Abstract
Purpose :
Retinal pigment epithelium (RPE) acts as the retina-blood barrier and plays an important role in visual function. Membrane proteins, functioning as receptors, enzymes, and transporters, play a central role in the cellular functions of RPE. Both donor-derived adult RPE (aRPE) and pluripotent stem cell-derived RPE (PSC-RPE) are candidates for cell replacement strategy in diseases with RPE loss. We hypothesize that identifying surface proteins common to and differentially expressed between aRPE and PSC-RPE can help identify targets to minimize immune response to graft tissue and improve graft integration.
Methods :
We used the cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) technique utilizing barcoded antibodies to label cells. Barcodes were captured and identified using the well-based iCELL8 single-cell RNA sequencing technology. This technique allowed us to simultaneously obtain surface protein composition and transcriptome profiles.
Results :
Single-cell sequencing using surface proteins revealed different subpopulations in both aRPE and PSC-RPE suggesting cellular heterogeneity. Transcriptome profiles confirmed expression of key RPE markers MITF, RPE65, and BEST1 in all subpopulations. A fraction of surface proteins, Podoplanin and CD63, are universally expressed while proteins involved in immune function such as CD270 and CD200 are specific to distinct subpopulations and cell source, suggesting functional heterogeneity across RPE subpopulations and cell origin. CD270 expression is higher in aRPE than in PSC-RPE, whereas CD200 is expressed in cells with differing morphology between aRPE and PSC-PRE. Another example of cell source heterogeneity is observed in our cluster 2 subpopulation, which across cell origins expressed Class I MHC complex. However, within this cluster, aRPE and PSC-RPE demonstrate a subset of distinct cell surface profiles indicative of altered function.
Conclusions :
Identification of subpopulations based on surface proteins suggests that functional heterogeneity is common to aRPE and PSC-RPE. Subpopulation-based differences between PSC-RPE and aRPE can inform strategies to improve transplant efficacy. Universally expressed surface proteins can be used as standardized markers for PSC-RPE across differentiation strategies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.