Abstract
Purpose :
Since the advent of induced pluripotent stem cell (iPSC) technology over a decade ago, scientists have harnessed it to develop differentiation processes that can be applied to clinical therapeutics for better patient outcomes. To that end, we have developed a protocol to differentiate iPSCs into fully polarized and functional retinal pigment epithelium (RPE) cells in a way that recapitulates endogenous biological development of RPE. Verifying that our protocol mirrors natural differentiation is a vital step towards developing next-generation therapeutics for diseases that affect RPE.
Methods :
iPSC lines were acquired from Allen Institute with GFP tags for TUBA1B and F-ACTIN. Dual SMAD inhibition was used to generate neuroectoderm which was then directed towards RPE progenitors. Inhibition of the FGF and TGF pathways with addition of activin-A then induced the RPE progenitors into committed RPE cells. This differentiation was staged on iBidi chamber slides and lines were fixed at critical stages of differentiation and maturation. The fixed lines were then stained with antibodies for marker proteins critical for development and imaged for fluorescence analysis.
Results :
TRA-1-81 and SOX2, proteins expressed most in pluripotent stem cells, decreased during the differentiation process, confirming the cells’ transformation from a pluripotent to a non-pluripotent state. Randomly organized intracellular actin filaments in differentiating cells rearranged into polygonal shapes to provide the hexagonal architecture characteristic of mature RPE cells. Cilia, another sign of RPE maturity, was observed with the TUBA1B GFP tag during the start of RPE maturation. MITF expression increased during differentiation, marking RPE lineage. Concomitantly, expression of TYRP1, needed for melanosome formation also increased. Absorbance of light by the cells also increased with maturation, suggesting increasing pigmentation. Additionally, PAX6, OTX2 and RPE65, which are marker proteins for RPE, increase with time.
Conclusions :
Our differentiation protocol is successful in developing RPE cells from human-derived iPSC lines. These cells recapitulate the hallmark features and expression patterns characteristic of human RPE cells. This study has provided a more robust understanding of RPE differentiation process and advances us towards a biologically capable clinical therapeutic for RPE disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.