Activation of the urotensin-II receptor by remdesivir induces cardiomyocyte dysfunction.

Remdesivir is an antiviral drug used for COVID-19 treatment worldwide. Cardiovascular side effects have been associated with remdesivir; however, the underlying molecular mechanism remains unknown. Here, we performed a large-scale G-protein-coupled receptor screening in combination with structural modeling and found that remdesivir is a selective, partial agonist for urotensin-II receptor (UTS2R) through the Gαi/o-dependent AKT/ERK axis. Functionally, remdesivir treatment induced prolonged field potential and APD90 in human induced pluripotent stem cell (iPS)-derived cardiomyocytes and impaired contractility in both neonatal and adult cardiomyocytes, all of which mirror the clinical pathology. Importantly, remdesivir-mediated cardiac malfunctions were effectively attenuated by antagonizing UTS2R signaling. Finally, we characterized the effect of 110 single-nucleotide variants in UTS2R gene reported in genome database and found four missense variants that show gain-of-function effects in the receptor sensitivity to remdesivir. Collectively, our study illuminates a previously unknown mechanism underlying remdesivir-related cardiovascular events and that genetic variations of UTS2R gene can be a potential risk factor for cardiovascular events during remdesivir treatment, which collectively paves the way for a therapeutic opportunity to prevent such events in the future.

Modeling Heart Diseases on a Chip: Advantages and Future Opportunities.

Heart disease is a significant burden on global health care systems and is a leading cause of death each year. To improve our understanding of heart disease, high quality disease models are needed. These will facilitate the discovery and development of new treatments for heart disease. Traditionally, researchers have relied on 2D monolayer systems or animal models of heart disease to elucidate pathophysiology and drug responses. Heart-on-a-chip (HOC) technology is an emerging field where cardiomyocytes among other cell types in the heart can be used to generate functional, beating cardiac microtissues that recapitulate many features of the human heart. HOC models are showing great promise as disease modeling platforms and are poised to serve as important tools in the drug development pipeline. By leveraging advances in human pluripotent stem cell-derived cardiomyocyte biology and microfabrication technology, diseased HOCs are highly tuneable and can be generated via different approaches such as: using cells with defined genetic backgrounds (patient-derived cells), adding small molecules, modifying the cells' environment, altering cell ratio/composition of microtissues, among others. HOCs have been used to faithfully model aspects of arrhythmia, fibrosis, infection, cardiomyopathies, and ischemia, to name a few. In this review, we highlight recent advances in disease modeling using HOC systems, describing instances where these models outperformed other models in terms of reproducing disease phenotypes and/or led to drug development.

SARS-CoV-2 Infection of Human Neurons Is TMPRSS2 Independent, Requires Endosomal Cell Entry, and Can Be Blocked by Inhibitors of Host Phosphoinositol-5 Kinase.

2019 coronavirus disease (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms lasting from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to an infection of brain cells. We found that a small fraction of human induced pluripotent stem cell (iPSC)-derived neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), despite very low levels of its expression in neurons. The presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout infected cells, and an increase in the level of viral RNA in the culture medium (viral release) within the first 48 h of infection suggested that the infection was productive. Productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of the viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface spike protein, which can occur at the plasma membrane or from endosomes or lysosomes. We found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases, including transmembrane serine protease 2 (TMPRSS2). Inhibition of cathepsin L also did not significantly block infection. In contrast, the neuronal infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K), which regulates early to late endosome maturation. IMPORTANCE COVID-19 is a disease caused by the coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. SARS-CoV-2 infection of the human brain has been documented, but it is unclear whether the observed neurological symptoms are linked to direct brain infection. The mechanism of virus entry into neurons has also not been characterized. Here, we investigated SARS-CoV-2 infection by using a human iPSC-derived neural cell model and found that a small fraction of cortical-like neurons was naturally susceptible to infection. The productive infection was ACE2 dependent and TMPRSS2 independent. We also found that the virus used the late endosomal and lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of cellular PIK5K.

iPSC-derived three-dimensional brain organoid models and neurotropic viral infections.

