Abstract
Purpose:
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.
Methods:
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).
Results:
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.
Conclusions:
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.
Keywords: 721 stem cells •
698 retinal development •
500 differentiation