ABSTRACT
Single-cell RNA sequencing (scRNA-seq) has enabled the identification of novel gene signatures and cell heterogeneity in numerous tissues and diseases. Here we review the use of this technology for Fuchs' Endothelial Corneal Dystrophy (FECD). FECD is the most common indication for corneal endothelial transplantation worldwide. FECD is challenging to manage because it is genetically heterogenous, can be autosomal dominant or sporadic, and progress at different rates. Single-cell RNA sequencing has enabled the discovery of several FECD subtypes, each with associated gene signatures, and cell heterogeneity. Current FECD treatments are mainly surgical, with various Rho kinase (ROCK) inhibitors used to promote endothelial cell metabolism and proliferation following surgery. A range of emerging therapies for FECD including cell therapies, gene therapies, tissue engineered scaffolds, and pharmaceuticals are in preclinical and clinical trials. Unlike conventional disease management methods based on clinical presentations and family history, targeting FECD using scRNA-seq based precision-medicine has the potential to pinpoint the disease subtypes, mechanisms, stages, severities, and help clinicians in making the best decision for surgeries and the applications of therapeutics. In this review, we first discuss the feasibility and potential of using scRNA-seq in clinical diagnostics for FECD, highlight advances from the latest clinical treatments and emerging therapies for FECD, integrate scRNA-seq results and clinical notes from our FECD patients and discuss the potential of applying alternative therapies to manage these cases clinically.
ABSTRACT
Adults with no knowledge of sign languages can perceive distinctive markers that signal event boundedness (telicity), suggesting that telicity is a cognitively natural semantic feature that can be marked iconically (Strickland et al., 2015). This study asks if non-signing children (5-year-olds) can also link telicity to iconic markers in sign. Experiment 1 attempted three close replications of Strickland et al. (2015) and found only limited success. However, Experiment 2 showed that children can both perceive the relevant visual feature and can succeed at linking the visual property to telicity semantics when allowed to filter their answer through their own linguistic choices. Children's performance demonstrates the cognitive naturalness and early availability of the semantics of telicity, supporting the idea that telicity helps guide the language acquisition process.
Subject(s)
Sign Language , Humans , Male , Female , Child, Preschool , Semantics , Language DevelopmentABSTRACT
Embracing a "more global" urban comparison in scientific assessments of climate actions by cities is essential to drive greater and more inclusive participation in global efforts to curb climate change. This comparison needs to engage cities irrespective of their size and status: when we do so, distinctive patterns of urban climate mitigation actions across a diverse range of cities emerge. Employing K-means clustering as a pattern recognition method, this study compares cities based on selected aspects of their reported mitigation actions to the Carbon Disclosure Project. It explores whether the identified clusters facilitate the comparison of a socio-spatially diverse range of cities. The study identifies five clusters within two themes, namely the nature and finance-implementation of actions, shedding light on shared and distinct governance aspects of mitigation actions by cities. Notably, the study underscores how governance patterns transcend city size and global status. These findings offer valuable insights for broadening the comparative imagination of cities and inter-city networking opportunities.
Subject(s)
Cities , Climate Change , Cluster Analysis , HumansABSTRACT
The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGß4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGß4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.
Subject(s)
Epithelium, Corneal , Limbus Corneae , Humans , Stem Cell Niche , Limbal Stem Cells , Cornea , Epithelium, Corneal/metabolism , Gene Expression ProfilingABSTRACT
Necroptosis, a type of lytic cell death executed by the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) has been implicated in the detrimental inflammation caused by SARS-CoV-2 infection. We minimally and extensively passaged a single clinical SARS-CoV-2 isolate to create models of mild and severe disease in mice allowing us to dissect the role of necroptosis in SARS-CoV-2 disease pathogenesis. We infected wild-type and MLKL-deficient mice and found no significant differences in viral loads or lung pathology. In our model of severe COVID-19, MLKL-deficiency did not alter the host response, ameliorate weight loss, diminish systemic pro-inflammatory cytokines levels, or prevent lethality in aged animals. Our in vivo models indicate that necroptosis is dispensable in the pathogenesis of mild and severe COVID-19.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Mice , SARS-CoV-2/metabolism , Necroptosis/physiology , Protein Kinases/metabolism , Disease Models, Animal , Receptor-Interacting Protein Serine-Threonine Kinases/metabolismABSTRACT
The diversity of COVID-19 disease in otherwise healthy people, from seemingly asymptomatic infection to severe life-threatening disease, is not clearly understood. We passaged a naturally occurring near-ancestral SARS-CoV-2 variant, capable of infecting wild-type mice, and identified viral genomic mutations coinciding with the acquisition of severe disease in young adult mice and lethality in aged animals. Transcriptomic analysis of lung tissues from mice with severe disease elucidated a host antiviral response dominated mainly by interferon and IL-6 pathway activation in young mice, while in aged animals, a fatal outcome was dominated by TNF and TGF-ß signaling. Congruent with our pathway analysis, we showed that young TNF-deficient mice had mild disease compared to controls and aged TNF-deficient animals were more likely to survive infection. Emerging clinical correlates of disease are consistent with our preclinical studies, and our model may provide value in defining aberrant host responses that are causative of severe COVID-19.
