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Home / Equine Research Bank / Can J Vet Res / Osteogenic potential of sorted equine mesenchymal stem cell ...
Canadian journal of veterinary research = Revue canadienne de recherche veterinaire2015; 79(2); 101-108;

Osteogenic potential of sorted equine mesenchymal stem cell subpopulations.

Abstract: The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow aspirates of the fourth and fifth sternebrae. Aliquots of 800 × 10(3) MSCs from each tissue source were sorted into 5 fractions using non-equilibrium GrFFF (GrFFF proprietary system). Pooled fractions were cultured and expanded for use in osteogenic assays, including flow cytometry, histochemistry, bone nodule assays, and real-time quantitative polymerase chain reaction (qPCR) for gene expression of osteocalcin (OCN), RUNX2, and osterix. Equine MMSCs and BMSCs were consistently sorted into 5 fractions that remained viable for use in further osteogenic assays. Statistical analysis confirmed strongly significant upregulation of OCN, RUNX2, and osterix for the BMSC fraction 4 with P < 0.00001. Flow cytometry revealed different cell size and granularity for BMSC fraction 4 and MMSC fraction 2 compared to unsorted controls and other fractions. Histochemisty and bone nodule assays revealed positive staining nodules without differences in average nodule area, perimeter, or stain intensity between tissues or fractions. As there are different subpopulations of MSCs with different osteogenic capacities within equine muscle- and bone marrow-derived sources, these differences must be taken into account when using equine stem cell therapy to induce bone healing in veterinary medicine. Les objectifs de la présente étude étaient d’utiliser une méthode non-équilibrée de fractionnement par flot sous champs gravitationnel (GrFFF), une méthode sans marquage immunologique de séparation des cellules souches mésenchymateuses (MSCs), afin de séparer les cellules souches mésenchymateuses dérivées du tissu musculaire équin (MMSCs) et les cellules souches mésenchymateuses provenant de la moelle osseuse (BMSCs) en sous-populations et de réaliser des essais afin de comparer leurs capacités ostéogéniques. Des cellules provenant d’un jeune cheval adulte furent isolées du muscle semi-tendineux gauche et d’aspirations de la moelle osseuse de la quatrième et cinquième strernèbre. Des aliquotes de 800 × 103 MSCs provenant de chaque source de tissu furent séparés en 5 fractions par GrFFF non-équilibré (système breveté GrFFF). Des fractions regroupées ont été mises en culture afin de proliférer pour utilisation dans des essais ostéogéniques, incluant la cytométrie en flux, l’histochimie, des essais de nodules osseux, et l’amplification en chaine quantitative par la polymérase (qPCR) pour l’expression des gènes de l’ostéocalcine (OCN), RUNX2, et osterix. Les MMSCs et BMSCs équins étaient séparés de manière constante en 5 fractions qui demeuraient viables pour utilisation dans des essais ostéogéniques additionnels. Les analyses statistiques ont confirmé une régulation à la hausse très significative pour OCN, RUNX2 et osterix pour la fraction 4 des BMSC (P < 0,00001). La cytométrie en flux a révélé une taille et une granularité différente pour la fraction 4 des BMSCs et la fraction 2 des MMSCs comparativement aux témoins non-séparés et aux autres fractions. L’histochimie et les essais de nodules osseux ont révélé des nodules se colorant positivement sans différence pour les tissus ou les fractions dans la moyenne de la surface du nodule, du périmètre, ou de l’intensité de la coloration. Étant donné qu’il y a différentes sous-populations de MSCs avec différentes capacités ostéogéniques parmi les sources dérivées du muscle et de la moelle osseuse, ces différences doivent être prises en compte lors de l’utilisation thérapeutique en médecine vétérinaire des cellules souches pour induire la guérison osseuse.(Traduit par Docteur Serge Messier).
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Publication Date: 2015-04-09 PubMed ID: 25852225PubMed Central: PMC4365701
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research investigates the use of gravitational field-flow fractionation to sort horse muscle and bone marrow-derived stem cells into different groups and evaluate their bone-building properties. The study identifies distinct mesenchymal stem cell subpopulations with different osteogenic capabilities important for bone healing in veterinary medicine.

