In the dog, mesenchymal stem cells (MSCs) have been shown to reside in the bone marrow (bone marrow-derived mesenchymal stem cells: BM-MSCs) as well as in the adipose tissue (adipose tissue-derived stem cells: ADSCs). Potential application fields for these multipotent MSCs in small animal practice are joint diseases as MSCs of both sources have shown to possess chondrogenic differentiation ability. However, it is not clear whether the chondrogenic differentiation potential of cells of these two distinct tissues is truly equal. Therefore, we compared MSCs of both origins in this study in terms of their chondrogenic differentiation ability and suitability for clinical application. BM-MSCs harvested from the femoral neck and ADSCs from intra-abdominal fat tissue were examined for their morphology, population doubling time (PDT) and CD90 surface antigen expression. RT-PCR served to assess expression of pluripotency marker Oct4 and early differentiation marker genes. Chondrogenic differentiation ability was compared and validated using histochemistry, transmission electron microscopy (TEM) and quantitative RT-PCR. Both cell populations presented a highly similar morphology and marker expression in an undifferentiated stage except that freshly isolated ADSCs demonstrated a significantly faster PDT than BM-MSCs. In contrast, BM-MSCs revealed a morphological superior cartilage formation by the production of a more abundant and structured hyaline matrix and higher expression of lineage specific genes under the applied standard differentiation protocol. However, further investigations are necessary in order to find out if chondrogenic differentiation can be improved in canine ADSCs using different protocols and/or supplements.

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.

BackgroundTherapy with mesenchymal stem cells (MSCs) has been reported to provide beneficial effects in the treatment of neurological and orthopaedic disorders in dogs. The exact mechanism of action is poorly understood. Magnetic resonance imaging (MRI) gives the opportunity to observe MSCs after clinical administration. To visualise MSCs with the help of MRI, labelling with an MRI contrast agent is necessary. However, it must be clarified whether there is any negative influence on cell function and viability after labelling prior to clinical administration.ResultsFor the purpose of the study, seven samples with canine adipose-derived stem cells were incubated with superparamagnetic iron oxide nanoparticles (SPIO: 319.2 μg/mL Fe) for 24 h. The internalisation of the iron particles occurred via endocytosis. SPIO particles were localized as free clusters in the cytoplasm or within lysosomes depending on the time of investigation. The efficiency of the labelling was investigated using Prussian blue staining and MACS assay. After 3 weeks the percentage of SPIO labelled canine stem cells decreased. Phalloidin staining showed no negative effect on the cytoskeleton. Labelled cells underwent osteogenic and adipogenic differentiation. Chondrogenic differentiation occurred to a lesser extent compared with a control sample. MTT-Test and wound healing assay showed no influence of labelling on the proliferation. The duration of SPIO labelling was assessed using a 1 Tesla clinical MRI scanner and T2 weighted turbo spin echo and T2 weighted gradient echo MRI sequences 1, 2 and 3 weeks after labelling. The hypointensity caused by SPIO lasted for 3 weeks in both sequences.ConclusionsAn Endorem labelling concentration of 319.2 μg/mL Fe (448 μg/mL SPIO) had no adverse effects on the viability of canine ASCs. Therefore, this contrast agent could be used as a model for iron oxide labelling agents. However, the tracking ability in vivo has to be evaluated in further studies.

Adipose tissue-derived stem cells (ADSCs) represent a promising subpopulation of adult stem cells for tissue engineering applications in veterinary medicine. In this study we focused on the morphological and molecular biological properties of the ADSCs. The expression of stem cell markers Oct4, Nanog and the surface markers CD90 and CD105 were detected using RT-PCR. ADSCs showed a proliferative potential and were capable of adipogenic and osteogenic differentiation. Expression of Alkaline phosphatase (AP), phosphoprotein (SPP1), Runx2 and osteocalcin (OC) mRNA were positive in osteogenic lineages and peroxisome proliferator activated receptor (Pparγ2) mRNA was positive in adipogenic lineages. ADSCs show stem cell and surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow-derived mesenchymal stem cells (BM-MSCs). The availability of an easily accessible and reproducible cell source may greatly facilitate the development of stem cell based tissue engineering and therapies for regenerative equine medicine.