Subject(s)
COVID-19 , SARS-CoV-2 , Young Adult , Humans , Mice , Animals , Aged , SARS-CoV-2/genetics , COVID-19/genetics , Virulence/genetics , Mutation , Disease Models, AnimalABSTRACT
BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.
Subject(s)
COVID-19 , Carrier Proteins , Cricetinae , Humans , Mice , Rats , Animals , COVID-19 Vaccines , SARS-CoV-2 , Protein Subunits , COVID-19/prevention & control , Australia , Adjuvants, Immunologic , Antibodies, Neutralizing , Antibodies, ViralABSTRACT
The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants.
ABSTRACT
HORMAD1 expression is usually restricted to germline cells, but it becomes mis-expressed in epithelial cells in ~60% of triple-negative breast cancers (TNBCs), where it is associated with elevated genomic instability (1). HORMAD1 expression in TNBC is bimodal with HORMAD1-positive TNBC representing a biologically distinct disease group. Identification of HORMAD1-driven genetic dependencies may uncover novel therapies for this disease group. To study HORMAD1-driven genetic dependencies, we generated a SUM159 cell line model with doxycycline-inducible HORMAD1 that replicated genomic instability phenotypes seen in HORMAD1-positive TNBC (1). Using small interfering RNA screens, we identified candidate genes whose depletion selectively inhibited the cellular growth of HORMAD1-expressing cells. We validated five genes (ATR, BRIP1, POLH, TDP1 and XRCC1), depletion of which led to reduced cellular growth or clonogenic survival in cells expressing HORMAD1. In addition to the translesion synthesis (TLS) polymerase POLH, we identified a HORMAD1-driven dependency upon additional TLS polymerases, namely POLK, REV1, REV3L and REV7. Our data confirms that out-of-context somatic expression of HORMAD1 can lead to genomic instability and reveals that HORMAD1 expression induces dependencies upon replication stress tolerance pathways, such as translesion synthesis. Our data also suggest that HORMAD1 expression could be a patient selection biomarker for agents targeting replication stress.
Subject(s)
Triple Negative Breast Neoplasms , Cell Cycle Proteins/genetics , DNA Damage/genetics , DNA Repair , DNA Replication/genetics , DNA-Binding Proteins/genetics , DNA-Directed DNA Polymerase/genetics , Genomic Instability/genetics , Humans , Nucleotidyltransferases/genetics , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , X-ray Repair Cross Complementing Protein 1/geneticsABSTRACT
Classical quantifiers (like "all," "some," and "none") express relationships between two sets, allowing us to make generalizations (like "no elephants fly"). Devices like these appear to be universal in human languages. Is the ubiquity of quantification due to a universal property of the human mind or is it attributable to more gradual convergence through cultural evolution? We investigated whether classical quantifiers are present in a new language emerging in isolation from other languages, Nicaraguan Sign Language (NSL). An observational study of historical data collected in the 1990s found evidence of potential quantifier forms. To confirm the quantificational meaning of these signs, we designed a production study that elicited, from three age cohorts of NSL signers (N = 17), three types of quantifiers: universal (all), existential (some), and negative (none). We find evidence for these classical quantifiers in the very first generation of signers, suggesting they may reflect a universal property of human cognition or a very rapid construction process.
Subject(s)
Cultural Evolution , Semantics , Humans , Language , Language Development , Sign LanguageABSTRACT
Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.
