Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model

Long non-coding RNAs: a potential novel class of cancer biomarkers

Long noncoding RNAs (lncRNAs) are a novel class of RNA molecules defined as transcripts longer than 200 nucleotides that lack protein coding potential. They constitute a major, but still poorly characterized part of human transcriptome, however evidence is growing that they are important regulatory molecules involved in various cellular processes. It is becoming increasingly clear that many lncRNAs are deregulated in cancer and some of them can be important drivers of malignant transformation. On the one hand, some lncRNAs can have highly specific expression in particular types of cancer making them a promising tool for diagnosis. The expression of other lncRNAs can correlate with different pathophysiological features of tumor growth and with patient survival, thus making them convenient biomarkers for prognosis. In this review we outline the current state of knowledge about the fast growing field of application of lncRNAs as tumor biomarkers

Sense-antisense gene-pairs in breast cancer and associated pathological pathways

More than 30% of human protein-coding genes form hereditary complex genome architectures composed of sense-antisense (SA) gene pairs (SAGPs) transcribing their RNAs from both strands of a given locus. Such architectures represent important novel components of genome complexity contributing to gene expression deregulation in cancer cells. Therefore, the architectures might be involved in cancer pathways and, in turn, be used for novel drug targets discovery. However, the global roles of SAGPs in cancer pathways has not been studied. Here we investigated SAGPs associated with breast cancer (BC)-related pathways using systems biology, prognostic survival and experimental methods. Gene expression analysis identified 73 BC-relevant SAGPs that are highly correlated in BC. Survival modelling and metadata analysis of the 1161 BC patients allowed us to develop a novel patient prognostic grouping method selecting the 12 survival-significant SAGPs. The qRT-PCR-validated 12-SAGP prognostic signature reproducibly stratified BC patients into low- and high-risk prognostic subgroups. The 1381 SAGP-defined differentially expressed genes common across three studied cohorts were identified. The functional enrichment analysis of these genes revealed the GABPA gene network, including BC-relevant SAGPs, specific gene sets involved in cell cycle, spliceosomal and proteasomal pathways. The co-regulatory function of GABPA in BC cells was supported using siRNA knockdown studies. Thus, we demonstrated SAGPs as the synergistically functional genome architectures interconnected with cancer-related pathways and associated with BC patient clinical outcomes. Taken together, SAGPs represent an important component of genome complexity which can be used to identify novel aspects of coordinated pathological gene networks in cancers.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

Generation of two isogenic human induced pluripotent stem cell lines from a 15 year-old female patient with MERRF syndrome and A8344G mutation of mitochondrial DNA

MERRF syndrome is predominantly caused by A8344G mutation in the mitochondrial DNA (mtDNA), affecting MT-TK gene, which impairs the mitochondrial electron transport chain function. Here, we report the generation of two isogenic induced pluripotent stem cell (iPSC) lines, TVGH-iPSC-MRF-Mlow and TVGH-iPSC-MRF-Mhigh, from the skin fibroblasts of a female MERRF patient harboring mtDNA A8344G mutation by using retrovirus transduction system. Both cell lines share the same genetic background except containing different proportions of mtDNA with the A8344G mutation. Both cell lines exhibited the pluripotency and capacity to differentiate into three germ layers

Expression profiling of cell-intrinsic regulators in the process of differentiation of human iPSCs into retinal lineages

