Comparing Three Methods of Co-culture of Retinal Pigment Epithelium with Progenitor Cells Derived Human Embryonic Stem Cells

摘要

Background: Close interaction between retinal pigment epithelium (RPE) and photoreceptors plays an essential role in visual function. The objective of this study is to determine the effects of RPE cells in the differentiation of progenitor derived human embryonic stem cells (hESC) into retinal cells; we developed in vitro co-culture models and compare these models to investigate in which model the expression of photoreceptor markers is superior. It seems the effects of RPE cells on differentiation of retinal progenitor cells (RPCs) through the cell-to-cell contact or with the use of insert and compare of these methods has not been reported yet. Methods: Initially, retinal progenitors (RPs) were differentiated from hESC. After isolation of RPE sheet from rabbit eyes, demonstrated these cells maintains the integrity and feature after 2 weeks. Next, we examined the induction of photoreceptors by the co-culture of RPE through insert in 1 week and 2 weeks (indirect) or without insert by the cell-to-cell contact (direct). The differentiation of retinal cells was verified by protein and gene expression in these three methods. The adherent cells were morphologically examined using phase contrast microscopy and characterized by immunofluorescent staining and reverse transcription.polymerase chain reaction (RT-PCR) Results: Evaluation of immunostaining showed that hESC, highly (80%) can be directed to the RPs fate. Upon co-culture of RPCs with RPE sheet using insert for 2 weeks or by the cell-to-cell contact, these cells differentiated to neural retina and expressed photoreceptor-specific markers. However, in direct co-culture, some mature photoreceptor markers like arrestin expressed in compare with indirect co-culture. Conclusions: The expression of late photoreceptor marker could be improved when RPE cells seeded on RPCs in compare with the use of insert.