Induced Pluripotent Stem Cell-Derived Organoids: Their Implication in COVID-19 Modeling.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a significant global health issue. This novel virus's high morbidity and mortality rates have prompted the scientific community to quickly find the best COVID-19 model to investigate all pathological processes underlining its activity and, more importantly, search for optimal drug therapy with minimal toxicity risk. The gold standard in disease modeling involves animal and monolayer culture models; however, these models do not fully reflect the response to human tissues affected by the virus. However, more physiological 3D in vitro culture models, such as spheroids and organoids derived from induced pluripotent stem cells (iPSCs), could serve as promising alternatives. Different iPSC-derived organoids, such as lung, cardiac, brain, intestinal, kidney, liver, nasal, retinal, skin, and pancreatic organoids, have already shown immense potential in COVID-19 modeling. In the present comprehensive review article, we summarize the current knowledge on COVID-19 modeling and drug screening using selected iPSC-derived 3D culture models, including lung, brain, intestinal, cardiac, blood vessels, liver, kidney, and inner ear organoids. Undoubtedly, according to reviewed studies, organoids are the state-of-the-art approach to COVID-19 modeling.

Generation of four human induced pluripotent stem cell lines from COVID-19 hospitalized patients with increased levels of cardiac Troponin in the acute infection phase developing or not myocarditis.

Coronavirus disease (COVID-19) is an infectious disease caused by SARS-CoV-2 virus, leading to mild to severe respiratory symptoms. Cardiovascular involvement is frequent and mainly manifests with myocarditis, arrhythmias, cardiac arrests, heart failure and coagulation abnormality. We generated human induced pluripotent stem cells (hiPSCs) from four COVID-19 patients, all characterized by increased levels of high-sensitivity Troponin I (hsTnI) during the infection acute phase, who developed (n = 2) or not (n = 2) severe myocarditis, as COVID-19 complication. The established hiPSCs were characterized for pluripotency and genomic stability, and constitute a useful resource for studying the mechanisms underlying the variability in COVID-19 severe cardiac manifestations.

[Clinical applications of iPS cell-derived platelets].

The COVID-19 pandemic has cast a shadow over transfusion medicine based on the blood donation system. However, managing alloimmune platelet transfusion refractoriness (allo-PTR) has already been difficult. As a first step toward resolving this issue using induced pluripotent stem cell-derived platelet products (iPSC-PLTs), a clinical trial of autologous products (iPLAT1) was conducted in a patient with allo-PTR caused by anti-HPA-1a antibodies who had no compatible donor, and safety was confirmed. To produce iPSC-PLTs, a master cell bank (MCB) of expandable megakaryocyte lines (imMKCLs) is established from iPSCs. From this MCB, iPSC-PLTs are manufactured using a newly developed turbulent-type bioreactor and various compounds. Their quality, safety, and efficacy are confirmed by extensive preclinical studies. Based on the findings of the iPLAT1 study, a clinical trial of allo-transfusion of HLA homozygous iPSC-PLTs is currently ongoing and HLA class I-deficient O-type universal iPSC-PLTs are also being developed. iPSC-PLTs are expected to solve various problems, including allo-PTR in platelet transfusion, and greatly contribute to the advancement of transfusion medicine.

Identification of the Hub Genes and the Signaling Pathways in Human iPSC-Cardiomyocytes Infected by SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) is an enveloped single-stranded RNA virus that can lead to respiratory symptoms and damage many organs such as heart, kidney, intestine, brain and liver. It has not been clearly documented whether myocardial injury is caused by direct infection of cardiomyocytes, lung injury, or other unknown mechanisms. The gene expression profile of GSE150392 was obtained from the Gene Expression Omnibus (GEO) database. The processing of high-throughput sequencing data and the screening of differentially expressed genes (DEGs) were implemented by R software. The R software was employed to analyze the Gene Ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The protein-protein interaction (PPI) network of the DEGs was constructed by the STRING website. The Cytoscape software was applied for the visualization of PPI network and the identification of hub genes. The statistical analysis was performed by the GraphPad Prism software to verify the hub genes. A total of 516 up-regulated genes and 191 down-regulated genes were screened out. The top 1 enrichment items of GO in biological process (BP), Cellular Component (CC), and Molecular Function (MF) were type I interferon signaling pathway, sarcomere, and receptor ligand activity, respectively. The top 10 enrichment pathways, including TNF signaling pathway, were identified by KEGG enrichment analysis. A PPI network was established, consisting of 613 nodes and 3,993 edges. The 12 hub genes were confirmed as statistically significant, which was verified by GSE151879 dataset. In conclusion, the hub genes of human iPSC-cardiomyocytes infected with SARS-CoV-2 were identified through bioinformatics analysis, which may be used as biomarkers for further research.

