Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 μM BrdU and 10 μM BrdU. The cells were then cultured for 6 d or passaged (1-3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3 ± 1.6% PKH67+ and 98.4 ± 1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2 ± 4.6% 10 μM BrdU+ and 77.6 ± 4.6% 5 μM BrdU+), which remained high for 6 d. Viability of labeled cells was 91 ± 3.8% PKH67+, 90 ± 1.5% DiI+, 91 ± 0.8% 5 μM BrdU+, and 76.9 ± 0.9% 10 μM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.

• Canine bone marrow-derived mesenchymal stem (BMMSC) express genes for differentiation toward osteo-, chondro-, and tendo-genic directions. • Canine BMMSC express genes associated with angiogenic, neurotrophic, and immunomodulatory properties. • Dynamic of BMMSC conditioned medium (CM) proteome revealed transcription and translation factors, osteogenic, growth, angiogenic, and neurotrophic factors. • BMMSC CM express angiogenic activity in the chorioallantoic membrane (CAM) assay.

Spinal cord injury (SCI) involves nerve damage and often leads to motor, sensory and autonomic dysfunctions. In the present study, we have designed a clinical protocol to assess the feasibility of systemic delivery of allogenic canine bone marrow tissue-derived mesenchymal stem cell conditioned medium (BMMSC CM) to dogs with SCI. Four client-owned dogs with chronic SCI lasting more than six months underwent neurological and clinical evaluation, MRI imaging and blood tests before being enrolled in this study. All dogs received four intravenous infusions with canine allogenic BMMSC CM within one month. Between the infusions the dogs received comprehensive physiotherapy, which continued for three additional months. No adverse effects or complications were observed during the one, three and six months follow-up periods. Neither blood chemistry panel nor hematology profile showed any significant changes. All dogs were clinically improved as assessed using Olby locomotor scales after one, three and six months of BMMSC CM treatment. Furthermore, goniometric measurements revealed partial improvement in the range of joint motion. Bladder function improved in two disabled dogs. We conclude that multiple delivery of allogenic cell-derived conditioned medium to dogs with chronic SCI is feasible, and it might be clinically beneficial in combination with physiotherapy.

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The aim of this study was to detect vertebral malformations among French Bulldogs admitted between the years 2011 -2016 due to the high popularity of the breed and the intentions to increase the breed welfare by reducing the occurrence of congenital anomalies. Besides, we aimed to look for gender predisposition, possible vertebral predisposition, occurrence of clinical symptoms and radiographic findings.