Abstract Background Differentiation of human induced pluripotent stem cells (hiPSCs) into retinal lineages offers great potential for medical application. Therefore, it is of crucial importance to know the key intrinsic regulators of differentiation and the specific biomarker signatures of cell lineages. Methods In this study, we used microarrays to analyze transcriptomes of terminally differentiated retinal ganglion cell (RGC) and retinal pigment epithelium (RPE) lineages, as well as intermediate retinal progenitor cells of optic vesicles (OVs) derived from hiPSCs. In our analysis, we specifically focused on the classes of transcripts that encode intrinsic regulators of gene expression: the transcription factors (TFs) and epigenetic chromatin state regulators. We applied two criteria for the selection of potentially important regulators and markers: firstly, the magnitude of fold-change of upregulation; secondly, the contrasted pattern of differential expression between OV, RGC and RPE lineages. Results We found that among the most highly overexpressed TF-encoding genes in the OV/RGC lineage were three members of the Collier/Olfactory-1/Early B-cell family: EBF1, EBF2 and EBF3. Knockdown of EBF1 led to significant impairment of differentiation of hiPSCs into RGCs. EBF1 was shown to act upstream of ISL1 and BRN3A, the well-characterized regulators of RGC lineage specification. TF-encoding genes DLX1, DLX2 and INSM1 were the most highly overexpressed genes in the OVs, indicating their important role in the early stages of retinal differentiation. Along with MITF, the two paralogs, BHLHE41 and BHLHE40, were the most robust TF markers of RPE cells. The markedly contrasted expression of ACTL6B, encoding the component of chromatin remodeling complex SWI/SNF, discriminated hiPSC-derived OV/RGC and RPE lineages. Conclusions We identified novel, potentially important intrinsic regulators of RGC and RPE cell lineage specification in the process of differentiation from hiPSCs. We demonstrated the crucial role played by EBF1 in differentiation of RGCs. We identified intrinsic regulator biomarker signatures of these two retinal cell types that can be applied with high confidence to confirm the cell lineage identities

Establishing Liposome-Immobilized Dexamethasone-Releasing PDMS Membrane for the Cultivation of Retinal Pigment Epithelial Cells and Suppression of Neovascularization

Age-related macular degeneration (AMD) is the eye disease with the highest epidemic incidence, and has great impact on the aged population. Wet-type AMD commonly has the feature of neovascularization, which destroys the normal retinal structure and visual function. So far, effective therapy options for rescuing visual function in advanced AMD patients are highly limited, especially in wet-type AMD, in which the retinal pigmented epithelium and Bruch’s membrane structure (RPE-BM) are destroyed by abnormal angiogenesis. Anti-VEGF treatment is an effective remedy for the latter type of AMD; however, it is not a curative therapy. Therefore, reconstruction of the complex structure of RPE-BM and controlled release of angiogenesis inhibitors are strongly required for sustained therapy. The major purpose of this study was to develop a dual function biomimetic material, which could mimic the RPE-BM structure and ensure slow release of angiogenesis inhibitor as a novel therapeutic strategy for wet AMD. We herein utilized plasma-modified polydimethylsiloxane (PDMS) sheet to create a biomimetic scaffold mimicking subretinal BM. This dual-surface biomimetic scaffold was coated with laminin and dexamethasone-loaded liposomes. The top surface of PDMS was covalently grafted with laminin and used for cultivation of the retinal pigment epithelial cells differentiated from human induced pluripotent stem cells (hiPSC-RPE). To reach the objective of inhibiting angiogenesis required for treatment of wet AMD, the bottom surface of modified PDMS membrane was further loaded with dexamethasone-containing liposomes via biotin-streptavidin linkage. We demonstrated that hiPSC-RPE cells could proliferate, express normal RPE-specific genes and maintain their phenotype on laminin-coated PDMS membrane, including phagocytosis ability, and secretion of anti-angiogenesis factor PEDF. By using in vitro HUVEC angiogenesis assay, we showed that application of our membrane could suppress oxidative stress-induced angiogenesis, which was manifested in decreased secretion of VEGF by RPE cells and suppression of vascularization. In conclusion, we propose modified biomimetic material for dual delivery of RPE cells and liposome-enveloped dexamethasone, which can be potentially applied for AMD therapy

Circular RNA hsa_circ_0000190 Facilitates the Tumorigenesis and Immune Evasion by Upregulating the Expression of Soluble PD-L1 in Non-Small-Cell Lung Cancer

