The retina is a widely profiled tissue in multiple species by single-cell RNA sequencing studies. However, integrative research of the retina across species is lacking. Here, we construct the first ...single-cell atlas of the human and porcine ocular compartments and study inter-species differences in the retina. In addition to that, we identify putative adult stem cells present in the iris tissue. We also create a disease map of genes involved in eye disorders across compartments of the eye. Furthermore, we probe the regulons of different cell populations, which include transcription factors and receptor-ligand interactions and reveal unique directional signalling between ocular cell types. In addition, we study conservation of regulons across vertebrates and zebrafish to identify common core factors. Here, we show perturbation of KLF7 gene expression during retinal ganglion cells differentiation and conclude that it plays a significant role in the maturation of retinal ganglion cells.
Abstract
Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development ...of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications.
Retinal pigment epithelial (RPE) cell dysfunction is a key driving force of AMD. RPE cells form a metabolic interface between photoreceptors and choriocapillaris, performing essential functions for ...retinal homeostasis. Through their multiple functions, RPE cells are constantly exposed to oxidative stress, which leads to the accumulation of damaged proteins, lipids, nucleic acids, and cellular organelles, including mitochondria. As miniature chemical engines of the cell, self-replicating mitochondria are heavily implicated in the aging process through a variety of mechanisms. In the eye, mitochondrial dysfunction is strongly associated with several diseases, including age-related macular degeneration (AMD), which is a leading cause of irreversible vision loss in millions of people globally. Aged mitochondria exhibit decreased rates of oxidative phosphorylation, increased reactive oxygen species (ROS) generation, and increased numbers of mitochondrial DNA mutations. Mitochondrial bioenergetics and autophagy decline during aging because of insufficient free radical scavenger systems, the impairment of DNA repair mechanisms, and reductions in mitochondrial turnover. Recent research has uncovered a much more complex role of mitochondrial function and cytosolic protein translation and proteostasis in AMD pathogenesis. The coupling of autophagy and mitochondrial apoptosis modulates the proteostasis and aging processes. This review aims to summarise and provide a perspective on (i) the current evidence of autophagy, proteostasis, and mitochondrial dysfunction in dry AMD; (ii) current in vitro and in vivo disease models relevant to assessing mitochondrial dysfunction in AMD, and their utility in drug screening; and (iii) ongoing clinical trials targeting mitochondrial dysfunction for AMD therapeutics.
Background Retinal regenerative therapies hold great promise for the treatment of inherited retinal degenerations (IRDs). Studies in preclinical lower mammal models of IRDs have suggested visual ...improvement following retinal photoreceptor precursors transplantation, but there is limited evidence on the ability of these transplants to rescue retinal damage in higher mammals. The purpose of this study was to evaluate the therapeutic potential of photoreceptor precursors derived from clinically compliant induced pluripotent stem cells (iPSCs). Methods Photoreceptor precursors were sub-retinally transplanted into non-human primates (Macaca fascicularis). The cells were transplanted both in naïve and cobalt chloride-induced retinal degeneration models who had been receiving systemic immunosuppression for one week prior to the procedure. Optical coherence tomography, fundus autofluorescence imaging, electroretinography, ex vivo histology and immunofluorescence staining were used to evaluate retinal structure, function and survival of transplanted cells. Results There were no adverse effects of iPSC-derived photoreceptor precursors on retinal structure or function in naïve NHP models, indicating good biocompatibility. In addition, photoreceptor precursors injected into cobalt chloride-induced retinal degeneration NHP models demonstrated an ability both to survive and to mature into cone photoreceptors at 3 months post-transplant. Optical coherence tomography showed restoration of retinal ellipsoid zone post-transplantation. Conclusions These findings demonstrate the safety and therapeutic potential of clinically compliant iPSC-derived photoreceptor precursors as a cell replacement source for future clinical trials. Keywords: Cell therapy, Induced pluripotent stem cells, Non-human primates, Photoreceptor precursors
Natural polysaccharide pectin has for the first time been grafted with polyhydroxybutyrate (PHB) via ring-opening polymerization of β-butyrolactone. This copolymer, pectin-polyhydroxybutyrate ...(pec-PHB), was blended with PHB in various proportions and electrospun to produce nanofibers that exhibited uniform and bead-free nanostructures, suggesting the miscibility of PHB and pec-PHB. These nanofiber blends exhibited reduced fiber diameters from 499 to 336–426 nm and water contact angles from 123.8 to 88.2° on incorporation of pec-PHB. They also displayed 39–335% enhancement of elongation at break relative to pristine PHB nanofibers. pec-PHB nanofibers were found to be noncytotoxic and biocompatible. Human retinal pigmented epithelium (ARPE-19) cells were seeded onto pristine PHB and pec-PHB nanofibers as scaffold and showed good proliferation. Higher proportions of pec-PHB (pec-PHB10 and pec-PHB20) yielded higher densities of cells with similar characteristics to normal RPE cells. We propose, therefore, that nanofibers of pec-PHB have significant potential as retinal tissue engineering scaffold materials.
