An in vitro comparison of the osteogenic potential of equine stem cell populations and subpopulations from multiple tissue sources

Abstract

An in vitro comparison of the osteogenic potential of equine stem cell populations and subpopulations from multiple tissue sources was made to identify the ideal equine donor tissue as a source of MSCs to promote bone healing. Equine muscle tissue– and periosteal tissue–derived cells where characterized as mesenchymal stem cells (MSCs) and their proliferation capacity and osteogenic potential was assessed in comparison with bone marrow– and adipose tissue–derived MSCs. Cells were isolated from skeletal muscle, periosteal, and adipose tissues, and sternal bone marrow aspirates. Morphology, adherence to plastic, trilineage differentiation, and detection of stem cell surface markers CD44 and CD90 were used to characterize cells as MSCs. Osteogenic potential of MSCs was measured by osteocalcin gene expression. Mesenchymal stromal cell cultures were counted at 24, 48, 72, and 96 hours to determine tissue-specific MSC proliferative capacity. Muscle MSCs (MMSCs), periosteum MSCs (PMSCs), and adipose MSCs (AMSCs) proliferated significantly faster than did bone marrow MSCs (BMSCs) at 72 and 96 hours. Non-equilibrium gravitational field-flow fractionation (GrFFF) was validated as a method for sorting MSCs from four donor sources (muscle, periosteum, bone marrow, and adipose tissue) into subpopulations. Aliquots of MSCs from each tissue source were consistently separated into 6 fractions by continuous flow (GrFFF proprietary system) and these fractions remained viable for use in further assays. Absorbencies (OD) were compared, and trilineage assays performed. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of <0.05 when fraction 2 and 3 were compared to fractions 1, 4, 5, and 6. GrFFF was used to sort MMSCs and BMSCs into subpopulations and perform assays allowing comparison of their osteogenic capabilities. Aliquots of MMSCs and BMSCs were sorted into 5 fractions using non-equilibrium GrFFF. Pooled fractions were cultured and expanded for assays including: flow cytometry, histochemistry, bone nodule assays, and real time PCR to identify upregulation of osteocalcin, RUNX2, and osterix. There was significant upregulation of osteocalcin, RUNX2, and osterix for the BMSC fraction 4 with P<0.00001 indicating high osteogenic potential. Flow cytometry revealed different cell size and granularity for BMSC fraction 4 and MMSC fraction 2 when compared with unsorted controls and other fractions. Histochemistry and bone nodule assays revealed positive staining nodules but no significant differences between tissues or fractions. It was concluded that 1) equine muscle and periosteum are sources of MSCs that have osteogenic potential comparable to that of equine adipose- and bone marrow–derived MSCs, 2) non-equilibrium GrFFF is a valid method for sorting equine MMSCs, PMSCs, BMSCs, AMSCs into subpopulations that remain viable and 3) subpopulations of MSCs exist and have different osteogenic capacities within equine muscle and bone marrow derived sources. These findings are important contributions to equine stem cell therapy and bone healing in veterinary medicine