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Michael Young, Andrew Michaelson, Petr Baranov, Rebecca Carrier; Hybrid interphotoreceptor matrix - poly(caprolactone) scaffolds for human retinal progenitor cell culture and differentiation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4699.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cell therapy is a burgeoning technology for degenerative disorders affecting organs with limited regenerative capacity, such as the nervous system, including the retina. Although experimental studies highlight the potential of stem cells or precursors/progenitors delivered as a single-cell suspension, a tissue engineering strategy is a promising approach. Previously we utilized fibronectin coating for poly(caprolactone) (PCL) scaffolds to induce photoreceptor differentiation from human retinal progenitor cells (hRPCs). One alternative approach is the functionalization of the polymer with extracellular matrix. Ideally, the incorporated molecules should provide adhesive and differentiation stimuli to the cells. Here we describe the differentiation of hRPCs on a hybrid interphotoreceptor matrix-PCL film.
IPM was isolated from fresh bovine retinas, dissolved in dichloromethane and mixed with 10% PCL. The scaffolds were prepared using a previously described spinning method. hRPCs were isolated from human fetal neural retina at 16 weeks of gestation and expanded up to passage 9.The effect of IPM-PCL scaffold on hRPC morphology was assessed by scanning electron microscopy and differentiation was assessed by flow cytometry analysis for mature photoreceptor markers after 7 days in culture.
Lectin staining revealed that IPM lost its “honeycomb” structure during incorporation into PCL, although the adhesive ability of the scaffold was greatly increased (70% vs 15%). However, we were not able to achieve the rate of adhesion found for the current standard of fibronectin coating (95%). The rate of proliferation of hRPCs on the IPM-PCL hybrid matrix was lower compared to fibronectin. Flow cytometry revealed the increased expression of developing photoreceptor transcription factors (Crx, Nrl) and mature photoreceptor markers (Opsin Blue, Opsin Red/Green, Rhodopsin) after 7 days of differentiation on PCL-IPM, a result that was equivalent to that obtained with fibronectin.
The approach we describe allows one to overcome both the inherent lack of cell adhesion (specific for hydrophobic PCL) and the water-insolubility of IPM (which complicates its usage in vitro). We have shown that these hybrid scaffolds drive the hRPC fate towards photoreceptors, thereby providing a new tool in the development of retinal repair strategies.
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