Human Embryonic Stem Cell-derived Lung Organoids: a Model for SARS-CoV-2 Infection and Drug Test

AbstractThe 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 demonstrated that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids. Ciliated cells, alveolar type 2 (AT2) cells and rare club cells were virus target cells. Electron microscopy captured typical replication, assembly and release ultrastructures and revealed the presence of viruses within lamellar bodies in AT2 cells. Virus infection induced more severe cell death in alveolar organoids than in airway organoids. Additionally, RNA-seq revealed early cell response to SARS-CoV-2 infection and an unexpected downregulation of ACE2 mRNA. Further, compared to the transmembrane protease, serine 2 (TMPRSS2) inhibitor camostat, the nucleotide analog prodrug Remdesivir potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model for SARS-CoV-2 infection and drug discovery.

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Host metabolism dysregulation and cell tropism identification in human airway and alveolar organoids upon SARS-CoV-2 infection

Protein & Cell ◽

10.1007/s13238-020-00811-w ◽

2020 ◽

Cited By ~ 1

Author(s):

Rongjuan Pei ◽

Jianqi Feng ◽

Yecheng Zhang ◽

Hao Sun ◽

Lian Li ◽

...

Keyword(s):

Cell Lines ◽

Three Dimensional ◽

Embryonic Stem ◽

Neutralizing Antibody ◽

Underlying Mechanism ◽

Alveolar Type ◽

Physiological Conditions ◽

Therapeutic Drugs ◽

Distal Airway

AbstractThe 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.

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Derivation of goat embryonic stem cell-like cell lines from in vitro produced parthenogenetic blastocysts

Small Ruminant Research ◽

10.1016/j.smallrumres.2013.01.018 ◽

2013 ◽

Vol 113 (1) ◽

pp. 145-153 ◽

Cited By ~ 1

Author(s):

Arun Kumar De ◽

Shweta Garg ◽

Dinesh Kumar Singhal ◽

Hrudananda Malik ◽

Ayan Mukherjee ◽

...

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Embryonic Stem

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The embryonic stem cell test (EST), an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells

Ersatz- und Ergänzungsmethoden zu Tierversuchen - Forschung ohne Tierversuche 1997 ◽

10.1007/978-3-7091-7500-2_69 ◽

1998 ◽

pp. 289-290 ◽

Cited By ~ 4

Author(s):

H. Spielmann ◽

I. Pohl ◽

B. Döring ◽

M. Liebsch ◽

F. Moldenhauer

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Embryonic Stem ◽

Mouse Cell ◽

3T3 Fibroblasts ◽

Cell Test

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296 PRODUCTION OF HEMIZYGOUS AND HOMOZYGOUS EMBRYONIC STEM CELL-DERIVED NEURAL PROGENITOR CELLS FROM THE TRANSGENIC ALZHEIMER GÖTTINGEN MINIPIG

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab296 ◽

2011 ◽

Vol 23 (1) ◽

pp. 245

Author(s):

