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1.
Stem Cell Res Ther ; 11(1): 448, 2020 10 23.
Article in English | MEDLINE | ID: covidwho-1388825

ABSTRACT

Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)-a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research.


Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Human Embryonic Stem Cells/cytology , Lung Diseases/therapy , Organoids/cytology , Cell Line , Dependovirus/immunology , Gene Transfer Techniques , Genetic Vectors/genetics , Humans , Lung/metabolism , Models, Biological , Parenchymal Tissue/cytology
2.
Commun Biol ; 4(1): 926, 2021 07 29.
Article in English | MEDLINE | ID: covidwho-1387497

ABSTRACT

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Human Embryonic Stem Cells/cytology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/virology , SARS-CoV-2/physiology , Benztropine/pharmacology , Humans , Myocytes, Cardiac/cytology , Peptides/pharmacology
3.
Stem Cell Reports ; 16(5): 1156-1164, 2021 05 11.
Article in English | MEDLINE | ID: covidwho-1225409

ABSTRACT

Coronavirus disease 2019 (COVID-19) patients have manifested a variety of neurological complications, and there is still much to reveal regarding the neurotropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human stem cell-derived brain organoids offer a valuable in vitro approach to study the cellular effects of SARS-CoV-2 on the brain. Here we used human embryonic stem cell-derived cortical organoids to investigate whether SARS-CoV-2 could infect brain tissue in vitro and found that cortical organoids could be infected at low viral titers and within 6 h. Importantly, we show that glial cells and cells of the choroid plexus were preferentially targeted in our model, but not neurons. Interestingly, we also found expression of angiotensin-converting enzyme 2 in SARS-CoV-2 infected cells; however, viral replication and cell death involving DNA fragmentation does not occur. We believe that our model is a tractable platform to study the cellular effects of SARS-CoV-2 infection in brain tissue.


Subject(s)
COVID-19/pathology , Choroid Plexus/pathology , Human Embryonic Stem Cells/cytology , Neuroglia/virology , Organoids/innervation , Organoids/pathology , Cells, Cultured , Choroid Plexus/cytology , Choroid Plexus/virology , Humans , Neuroglia/pathology , Neurons/virology , Organoids/cytology , SARS-CoV-2/pathogenicity
4.
Protein Cell ; 12(9): 717-733, 2021 09.
Article in English | MEDLINE | ID: covidwho-973695

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.


Subject(s)
Alveolar Epithelial Cells/metabolism , COVID-19/pathology , Lung/metabolism , SARS-CoV-2/physiology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/virology , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/therapeutic use , COVID-19/drug therapy , COVID-19/immunology , COVID-19/virology , Down-Regulation , Drug Discovery , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Immunity , Lipid Metabolism , Lung/cytology , Lung/virology , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Virus Replication/drug effects
7.
Curr Protoc Stem Cell Biol ; 54(1): e118, 2020 09.
Article in English | MEDLINE | ID: covidwho-635380

ABSTRACT

The normal development of the pulmonary system is critical to transitioning from placental-dependent fetal life to alveolar-dependent newborn life. Human lung development and disease have been difficult to study due to the lack of an in vitro model system containing cells from the large airways and distal alveolus. This article describes a system that allows human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to differentiate and form three-dimensional (3D) structures that emulate the development, cytoarchitecture, and function of the lung ("organoids"), containing epithelial and mesenchymal cell populations, and including the production of surfactant and presence of ciliated cells. The organoids can also be invested with mesoderm derivatives, differentiated from the same human pluripotent stem cells, such as alveolar macrophages and vasculature. Such lung organoids may be used to study the impact of environmental modifiers and perturbagens (toxins, microbial or viral pathogens, alterations in microbiome) or the efficacy and safety of drugs, biologics, and gene transfer. © 2020 Wiley Periodicals LLC. Basic Protocol: hESC/hiPSC dissection, definitive endoderm formation, and lung progenitor cell induction.


Subject(s)
Coronavirus Infections/pathology , Lung/cytology , Organoids/cytology , Pneumonia, Viral/pathology , Respiratory Tract Infections/pathology , Betacoronavirus , COVID-19 , Cell Culture Techniques , Cell Differentiation , Coronavirus Infections/therapy , Endoderm/cytology , Human Embryonic Stem Cells/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Lung/growth & development , Lung/physiology , Models, Biological , Pandemics , Patient-Specific Modeling , Pneumonia, Viral/therapy , Respiratory Tract Infections/therapy , SARS-CoV-2 , Time-Lapse Imaging
8.
Cell Res ; 30(9): 794-809, 2020 09.
Article in English | MEDLINE | ID: covidwho-601806

ABSTRACT

Lung injury and fibrosis represent the most significant outcomes of severe and acute lung disorders, including COVID-19. However, there are still no effective drugs to treat lung injury and fibrosis. In this study, we report the generation of clinical-grade human embryonic stem cells (hESCs)-derived immunity- and matrix-regulatory cells (IMRCs) produced under good manufacturing practice requirements, that can treat lung injury and fibrosis in vivo. We generate IMRCs by sequentially differentiating hESCs with serum-free reagents. IMRCs possess a unique gene expression profile distinct from that of umbilical cord mesenchymal stem cells (UCMSCs), such as higher expression levels of proliferative, immunomodulatory and anti-fibrotic genes. Moreover, intravenous delivery of IMRCs inhibits both pulmonary inflammation and fibrosis in mouse models of lung injury, and significantly improves the survival rate of the recipient mice in a dose-dependent manner, likely through paracrine regulatory mechanisms. IMRCs are superior to both primary UCMSCs and the FDA-approved drug pirfenidone, with an excellent efficacy and safety profile in mice and monkeys. In light of public health crises involving pneumonia, acute lung injury and acute respiratory distress syndrome, our findings suggest that IMRCs are ready for clinical trials on lung disorders.


Subject(s)
Human Embryonic Stem Cells/immunology , Lung Injury/therapy , Lung/pathology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/immunology , Animals , Cells, Cultured , Female , Fibrosis , Haplorhini , Human Embryonic Stem Cells/cytology , Humans , Immunity , Immunomodulation , Lung/immunology , Lung Injury/immunology , Lung Injury/pathology , Male , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred C57BL
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