부산대학교 도서관

PURPOSE OF STUDY: Lethal neonatal respiratory distress syndrome can be caused by a rare, recessive mutation in the surfactant protein-B (SFTPB) gene (P133ins2) and the only treatment option is lung transplantation. Using human induced pluripotent stem cell (hiPSC) technologies to differentiate them into organ specific cells and correct the mutations with cell based gene therapies, patient specific diseases can be investigated and treated. Our hypothesis is that alveolar type II (ATII) cells can be derived through directed differentiation of hiPSCs from individuals genetically deficient in pulmonary SPFTB and function can be restored through lentiviral mediated over-expression of SFTPB. METHODS USED: We used a lentivirus vector containing the SFTPB wild type sequence downstream from a constitutively expressed promotor and a GFP-puromycin reporter sequence for infection verification. We infected hiPSCs derived from patient specific SFTPB deficient fibroblasts and then differentiated the SFTPB-rescued hiPSCs into 3D lung organoids made up of ATII cells. SUMMARY OF RESULTS: The SFTPB deficient hiPSCs were successfully infected with the lentivirus and expression levels of SFTPB were elevated in the SFTPB-rescued hiPSCs compared with the SFTPB deficient hiPSCs with QPCR and western blots. Using a novel directed differentiation protocol, the 3D organoids derived from the SFTPB-rescued hiPSCs showed gene and protein expression ATII cells. Lamellar bodies, representing surfactant storage in ATII cells were detected through electron microscopy in the SFTPB-rescued cells. CONCLUSIONS: This represents the first step in using cell based gene therapy to overexpress a non-functional gene in surfactant deficiency which will hopefully lead to a possible cure for newborn babies.