Chondrogenesis of Mesenchymal Stem Cell Derived from Canine Adipose Tissue / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Cartilage reconstruction is one of the important medical issues studied in otolaryngology today. Tissue engineering is presently being utilized in parts of cartilage repair. Sources of cells for tissue engineering are chondrocytes from mature cartilage and bone marrow mesenchymal stem cells that are able to differentiate into chondrocytes. Recent studies have shown that adipose tissue have mesenchymal stem cells which can differentiate into adipogenic, chondrogenic myogenic, osteogenic cells and neural cells in vitro. In this study, we have examined chondrogenic potential of the canine adipose tissue derived mesenchymal stem cell (ATSC). MATERIALS AND METHOD: We harvested canine adipose tissue from the inguinal area. ATSCs were enzymatically released from canine adipose tissue. Under appropriate culture conditions, ATSCs were induced to differentiate into chondrocyte lineages using micromass culture technique. We used immunostain to type II collagen and toluidine blue stain to confirm chondrogenic differentiation of ATSCs. RESULTS: We could isolate ATSCs from canine adipose tissue. ATSCs expressed CD29 and CD44 which are specific surface markers of mesenchymal stem cell. ATSCs differentiated into micromass that has positive response to immunostain of type II collagen and toluidine blue stain. CONCLUSION: In vitro, ATSCs differentiated into cells that have characteristic cartilage matrix molecules in the presence of lineage-specific induction factors. Adipose tissue may represent an alternative source to bone marrow-derived MSCs.

Extracellular ATP stimulates Na+ and Cl- transport through the activation of multiple purinergic receptors on the apical and basolateral membranes in M-1 mouse cortical collecting duct cells

The mammalian cortical collecting duct (CCD) plays a major role in regulating renal NaCl reabsorption, which is important in Na+ and Cl- homeostasis. The M-1 cell line, derived from the mouse cortical collecting duct, has been used as a mammalian model of the study on the electrolytes transport in CCD. M-1 cells were grown on collagen-coated permeable support and short circuit current (Isc) was measured. M-1 cells developed amiloride-sensitive current 5apprx7 days after seeding. Apical and basolateral addition of ATP induced increase in Isc in M-1 cells, which was partly retained in Na+/-free or Cl--free solution, indicating that ATP increased Na+ absorption and Cl- secretion in M-1 cells. Cl- secretion was mediated by the activation of apical cystic fibrosis transmembrane regulator (CFTR) chloride channels and Ca2+/-activated chloride channels, but Na+ absorption was not mediated by activation of epithelal sodium channel (ENaC). ATP increased cAMP content in M-1 cells. The RT-PCR analysis demonstrated that M-1 cells express P2Y2, P2X3 and P2Y4 receptors. These results showed that ATP regulates Na+ and Cl- transports via multiple P2 purinoceptors on the apical and basolateral membranes in M-1 cells.

Molecular analysis of AQP2 promoter. I. cAMP-dependent regulation of mouse AQP2 gene

To determine molecular mechanisms of Aquaporin-CD (AQP2) gene regulation, the promoter region of the AQP2 gene was examined by transiently transfecting a promoter-luciferase reporter fusion gene into mouse renal collecting duct cell lines such as mIMCD-3, mIMCD-K2, and M-1 cells, and NIH3T3 mouse embryo fibroblast cells. PCR-Southern analysis reveals that mIMCD-3 and mIMCD-K2 cells express AQP2, but M-1 and NIH3T3 cells do not, and that the treatment with cpt-cAMP (400 muM) or forskolin/isobutylmethylxanthine (IBMX) increased the AQP2 expression in IMCD cells. In both IMCD and NIH3T3 cells, the constructs containing the promoter of AQP2 gene showed promoter activities, indicating lack of tissue-specific element in the 1.4 kb 5'-flanking region of the mouse AQP2 gene. Luciferase activity in the IMCD cells transfected with the construct containing 5-flanking region showed responsiveness to cpt-cAMP, indicating that the 1.4 kb 5'-flanking region contains the element necessary for the regulatory mechanism by cAMP. The promoter-luciferase constructs which do not have a cAMP-responsible element (CRE) still showed the cAMP responsiveness in IMCD cells, but not in NIH3T3 cells. Increase in medium osmolarity did not affect AQP2 promoter activity in mIMCD-K2 cells. These results demonstrate that AQP2 gene transcription is increased with cAMP treatment through multiple motifs including CRE in the 5'-flanking region of the gene in vitro, and the regulatory mechanism may be important for in vivo regulation of AQP2 expression.