303 IN VITRO NEURONAL DIFFERENTIATION OF MESENCHYMAL STEM CELLS DERIVED FROM CANINE EAR SKIN

Abstract

The canine has been a useful animal model for the study of fundamental mechanisms and the testing of new therapies for several human pathologies using mesenchymal stem cells (MSC). For preclinical applications, the most commonly used source of canine MSC is bone marrow. Because the amount of autologous bone marrow that can be obtained is limited, skin tissue could supply a noninvasive alternative with large quantities available for the establishment of MSC. In this study, we isolated canine MSC (cMSC) from ear skin and evaluated the expression of transcriptional factors and in vitro differentiation into multiple mesenchymal lineages. The cMSC isolated from the ear skin of a female beagle dog (6 years old) were cultured in advanced-DMEM/F12 (1:1, v/v) supplemented with 10% serum replacement at 37°C, 5% CO₂ in a humidified atmosphere. The cMSC at passage 3 were analysed for expression of surface markers (CD44, CD90, and CD105) and transcriptional factors (Oct-4, Nanog, and Sox2) using flow cytometry, immunohistochemical staining and RT-PCR, respectively. Differentiations into adipocytes and osteocytes of cMSC were carried out under controlled conditions for 2 and 4 weeks and evaluated by staining (Oil Red O, von Kossa and Alizarin Red S, respectively). The cMSC were induced to differentiate into neural cells in the controlled condition for 6 h. Neuronal differentiated cMSC were evaluated by immunohistochemical staining, RT-PCR, and Western blot of specific markers of neuron, such as β-tubulin, microtubule associated protein (MAP-2), neuronfilament M (NF-M), nerve growth factor (NGF), and nestin. The MSC surface markers such as CD44, 90, and 105 were highly detected, and transcriptional factors (Oct-4, Nanog, and Sox2) were expressed in cMSC. Adipocyte induced cells were positive by staining with Oil Red O, and osteocytes were stained by von Kossa and Alizarin Red S. Neuronal specific markers such as β-tubulin, MAP-2, NF-M, NGF, and nestin were expressed in the neuron induced cMSC. In conclusion, canine ear-skin-derived MSC have the capacity for differentiation into multiple lineages and have a confirmed great capability for neuronal differentiation. Hence, canine ear skin tissue could be considered a source for applications of MSC for neuronal regeneration therapy of canine and a preclinical research model for human.