OBrien, Email: ude.nmu@400eirbo. Kuldeep S. Methods We isolated MSCs from the lungs (L-MSCs) of 4C6-week-old germ-free pigs. We determined the self-renewal, proliferation and differentiation potential of L-MSCs. We also examined the mechanisms of immunoregulation by porcine L-MSCs. Results MSCs isolated from porcine lungs showed spindle-shaped morphology and proliferated actively in culture. Porcine L-MSCs expressed mesenchymal markers CD29, CD44, CD90 and CD105 and lacked the expression of hematopoietic markers CD34 and CD45. These cells were multipotent and differentiated into adipocytes, osteocytes and epithelial cells. Like human MSCs, L-MSCs possessed immunoregulatory properties and inhibited proliferation of T cells and interferon- and tumor necrosis factor- production by T cells and dendritic cells, respectively, and increased the production of T-helper 2 cytokines interleukin (IL)-4 and IL-13 by T cells. L-MSCs induced the Apigenin-7-O-beta-D-glucopyranoside production of prostaglandin E2 (PGE2) in MSCCT cell co-cultures and inhibition of PGE2 significantly restored (not completely) the immune modulatory effects of L-MSCs. Conclusions Here, we demonstrate that MSCs can be isolated from porcine lung and that these cells, similar to human lung MSCs, possess in vitro proliferation, differentiation and immunomodulatory functions. Thus, these cells may serve as a model system to evaluate the contribution of lung MSCs in modulating the immune response, interactions with resident epithelial cells and tissue repair in a pig model of human lung diseases. value <0.05 was considered to be statistically significant. Results Isolation of plastic-adherent porcine L-MSCs MSCs were successfully isolated from the lungs of all six pigs. These MSCs showed characteristic features of MSCs, such as adherence to plastic surface and fibroblast-like morphology (Fig.?1a). Open in a separate window Fig. 1 Characteristics of porcine L-MSCs. a Morphology of porcine L-MSCs. Porcine L-MSCs exhibit characteristic fibroblast-like morphology. b Colony forming unit-fibroblast assay. L-MSCs were cultured Apigenin-7-O-beta-D-glucopyranoside at 100 cells/well in a six-well plate. Single cells proliferated and formed colonies as shown by Giemsa staining. c In vitro proliferation potential of L-MSCs. L-MSCs were suspended in DMEM containing 10 %10 % FBS and cultured in a 96-well plate. At indicated intervals, cell proliferation was measured by MTT assay. Optical density (isotype control, antibody staining. (c) Expression of Oct4 on L-MSCs. L-MSCs were examined for the expression of the pluripotency marker, Oct4, by IFA. BM-MSCs were included as positive control. Bone marrow mesenchymal stem cell, Lung mesenchymal stem cell, Swine leucocyte antigen L-MSCs were also examined for the expression of the pluripotency marker Oct4 (Fig.?2c). The expression of Oct4 was mainly detected in the cell nuclei of L-MSCs. Porcine L-MSCs can differentiate into adipocytes, osteocytes and epithelial cells MSCs from BM and other anatomical locations demonstrate mutilineage differentiation potential. L-MSCs also demonstrated mutilineage differentiation potential. L-MSCs when cultured in adipocyte induction media for 21 days differentiated into adipocytes. Differentiated cells contained multiple lipid vacuoles as demonstrated by staining with Oil Red O Rabbit Polyclonal to DCC (Fig.?3a). Incubation of L-MSCs in osteogenic media for 3 weeks demonstrated tightly packed nodule-like structures. Calcium deposition in differentiated cells was detected by Von Kossa staining (Fig.?3c). Open in a separate window Fig. 3 Differentiation potential of porcine L-MSCs. a Adipocyte differentiation. L-MSCs when cultured in adipogenic medium for 21 days showed lipid droplets in the cytoplasm of differentiated cells. b No adipocyte differentiation was detected in cells cultured in DMEM. c Osteocyte differentiation. L-MSCs cultured in osteogenic medium for 21 days showed calcium deposition as detected by Von Kossa staining. d No osteogenic differentiation was observed in cells cultured in DMEM. eCh Epithelial differentiation. L-MSCs cultured in epithelial differentiation medium for 10 days exhibited cuboidal morphology (e) and were found to express epithelial markers pan-cytokeratin (g) and cytokeratin-18 (i) whereas L-MSCs cultured in DMEM displayed normal spindle-shaped morphology (f), and expression of pan-cytokeratin (h) and cytokeratin-18 (j) was not detected on undifferentiated L-MSCs L-MSCs also differentiated into epithelial cells. L-MSCs cultured in epithelial cell differentiation media for 10 days exhibited cuboidal-like morphology (Fig.?3e) and Apigenin-7-O-beta-D-glucopyranoside positive staining for epithelial cell markers pancytokeratin and cytokeratin-18 (Fig.?3g and i). Immunomodulation by L-MSCs L-MSCs inhibit TNF- secretion by DCs L-MSCs were co-cultured with BM-derived DCs at a ratio of 1 1:10 and stimulated with LPS overnight. Data are expressed as percent change in TNF- production in DCs in the presence or absence of MSCs (Fig.?4). There was more than a.