V. J. Hall ◽

J. Jakobsen ◽

A. Gunnarsson ◽

M. Schmidt ◽

A. Lund Jørgensen ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Real Time Pcr ◽

Embryonic Stem ◽

Neural Progenitor ◽

Novel Drugs

Alzheimer’s disease is the most prevalent cause of dementia and afflicts ∼26 million people worldwide. There are currently no cures for this disease. Production of in vitro models of the disease would be extremely useful for studying disease mechanisms and for potential screening of novel drugs. In this study we produced 2 hemizygote and 2 homozygote embryonic stem cell-derived neural progenitor cell lines from Day 8 transgenic blastocysts carrying a human gene linked to early-onset Alzheimer’s disease [Swedish mutation of the amyloid precursor protein (hAPPsw)]. Following onset of spontaneous oestrus, a mating of hAPPsw± × hAPPsw± Göttingen transgenic progeny was performed. Eight days after the first of 2 matings, embryos were flushed from the tip of both cornuas of the gilt under surgical anaesthesia. A total of 6 blastocysts were obtained and 7 corpora lutei recorded. Blastocysts were transported for 4 h in porcine zygote medium 3 (PZM-3) in hypoxic, humidified conditions at 39°C to the cell laboratory. Compact epiblasts were mechanically isolated from the embryo using insulin needles and cultured on inactivated mouse embryonic fibroblasts in embryonic stem cell medium, supplemented with 20 ng mL–1 human recombinant basic fibroblast growth factor (Prospec) and 20 ng mL–1 human recombinant Activin A (Prospec), for a period of 5 days in hypoxic conditions at 39°C. Five of the 6 epiblasts expanded to form embryonic stem-cell-like outgrowth colonies. These were cut into small colonies and plated on MS5 murine stromal cells to induce spontaneous neural differentiation in DMEM medium containing 15% knockout serum replacement. Neuronal rosette-like structures were identified from Day 10 of differentiation onward. Six rosette structures were mechanically isolated from 4 outgrowths and plated in serum-free conditions on Matrigel-coated dishes. Two of the 6 lines failed to proliferate beyond passage 2. The 4 remaining cell lines have currently been cultured to passage 7. These lines were analysed at passage 5 by comparative real-time PCR and found to be positive for the neural progenitor markers VIMENTIN, SOX2, NESTIN PAX6, MUSASHI; other neural markers BETAIIITUBULIN and NCAM; and the astrocyte marker, GFAP. These lines were also subjected to analysis by immunocytochemistry and found to express SOX2, VIMENTIN, and NESTIN. Further genotyping by comparative real-time PCR using primers designed to target the hAPPsw gene revealed that 2 lines carried a single copy of hAPPsw and 2 lines carried 2 copies of hAPPsw. The expression levels of the hAPPsw transgene in these cell lines were determined using quantitative PCR. These cell lines are currently being investigated for their ability to differentiate into cholinergic neurons and for their expression of hyperphosphorylated TAU and β-Amyloid secretion. These cell lines will be potentially relevant for the in vitro study of amyloid precursor protein accumulation in neural cells and its role in cell death, as well as for potential screening of novel drugs for Alzheimer’s disease.

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In Vitro Generation of Three‐Dimensional Substrate‐Adherent Embryonic Stem Cell–Derived Neural Aggregates for Application in Animal Models of Neurological Disorders

Current Protocols in Stem Cell Biology ◽

10.1002/9780470151808.sc02d11s21 ◽

2012 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Gunnar Hargus ◽

Yi‐Fang Cui ◽

Marcel Dihné ◽

Christian Bernreuther ◽

Melitta Schachner

Keyword(s):

Stem Cell ◽

Animal Models ◽

Embryonic Stem Cell ◽

Neurological Disorders ◽

Three Dimensional ◽

Embryonic Stem

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Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin

Human Molecular Genetics ◽

10.1093/hmg/ddr506 ◽

2011 ◽

Vol 21 (4) ◽

pp. 751-764 ◽

Cited By ~ 29

Author(s):

Silvia V. Diaz Perez ◽

Rachel Kim ◽

Ziwei Li ◽

Victor E. Marquez ◽

Sanjeet Patel ◽

...

Keyword(s):

Stem Cell ◽

Chemical Modification ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Human Embryonic Stem Cell ◽

Embryonic Stem ◽

Stem Cell Lines

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SOX17 Is Not Required for the Derivation and Maintenance of Mouse Extraembryonic Endoderm Stem Cell Lines

10.1101/2022.01.09.475525 ◽

2022 ◽

Author(s):

Xudong Dong ◽

Ailing Ding ◽

Jiangwei Lin

Keyword(s):

Stem Cell ◽

Cell Lines ◽

Embryonic Stem ◽

Cell Lineage ◽

Preimplantation Embryos ◽

Primitive Endoderm ◽

Extraembryonic Endoderm ◽

Gene Expression Analyses ◽

Stem Cell Lines

Extraembryonic endoderm stem (XEN) cell lines can be derived and maintained in vitro and reflect the primitive endoderm cell lineage. SOX17 is thought to be required for the derivation and maintenance of mouse XEN cell lines. Here we have re-evaluated this requirement for SOX17. We derived multiple SOX17-deficient XEN cell lines from preimplantation embryos of a SOX17-Cre knockout strain and chemically converted multiple SOX17-deficient embryonic stem cell lines into XEN cell lines by transient culturing with retinoic acid and Activin A. We confirmed the XEN profile of SOX17-deficient cell lines by immunofluorescence with various markers, by NanoString gene expression analyses, and by their contribution to the extraembryonic endoderm of chimeric embryos produced by injecting these cells into blastocysts. Thus, SOX17 is not required for the derivation and maintenance of XEN cell lines.

