Abstract
Purpose::
Dysfunction, injury, and loss of retinal pigment epithelium (RPE) cells is a prominent feature of Age Related Macular Degeneration (AMD) and also occurs in subtypes of Retinitis Pigmentosa (RP). Our objective was to evaluate the ability of human embryonic stem cells (hESC) to give rise to RPE cells in-vitro and to examine their survival in-vivo following intraocular transplantation into rodent eyes.
Methods::
Differentiation of hESC was induced by culturing embryonic bodies (EBs) in suspension for at least 4 weeks. Clusters of pigmented cells within the EBs were mechanically dissected, plated and further cultured on laminin-coated slides. Phenotype of the cells was characterized by immunostaining and PCR for RPE-associated markers. For in-vivo transplantation, a cell suspension enriched with pigmented cells was derived from the cultures and delivered into the sub-retinal or intravitreal space of RCS rats which manifest an RPE-specific mutation and in albino Sprague-Dawley rats. Survival of engrafted cells and expression of RPE-specific markers were examined by immunohistology 3.5 weeks after transplantation.
Results::
Prolonged differentiation of hESC within EBs gave rise to clusters of pigmented cells. Nearly 70% of EBs contained such pigmented clusters. Expression of the RPE-specific transcription factor MiTF-A was increased in pigmented EBs from the fifth week of incubation. Following plating on laminin, pigmented cells assumed a characteristic polygonal RPE-like shape and expressed the RPE-associated markers CRALBP, RPE65, MiTF-A, Bestrophin, and ZO-1. Following intraocular transplantation, hESC-derived pigmented cells survived and were present in intraviteal, intraretinal, and subretinal grafts. A large fraction of transplanted cells were pigmented, and expressed mature RPE markers such as RPE65 and ZO-1.
Conclusions::
hESC cells can give rise to pigmented cells in-vitro which demonstrate an RPE-like morphology and express RPE-specific markers. Following intraocular transplantation, the cells survive for at least 3.5 weeks and maintain their RPE-like qualities. This may be a first step towards the future use of hESC as an unlimited source for renewing or replacing degenerating RPE cells.
Keywords: retinal pigment epithelium • differentiation • transplantation