Purchase this article with an account.
Furong Gao, Zongyi Li, Weiye Li, Lixia Lu, Guotong Xu; Efficient generation of retinal pigment epithelium cells from human embryonic stem cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1379.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) disorders usually cause problems with vision. RPE derived from human embryonic stem cells (hESCs) may become a promising therapeutic option for transplantation in these retinal diseases. However, induction of hESCs to RPE cells often takes several months with a low frequency. The purpose of this study was to establish an improved method that takes a short time and with a high efficiency.
ShhES2 cells were induced to differentiate into RPE by the embryoid body (EB) formation method. We treated embryoid bodies with a combination of CKI-7, SB431542, and two other factors in N2B27 medium with or without PJ34. The EBs were cultured for 3 days and then transferred to matrigel-coated dishes for attached growth for two weeks. At last, the cells were maintained in hESCs medium without bFGF for an additional 2 weeks. Then, the cells were passaged for further differentiation. Assessment of differentiation was performed using pigmentation formation, mRNA and immunocytochemistry. The abilities of hESCs-derived RPE cells in rescuing retinal structure and visual function of RCS rats after subretinal transplantation were evaluated by electroretinogram (ERG), histology (HE staining) and immunohistology (TUNEL assay).
Compared to the four factors group, PJ34, in combination with the four factors increased the expression of transcripts of the RPE cell markers MITF and RPE65 after 4 weeks of differentiation, but decreased photoreceptor markers Rx and CHX10. In addition, in the PJ34 group, pigmented areas with a cobblestone appearance began to appear within the differentiating clusters after 4 weeks, and by 6 weeks, almost all the ShhES2 differentiating cells contained pigment granules and had a cobblestone appearance. In the absence of PJ34 group, much less pigmented areas were observed. Furthermore, the transplanted hESCs-derived RPE cell can improve the retinal structure and function of RCS rats.
This study demonstrated that PJ34 can promote the differentiation of hESCs toward an RPE fate at a high efficiency in a short time. The subretinal transplantation of hESC-derived RPE appears to improve the structure and function of degenerative retina of RCS rats. These findings indicate a new short-term and efficient protocol for differentiation of hESCs to RPE cells and thus may be useful in the treatment of retinal degenerations.
This PDF is available to Subscribers Only