Ezati Razie, Etemadzadeh Azadeh, Soheili Zahra-Soheila, Samiei Shahram, Ranaei Pirmardan Ehsan, Davari Malihe, Najafabadi Hoda Shams
Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
J Cell Physiol. 2018 Feb;233(2):1222-1235. doi: 10.1002/jcp.25991. Epub 2017 Jun 22.
Cell replacement is a promising therapy for degenerative diseases like age-related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed toward persuading for cells that can differentiate into retinal neurons. In this report, we have investigated reprogramming of the human RPE cells and concerned the effect of donor age on the cellular fate as a critical determinant in reprogramming competence. We evaluated the effect of SOX2 over-expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into adeno-associated virus (AAV2) and primary culture of human neonatal/adult RPE cells were infected by recombinant virus. De-differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real-time PCR and ICC for neural/retinal progenitor cells' markers. Gene expression analysis showed 80-fold and 12-fold over-expression for SOX2 gene in infected neonatal and adult hRPE cells, respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8-fold and 2.5-fold, respectively. PAX6 expression was increased threefold and 2.5-fold in neonatal/adult treated cultures. Howbeit, we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to de-differentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1, and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro-retinal terminally differentiated cells.
细胞替代疗法是治疗诸如年龄相关性黄斑变性（AMD）等退行性疾病的一种有前景的方法。由于人类视网膜缺乏再生能力，因此人们将大量注意力转向诱导能够分化为视网膜神经元的细胞。在本报告中，我们研究了人类视网膜色素上皮（RPE）细胞的重编程，并关注供体年龄对细胞命运的影响，这是重编程能力的一个关键决定因素。我们评估了SOX2过表达在培养的人类新生儿和成人RPE细胞中的作用。将人类SOX2基因的编码区克隆到腺相关病毒（AAV2）中，并用重组病毒感染人类新生儿/成人RPE细胞的原代培养物。通过定量实时PCR和免疫细胞化学（ICC）检测神经/视网膜祖细胞标志物，研究RPE向神经/视网膜祖细胞的去分化。基因表达分析显示，在感染的新生儿和成人hRPE细胞中，SOX2基因的过表达分别为80倍和12倍。新生儿和成人hRPE细胞中巢蛋白（Nestin）的增加倍数分别为3.8倍和2.5倍。在新生儿/成人处理的培养物中，PAX6的表达增加了3倍和2.5倍。然而，我们在新生儿hRPE培养物中未检测到视紫红质和CHX10的表达，在成人hRPE细胞中也未检测到视紫红质的表达。结果表明，SOX2诱导人类新生儿/成人RPE细胞向视网膜祖细胞去分化。然而，在成人hRPE处理的培养物中，PAX6、CHX10、Thy1和视紫红质阳性细胞数量的增加清楚地表明了大量神经视网膜终末分化细胞的产生。