Lung cancer is the leading cause of death from cancer in Taiwan and throughout the world. Immunotherapy has revealed promising and significant efficacy in NSCLC, through immune checkpoint inhibition by blocking programmed cell death protein (PD)-1/PD-1 ligand (PD-L1) signaling pathway to restore patients’ T-cell immunity. One novel type of long, non-coding RNAs, circular RNAs (circRNAs), are endogenous, stable, and widely expressed in tissues, saliva, blood, urine, and exosomes. Our previous results revealed that the plasma level of hsa_circ_0000190 can be monitored by liquid-biopsy-based droplet digital PCR and may serve as a valuable blood-based biomarker to monitor the disease progression and the efficacy of immunotherapy. In this study, hsa_circ_0000190 was shown to increase the PD-L1 mRNA-mediated soluble PD-L1 (sPD-L1) expression, consequently interfering with the efficacy of anti-PD-L1 antibody and T-cell activation, which may result in immunotherapy resistance and poor outcome. Our results unraveled that hsa_circ_0000190 facilitated the tumorigenesis and immune evasion of NSCLC by upregulating sPD-L1 expression, potentially developing a different aspect in elucidating the molecular immunopathogenesis of NSCLC. Hsa_circ_0000190 upregulation can be an effective indicator for the progression of NSCLC, and hsa_circ_0000190 downregulation may possess a potential therapeutic value for the treatment of NSCLC in combination with immunotherapy

The Natural History of SARS-CoV-2-Incurred Disease: From Infection to Long COVID

The coronavirus SARS-CoV-2 is the causative pathogen of the COVID-19 pandemic that has been causing global upheaval since 2019. The widespread administration of vaccines has partially deterred the spread of SARS-CoV-2, yet the virus is mutating its genome to reduce its antigenicity and evade the human herd immunity. It seems that SARS-CoV-2 will co-exist with the human population for many decades to come. While most infected individuals only experience mild to moderate symptoms, some develop severe pulmonary and systemic disease that can result in hospitalization or even death. The natural history model of SARS-CoV-2 infection has been proposed which includes three sequential stages: the early infection stage, pulmonary stage, and hyper-inflammatory stage. Recently, it has been observed that many people who recovered from an acute infection still experience persistent symptoms for weeks or months, a condition known as long COVID. Furthermore, some COVID-19 patients display escalated rates of both macro- and micro-thrombosis due to endotheliopathy. Hence, we added the thrombosis and convalescent stages to the natural history model, encompassing the entire period from early infection to long COVID. The early infection stage is characterized by symptomatic or asymptomatic elevation of viral titers. Some patients progress to the pulmonary stage characterized by opacities in chest X-rays and computed tomography. The thrombosis stage is characterized by heightened rates of pulmonary thrombosis and consistently elevated D-dimer levels. The hyper-inflammatory stage is characterized by storms of cytokines, such as IL-6, IL-17, and interferons, which is a systemic effect. In the convalescent stage, some people recover completely, while others suffer from long COVID with persistent symptoms such as fatigue, shortness of breath, or brain fog. The natural history model of SARS-CoV-2 infection can be used to elucidate treatment and care

Generation of <i>GLA</i>-Knockout Human Embryonic Stem Cell Lines to Model Autophagic Dysfunction and Exosome Secretion in Fabry Disease-Associated Hypertrophic Cardiomyopathy

Fabry disease (FD) is a rare inherited disorder characterized by a wide range of systemic symptoms; it is particularly associated with cardiovascular and renal problems. Enzyme replacement therapy and pharmacological chaperone migalastat are the only approved and effective treatment strategies for FD patients. It is well documented that alpha-galactosidase A (GLA) enzyme activity deficiency causes globotriaosylceramide (Gb3) accumulation, which plays a crucial role in the etiology of FD. However, the detailed mechanisms remain unclear, and the lack of a reliable and powerful disease model is an obstacle. In this study, we created such a model by using CRISPR/Cas9-mediated editing of GLA gene to knockout its expression in human embryonic stem cells (hESCs). The cardiomyocytes differentiated from these hESCs (GLA-null CMs) were characterized by the accumulation of Gb3 and significant increases of cell surface area, the landmarks of FD-associated cardiomyopathy. Furthermore, we used mass spectrometry to compare the proteomes of GLA-null CMs and parental wild type CMs and found that the Rab GTPases involved in exocytotic vesicle release were significantly downregulated. This caused impairment of autophagic flux and protein turnover, resulting in an increase of reactive oxygen species and apoptosis. To summarize, we established a FD model which can be used as a promising tool to study human hypertrophic cardiomyopathy in a physiologically and pathologically relevant manner and to develop new therapies by targeting Rab GTPases signaling-related exosomal vesicles transportation