The effect of extracellular matrix on differentiation of adipose derived-stem cell into islet-like cells

• Main Authors: Tzu-Hsuan Huang, 黃子瑄
• Other Authors: 丁詩同
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/f2nuhr

碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 106 === Type 1 diabetes (T1D) is an autoimmune disease, apoptotic pancreatic islet cells in T1D patients are unable to secrete enough insulin to control blood glucose. Hyperglycemia increases the risk of ketoacidosis, fatigue, and long-term damage of vascular retinopathy. To substitute tedious life-long exogenous insulin injections, cell therapy is a potential treatment to improve life quality of T1D patients. Among the pluripotent stem cells, the unique advantage of easily accessible, abundant and multipoint adipose-derived stem cells (ADSCs) could be a promising source for transplantation and regenerative medicine. Stem cell fate is harnessed by systemic gene expression and microenvironment (niche) which includes soluble ligands, cell communications and extracellular matrixes (ECMs) that are mostly fibril glycoproteins secreted by cells. Not only acting as supporting structures, ECMs can communicate inside and outside information by binding integrins on the cell membrane with their specific motif. This interaction is critical for the determination of the pluripotent stem-cell fate, such as self-renewal, proliferation, pluripotency and differentiation. The porcine ADSC (pADSC) was isolated from the dorsal subcutaneous fat in 7 to 11- day-old Landrace x Yorkshire hybrid piglets (Sus scrofa domestics). pADSCs were induced to porcine islet-like clusters (pILC) of insulin producing cells by chemical induction medium developed in our lab on different coating dishes with regular polystyrene, chitosan, or chitosan+FGF-2. To get the ECM structure with cell removal, decellularizing solutions such as NH4OH, Triton X-100 and DNase were combined to minimize intracellular matrix and preserve ECM protein. Levels of nucleus elimination was examed by fluorescent DAPI staining. Cytoskeletal β-actin and one of the ECM protein fibronectin were detected by western blot. Scanning electron microscope were used to observe the outlook of ECM surface. These analyses showed nuclear and cytosolic proteins were dramatically reduced and the 3-D structure of ECM were well- preserved. Proteomics were also performed to compare pADSC-ECM and pILC-ECM, seven ECM proteins were identified and individual gene expression was confirmed by real-time qPCR. Finally, pADSCs were re-seeded on pADSC-ECM or pPILC-ECM to test whether ECMs promote differentiation of islet-like cells. Expression of pancreatic islet differentiation marker (GLUT2, ISL1, GCK1) was found to be up-regulated, especially on culture dishes with chitosan or chitosan+FGF-2 (P<0.05). In conclusion, decellularizing methods for pADSC or pILC were established. Furthermore, the composition of ECMs entails pancreatic islet differentiation and identified proteins of extracellular matrix in this research may provide a suitable niche assisting pADSC differentiation into insulin-producing cells.