Optimizing Retinal Progenitor Differentiation of hESC - Effect of RPE Co-Culture

Abstract

Purpose: : To optimize the differentiation of human embryonic stem cells (hESC) to retinal and photoreceptor progenitors for obtaining 3D sheets of retinal progenitors with RPE for transplantation.

Methods: : hESCs were differentiated according to modifications of Nistor et al. (2010), including delayed hyaluronic acid treatment, longer time in suspension culture, and a change in concentration and treatment time with transcription factors Dkk1 and LeftyA. Additional studies focused on variations in exposure time and concentration of FGF-8, Sonic Hedgehog (shh), and Activin A. Cell cultures were analyzed by immunohistochemistry for the transcription factors Chx10, CRX, NRL and various other cell markers.

Results: : Expression of photoreceptor progenitor markers could be improved compared to the original differentiation protocol by using a higher concentration of Dkk1/LeftyA in the beginning of the culture, starting hyaluronic acid treatment after 3 weeks, and maintaining the cultures in suspension until d 28-29. FGF8 and shh treatment did not improve photoreceptor progenitor differentiation. Early Avtivin A treatment together with RPE co-culture (d 44-58 of differentiation) dramatically increased expression of the photoreceptor progenitor markers CRX and NRL in more than 90% of the cells. However, no mature photoreceptor markers such as recoverin were found at d 58 of differentiation.

Conclusions: : Short treatment with transcription factors LeftyA and Dkk1 at high concentration (40-60 ng/ml) is sufficient to induce retinal differentiation. Early Activin A treatment, longer time in suspension culture, delayed hyaluronic acid and co-culture with RPE improves expression of photoreceptor progenitor markers. The next step is to test these tissues in retinal degenerate nude rats.

Keywords: transcription factors • differentiation • retinal development