Following collagenase digestion, canine UC derived cells were plated with MSC culture medium. Spindle shaped plastic adherent cells were observed after 3 days of seeding and created a homogeneous monolayer on the culture flask. In subsequent passages the cells were able to maintain their fibroblast like morphology. In immunocytochemical staining, cells were found positive for the MSC-specific surface markers CD105, CD90 and CD73; and were negative for the hematopoietic cell marker CD45. The cells were induced to differentiate into osteocytes, chondrocytes, and adipocytes in standard in vitro differentiating conditions. Osteogenic differentiation was confirmed by the mineralized nodules of orange-red color in alizarin red staining, chondrogenic differentiation was confirmed by proteoglycan accumulation as blue color after Alcian blue staining and, lipid vacuoles were observed in red color in the adipogenic differentiated in Oil red- O staining (Fig. 1A-E).
The derivation of MSCs from umbilical cord is a non-invasive method with minimum ethical concern and therefore, considered a choice cell source for therapeutic applications. Besides human, the MSCs have been successfully isolated and characterized from caprine, bubaline, canine, equine and bovine umbilical cord matrix till date. Canine MSCs were found plastic adherent small spindle shaped fibroblast like cells that express MSC-specific surface markers and, had the tri-lineage differentiation capability in vitro as noticed in our experiment (Das et al. 2017). Therefore, the cells isolated here from canine umbilical cord tissue meet the criterion of multipotent mesenchymal stem cells.
In gene expression analysis of these cells positive amplifications were noticed against the transcripts of GAPDH, CXCL8, PI3, HAMP, LCN2 and SLPI only which was confirmed by the agarose gel electrophoresis of the RT-PCR products (Fig. 1F). However, no expression was detected for the transcripts of CAMP, DEFB1, DEFB4A, DEFB103A, Lactoferrin and GNLY.
AMPs, at a low concentration, are not only naturally secreted by humans, but also by other species. Moreover, there is tissue and species-specific variations in the expression profile of each of the known AMPs. AMPs synthesized by MSCs of different species exhibit strong antimicrobial effects via direct and indirect mechanism (Meisel et al. 2011; Krasnodembskaya et al. 2010). AMPs like elafin, hepcidin and lipocalin 2; and other immunomodulatory molecules CXCL8 ligand, SLPI and lactoferrin are actively involved in innate immunity although they serve other functions too. A recent study showed an expression of AMPs, Apolipoprotein B and D, amyloid-β peptide, Cathepsin B, Protein S100-A4 in the conditioned medium (CM) of canine bone marrow derived MSCs (Bujňáková et al. 2020). Previously Elafin expression was observed in horse peripheral blood MSC-CM, canine amniotic membrane and in human amniotic epithelial cells (Harman et al. 2017). Elafin attenuates several key processes in the inflammatory cascade by inhibiting neutrophil elastase and proteinase 3 (Shaw and Wiedow 2011). SLPI is a member of innate immunity associated proteins and, it has been noticed that SLPI along with the Elafin are considered as the members of trapping gene family (Doumas et al. 2005). Therefore, it can be said that if AMP Elafin gets expressed in an antimicrobial interaction within the host system, the expression of SLPI might be seen. The CXCL8 ligand helps in recruiting neutrophils at the site of infection (de Oliveira et al. 2013); Hepcidin acts as a regulator of systemic iron homeostasis (El Gendy et al. 2019). In neutrophils, lipocalin-2 is produced at stages of granulopoiesis in the bone marrow, stored in the secondary granules and have bacteriostatic effect (Dahl et al. 2018). The expression of BD family genes cBD 1, cBD 102, cBD 103 etc. have been observed in canine amniotic membrane, skin, trachea and testis (Lohajaroensub et al. 2022). However, in our study with canine umbilical cord derived MSC we did not find any expression of BD family genes. It is important to note that the expressions of AMPs vary during in vitro culture of MSCs of different species and, source tissue also carry a significant role in expressing different kinds of AMPs.
The canine umbilical cord tissue derived MSCs were isolated and characterized. During standard in vitro propagation a screening was done to check the expression of few commonly known AMPs in these cells. It has been found for the first time that canine MSCs naturally express few AMPs like CXCL8, Elafin, Hepcidin, Lipocalin 2 and SLPI. However, their relation with antimicrobial effect of canine MSCs needs to be explored.