Efficient differentiation of human embryonic stem cells to retinal pigment epithelium under defined conditions.

Age-related macular degeneration (AMD) is a highly prevalent form of blindness caused by loss death of cells of the retinal pigment epithelium (RPE). Transplantation of pluripotent stem cell (PSC)-derived RPE cells is considered a promising therapy to regenerate cell function and vision. OBJECTIVE: The objective of this study is to develop a rapid directed differentiation method for production of RPE cells from PSC which is rapid, efficient, and fully defined and produces cells suitable for clinical use. DESIGN: A protocol for cell growth and differentiation from hESCs was developed to induce differentiation through screening small molecules which regulated a primary stage of differentiation to the eyefield progenitor, and then, a subsequent set of molecules to drive differentiation to RPE cells. Methods for cell plating and maintenance have been optimized to give a homogeneous population of cells in a short 14-day period, followed by a procedure to support maturation of cell function. RESULTS: We show here the efficient production of RPE cells from human embryonic stem cells (hESCs) using small molecules in a feeder-free system using xeno-free/defined medium. Flow cytometry at day 14 showed ~ 90% of cells expressed the RPE markers MITF and PMEL17. Temporal gene analysis confirmed differentiation through defined cell intermediates. Mature hESC-RPE cell monolayers exhibited key morphological, molecular, and functional characteristics of the endogenous RPE. CONCLUSION: This study identifies a novel cell differentiation process for rapid and efficient production of retinal RPE cells directly from hESCs. The described protocol has utility for clinical-grade cell production for human therapy to treat AMD.

Dorsal-Ventral Differences in Retinal Structure in the Pigmented Royal College of Surgeons Model of Retinal Degeneration.

Retinitis pigmentosa is a family of inherited retinal degenerations associated with gradual loss of photoreceptors, that ultimately leads to irreversible vision loss. The Royal College of Surgeon's (RCS) rat carries a recessive mutation affecting mer proto-oncogene tyrosine kinase (merTK), that models autosomal recessive disease. The aim of this study was to understand the glial, microglial, and photoreceptor changes that occur in different retinal locations with advancing disease. Pigmented RCS rats (RCS-p+/LAV) and age-matched isogenic control rdy (RCS-rdy +p+/LAV) rats aged postnatal day 18 to 6 months were evaluated for in vivo retinal structure and function using optical coherence tomography and electroretinography. Retinal tissues were assessed using high resolution immunohistochemistry to evaluate changes in photoreceptors, glia and microglia in the dorsal, and ventral retina. Photoreceptor dysfunction and death occurred from 1 month of age. There was a striking difference in loss of photoreceptors between the dorsal and ventral retina, with a greater number of photoreceptors surviving in the dorsal retina, despite being adjacent a layer of photoreceptor debris within the subretinal space. Loss of photoreceptors in the ventral retina was associated with fragmentation of the outer limiting membrane, extension of glial processes into the subretinal space that was accompanied by possible adhesion and migration of mononuclear phagocytes in the subretinal space. Overall, these findings highlight that breakdown of the outer limiting membrane could play an important role in exacerbating photoreceptor loss in the ventral retina. Our results also highlight the value of using the RCS rat to model sectorial retinitis pigmentosa, a disease known to predominantly effect the inferior retina.

Advancing a Stem Cell Therapy for Age-Related Macular Degeneration.

The retinal pigment epithelium (RPE) is a multifunctional monolayer located at the back of the eye required for the survival and function of the light-sensing photoreceptors. In Age-related Macular Degeneration (AMD), the loss of RPE cells leads to photoreceptor death and permanent blindness. RPE cell transplantation aims to halt or reverse vision loss by preventing the death of photoreceptor cells and is considered one of the most viable applications of stem cell therapy in the field of regenerative medicine. Proof-of-concept of RPE cell transplantation for treating retinal degenerative disease, such as AMD, has long been established in animal models and humans using primary RPE cells, while recent research has focused on the transplantation of RPE cells derived from human pluripotent stem cells (hPSC). Early results from clinical trials indicate that transplantation of hPSC-derived RPE cells is safe and can improve vision in AMD patients. Current hPSC-RPE cell production protocols used in clinical trials are nevertheless inefficient. Treatment of large numbers of AMD patients using stem cellderived products may be dependent on the ability to generate functional cells from multiple hPSC lines using robust and clinically-compliant methods. Transplantation outcomes may be improved by delivering RPE cells on a thin porous membrane for better integration into the retina, and by manipulation of the outcome through control of immune rejection and inflammatory responses.

Platypus Induced Pluripotent Stem Cells: The Unique Pluripotency Signature of a Monotreme.

The platypus (Ornithorhynchus anatinus) is an egg-laying monotreme mammal whose ancestors diverged ∼166 million years ago from the evolutionary pathway that eventually gave rise to both marsupial and eutherian mammals. Consequently, its genome is an extraordinary amalgam of both ancestral reptilian and derived mammalian features. To gain insight into the evolution of mammalian pluripotency, we have generated induced pluripotent stem cells from the platypus (piPSCs). Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors POU5F1/OCT4, SOX2, and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 and 166 million years ago, primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. Using the RNAseq data, we were also able to demonstrate that in both fibroblasts and piPSCs, the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1, indicating that there is no upregulation of X-linked genes. Finally, the RNAseq data also allowed us to explore the process of X-linked gene inactivation in the platypus, where we determined that for any given gene, there is no preference for silencing of the maternal or paternal allele; that is, within a population of cells, the silencing of X-linked genes is not imprinted.

Design, development and characterization of synthetic Bruch's membranes.

Age-related macular degeneration (AMD) is a leading cause of blindness, and dry AMD has no effective treatment. Retinal constructs comprising retinal pigment epithelium (RPE) cells supported by electrospun scaffolds have been investigated to treat dry AMD. However, electrospun scaffolds studied to-date do not mimic the structural microenvironment of human Bruch's membrane (BM), essential for native-like RPE monolayers. The aim of this study was to develop a structurally biomimetic scaffold designed to support a functional RPE monolayer, comprising porous, electrospun nanofibrous membranes (ENMs), coated with laminin, mimicking the inner collagenous layer (ICL) and basal RPE lamina respectively, the cell supporting layers of the BM. In vitro evaluation showed 70nm PLLA ENMs adsorbed high amounts of laminin and supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. 70nm PLLA ENMs were successfully implanted into the subretinal space of RCS-rdy+p+/LAV rats, also commonly know as rdy rats. At week 4, in the absence of immunosuppressants, implanted PLLA ENMs were surrounded by a significantly low number of activated microglial cells, compared to week 1, indicating no adverse long-term immune response. In conclusion, we successfully designed and tested ENMs emulating the RPE cell supporting layers of the BM, and found 70nm PLLA ENMs to be best suited as scaffolds for fabricating retinal constructs. STATEMENT OF SIGNIFICANCE: Age related macular degeneration (AMD) is a leading cause of vision loss in the developed world, with an increasing number of people suffering from blindness or severe visual impairment. Transplantation of retinal pigment epithelium (RPE) cells supported on a synthetic, biomimetic-like Bruch's membrane (BM) is considered a promising treatment. However, the synthetic scaffolds used do not mimic the microenvironment of the RPE cell supporting layers, required for the development of a functional RPE monolayer. This study indicated that porous, laminin coated, 70nm PLLA ENMs supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. These findings indicate the potential clinical use of porous, laminin coated, 70nm PLLA ENMs in fabricating retinal constructs aimed at treating dry AMD.