Comparison of culture requirements for canine and human umbilical cord-derived mesenchymal stromal cells

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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Burdzińska, Anna (Department of Immunology, Transplantology and Internal Diseases, Medical University of Warsaw )


Treating immune-related diseases, which incidence is steadily growing, is still a challenge. Among the new treatments under development, mesenchymal stromal cell (MSC) transplants seem to be one of the most promising. Although MSC-based therapies are being implemented into the clinic, many aspects regarding these procedures requires further optimization. A new trend in translational medicine is to treat veterinary medicine as a bridge between research on diseases models in laboratory animals and human clinical trials. Dogs suffer from immune-related diseases which are similar to those in humans. One of them is anal furunculosis, which is considered to be a model of anal fistulas in Crohn’s disease in humans. The objective of this study was to optimize the culture conditions of canine mesenchymal stromal cells isolated from umbilical cords (cUC-MSC) and to compare their requirements to human counterparts (hUC-MSC). Cells isolated from 4 canine UC and 4 human UC were used in this study. Human cells met standard identification requirements for human MSCs. Isolated canine cells expressed MSC markers like CD90, CD44 and did not express hematopoietic markers like CD45 and CD11b (flow cytometry). Additionally they expressed vimentin (immunocytochemistry). In standard growth medium (GM, composed of DMEM+10% foetal bovine serum) and on standard uncoated culture surface canine cells underwent rapid senescence. After the 3rd passage a mean of 50.9% of cells displayed features of in vitro aging (β-galactosidase assay, β-Gal). In contrast, only 8.7% of human cells were senescent in the same conditions after 3rd passage, p=0.008 in comparison to canine cells. The metabolic activity (MTT assay) under these basic growth conditions was also significantly higher in human cells than in canine cells (p=0.001). The addition of basic fibroblast growth factor (bFGF, 5 ng/mL), coating culture dishes with porcine gelatin (GEL) and combination of both factors (bFGF+GEL) were tested to establish favorable growth conditions for canine cells. The highest growth rate was observed in bFGF+GEL conditions. The average population doubling time amounted 4.5, 1.7, 2.9, and 1.3 days for cells cultured in GM, bFGF, GEL and bFGF+GEL, respectively. The β-Gal assay performed in canine cells cultured in bFGF+GEL conditions showed that, on average, 6.2% of the cells after 3rd passage were senescent, and this level was 8.1 times lower than that of cells grown in GM (p=0.02). To assess the tolerogenic activity, cUC-MSCs and hUC-MSCs were co-cultured with allo-activated human peripheral blood mononuclear cells. Both cell types increased the proportion of T regulatory cells (FoxP3 expressing cells) in the population of CD3+CD4+ cells. Obtained results demonstrate that cells isolated from canine umbilical cords have different culture requirements than human UC-MSCs. However, when cultured in the presence of bFGF and on gelatin-coated dishes, a sufficient number of cells from one canine umbilical cord can be obtained for transplantation into at least one canine patient. These results open up the possibility of testing the effects of cell transplantation in dogs with naturally occurring immune-mediated diseases. Such veterinary clinical trials might also help to improve MSC-based techniques in humans.


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