Human induced pluripotent stem cell-derived salivary gland organoids model SARS-CoV-2 infection and replication.

Salivary glands act as virus reservoirs in various infectious diseases and have been reported to be targeted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the mechanisms underlying infection and replication in salivary glands are still enigmatic due to the lack of proper in vitro models. Here, we show that human induced salivary glands (hiSGs) generated from human induced pluripotent stem cells can be infected with SARS-CoV-2. The hiSGs exhibit properties similar to those of embryonic salivary glands and are a valuable tool for the functional analysis of genes during development. Orthotopically transplanted hiSGs can be engrafted at a recipient site in mice and show a mature phenotype. In addition, we confirm SARS-CoV-2 infection and replication in hiSGs. SARS-CoV-2 derived from saliva in asymptomatic individuals may participate in the spread of the virus. hiSGs may be a promising model for investigating the role of salivary glands as a virus reservoir.

A human iPSC-array-based GWAS identifies a virus susceptibility locus in the NDUFA4 gene and functional variants.

Population-based studies to identify disease-associated risk alleles typically require samples from a large number of individuals. Here, we report a human-induced pluripotent stem cell (hiPSC)-based screening strategy to link human genetics with viral infectivity. A genome-wide association study (GWAS) identified a cluster of single-nucleotide polymorphisms (SNPs) in a cis-regulatory region of the NDUFA4 gene, which was associated with susceptibility to Zika virus (ZIKV) infection. Loss of NDUFA4 led to decreased sensitivity to ZIKV, dengue virus, and SARS-CoV-2 infection. Isogenic hiPSC lines carrying non-risk alleles of SNPs or deletion of the cis-regulatory region lower sensitivity to viral infection. Mechanistic studies indicated that loss/reduction of NDUFA4 causes mitochondrial stress, which leads to the leakage of mtDNA and thereby upregulation of type I interferon signaling. This study provides proof-of-principle for the application of iPSC arrays in GWAS and identifies NDUFA4 as a previously unknown susceptibility locus for viral infection.

Investigating and Resolving Cardiotoxicity Induced by COVID-19 Treatments using Human Pluripotent Stem Cell-Derived Cardiomyocytes and Engineered Heart Tissues.

Coronavirus disease 2019 continues to spread worldwide. Given the urgent need for effective treatments, many clinical trials are ongoing through repurposing approved drugs. However, clinical data regarding the cardiotoxicity of these drugs are limited. Human pluripotent stem cell-derived cardiomyocytes (hCMs) represent a powerful tool for assessing drug-induced cardiotoxicity. Here, by using hCMs, it is demonstrated that four antiviral drugs, namely, apilimod, remdesivir, ritonavir, and lopinavir, exhibit cardiotoxicity in terms of inducing cell death, sarcomere disarray, and dysregulation of calcium handling and contraction, at clinically relevant concentrations. Human engineered heart tissue (hEHT) model is used to further evaluate the cardiotoxic effects of these drugs and it is found that they weaken hEHT contractile function. RNA-seq analysis reveals that the expression of genes that regulate cardiomyocyte function, such as sarcomere organization (TNNT2, MYH6) and ion homeostasis (ATP2A2, HCN4), is significantly altered after drug treatments. Using high-throughput screening of approved drugs, it is found that ceftiofur hydrochloride, astaxanthin, and quetiapine fumarate can ameliorate the cardiotoxicity of remdesivir, with astaxanthin being the most prominent one. These results warrant caution and careful monitoring when prescribing these therapies in patients and provide drug candidates to limit remdesivir-induced cardiotoxicity.

