BackgroundThe dog represents an excellent large animal model for translational cell-based studies. Importantly, the properties of canine multipotent stromal cells (cMSCs) and the ideal tissue source for specific translational studies have yet to be established. The aim of this study was to characterize cMSCs derived from synovium, bone marrow, and adipose tissue using a donor-matched study design and a comprehensive series of in-vitro characterization, differentiation, and immunomodulation assays.MethodsCanine MSCs were isolated from five dogs with cranial cruciate ligament rupture. All 15 cMSC preparations were evaluated using colony forming unit (CFU) assays, flow cytometry analysis, RT-PCR for pluripotency-associated genes, proliferation assays, trilineage differentiation assays, and immunomodulation assays. Data were reported as mean ± standard deviation and compared using repeated-measures analysis of variance and Tukey post-hoc test. Significance was established at p < 0.05.ResultsAll tissue samples produced plastic adherent, spindle-shaped preparations of cMSCs. Cells were negative for CD34, CD45, and STRO-1 and positive for CD9, CD44, and CD90, whereas the degree to which cells were positive for CD105 was variable depending on tissue of origin. Cells were positive for the pluripotency-associated genes NANOG, OCT4, and SOX2. Accounting for donor and tissue sources, there were significant differences in CFU potential, rate of proliferation, trilineage differentiation, and immunomodulatory response. Synovium and marrow cMSCs exhibited superior early osteogenic activity, but when assessing late-stage osteogenesis no significant differences were detected. Interestingly, bone morphogenic protein-2 (BMP-2) supplementation was necessary for early-stage and late-stage osteogenic differentiation, a finding consistent with other canine studies. Additionally, synovium and adipose cMSCs proliferated more rapidly, displayed higher CFU potential, and formed larger aggregates in chondrogenic assays, although proteoglycan and collagen type II staining were subjectively decreased in adipose pellets as compared to synovial and marrow pellets. Lastly, cMSCs derived from all three tissue sources modulated murine macrophage TNF-α and IL-6 levels in a lipopolysaccharide-stimulated coculture assay.ConclusionsWhile cMSCs from synovium, marrow, and adipose tissue share a number of similarities, important differences in proliferation and trilineage differentiation exist and should be considered when selecting cMSCs for translational studies. These results and associated methods will prove useful for future translational studies involving the canine model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0639-6) contains supplementary material, which is available to authorized users.

The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.

Tissue engineered bone grafts based on bone marrow mesenchymal stromal cells (MSCs) are being actively developed for craniomaxillofacial (CMF) applications. As for all tissue engineered implants, the bone-regenerating capacity of these MSC-based grafts must first be evaluated in animal models prior to human trials. Canine models have traditionally resulted in improved clinical translation of CMF grafts relative to other animal models. However, the utility of canine CMF models for evaluating MSC-based bone grafts rests on canine MSCs (cMSCs) responding in a similar manner to scaffold-based stimuli as human MSCs (hMSCs). Herein, cMSC and hMSC responses to polyethylene glycol (PEG)-based scaffolds were therefore compared in the presence or absence of osteoinductive polydimethylsiloxane (PDMS). Notably, the conjugation of PDMS to PEG-based constructs resulted in increases in both cMSC and hMSC osteopontin and calcium deposition. Based on these results, cMSCs were further used to assess the efficacy of tethered bone morphogenic protein 2 (BMP2) in enhancing PEG-PDMS scaffold osteoinductivity. Addition of low doses of tethered BMP2 (100 ng/mL) to PEG-PDMS systems increased cMSC expression of osterix and osteopontin compared to both PEG-PDMS and PEG-BMP2 controls. Furthermore, these increases were comparable to effects seen with up to five-times higher BMP2 doses noted in literature. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2382-2393, 2018.

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