Isolation and characterization of canine umbilical cord blood-derived mesenchymal stem cells

Abstract

Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence- activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III beta tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.

Fig. 1

Identification of the cumulative population doubling level (CPDL) and culture of canine umbilical cord blood (cUCB)-mesenchymal stem cells (MSCs). Cells were cultured in DMEM (with 20% FBS). A: Two bars in a graph indicate the CPDL increase. Both bars show a consistently increasing growth rate during the passages. Each bar increase originates from the CPDL cumulative values, which were two separated sampled cells. B: Phase-contrast image of cUCB-MSCs, ×200.

Fig. 2

Immunostaining of undifferentiated and neuronal differentiated cUCB-MSCs. cUCB-MSCs were immunostained with glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), neuronal class III β tubulin (Tuj-1), Nestin and neurofilament M (NF160). Negative control was confirmed with Alexa 488 (green) and Alexa 594 (red). A: The cells were cultured with basal cultured media. B: The cells were cultured with neuronal differentiation media. C-H: Comparing to basal culture condition (undifferentiation) with neuronal differentiation condition. C, E and G: Undifferentiation; D, F and H: Neuronal differentiation. Nestin, Tuj-1 and NF160 were green. GFAP and MAP2 were red. Scale bars = 50 µm.

Fig. 3

Osteogenic and chondrogenic differentiation of cUCB-MSCs. A-C: Osteogenic differentiation. A and B: Alizarin red S staining. A: Undifferentiation (UDF), B: Differentiation (DF), C: RT-PCR. (D-F) Chondrogenic differentiation. D: Pellet formation; E and F: Toluidine blue stain. A: ×200, B: ×200, E: ×100, F: ×200.