In vitro and in vivo differences in neurovirulence between D614G, Delta And Omicron BA.1 SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with various neurological complications. Although the mechanism is not fully understood, several studies have shown that neuroinflammation occurs in the acute and post-acute phase. As these studies have predominantly been performed with isolates from 2020, it is unknown if there are differences among SARS-CoV-2 variants in their ability to cause neuroinflammation. Here, we compared the neuroinvasiveness, neurotropism and neurovirulence of the SARS-CoV-2 ancestral strain D614G, the Delta (B.1.617.2) and Omicron BA.1 (B.1.1.529) variants using in vitro and in vivo models. The Omicron BA.1 variant showed reduced neurotropism and neurovirulence compared to Delta and D614G in human induced pluripotent stem cell (hiPSC)-derived cortical neurons co-cultured with astrocytes. Similar differences were obtained in Syrian hamsters inoculated with D614G, Delta and the Omicron BA.1 variant 5 days post infection. Replication in the olfactory mucosa was observed in all hamsters, but most prominently in D614G inoculated hamsters. Furthermore, neuroinvasion into the CNS via the olfactory nerve was observed in D614G, but not Delta or Omicron BA.1 inoculated hamsters. Furthermore, neuroinvasion was associated with neuroinflammation in the olfactory bulb of hamsters inoculated with D614G. Altogether, our findings suggest differences in the neuroinvasive, neurotropic and neurovirulent potential between SARS-CoV-2 variants using in vitro hiPSC-derived neural cultures and in vivo in hamsters during the acute phase of the infection.

Public Cord Blood Banks as a source of starting material for clinical grade HLA-homozygous induced pluripotent stem cells.

BACKGROUND: The increasing number of clinical trials for induced pluripotent stem cell (iPSC)-derived cell therapy products makes the production on clinical grade iPSC more and more relevant and necessary. Cord blood banks are an ideal source of young, HLA-typed and virus screened starting material to produce HLA-homozygous iPSC lines for wide immune-compatibility allogenic cell therapy approaches. The production of such clinical grade iPSC lines (haplolines) involves particular attention to all steps since donor informed consent, cell procurement and a GMP-compliant cell isolation process. METHODS: Homozygous cord blood units were identified and quality verified before recontacting donors for informed consent. CD34+ cells were purified from the mononuclear fraction isolated in a cell processor, by magnetic microbeads labelling and separation columns. RESULTS: We obtained a median recovery of 20.0% of the collected pre-freezing CD34+, with a final product median viability of 99.1% and median purity of 83.5% of the post-thawed purified CD34+ population. CONCLUSIONS: Here we describe our own experience, from unit selection and donor reconsenting, in generating a CD34+ cell product as a starting material to produce HLA-homozygous iPSC following a cost-effective and clinical grade-compliant procedure. These CD34+ cells are the basis for the Spanish bank of haplolines envisioned to serve as a source of cell products for clinical research and therapy.

Selection of O-negative induced pluripotent stem cell clones for high-density red blood cell production in a scalable perfusion bioreactor system.

OBJECTIVES: Large-scale generation of universal red blood cells (RBCs) from O-negative (O-ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year-round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high-density culture platform for large-scale generation of RBCs in vitro. MATERIALS AND METHODS: We generated 10 O-ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high-density cultures of erythroblasts in vitro. RESULTS: Based on their tri-lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small-scale generation of high-density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation. CONCLUSIONS: This study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume-controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Thrombosis-related circulating miR-16-5p is associated with disease severity in patients hospitalised for COVID-19.

