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Dawn Landis; RPE Differentiation from hESC and hiPSC on Electrospun Scaffolding. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4562.
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© ARVO (1962-2015); The Authors (2016-present)
A major objective of current research on stem cell-derived photoreceptors is to produce cells that are well differentiated and form outer segments(OS) for disease studies, drug discoveries, and donor tissues for transplantation. Absent in current 3D retinal differentiation systems is an RPE layer, which is critical for OS development and maintenance through nourishment of photoreceptors, buffering of ions, and production of visual compounds. We hypothesize an electrospun polymer that sharing physical characteristics with Bruch’s membrane may be a better substrate for RPE differentiation.
In order to support photoreceptors we have developed techniques using various factors and culture conditions to differentiate RPE from stem cells (H9) and patient iPSCs on matrigel (a previously established protocol) and electrospun polymer scaffolding made of slowly biodegradable material (PCL). RPE cultures were assessed by pigment production, IHC for tight junction markers, apical basal polarization, and mature RPE cellular markers, and morphological measurements.
We developed a PCL based electrospun scaffold mimicing the Bruch’s membrane to differentiate stem cells into RPE on. H9 ESCs differentiated into RPE on scaffolding mimic adult RPE (similarly to previously protocols with matrigel) evidenced through expression of typical proteins of mature RPE cells (MITF, CRALBP, EZRIN, & ZO-1), and distinctive RPE morphology such as: tight junctions between cells required for epithelial monolayer, hexagonal shape, and microvilli projections. iPSCs were also differentiated into RPE and developed similar characteristics as ESC. We tested patient iPSCs with a CRX mutation CRX for defects in differentiation and function. Control and CRX mutant iPSCs differentiated into RPE. Further experiments need to be done to determine if differences in RPE function exist.
We have differentiated RPE from ESC and iPSC cells from control and CRX mutant patients. Our culture system on the PCL based electrospun scaffold is comparable to current 2D matrigel protocols and may be more suitable for use in co-culture systems with 3D retinal differentiation. Using this system to test CRX mutant cells, it appears differentiation occurs similarly in control and mutant iPSCs although further tests are necessary. These culture systems could be beneficial in photoreceptor and RPE development assays, small molecule screening, and donor tissue.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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