TDK conference 2013

Mesenchymal stem cells (MSCs) also called as multipotent adult stem cells are present in almost all organs and tissues in dogs as well. MSCs are self-renewing, can differentiate toward adipogenic, osteogenic and chondrogenic lineages. The most available tissue sources of MSCs include the bone marrow, the adipose tissue, and the Wharton’s jelly of the umbilical cord. Regenerative medicine both in the human and veterinary fields often use adipose tissue originated stem cells for bone and cartilage defect replacements.

Aims of our studies were to establish methods for MSCs isolation, characterisation and differentiation and to prove their therapeutical potentials for the veterinary use. Furthermore we compared the in vitro growing and differentiating abilities of MSCs originated from dogs at different age.

Five to 20 gramm adipose tissue (AT) samples were collected from subcutaneous fat depots from the thoracic region of Beagle dogs using standard sterile surgical procedures. The AT was cut into small pieces, digested by collagenase enzyme, and separated the fat from the mixed population cell pellet by centrifugation. Only the cells adhered the bottom of culture flasks, passed further in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). The culture medium was replaced with osteo-inductive and chondro-inductive mediums, when the cells showed homogenous fibroblast-like morphology under light microscope. At the start, one and three weeks later the osteogenesis was detected by Alizarin red staining (calcium deposits) and 5-bromo-4-chloro-3'-indolyl phosphate / nitro blue tetrazolium test (BCIP/NBT, alkaline phophatase production) while the chondrogenesis was tested by dimethyl-methylblue staining.

cAD-MSCs originated from young dogs show faster multiplication in vitro before and after induction of osteo- and chondrogenesis. We strongly believe that canine adipose tissue is an available source of MSCs also in dogs and these cells with or without in vitro differentiation theoretically available for veterinary regenerative medicine. The canine model could be a useful tool for human stem cell research.