Progress in stem cell research has revolutionized the medical field for more than two decades. More recently, the discovery of induced pluripotent stem cells (iPSCs) has allowed for the development of advanced disease modeling and tissue engineering platforms. iPSCs are generated from adult somatic cells by reprogramming them into an embryonic-like state via the expression of transcription factors required for establishing pluripotency. In the context of the central nervous system (CNS), iPSCs have the potential to differentiate into a wide variety of brain cell types including neurons, astrocytes, microglial cells, endothelial cells, and oligodendrocytes. iPSCs can be used to generate brain organoids by using a constructive approach in three-dimensional (3D) culture in vitro. Recent advances in 3D brain organoid modeling have provided access to a better understanding of cell-to-cell interactions in disease progression, particularly with neurotropic viral infections. Neurotropic viral infections have been difficult to study in two-dimensional culture systems in vitro due to the lack of a multicellular composition of CNS cell networks. In recent years, 3D brain organoids have been preferred for modeling neurotropic viral diseases and have provided invaluable information for better understanding the molecular regulation of viral infection and cellular responses. Here we provide a comprehensive review of the literature on recent advances in iPSC-derived 3D brain organoid culturing and their utilization in modeling major neurotropic viral infections including HIV-1, HSV-1, JCV, ZIKV, CMV, and SARS-CoV2.

Human brain microphysiological systems in the study of neuroinfectious disorders.

Microphysiological systems (MPS) are 2D or 3D multicellular constructs able to mimic tissue microenvironments. The latest models encompass a range of techniques, including co-culturing of various cell types, utilization of scaffolds and extracellular matrix materials, perfusion systems, 3D culture methods, 3D bioprinting, organ-on-a-chip technology, and examination of tissue structures. Several human brain 3D cultures or brain MPS (BMPS) have emerged in the last decade. These organoids or spheroids are 3D culture systems derived from induced pluripotent cells or embryonic stem cells that contain neuronal and glial populations and recapitulate structural and physiological aspects of the human brain. BMPS have been introduced recently in the study and modeling of neuroinfectious diseases and have proven to be useful in establishing neurotropism of viral infections, cell-pathogen interactions needed for infection, assessing cytopathological effects, genomic and proteomic profiles, and screening therapeutic compounds. Here we review the different methodologies of organoids used in neuroinfectious diseases including spheroids, guided and unguided protocols as well as microglia and blood-brain barrier containing models, their specific applications, and limitations. The review provides an overview of the models existing for specific infections including Zika, Dengue, JC virus, Japanese encephalitis, measles, herpes, SARS-CoV2, and influenza viruses among others, and provide useful concepts in the modeling of disease and antiviral agent screening.

The original strain of SARS-CoV-2, the Delta variant, and the Omicron variant infect microglia efficiently, in contrast to their inability to infect neurons: Analysis using 2D and 3D cultures.

COVID-19 causes neurological damage, systemic inflammation, and immune cell abnormalities. COVID-19-induced neurological impairment may be caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which directly infects cells of the central nervous system (CNS) and exerts toxic effects. Furthermore, SARS-CoV-2 mutations occur constantly, and it is not well understood how the infectivity of the virus to cells of the CNS changes as the virus mutates. Few studies have examined whether the infectivity of cells of CNS - neural stem/progenitor cells (NS/PCs), neurons, astrocytes, and microglia - varies among SARS-CoV-2 mutant strains. In this study, therefore, we investigated whether SARS-CoV-2 mutations increase infectivity to CNS cells, including microglia. Since it was essential to demonstrate the infectivity of the virus to CNS cells in vitro using human cells, we generated cortical neurons, astrocytes, and microglia from human induced pluripotent stem cells (hiPSCs). We added pseudotyped lentiviruses of SARS-CoV-2 to each type of cells, and then we examined their infectivity. We prepared three pseudotyped lentiviruses expressing the S protein of the original strain (the first SARS-CoV-2 discovered in the world), the Delta variant, and the Omicron variant on their envelopes and analyzed differences of their ability to infect CNS cells. We also generated brain organoids and investigated the infectivity of each virus. The viruses did not infect cortical neurons, astrocytes, or NS/PCs, but microglia were infected by the original, Delta, and Omicron pseudotyped viruses. In addition, DPP4 and CD147, potential core receptors of SARS-CoV-2, were highly expressed in the infected microglia, while DPP4 expression was deficient in cortical neurons, astrocytes, and NS/PCs. Our results suggest that DPP4, which is also a receptor for Middle East respiratory syndrome-coronavirus (MERS-CoV), may play an essential role in the CNS. Our study is applicable to the validation of the infectivity of viruses that cause various infectious diseases in CNS cells, which are difficult to sample from humans.

SARS-CoV-2 spike protein-mediated cardiomyocyte fusion may contribute to increased arrhythmic risk in COVID-19.

