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Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs). These induced pluripotent stem (iPS) cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD), the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE). As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS) dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.

Microarray technology has become extremely useful in expediting the investigation of large libraries of materials in a variety of biomedical applications, such as in DNA chips, protein and cellular microarrays. In the development of cellular microarrays, traditional high-throughput printing strategies on stiff, glass substrates and non-covalent attachment methods are limiting. We have developed a facile strategy to fabricate multifunctional high-throughput microarrays embedded at the surface of a hydrogel substrate using thiol-ene chemistry. This user-friendly method provides a platform for the immobilization of a combination of bioactive and diagnostic molecules, such as peptides and dyes, at the surface of poly(ethylene glycol)-based hydrogels. The robust and orthogonal nature of thiol-ene chemistry allows for a range of covalent attachment strategies in a fast and reliable manner, and two complementary strategies for the attachment of active molecules are demonstrated. Many lab-on-a-chip applications use microarrays for the high-throughput screening of a range of materials, including biomolecules such as DNA and proteins, as well as living cells. To address some of the limitations of traditional printed microarrays, researchers have now developed robust hydrogel-based systems with thiol-ene chemistry that enables different covalent attachment strategies to be implemented in an orthogonal fashion.

This study compares maintenance of human embryonic stem cells (hESCs) and their differentiation into retinal pigmented epithelial cells (RPE) using Matrigel, Synthemax‐R, and Synthemax II‐SC. Expression of RPE‐specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium‐derived factor. Results suggest that Synthemax II‐SC is a suitable substrate for hESC culture and the xeno‐free derivation of RPE for cellular therapies. Age‐related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno‐free) are preferred for clinical translation. This avoids exposing AMD patients to animal‐derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II‐SC, which is a novel, synthetic animal‐derived component‐free, RGD peptide‐containing copolymer compliant with good manufacturing practices designed for xeno‐free stem cell culture. Cells on Synthemax II‐SC were compared with cultures grown with xenogeneic and xeno‐free control substrates. This report demonstrates that Synthemax II‐SC supports long‐term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE‐specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium‐derived factor. Both hESCs and hESC‐RPE maintained normal karyotypes after long‐term culture on Synthemax II‐SC. Furthermore, RPE generated on Synthemax II‐SC are functional when seeded onto parylene‐C scaffolds designed for clinical use. These experiments suggest that Synthemax II‐SC is a suitable, defined substrate for hESC culture and the xeno‐free derivation of RPE for cellular therapies.

The MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast \"feeder cells\". Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

Objective: To evaluate the feasibility of a new technique for the implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium (RPE) cells into the subretinal space of retina-degenerate Royal College of Surgeon (RCS) rats. Methods: A platform device was used for the implantation of 4-µm-thick parylene substrates containing a monolayer of human embryonic stem cell-derived RPE (hESC-RPE). Normal Copenhagen rats (n = 6) and RCS rats (n = 5) were used for the study. Spectral-domain optical coherence tomography (SD-OCT) scanning and histological examinations were performed to confirm placement location of the implant. hESC-RPE cells attached to the substrate before and after implantation were evaluated using standard cell counting techniques. Results: SD-OCT scanning and histological examination revealed that the substrates were precisely placed in the rat’s subretinal space. The hESC-RPE cell monolayer that covered the surface of the substrate was found to be intact after implantation. Cell counting data showed that less than 2% of cells were lost from the substrate due to the implantation procedure (preimplantation count 2,792 ± 74.09 cells versus postimplantation count 2,741 ± 62.08 cells). Detailed microscopic examination suggested that the cell loss occurred mostly along the edges of the implant. Conclusion: With the help of this platform device, it is possible to implant ultrathin substrates containing an RPE monolayer into the rat’s subretinal space. This technique can be a useful approach for stem cell-based tissue bioengineering techniques in retinal transplantation research.

The retina detects and converts light stimulus into an electrochemical signal that is sent to the brain via the optic nerve. Retinal ganglion cells (RGC) are the neurons that extend axons out of the retina to synapse with the brain, and improper axon extension can cause visual dysfunction. Following normal development, mature RGCs can degenerate in sight-threatening disorders that may be exacerbated by a robust immune response. Osteopontin (OPN) is a multi-functional glycoprotein with adhesive, chemoattractant, and immune response activities. Adult rodent RGCs express OPN and increased OPN mRNA has been reported in retinal disease. However, the function of OPN in the developing and diseased retina is unknown. In the present study, it was demonstrated that OPN is a novel, integrin-dependent substrate for embryonic RGC neurite outgrowth. OPN expression was detected in nascent RGCs, the axonal fiber layer, and the optic nerve and co-localized with integrins α4 and β1 at the temporal peak of axogenesis. In vitro, purified OPN supported RGC neurite outgrowth, which was inhibited with antibodies against the α4 and β1 subunits. OPN concentration in the adult retina was reported for the first time. However, histological studies of OPN null mice suggest that OPN is not necessary for normal retinal development. In retinal inflammation, OPN mRNA was previously shown to increase. However, the function and source of increased OPN was unidentified. In this work, leukocytes and microglia were identified as OPN-expressing cells. OPN null mice showed decreased incidence of experimental autoimmune uveitis and attenuated disease marked by decreases in serum IgG level, lymphocyte proliferation, vitreal infiltrates, and number of granulomas. Intraocular injection of OPN in normal mice was sufficient to cause EAU-like disease, inducing retinal disruption and leukocyte infiltration. OPN was also increased in a mouse model for glaucoma and microglia were identified as OPN-expressing cells. Further, OPN null mice induced with disease showed increased surviving RGCs compared to wild type. Taken together, these data indicate that OPN functions in the eye as a novel, integrin-dependent substrate for RGC neurite outgrowth and as a pro-inflammatory molecule that exacerbates the immune response in retinal inflammation and glaucoma.