Hospital. Mr. Braun's work was supported by the Rotary Club Reutlingen-Tübingen.

The authors thank Dr. Hans J. Bühring for technical support and Diane Blaurock for assistance.

Address correspondence to Dr. Aicher (Aicher@uni-tuebingen.de).