Subject(s)
COVID-19/immunology , Caspase 8/metabolism , Interferon-gamma/metabolism , Lymphohistiocytosis, Hemophagocytic/immunology , Macrophages/immunology , Mitochondria/metabolism , SARS-CoV-2/physiology , Animals , Caspase 8/genetics , Cells, Cultured , Cytotoxicity, Immunologic , Humans , Interferon-gamma/genetics , Macrophage Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide Synthase Type II/metabolism , Pathogen-Associated Molecular Pattern Molecules/immunology , Signal Transduction , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
An early event in lung oncogenesis is loss of the tumour suppressor gene LIMD1 (LIM domains containing 1); this encodes a scaffold protein, which suppresses tumorigenesis via a number of different mechanisms. Approximately 45% of non-small cell lung cancers (NSCLC) are deficient in LIMD1, yet this subtype of NSCLC has been overlooked in preclinical and clinical investigations. Defining therapeutic targets in these LIMD1 loss-of-function patients is difficult due to a lack of 'druggable' targets, thus alternative approaches are required. To this end, we performed the first drug repurposing screen to identify compounds that confer synthetic lethality with LIMD1 loss in NSCLC cells. PF-477736 was shown to selectively target LIMD1-deficient cells in vitro through inhibition of multiple kinases, inducing cell death via apoptosis. Furthermore, PF-477736 was effective in treating LIMD1-/- tumours in subcutaneous xenograft models, with no significant effect in LIMD1+/+ cells. We have identified a novel drug tool with significant preclinical characterisation that serves as an excellent candidate to explore and define LIMD1-deficient cancers as a new therapeutic subgroup of critical unmet need.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Intracellular Signaling Peptides and Proteins/deficiency , LIM Domain Proteins/deficiency , Lung Neoplasms/genetics , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Female , Humans , Lung Neoplasms/pathology , Mice , Mice, Inbred NOD , Proof of Concept Study , TransfectionABSTRACT
OBJECTIVE: To determine whether the use of acid suppression and thickened feeds impact laryngomalacia outcomes in infants, including supraglottoplasty risk, time to supraglottoplasty, and hospitalization risk. STUDY DESIGN: We performed a retrospective cohort study to compare risk and time with supraglottoplasty and frequency and duration of hospitalizations for infants diagnosed with laryngomalacia at Boston Children's Hospital between January 1 and December 31, 2017. The primary outcomes were supraglottoplasty requirement, time to supraglottoplasty, and hospitalization risk. Multivariate analyses were performed to determine predictors of supraglottoplasty and hospitalization risk after adjusting for laryngomalacia severity and comorbidities in addition to propensity score adjustment. Kaplan-Meier curves were created to determine the impact of acid suppression use on time to supraglottoplasty. RESULTS: In total, 236 subjects with mean age 62.6 ± 4 days were included in the analysis; 55% were treated with acid suppression. Subjects treated with acid suppression had a greater risk of supraglottoplasty (hazard ratio 3.36, 95% CI 1.36-8.29, P = .009), shorter time to supraglottoplasty (5.64 ± 0.92 vs 7.98 ± 1.92 months, P = .006), and increased respiratory hospitalization risk (relative risk 1.97, 95% CI 1.01-3.85, 0.047), even after adjustment for covariates. Subjects receiving thickening had fewer respiratory hospitalization nights and longer time to supraglottoplasty (9.3 ± 1.7 vs 4.56 ± 0.73 months, P = .004), even after adjustment. CONCLUSIONS: Acid suppression use does not reduce the frequency of supraglottoplasty and related hospitalizations compared with untreated subjects. However, patients treated with thickening have decreased hospitalization and longer time to supraglottoplasty, suggesting that thickening of feeds may be a preferred intervention over acid suppression.
Subject(s)
Anti-Ulcer Agents/administration & dosage , Deglutition Disorders/therapy , Gastroesophageal Reflux/prevention & control , Laryngomalacia/complications , Anti-Ulcer Agents/adverse effects , Deglutition Disorders/etiology , Female , Gastroesophageal Reflux/etiology , Glottis/surgery , Hospitalization , Humans , Infant , Laryngomalacia/surgery , Laryngomalacia/therapy , Male , Retrospective Studies , Severity of Illness Index , Treatment OutcomeABSTRACT
BACKGROUND: Esophageal growth using the Foker process (FP) for long-gap esophageal atresia (LGEA) has evolved over time. METHODS: Contemporary LGEA patients treated from 2014-2020 were compared to historical controls (2005 to <2014). RESULTS: 102 contemporary LGEA patients (type A 50%, B 18%, C 32%; 36% prior anastomotic attempt; 20 with esophagostomy) underwent either primary repair (n=23), jejunal interposition (JI; n = 14), or Foker process (FP; n = 65; 49 primary [p], 16 rescue [r]). The contemporary p-FP cohort experienced significantly fewer leaks on traction (4% vs 22%), bone fractures (2% vs 22%), anastomotic leak (12% vs 37%), and Foker failure (FPâJI; 0% vs 15%), when compared to historical p-FP patients (n = 27), all p ≤ 0.01. Patients who underwent a completely (n = 11) or partially (n = 11) minimally invasive FP experienced fewer median days paralyzed (0 vs 8 vs 17) and intubated (9 vs 15 vs 25) compared to open FP patients, respectively (all p ≤ 0.03), with equivalent leak rates (18% vs 9% vs 26%, p = 0.47). At one-year post-FP, most patients (62%) are predominantly orally fed. CONCLUSION: With continued experience and technical refinements, the Foker process has evolved with improved outcomes, less morbidity and maximal esophageal preservation.