SARS-CoV-2 tropism for the ACE2 receptor, along with the multifaceted inflammatory reaction, is likely to drive the generalized hypercoagulable and thrombotic state seen in patients with COVID-19. Using the original bioinformatic workflow and network medicine approaches we reanalysed four coronavirus-related expression datasets and performed co-expression analysis focused on thrombosis and ACE2 related genes. We identified microRNAs (miRNAs) which play role in ACE2-related thrombosis in coronavirus infection and further, we validated the expressions of precisely selected miRNAs-related to thrombosis (miR-16-5p, miR-27a-3p, let-7b-5p and miR-155-5p) in 79 hospitalized COVID-19 patients and 32 healthy volunteers by qRT-PCR. Consequently, we aimed to unravel whether bioinformatic prioritization could guide selection of miRNAs with a potential of diagnostic and prognostic biomarkers associated with disease severity in patients hospitalized for COVID-19. In bioinformatic analysis, we identified EGFR, HSP90AA1, APP, TP53, PTEN, UBC, FN1, ELAVL1 and CALM1 as regulatory genes which could play a pivotal role in COVID-19 related thrombosis. We also found miR-16-5p, miR-27a-3p, let-7b-5p and miR-155-5p as regulators in the coagulation and thrombosis process. In silico predictions were further confirmed in patients hospitalized for COVID-19. The expression levels of miR-16-5p and let-7b in COVID-19 patients were lower at baseline, 7-days and 21-day after admission compared to the healthy controls (p < 0.0001 for all time points for both miRNAs). The expression levels of miR-27a-3p and miR-155-5p in COVID-19 patients were higher at day 21 compared to the healthy controls (p = 0.007 and p < 0.001, respectively). A low baseline miR-16-5p expression presents predictive utility in assessment of the hospital length of stay or death in follow-up as a composite endpoint (AUC:0.810, 95% CI, 0.71-0.91, p < 0.0001) and low baseline expression of miR-16-5p and diabetes mellitus are independent predictors of increased length of stay or death according to a multivariate analysis (OR: 9.417; 95% CI, 2.647-33.506; p = 0.0005 and OR: 6.257; 95% CI, 1.049-37.316; p = 0.044, respectively). This study enabled us to better characterize changes in gene expression and signalling pathways related to hypercoagulable and thrombotic conditions in COVID-19. In this study we identified and validated miRNAs which could serve as novel, thrombosis-related predictive biomarkers of the COVID-19 complications, and can be used for early stratification of patients and prediction of severity of infection development in an individual.Abbreviations: ACE2, angiotensin-converting enzyme 2AF, atrial fibrillationAPP, Amyloid Beta Precursor ProteinaPTT, activated partial thromboplastin timeAUC, Area under the curveAß, amyloid betaBMI, body mass indexCAD, coronary artery diseaseCALM1, Calmodulin 1 geneCaM, calmodulinCCND1, Cyclin D1CI, confidence intervalCOPD, chronic obstructive pulmonary diseaseCOVID-19, Coronavirus disease 2019CRP, C-reactive proteinCV, CardiovascularCVDs, cardiovascular diseasesDE, differentially expressedDM, diabetes mellitusEGFR, Epithelial growth factor receptorELAVL1, ELAV Like RNA Binding Protein 1FLNA, Filamin AFN1, Fibronectin 1GEO, Gene Expression OmnibushiPSC-CMs, Human induced pluripotent stem cell-derived cardiomyocytesHSP90AA1, Heat Shock Protein 90 Alpha Family Class A Member 1Hsp90α, heat shock protein 90αICU, intensive care unitIL, interleukinIQR, interquartile rangelncRNAs, long non-coding RNAsMI, myocardial infarctionMiRNA, MiR, microRNAmRNA, messenger RNAncRNA, non-coding RNANERI, network-medicine based integrative approachNF-kB, nuclear factor kappa-light-chain-enhancer of activated B cellsNPV, negative predictive valueNXF, nuclear export factorPBMCs, Peripheral blood mononuclear cellsPCT, procalcitoninPPI, Protein-protein interactionsPPV, positive predictive valuePTEN, phosphatase and tensin homologqPCR, quantitative polymerase chain reactionROC, receiver operating characteristicSARS-CoV-2, severe acute respiratory syndrome coronavirus 2SD, standard deviationTLR4, Toll-like receptor 4TM, thrombomodulinTP53, Tumour protein P53UBC, Ubiquitin CWBC, white blood cells.

