Abstract
Purpose :
To engineer retinal pigment epithelium-like cells by direct reprogramming of human fibroblasts for disease modeling, drug screening, and cell replacement therapy for degenerative ocular disorders
Methods :
Human foreskin fibroblasts grown in culture plates were infected with a mixture of doxycycline-inducible lentiviral expression vectors encoding CRX, OTX2, MITF, SIX3, SMAD6, SOX9, PAX6, RAX, FOXD1, HNF4A AND PKNOX2 as well as an rtTA-expressing vector (from FUdeltaGW-rtTA). Cells were then cultured in RPE growth medium (MEM alpha with GlutaMAX, 5% FBS, 1% Penicillin/Streptomycin, 0.1 mM NEAA, 1x N1 supplement, 83 mg/ml taurine, 20 μg/ml hydrocortisone, 13 ng/ml triiodo-thyronin) containing doxycycline to induce transgene expression. Medium plus doxycycline was replaced every 3 days. The cultures were examined for RPE marker gene expression by RT-PCR and immunocytochemistry.
Results :
Ectopic expression of five transcription factors (OTX2, MITF, SIX3, SOX9 and SMAD6) in human foreskin fibroblasts induced a mesenchymal to epithelial transition during the early stage of reprogramming, and was sufficient to induce a cobblestone-shaped morphology characteristic of RPE cells. RPE cells markers, including Best1, Rpe65, Tyr, Tyrp1, Rlbp1, and Trmp3 were upregulated in the RPE-like cells as revealed by RT-PCR and/or immunostaining.
Conclusions :
Human fibroblasts can be directly converted into RPE-like cells by five defined transcription factors.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.