Rapid generation of purified human RPE from pluripotent stem cells using 2D cultures and lipoprotein uptake-based sorting

Abstract

Background: Despite increasing demand, current protocols for human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) remain time, labor, and cost intensive. Additionally, absence of robust methods for selective RPE purification and removal of non-RPE cell impurities prevents upscaling of clinical quality RPE production. We aimed to address these challenges by developing a simplified hPSC-derived RPE production and purification system that yields high-quality RPE monolayers within 90 days. Methods: Human pluripotent stem cells were differentiated into RPE using an innovative time and cost-effective protocol relying entirely on 2D cultures and minimal use of cytokines. Once RPE identity was obtained, cells were transferred onto permeable membranes to acquire mature RPE morphology. RPE differentiation was verified by electron microscopy, polarized VEGF expression, establishment of high transepithelial electrical resistance and photoreceptor phagocytosis assay. After 4 weeks on permeable membranes, RPE cell cultures were incubated with Dil-AcLDL (DiI-conjugated acetylated low-density lipoproteins) and subjected to fluorescence-activated cell sorting (FACS) for purification and subculture. Results: Using our 2D cytokine scarce protocol, hPSC-derived functional RPE cells can be obtained within 2 months. Nevertheless, at this stage, most samples contain a percentage of non-RPE/early RPE progenitor cells that make them unsuitable for clinical application. We demonstrate that functional RPE cells express high levels of lipoprotein receptors and that this correlates with their ability to uptake lipoproteins. Combining photoreceptor uptake assay with lipoprotein uptake assay further confirms that only functional RPE cells uptake AcLDL. Incubation of mixed RPE/non-RPE cell cultures with fluorophore conjugated AcLDL and subsequent FACS-based isolation of labeled cells allows selective purification of mature functional RPE. When subcultured, DiI-AcLDL-labeled cells rapidly form pure homogenous high-quality RPE monolayers. Conclusions: Pure functional RPE monolayers can be derived from hPSC within 90 days using simplified 2D cultures in conjunction with our RPE PLUS protocol (RPE Purification by Lipoprotein Uptake-based Sorting). The simplicity of this protocol makes it scalable, and the rapidity of production and purification allows for high-quality RPE to be produced in a short span of time making them ideally suited for downstream clinical and in vitro applications.

Update of application of olfactory ensheathing cells and stem cells/exosomes in the treatment of retinal disorders

“…Michele et al used two-dimensional culture combined with a classification method based on lipoprotein absorption to study pure RPE. This method could obtain purely functional RPE from iPSCs in a short time, which improved the clinical efficacy of treating retinal disorders [ 111 ]. Lingam et al injected iPSCs-derived photoreceptor precursors into the non-human primate model of retinal degeneration induced by cobalt chloride.…”

Where Are We with RPE Replacement Therapy? A Translational Review from the Ophthalmologist Perspective

“…Spontaneous differentiation is labor intensive and highly variable across different iPSC lines [ 34 ]. Directed differentiation protocols can significantly increase yields in hiPS-RPE cell production [ 35 , 36 , 37 , 38 ]. However, they often requires time-consuming preparation protocols and are incompatible with cell therapy due to the use of animal-derived undefined products [ 39 , 40 ].…”

“…They can be autologous, thus preventing rejection issues, and less ethical problems arise from their collection when compared to ESCs. The disadvantages include the mutation load of iPS cells, which pass through several stresses and long culture periods, and potential safety issues related to this [ 38 ]. These cells do indeed show inconstant ability to differentiate into the specific cell type required.…”

Shaping the Microglia in Retinal Degenerative Diseases Using Stem Cell Therapy: Practice and Prospects

“…A scalable protocol was published that facilitated the production of high-quality RPE cells in a short time span and convenient application for both investigative and clinical use to solve the problem of high cost and low purity when generating RPE cells. Pure functional RPE monolayers have been derived from hiPSCs within 90 days using simplified 2D cultures ( Buchholz et al, 2013 ; Maruotti et al, 2015 ; Michelet et al, 2020 ). However, no further studies have adopted this protocol to date.…”