Session Title: New drug treatment and technology
Session Date/Time: Friday 27/09/2013 | 14:30-16:00
Paper Time: 15:02
Venue: Hall 3 (Level 0)
First Author: Z.Soheili IRAN
Co Author(s): H. Rezanejad F. Haddad S. Samiei M. Matin
The goal of the present study was investigation of human adipose tissue-derived stem cells (hADSCs) differentiation into photoreceptors and retinal pigment epithelium (RPE) cells. We have employed over expression of human PAX6 (5a) by lentiviral expression vectors.
National Institute of Genetic Engineering and Biotechnology
Human adipose tissue was obtained from individuals undergoing abdominoplasty. Isolated ADSCs were confirmed by their differentiation potential into adipocytes and osteocytes, as well as their MSC’s surface markers profile by flowcytometery. The coding region of human Pax6 (5a) gene isoform was cloned into pLEX- MCS Lentiviral expression vector. Lentiviral vectors pseudotyped were produced by calcium-phosphate transient transfection of HEK 293T cells. To quantify the effect of transduction of Pax6 (5a) on hADSCs proliferation and death, ADSCs from passages 2 post transduction were analyzed with the cell proliferation and cell death ELISA kits. Moreover, differentiation of hADSCs into retinal progenitor, photoreceptor and RPE cells were examined by morphological characteristics, quantitative real-time RT-PCR and immunocytochemistry for retinal cell specific markers.
ADSCs were isolated successfully from adipose tissue with a high yield and purity. The isolated ADSCs were successfully differentiated to adipocytes and osteocytes which stained with Oil Red-O and Alizarin Red staining respectively. Flowcytometric analysis of surface markers indicates the high purity (99.8%) of isolated ADSCs. Proliferation capacity of cells was significantly decreased 2 days after transduction which might be because of starting the neural differentiation. After 30 hours post transduction, cells gradually showing the characteristic morphology of neuronal cells and little axon-like processes emerged. Results confirmd that Pax6 (5a)- transduced hADSCs expressed nestin as a neural progenitor marker, RPE marker RPE65, the early photoreceptor markers NRL and recoverin and the mature photoreceptor marker rhodopsin.
In conclusion, the data gathered in this study confirmed that hADSCs can differentiate into retinal precursor, photoreceptor and RPE cells trough over expression of PAX6(5a) master gene.