Subject(s)
Esophageal Atresia , Algorithms , Anastomosis, Surgical , Esophageal Atresia/surgery , Humans , Retrospective Studies , Treatment OutcomeABSTRACT
Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.
Subject(s)
Genetic Loci , Human Embryonic Stem Cells/metabolism , Multiprotein Complexes/metabolism , Nanog Homeobox Protein/metabolism , Co-Repressor Proteins/metabolism , Human Embryonic Stem Cells/cytology , HumansABSTRACT
Bimodal bilinguals sometimes use code-blending, simultaneous production of (parts of) an utterance in both speech and sign. We ask what spoken language material is blended with entity and handling depicting signs (DS), representations of action that combine discrete components with iconic depictions of aspects of a referenced event in a gradient, analog manner. We test a semantic approach that DS may involve a demonstration, involving a predicate which obligatorily includes a modificational demonstrational component, and adopt a syntactic analysis which crucially distinguishes between entity and handling DS. Given the model of bilingualism we use, we expect both DS can be produced with speech that occurs in the verbal structure, along with vocal gestures, but speech that includes a subject is only expected to be blended with handling DS, not entity. The data we report from three Codas, native bimodal bilinguals, from the United States and one from Brazil conform with this prediction.
ABSTRACT
The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1-6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.
Subject(s)
Cellular Reprogramming/genetics , Gene Expression Regulation , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Trophoblasts/cytology , Trophoblasts/metabolism , Adult , Chromatin/genetics , Chromatin/metabolism , Ectoderm/cytology , Ectoderm/metabolism , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Transcription, GeneticSubject(s)
Adaptation, Psychological , COVID-19/psychology , Pandemics/statistics & numerical data , Quarantine/psychology , Social Support , Transplant Recipients/psychology , Transplant Recipients/statistics & numerical data , Adult , Aged , Aged, 80 and over , COVID-19/epidemiology , Female , Humans , Male , Middle Aged , Southeastern United States , Stress, Psychological , Surveys and QuestionnairesABSTRACT
Dysfunction of retinal glial cells, particularly Müller cells, has been implicated in several retinal diseases. Despite their important contribution to retinal homeostasis, a specific way to differentiate retinal glial cells from human pluripotent stem cells has not yet been described. Here, we report a method to differentiate retinal glial cells from human embryonic stem cells (hESCs) through promoting the Notch signaling pathway. We first generated retinal progenitor cells (RPCs) from hESCs then promoted the Notch signaling pathway using Notch ligands, including Delta-like ligand 4 and Jagged-1. We validated glial cell differentiation with qRT-PCR, immunocytochemistry, western blots and fluorescence-activated cell sorting as we promoted Notch signaling in RPCs. We found that promoting Notch signaling in RPCs for 2 weeks led to upregulation of glial cell markers, including glial fibrillary acidic protein (GFAP), glutamine synthetase, vimentin and cellular retinaldehyde-binding protein (CRALBP). Of these markers, we found the greatest increase in expression of the pan glial cell marker, GFAP. Conversely, we also found that inhibition of Notch signaling in RPCs led to upregulation of retinal neuronal markers including cone-rod homeobox (CRX) and orthodenticle homeobox 2 (OTX2) but with little expression of GFAP. This retinal glial differentiation method will help advance the generation of stem cell disease models to study the pathogenesis of retinal diseases associated with glial dysfunction such as macular telangiectasia type 2. This method may also be useful for the development of future therapeutics such as drug screening and gene editing using patient-derived retinal glial cells.