221 Isolation and characterization of mesenchymal stem cells from adipose tissue for their application on regenerative therapies in wild avian species

Mesenchymal stem cells (MSC) are adult stem cells that can mainly originate derivatives of mesoderm lineage such as osteocytes, adipocytes and chondrocytes. These cells have been isolated from several tissues (fat, bone marrow, among others) and applied in cellular therapies for treating joint and bone injuries, resulting in an improved recovery rate across diverse livestock species. In domestic birds, there are only few reports that have successfully isolated MSC from adipose tissue (aMSC), whereas no reports are available in wild avian species. Therefore, in this study we aimed to isolate, culture, and characterise aMSC from avian species with the ultimate goal of developing cellular therapies and contributing to wildlife conservation. Different amounts of fat tissue were isolated from domestic (Gallus gallus domesticus) or wild birds (Pavo cristatus and Parabuteo unicinctus) and incubated for 20 minutes at 37°C with 1 mg mL⁻¹ collagenase type I. Partially digested fat tissue was cultured to obtain aMSC. The culture media Dulbecco’s Modified Eagle Medium (DMEM) F12, and DMEM with high or low glucose were tested to determine the optimal basal media based on cell population doubling time (PDT). The molecular profile of aMSC was assessed by determining the expression of CD29, CD90, CD44, and CD34 markers by retrotranscription followed by conventional PCR. Finally, aMSC potential was evaluated qualitatively by inducing their differentiation into osteocytes, adipocytes and chondrocytes using specific growth factors and inhibitors, and stained with Alizarin red, Oil red O and Alcian blue to visualise calcium deposits, lipid drops and glycosaminoglycans, respectively. Three independent aMSCs lines in low and high passages were evaluated. During this study, a novel method that involves the culture of partially digested fat tissue was developed, enabling the retrieval of viable cells from 0.02 g of fat tissue. The aMSCs from domestic and wild birds exhibited a fibroblast-like growth morphology that remained stable for more than 10 passages. Based on PDT, culturing aMSC in DMEM F12 significantly improved cell proliferation compared with DMEM with high or low glucose (27 h, 32 h, and 37 h, respectively) (P < 0.05, one-way analysis of variance and Tukey’s post hoc test). Molecular profile showed that avian cells were positive for the stem cells markers CD29, CD44 and CD90, while negative for the haematopoietic marker CD34. Finally, aMSCs were capable of differentiating into osteocytes, adipocytes and chondrocytes, demonstrating their multipotency. Molecular profile and multipotency were maintained after multiple passages (+10), indicating a stable cellular phenotype under culture conditions. These findings underscore for the first time the optimized isolation of aMSCs from Gallus gallus domesticus, Pavo cristatus and Parabuteo unicinctus, allowing their future application in regenerative therapies.