Using human iPSC-derived kidney organoids to decipher SARS-CoV-2 pathology on single cell level.

We describe a protocol for single-cell RNA sequencing of SARS-CoV-2-infected human induced pluripotent stem cell (iPSC)-derived kidney organoids. After inoculation of kidney organoids with virus, we use mechanical and enzymatic disruption to obtain single cell suspensions. Next, we process the organoid-derived cells into sequencing-ready SARS-CoV-2-targeted libraries. Subsequent sequencing analysis reveals changes in kidney cells after virus infection. The protocol was designed for kidney organoids cultured in a 6-well transwell format but can be adapted to organoids with different organ backgrounds. For complete details on the use and execution of this protocol, please refer to Jansen et al. (2022).

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells.

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as "matrix"), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

Identification of critical genes and molecular pathways in COVID-19 myocarditis and constructing gene regulatory networks by bioinformatic analysis.

BACKGROUND: There is growing evidence of a strong relationship between COVID-19 and myocarditis. However, there are few bioinformatics-based analyses of critical genes and the mechanisms related to COVID-19 Myocarditis. This study aimed to identify critical genes related to COVID-19 Myocarditis by bioinformatic methods, explore the biological mechanisms and gene regulatory networks, and probe related drugs. METHODS: The gene expression data of GSE150392 and GSE167028 were obtained from the Gene Expression Omnibus (GEO), including cardiomyocytes derived from human induced pluripotent stem cells infected with SARS-CoV-2 in vitro and GSE150392 from patients with myocarditis infected with SARS-CoV-2 and the GSE167028 gene expression dataset. Differentially expressed genes (DEGs) (adjusted P-Value <0.01 and |Log2 Fold Change| ≥2) in GSE150392 were assessed by NetworkAnalyst 3.0. Meanwhile, significant modular genes in GSE167028 were identified by weighted gene correlation network analysis (WGCNA) and overlapped with DEGs to obtain common genes. Functional enrichment analyses were performed by using the "clusterProfiler" package in the R software, and protein-protein interaction (PPI) networks were constructed on the STRING website (https://cn.string-db.org/). Critical genes were identified by the CytoHubba plugin of Cytoscape by 5 algorithms. Transcription factor-gene (TF-gene) and Transcription factor-microRibonucleic acid (TF-miRNA) coregulatory networks construction were performed by NetworkAnalyst 3.0 and displayed in Cytoscape. Finally, Drug Signatures Database (DSigDB) was used to probe drugs associated with COVID-19 Myocarditis. RESULTS: Totally 850 DEGs (including 449 up-regulated and 401 down-regulated genes) and 159 significant genes in turquoise modules were identified from GSE150392 and GSE167028, respectively. Functional enrichment analysis indicated that common genes were mainly enriched in biological processes such as cell cycle and ubiquitin-protein hydrolysis. 6 genes (CDK1, KIF20A, PBK, KIF2C, CDC20, UBE2C) were identified as critical genes. TF-gene interactions and TF-miRNA coregulatory network were constructed successfully. A total of 10 drugs, (such as Etoposide, Methotrexate, Troglitazone, etc) were considered as target drugs for COVID-19 Myocarditis. CONCLUSIONS: Through bioinformatics method analysis, this study provides a new perspective to explore the pathogenesis, gene regulatory networks and provide drug compounds as a reference for COVID-19 Myocarditis. It is worth highlighting that critical genes (CDK1, KIF20A, PBK, KIF2C, CDC20, UBE2C) may be potential biomarkers and treatment targets of COVID-19 Myocarditis for future study.

Stem cells, cell therapies, and bioengineering in lung biology and disease 2021.