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182 Establishment of expanded potential embryonic stem cell lines from porcine embryos

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab182 ◽

2019 ◽

Vol 31 (1) ◽

pp. 215

Author(s):

M. Nowak-Imialek ◽

X. Gao ◽

P. Liu ◽

H. Niemann

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Differentiation Potential ◽

Germ Layers ◽

Embryonic Tissues

The domestic pig is an excellent large animal in biomedical medicine and holds great potential for testing the clinical safety and efficacy of stem cell therapies. Previously, numerous studies reported the derivation of porcine embryonic stem cell (ESC)-like lines, but none of these lines fulfilled the stringent criteria for true pluripotent germline competent ESC. Here, we report the first establishment of porcine expanded potential stem cells (pEPSC) from parthenogenetic and in vivo-derived blastocysts. A total of 12 cell lines from parthenogenetic blastocysts from Day 7 (12/24) and 26 cell lines from in vivo-derived blastocysts from Day 5 (26/27) were established using defined stem cell culture conditions. These cells closely resembled mouse ESC with regard to morphology, formed compact colonies with high nuclear/cytoplasmic ratios, and could be maintained in vitro for more than 40 passages with a normal karyotype. The pEPSC expressed key pluripotency genes, including OCT4, NANOG, SOX2, and SALL4 at similar levels as porcine blastocysts. Immunostaining analysis confirmed expression of critical cell surface markers SSEA-1 and SSEA-4 in pEPSC. The EPSC differentiated in vitro into tissues expressing markers of the 3 germ layers: SOX7, AFP, T, DES, CRABP2, α-SMA, β-tubulin, PAX6, and, notably, the trophoblast markers HAND1, GATA3, PGF, and KRT7. After injection into immunocompromised mice, the pEPSC formed teratomas with derivatives of the 3 germ layers and placental lactogen-1 (PL-1)-positive trophoblast-like cells. Additionally, pEPSC cultured in vitro under conditions specific for germ cells formed embryoid bodies, which contained ~9% primordial germ cell (PGC)-like cells (PGCLC) that expressed PGC-specific genes, including NANOS3, BLIMP1, TFAP2C, CD38, DND1, KIT, and OCT4 as detected by quantitative RT-PCR and immunostaining. Next, we examined the in vivo differentiation potential of pEPSC and injected pEPSC stably expressing the CAG-H2B-mCherry transgene reporter into porcine embryos. The donor cells proliferated and were localised in both the trophectoderm and inner cell mass of the blastocysts cultured in vitro. After transfer to 3 recipient sows, chimeric embryos implanted and a total of 45 fetuses were recovered on Days 26 to 28. Flow cytometry of single cells collected from embryonic and extraembryonic tissues of the fetuses revealed mCherry+ cells in 7 conceptuses, in both the placenta and embryonic tissues; in 3 chimeric conceptuses, mCherry+ cells were exclusively found in embryonic tissues; and in 2 conceptuses, mCherry+ cells were exclusively localised in the placenta. The contribution of the mCherry+ cells was low (0.4-1.7%), but they were found and co-detected in multiple porcine embryonic tissues using tissue lineage-specific markers, including SOX2, TUJ1, GATA4, SOX17, AFP, α-SMA, and trophoblast markers PL-1 and KRT7 in the placental cells. The successful establishment of pEPSC represents a major step forward in stem cell research and provides cell lines with the unique state of cellular potency useful for genetic engineering and unravelling pluripotency regulation in pigs.

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