Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

摘要

Highlights ? Xeno-free and defined differentiation of hES-RPE cells using recombinant laminin-521 ? Functional monolayer integration of hES-RPE cells in a novel large-eyed disease model ? Rescue of photoreceptors from induced degeneration by transplanted hES-RPE cells Summary Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in?vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model. Keywords human embryonic stem cells ; differentiation ; retinal pigment epithelium ; laminin ; xeno-free ; cellular therapy ; rabbit ; transplantation ; optical coherence tomography prs.rt("abs_end"); Introduction Age-related macular degeneration (AMD), the most common cause of severe vision loss in the Western world, occurs in wet (neovascular) and dry (degenerative) forms. In?early dry AMD, the retinal pigment epithelium (RPE) becomes dysfunctional, whereas end-stage disease, geographic atrophy (GA), is characterized by degeneration of RPE and photoreceptors ( Bhutto and Lutty, 2012 ). The RPE is a monolayer of polarized cells that constitutes the outer blood-retina barrier and performs central tasks in the eye, e.g., light adsorption, secretion of growth factors, and phagocytosis of photoreceptor outer segments (POS) ( Sparrow et?al., 2010 ). The apical surface harbors microvilli that interact with the light-sensitive POS, whereas the basolateral surface adheres to Bruch's membrane (BM), which in turn separates the RPE from the underlying choroid. Subretinal transplantation of RPE cells derived from human embryonic stem cells (hESC) could potentially be used as replacement therapy in GA ( Schwartz et?al., 2012 and Schwartz et?al., 2015 ). However, a critical question is whether donor cells integrate into the host RPE and support the overlying photoreceptors. Experimental transplantations of hESC-RPE have only been conducted in small-eyed rodent models ( Carido et?al., 2014 , Idelson et?al., 2009 , Lund et?al., 2006 and Vugler et?al., 2008 ). Functional effects in these models are however non-specific and surgical techniques, instrumentation, and imaging methods differ from those applied in humans, limiting their use as preclinical models (e.g., Pinilla et?al., 2009 ). We have recently described a damage model in the large-eyed rabbit that exhibits typical GA changes including photoreceptor loss and RPE alterations ( Bartuma et?al., 2015 ). Several hESC-RPE derivation protocols have been described with the common limitation of relying on culture steps that involve xeno- or human-feeder cells or use medium components that are either undefined or not xeno-free ( Klimanskaya et?al., 2004 , Lane et?al., 2014 , Osakada et?al., 2009 , Pennington et?al., 2015 and Vaajasaari et?al., 2011 ). Recently, we described a defined and xeno-free clonal culture of hESC using recombinant human laminin (rhLN) and E-cadherin ( Rodin et?al., 2014a and Rodin et?al., 2014b ). Encouraged by this work, we set out to evaluate whether rhLN-matrix could support efficient hESC-RPE differentiation. BM, the RPE basement membrane, contains four LNs, LN-111, LN-332, LN-511, and LN-521, that adhere to the RPE via specific integrins ( Aisenbrey et?al., 2006 ). In the present study, we show that rhLNs are more effective in supporting xeno-free and defined differentiation compared with exogenous matrix such as gelatin, used in ongoing clinical studies ( Schwartz et?al., 2012 ). Moreover, we demonstrate that suspension transplantations of rhLN-521-hESC-RPE integrate as polarized subretinal monolayers that rescue overlying photoreceptors from induced damage. We conclude that rhLN-521 effectively supports differentiation of clinically compliant hESC-RPE, and presents evidence of efficient long-term functional integration of hESC-RPE in a large-eyed disease model. Results Robust Induction of Primary Pigmented Cells Using Suspension Differentiation in Defined Medium We have recently developed a xeno-free hESC derivation and culture methodology, using rhLN-521-based matrix and a xeno-