The 9th biennial conference titled "Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases" was hosted virtually, due to the ongoing COVID-19 pandemic, in collaboration with the University of Vermont Larner College of Medicine, the National Heart, Lung, and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, and the International Society for Cell & Gene Therapy. The event was held from July 12th through 15th, 2021 with a pre-conference workshop held on July 9th. As in previous years, the objectives remained to review and discuss the status of active research areas involving stem cells (SCs), cellular therapeutics, and bioengineering as they relate to the human lung. Topics included 1) technological advancements in the in situ analysis of lung tissues, 2) new insights into stem cell signaling and plasticity in lung remodeling and regeneration, 3) the impact of extracellular matrix in stem cell regulation and airway engineering in lung regeneration, 4) differentiating and delivering stem cell therapeutics to the lung, 5) regeneration in response to viral infection, and 6) ethical development of cell-based treatments for lung diseases. This selection of topics represents some of the most dynamic and current research areas in lung biology. The virtual workshop included active discussion on state-of-the-art methods relating to the core features of the 2021 conference, including in situ proteomics, lung-on-chip, induced pluripotent stem cell (iPSC)-airway differentiation, and light sheet microscopy. The conference concluded with an open discussion to suggest funding priorities and recommendations for future research directions in basic and translational lung biology.

Modeling Nonischemic Genetic Cardiomyopathies Using Induced Pluripotent Stem Cells.

PURPOSE OF REVIEW: The advent of induced pluripotent stem cells (iPSC) has paved the way for new in vitro models of human cardiomyopathy. Herein, we will review existing models of disease as well as strengths and limitations of the system. RECENT FINDINGS: Preclinical studies have now demonstrated that iPSCs generated from patients with both acquired or heritable genetic diseases retain properties of the disease in vitro and can be used as a model to study novel therapeutics. iPSCs can be differentiated in vitro into the cardiomyocyte lineage into cells resembling adult ventricular myocytes that retain properties of cardiovascular disease from their respective donor. iPSC pluripotency allows for them to be frozen, stored, and continually used to generate iPSC-derived myocytes for future experiments without need for invasive procedures or repeat myocyte isolations to obtain animal or human cardiac tissues. While not without their limitations, iPSC models offer new ways for studying patient-specific cardiomyopathies. iPSCs offer a high-throughput avenue for drug development, modeling of disease pathophysiology in vitro, and enabling experimental repair strategies without need for invasive procedures to obtain cardiac tissues.

Bioinformatics analysis of potential therapeutic targets for COVID-19 infection in patients with carotid atherosclerosis.

BACKGROUND: COVID-19 is a new coronavirus that constitutes a great challenge to human health. At this stage, there are still cases of COVID-19 infection in some countries and regions, in which ischemic stroke (IS) is a risk factor for new coronavirus pneumonia, and patients with COVID-19 infection have a dramatically elevated risk of stroke. At the same time, patients with long-term IS are vulnerable to COVID-19 infection and have more severe disease, and carotid atherosclerosis is an early lesion in IS. METHODS: This study used human induced pluripotent stem cell (hiPSC)-derived monolayer brain cell dataset and human carotid atherosclerosis genome-wide dataset to analyze COVID-19 infection and carotid atherosclerosis patients to determine the synergistic effect of new coronavirus infection on carotid atherosclerosis patients, to clarify the common genes of both, and to identify common pathways and potential drugs for carotid atherosclerosis in patients with COVID-19 infection RESULTS: Using several advanced bioinformatics tools, we present the causes of COVID-19 infection leading to increased mortality in carotid atherosclerosis patients and the susceptibility of carotid atherosclerosis patients to COVID-19. Potential therapeutic agents for COVID-19 -infected patients with carotid atherosclerosis are also proposed. CONCLUSIONS: With COVID-19 being a relatively new disease, associations have been proposed for its connections with several ailments and conditions, including IS and carotid atherosclerosis. More patient-based data-sets and studies are needed to fully explore and understand the relationship.

