成果摘要 |
Millions of people suffer from organ and tissue loss and damage every year from accidents, birth defects, and diseases. Stem cells, such as human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells, are attractive prospects for translational medicine and drug discovery. Four main diseases are targeted for the clinical trials for stem cell therapy using hPS (human pluripotent stem) cells (mainly hES cells): (1) macular degeneration [age-related macular degeneration (AMD) and Stargardt’s macular dystrophy], (2) acute myocardial infarction (MI), (3) diabetes, and (4) spinal cord injury. However, efficient differentiation method of hPS cells into retinal pigment epithelium (RPE, for treatment of macular degeneration) has not yet investigated. It is unclear what kinds of molecular, physical, and biological design of cell culture biomaterials are preferable for differentiation of hPS cells into RPE. Therefore, we developed optimal cell culture biomaterials for differentiation of hPS cells into RPE in xeno-free and chemically-defined conditions in this study. We investigated (i) what kind of extracellular matrix (ECM) and ECM-derived nanosegments should be immobilized on cell culture biomaterials (biological cues) and (ii) which elasticity (physical cues) was the optimal for hPS cells to differentiate into RPE. We developed cell culture biomaterials grafted with ECM and ECM-derived oligopeptides having optimal elasticity, which were suitable for clinical usage in future. Poly(vinylalcohol-co-itaconic acid), PVA-IA, hydrogels were selected as the base biomaterials because PVA-IA hydrogels having different elasticity (i.e., different water content) can be prepared by controlling the crosslinking amount and PVA-IA hydrogels are known to be bioinert. Several ECM proteins (Matrigel, recombinant vitronectin, laminin-511 and laminin-521) and ECM-derived oligopeptides were grafted on the PVA-IA hydrogels having different elasticity in this project. hiPS cells were cultured and were differentiated into RPE on the PVA-IA hydrogels grafted with ECM and ECM-derived oligopeptides using differentiation protocols developed in this study. We evaluated the efficiency of RPE differentiation from the data and discussed the optimal cell culture biomaterials for specific differentiation of hPS cells into RPE. Human PSCs were differentiated into RPE cells on different extracellular matrix (ECM) protein-coated surface using different some protocols (i.e., NIC84 and Activin A protocols) in order to investigate the optimal differentiation conditions into RPE. We observed the polygonal morphologies of hiPSCs (HPS0077)-derived RPE, which expressed RPE specific marker (ZO-1 and RPE65) by flow cytometry and immunostaining analysis using both protocols. Finally, hiPSCs-derived RPE showed brown color (pigmented) cells. Matrigel-, Laminin-521- and Laminin-511-coated surfaces could support the differentiation of hPSCs into RPE efficiently, which were compared with Synthemax II- and Recombinant vitronectin-coated surfaces. This is explained that RPE cells are ectodermal lineage of the cells where Laminin is preferably support ectodermal cells via integrin α6β1 rather than integrin αVβ5, which is the main binding site of vitronectin. |