BACKGROUND: SARS-CoV-2-mediated COVID-19 may cause sudden cardiac death (SCD). Factors contributing to this increased risk of potentially fatal arrhythmias include thrombosis, exaggerated immune response, and treatment with QT-prolonging drugs. However, the intrinsic arrhythmic potential of direct SARS-CoV-2 infection of the heart remains unknown. OBJECTIVE: To assess the cellular and electrophysiological effects of direct SARS-CoV-2 infection of the heart using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS: hiPSC-CMs were transfected with recombinant SARS-CoV-2 spike protein (CoV-2 S) or CoV-2 S fused to a modified Emerald fluorescence protein (CoV-2 S-mEm). Cell morphology was visualized using immunofluorescence microscopy. Action potential duration (APD) and cellular arrhythmias were measured by whole cell patch-clamp. Calcium handling was assessed using the Fluo-4 Ca2+ indicator. RESULTS: Transfection of hiPSC-CMs with CoV-2 S-mEm produced multinucleated giant cells (syncytia) displaying increased cellular capacitance (75±7 pF, n = 10 vs. 26±3 pF, n = 10; P<0.0001) consistent with increased cell size. The APD90 was prolonged significantly from 419±26 ms (n = 10) in untransfected hiPSC-CMs to 590±67 ms (n = 10; P<0.05) in CoV-2 S-mEm-transfected hiPSC-CMs. CoV-2 S-induced syncytia displayed delayed afterdepolarizations, erratic beating frequency, and calcium handling abnormalities including calcium sparks, large "tsunami"-like waves, and increased calcium transient amplitude. After furin protease inhibitor treatment or mutating the CoV-2 S furin cleavage site, cell-cell fusion was no longer evident and Ca2+ handling returned to normal. CONCLUSION: The SARS-CoV-2 spike protein can directly perturb both the cardiomyocyte's repolarization reserve and intracellular calcium handling that may confer the intrinsic, mechanistic substrate for the increased risk of SCD observed during this COVID-19 pandemic.

SARS-CoV-2 Delta (B.1.617.2) variant replicates and induces syncytia formation in human induced pluripotent stem cell-derived macrophages.

Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity.

The molecular mechanism of cardiac injury in SARS-CoV-2 infection: Focus on mitochondrial dysfunction.

BACKGROUND: Coronavirus disease 2019(COVID-19) caused a large number of infections worldwide. Although some patients recovered from the disease, some of the other problems that accompanied it, such as cardiac injury, could affect the patient's subsequent quality of life and prognosis. OBJECTIVES: To clarify the molecular mechanism of cardiac injury in SARS-CoV-2 Infection. METHODS: The RNA-Seq dataset (GSE184715) comparing expression profiling of Mock human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and SARS-CoV-2-infected hiPSC-CMs was downloaded from Gene Expression Omnibus (GEO). Differentially expressed genes(DEGs) were performed by the R software. Degs were analyzed by enrichment analysis to clarify the affected pathways. Hub genes were screened out by a PPI network constructed from Degs. Finally, Connectivity Map was used to screen for the treatment of COVID-19 induced cardiac injury. RESULTS: 2705 differentially expressed genes were identified. Enrichment analysis confirmed that mitochondrial dysfunction was caused by SARS-CoV-2, meanwhile, cardiac muscle contraction was suppressed and NF-κB was activated. Based on the PPI network, 15 hub genes were identified. These 15 down-regulated hub genes were mainly involved in the reduced activity of complexes in the mitochondrial respiratory chain associated with mitochondrial dysfunction. Moreover, 5 candidate drugs were identified to treat cardiac injury. CONCLUSION: In conclusion, SARS-CoV-2 infection of cardiomyocytes causes mitochondrial dysfunction, including reduced mitochondrial respiratory chain complex activity and decreased ATP synthesis, leading to cardiomyocyte apoptosis, while the activated NF-κB also induced cytokine storms, ultimately resulting in cardiac injury.

Pluripotent Stem Cells of Order Carnivora: Technical Perspective.

Human and mouse induced pluripotent stem cells (PSCs) are widely used for studying early embryonic development and for modeling of human diseases. Derivation and studying of PSCs from model organisms beyond commonly used mice and rats may provide new insights into the modeling and treating human diseases. The order Carnivora representatives possess unique features and are already used for modeling human-related traits. This review focuses on the technical aspects of derivation of the Carnivora species PSCs as well as their characterization. Current data on dog, feline, ferret, and American mink PSCs are summarized.

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.