The traditional intravitreal injection delivery of antivascular endothelial growth factor (anti‐VEGF) to the posterior segment of the eye for treatment of retinal diseases is invasive and associated ...with sight‐threatening complications. To avoid such complications, there has been significant interest in developing polymers for topical drug delivery to the retina. This study reports a nanomicelle drug delivery system made of a copolymer EPC (nEPCs), which is capable of delivering aflibercept to the posterior segment topically through corneal–scleral routes. EPC is composed of poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), and polycaprolactone (PCL) segments. In this study, aflibercept‐loaded nEPCs (nEPCs + A) are capable of penetrating the cornea in ex vivo porcine eye models and deliver a clinically significant amount of aflibercept to the retina in laser‐induced choroidal neovascularization (CNV) murine models, causing CNV regression. nEPCs + A also demonstrate biocompatibility in vitro and in vivo. Interestingly, this study also suggests that nEPCs have intrinsic antiangiogenic properties. The ability to deliver anti‐VEGF drugs and the intrinsic antiangiogenic properties of nEPCs may result in synergistic effects, which can be harnessed for effective therapeutics. nEPCs may be a promising topical anti‐VEGF delivery platform for the treatment of retinal diseases.
nEPCs + A are aflibercept‐loaded nanomicelles formed by encapsulating aflibercept in an EPC copolymer solution. In this study, the ability of nEPCs + A to deliver clinically significant amounts of aflibercept to the retina for control of choroidal neovascularization in mice is described. Interestingly, nEPCs also demonstrate intrinsic antiangiogenic activity, which synergizes with their drug delivery capability for the treatment of neovascular retinal diseases.
Human organoids mimic the physiology and tissue architecture of organs and are of great significance for promoting the study of human diseases. Traditionally, organoid cultures rely predominantly on ...animal or tumor‐derived extracellular matrix (ECM), resulting in poor reproducibility. This limits their utility in for large‐scale drug screening and application for regenerative medicine. Recently, synthetic polymeric hydrogels, with high biocompatibility and biodegradability, stability, uniformity of compositions, and high throughput properties, have emerged as potential materials for achieving 3D architectures for organoid cultures. Compared to conventional animal or tumor‐derived organoids, these newly engineered hydrogel‐based organoids more closely resemble human organs, as they are able to mimic native structural and functional properties observed in‐situ. In this review, recent developments in hydrogel‐based organoid culture will be summarized, emergent hydrogel technology will be highlighted, and future challenges in applying them to organoid culture will be discussed.
Synthetic polymeric hydrogels are emerging materials for organoid cultures that can mimic in vivo 3D environment to offer a sophisticated model for various disease investigation, drug testing, or targeted gene editing, etc.
Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical ...trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (
,
, and
) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.
Hydrogels with more than one mode of crosslinking have gained interest due to improved control over hydrogel properties such as mechanical strength using multiple stimuli. In this work, sodium ...alginate was covalently conjugated onto thermoresponsive polyurethanes to prepare hybrid polymers (EPC-Alg) that are responsive to both temperature and Ca2+, forming orthogonally crosslinked hydrogels which are non-toxic to cells. Notably, the crosslinks are fully reversible, allowing for gel strength to be modulated via selective removal of either stimulus, or complete deconstruction of the hydrogel network by removing both stimuli. Higher alginate fractions increased the hydrophilicity and Ca2+ response of the EPC-Alg hydrogel, enabling tunable modulation of the thermal stability, stiffness and gelation temperatures. The EPC-Alg hydrogel could sustain protein release for a month and encapsulate neural spheroids with high cell viability after 7-day culture, demonstrating feasibility towards 3D cell encapsulation in cell-based biomedical applications such as cell encapsulation and cell therapy.
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Present day strategies for delivery of wireless photodynamic therapy (PDT) to deep-seated targets are limited by the inadequacy of irradiance and insufficient therapeutic depth. Here we report the ...design and preclinical validation of a flexible wireless upconversion nanoparticle (UCNP) implant (SIRIUS) that is capable of large field, high intensity illumination for PDT of deep-seated tumors. The implant achieves this by incorporating submicrometer core–shell–shell NaYF4 UCNPs into its design, which significantly enhances upconversion efficiency and mitigates light loss from surface quenching. We demonstrate the efficacy of SIRIUS UCNP implant mediated PDT in preclinical breast cancer disease models. In our in vitro experiments, SIRIUS directed 5-Aminolevulinic Acid (5-ALA) based wireless PDT leads to significant reactive oxygen species (ROS) generation and tumor apoptosis in hormonal receptor+/HER2+ (MCF7) and triple-negative (MDA-MB-231) breast cancer cell lines. In our in vivo rodent model, SIRIUS-driven PDT is shown to be significant in regressing tumors when applied to orthotopically inoculated breast tumors. Following successful preclinical validation, we also describe a clinical prototype of UCNP breast implant with potential dual cosmetic and onco-therapeutic functions. SIRIUS is an upconversion breast implant for wireless PDT that fulfils all the design prerequisites necessary for seamless clinical translation.
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