MESENCHYMAL STEM CELLS OF THE CANINE UMBILICAL CORD: ISOLATION, CHARACTERIZATION AND CLINICAL EVALUATION IN DOGS WITH NEUROLOGICAL SEQUELS OF DISTEMPER

Background Distemper is a viral multisystem disease that affects dogs of all ages. So far, there are only palliative treatments and the consequence of the disease is decreasing the quality of life of the animals and in more severe cases is euthanasia. Mesenchymal stem cells (MSCs) are potential candidates for the treatment of distemper due mainly to their immunomodulatory and anti-inflammatory characteristics. Although the most explored source for the isolation of MSC in dogs is the adipose tissue, the collection results in invasive procedures, and the cells may be influenced by the age and physical conditions of the donor, so there is great interest in seeking alternative sources of MSC. Our objectives are to validate and define an optimized isolation protocol for canine umbilical cord MSCs for application in dogs diagnosed with neurological sequels of distemper. Methods The umbilical cords obtained in elective cesarean sections were processed according to the following protocols: Group 1 - 1 hour in collagenase 1 mg/ml, group 2 - 1 hour in trypsin 0.125% + 1 hour in collagenase 1 mg/ml, and group 3 - tissue explants. Following isolation, the cells were cultured in the same way, in DMEM culture medium, and a CO2 incubator at 37°C. After cultivation, the groups were subjected to evaluation of specific membrane markers by flow cytometry (CD90, CD44, CD29, CD45, CD34, CD14, and HLA-DR). Later in this project, we will carry out cell differentiation tests and the therapeutic evaluation will be carried out through the transplantation of umbilical cord MSCs in dogs diagnosed with neurological sequels of distemper. Results Using the same amount of starting material, groups 1 and 2 showed more adhered cells (at least ten times greater) than group 3, in addition to having greater expansion capacity along the passages. Groups 1 and 2 were similar in the number of cells obtained, with group 2 showing more homogeneous morphology from passage 2. In a preliminary study of membrane markers by cytometry, we observed that groups 1 and 2 show expression of the characteristic markers of MSC, however, group 1 showed a greater number of cells positive for CD90 and CD44 and negative for CD34 and HLA-DR. Conclusions So far, we have observed that the canine umbilical cord is a very relevant source of MSC, and that isolation by enzymatic methods is more effective than obtaining by explants. Distemper is a viral multisystem disease that affects dogs of all ages. So far, there are only palliative treatments and the consequence of the disease is decreasing the quality of life of the animals and in more severe cases is euthanasia. Mesenchymal stem cells (MSCs) are potential candidates for the treatment of distemper due mainly to their immunomodulatory and anti-inflammatory characteristics. Although the most explored source for the isolation of MSC in dogs is the adipose tissue, the collection results in invasive procedures, and the cells may be influenced by the age and physical conditions of the donor, so there is great interest in seeking alternative sources of MSC. Our objectives are to validate and define an optimized isolation protocol for canine umbilical cord MSCs for application in dogs diagnosed with neurological sequels of distemper. The umbilical cords obtained in elective cesarean sections were processed according to the following protocols: Group 1 - 1 hour in collagenase 1 mg/ml, group 2 - 1 hour in trypsin 0.125% + 1 hour in collagenase 1 mg/ml, and group 3 - tissue explants. Following isolation, the cells were cultured in the same way, in DMEM culture medium, and a CO2 incubator at 37°C. After cultivation, the groups were subjected to evaluation of specific membrane markers by flow cytometry (CD90, CD44, CD29, CD45, CD34, CD14, and HLA-DR). Later in this project, we will carry out cell differentiation tests and the therapeutic evaluation will be carried out through the transplantation of umbilical cord MSCs in dogs diagnosed with neurological sequels of distemper. Using the same amount of starting material, groups 1 and 2 showed more adhered cells (at least ten times greater) than group 3, in addition to having greater expansion capacity along the passages. Groups 1 and 2 were similar in the number of cells obtained, with group 2 showing more homogeneous morphology from passage 2. In a preliminary study of membrane markers by cytometry, we observed that groups 1 and 2 show expression of the characteristic markers of MSC, however, group 1 showed a greater number of cells positive for CD90 and CD44 and negative for CD34 and HLA-DR. So far, we have observed that the canine umbilical cord is a very relevant source of MSC, and that isolation by enzymatic methods is more effective than obtaining by explants.