Ps-89: Adipose Tissue-Derived Mesenchymal Stem Cells Differentiation into Neural Retinal Cells through PAX6 Gene Over-Expression


Rezanejad Bardejee H *, Soheili ZS , Haddad F , Samiei S , Matin M , Manafi A ,

0

Objective: Adipose- derived mesenchymal stem cells (ADSCs) are a kind of mesenchymal stem cells which have revealed many advantages over other stem cellsources. Pax6 gene is highly conserved among species and plays an important role in eye morphogenesis. Here we studied the ADSCs differentiation into neural retina cells as a tool for cell therapy in retinal degenerative disorders. Materials and Methods: The coding region of human Pax6 (+5a) gene isoform was synthesized and cloned into pUC57 cloning vector. The coding sequence of Pax6 (+5a) was then digested by BamHI and XhoI restriction enzymes and subsequently subcloned into pLEX- IRES- Pur Lentiviral vector. The recombinant construct was confirmed by PCR, digestion and finally sequencing. For isolation of ADSCs, human adipose tissue was obtained from abdominal subcutaneous tissues of patients undergoing abdominoplasty. Isolated ADSCs were characterized by their differentiation potential into adipocytes and osteocytes, moreover their surface markers including CD34, CD45, CD73, CD 90 and CD105 were examined by flowcytometery. The lentiviral particles were produced in HEK293T cells and subsequently the ADSCs were transducted. The successful transducted cells were selected by puromycin resistance. Results: We constructed a vector expressing Pax6 protein. 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 differentiated cells was decreased strongly and cell death was observed. After 5 days culture in the presence of puromycin, cells developed a neuron-like configuration. Pax6 and Nestin expression were confirmed by immunocytochemistry(ICC). ICC for other retinal cell markers and Real-time PCR tests are undergoing. Conclusion: The ADSCs were differentiated to some cell types of neural retina. Although more researches are under investigation.