Sars-Cov-2 Spike Protein-Induced Damage of hiPSC-Derived Cardiomyocytes.

Sars-Cov-2 may trigger molecular and functional alterations of cardiomyocytes (CMs) of the heart due to the presence of receptor angiotensin-converting enzyme 2 (ACE2) of the host cells. While the endocytic itinerary of the virus via cleavage of the spike protein of Sars-Cov-2 is well understood, the role of the remaining part of the spike protein subunit and ACE2 complex is still elusive. Herein, the possible effects of this complex are investigated by using synthetic spike proteins of Sars-Cov-2, human-induced pluripotent stem cells (hiPSC), and a culture device made of an arrayed monolayer of cross-linked nanofibers. hiPSCs are first differentiated into CMs that form cardiac tissue-like constructs with regular beating and expression of both ACE2 and gap junction protein Connexin 43. When incubated with the spike proteins, the hiPSC-CMs undergo a rhythmic fluctuation with overstretched sarcomere structures and dispersed gap junction proteins. When incubated with the spike proteins and supplementary angiotensin II, the damage of the spike protein on hiPSC-CMs is enhanced due to downregulated ACE2, chromatin margination, altered Connexin 43 expression, sarcomere disruption, and beating break. This discovery may imply latent effects of the spike proteins on the heart.

SARS-CoV-2 induces barrier damage and inflammatory responses in the human iPSC-derived intestinal epithelium.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread and led to global health crises. COVID-19 causes well-known respiratory failure and gastrointestinal symptoms, such as diarrhea, nausea, and vomiting. Thus, human gastrointestinal cell models are urgently needed for COVID-19 research; however, it is difficult to obtain primary human intestinal cells. In this study, we examined whether human induced pluripotent stem cell (iPSC)-derived small intestinal epithelial cells (iPSC-SIECs) could be used as a SARS-CoV-2 infection model. We observed that iPSC-SIECs, such as absorptive and Paneth cells, were infected with SARS-CoV-2, and remdesivir treatment decreased intracellular SARS-CoV-2 replication in iPSC-SIECs. SARS-CoV-2 infection decreased expression levels of tight junction markers, ZO-3 and CLDN1, and transepithelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics. In addition, SARS-CoV-2 infection increased expression levels of proinflammatory genes, which are elevated in patients with COVID-19. These findings suggest iPSC-SIECs as a useful in vitro model for elucidating COVID-19 pathology and drug development.

SARS-CoV-2 infection of human-induced pluripotent stem cells-derived lung lineage cells evokes inflammatory and chemosensory responses by targeting mitochondrial pathways.

The COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily affects the lung, particularly the proximal airway and distal alveolar cells. NKX2.1+ primordial lung progenitors of the foregut (anterior) endoderm are the developmental precursors to all adult lung epithelial lineages and are postulated to play an important role in viral tropism. Here, we show that SARS-CoV-2 readily infected and replicated in human-induced pluripotent stem cell-derived proximal airway cells, distal alveolar cells, and lung progenitors. In addition to the upregulation of antiviral defense and immune responses, transcriptomics data uncovered a robust epithelial cell-specific response, including perturbation of metabolic processes and disruption in the alveolar maturation program. We also identified spatiotemporal dysregulation of mitochondrial heme oxygenase 1 (HMOX1), which is associated with defense against antioxidant-induced lung injury. Cytokines, such as TNF-α, INF-γ, IL-6, and IL-13, were upregulated in infected cells sparking mitochondrial ROS production and change in electron transport chain complexes. Increased mitochondrial ROS then activated additional proinflammatory cytokines leading to an aberrant cell cycle resulting in apoptosis. Notably, we are the first to report a chemosensory response resulting from SARS-CoV-2 infection similar to that seen in COVID-19 patients. Some of our key findings were validated using COVID-19-affected postmortem lung tissue sections. These results suggest that our in vitro system could serve as a suitable model to investigate the pathogenetic mechanisms of SARS-CoV-2 infection and to discover and test therapeutic drugs against COVID-19 or its consequences.