Research Methodology

  • The study involved sourcing cells from a young adult horse, specifically from the left semitendinosus muscle tissue and bone marrow aspirates of the fourth and fifth sternebrae.
  • A method called non-equilibrium gravitational field-flow fractionation (GrFFF) was utilized to separate these mesenchymal stem cells (MSCs) into five fractions, without the use of an immunotag.
  • These fractions were subsequently cultured and enlarged for further osteogenic assays or tests, which included flow cytometry, histochemistry, and bone nodule assays.
  • Real-time quantitative polymerase chain reaction (qPCR) was also used to measure the gene expression levels of osteocalcin (OCN), RUNX2, and osterix—genes which have significant roles in bone formation and osteogenic mechanisms.

Pertinent Findings

  • It was identified that equine mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSCs) could regularly be separated into five viable subpopulations for further osteogenic analysis.
  • A statistically significant upregulation of the osteogenic genes (OCN, RUNX2, osterix) was observed, especially for the BMSC fraction 4 (P < 0.00001).
  • Flow cytometry indicated different cell sizes and granularity for BMSC fraction 4 and MMSC fraction 2 when compared to unsorted controls and other fractions.
  • Histochemical analysis and bone nodule assays revealed positively stained nodules, but without differences between tissues or fractions in average nodule area, perimeter, or stain intensity.

Conclusions and Implications

  • The research confirmed that different subpopulations of MSCs derived from equine muscle and bone marrow have varying osteogenic properties.
  • This underscores the need to account for these differences when using equine stem cell therapy for bone healing in veterinary medicine, as the healing capacity is intermediary to the osteogenic ability of the stem cell source.

Cite This Article

APA
Radtke CL, Nino-Fong R, Rodriguez-Lecompte JC, Esparza Gonzalez BP, Stryhn H, McD￮ LA. (2015). Osteogenic potential of sorted equine mesenchymal stem cell subpopulations. Can J Vet Res, 79(2), 101-108.

Publication

Canadian journal of veterinary research = Revue canadienne de recherche veterinaire
ISSN: 1928-9022
NlmUniqueID: 8607793
Country: Canada
Language: English
Volume: 79
Issue: 2
Pages: 101-108

Researcher Affiliations

Radtke, Catherine L
  • Department of Health Management (Radtke, Nino-Fong, Esparza Gonzalez, Stryhn, McD￮), and Department of Pathology and Microbiology (Rodriguez-Lecompte), Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3.
Nino-Fong, Rodolfo
  • Department of Health Management (Radtke, Nino-Fong, Esparza Gonzalez, Stryhn, McD￮), and Department of Pathology and Microbiology (Rodriguez-Lecompte), Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3.
Rodriguez-Lecompte, Juan Carlos
  • Department of Health Management (Radtke, Nino-Fong, Esparza Gonzalez, Stryhn, McD￮), and Department of Pathology and Microbiology (Rodriguez-Lecompte), Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3.
Esparza Gonzalez, Blanca P
  • Department of Health Management (Radtke, Nino-Fong, Esparza Gonzalez, Stryhn, McD￮), and Department of Pathology and Microbiology (Rodriguez-Lecompte), Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3.
Stryhn, Henrik
  • Department of Health Management (Radtke, Nino-Fong, Esparza Gonzalez, Stryhn, McD￮), and Department of Pathology and Microbiology (Rodriguez-Lecompte), Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3.
McD￮, Laurie A
  • Department of Health Management (Radtke, Nino-Fong, Esparza Gonzalez, Stryhn, McD￮), and Department of Pathology and Microbiology (Rodriguez-Lecompte), Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3.

MeSH Terms

  • Animals
  • Biomarkers
  • Bone Marrow Cells
  • Flow Cytometry
  • Gene Expression Regulation
  • Horses
  • Mesenchymal Stem Cells / classification
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / physiology
  • Muscle, Skeletal / cytology
  • Osteogenesis / physiology
  • Real-Time Polymerase Chain Reaction

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