Coronavirus in an organoid
Scientists have created a biological lung model to study coronavirus infection
Yakov Kard, N+1
Scientists have grown model alveoli from human lung stem cells. They are suitable for studying coronavirus infection: the authors investigated the endogenous immune response in the early stages of the disease and showed that only one viral particle can be enough to infect a cell. The article has been accepted for publication in Cell Stem Cell (Youk et al., Three-dimensional human alveolar stem cell culture models reveal infection response to SARS-CoV-2).
SARS-CoV-2 is the virus responsible for the pandemic, from which more than a million people have already died worldwide (including more than twenty-five thousand in Russia). Studying the course of COVID-19 disease at the cellular level is extremely important for understanding the mechanisms of infection, searching for and modifying therapeutic approaches.
The infection is most acute in the lungs, where the coronavirus infects alveolar cells of the second type. These cells are difficult to cultivate in the laboratory, and therefore the existing models of the disease, with the exception of one, were created on the basis of other human and animal cells.
The recently described model, which uses alveolar cells of the second type, also has significant limitations. Due to the fact that pluripotent stem cells (and not donor lung cells) were used to create it, it is impossible to study the influence of individual factors, such as the patient's age or health status, on it.
A group of scientists from thirteen institutes in South Korea and the UK managed to create a three-dimensional long-lived cell culture of human alveolar cells of the second type and study its reaction to coronavirus infection. The main contribution to the work was made by Jeonghwan Youk, Taewoo Kim, Kelly Evans, Yong-i Jeong (Young-Il Jeong) and Yongsuk Hur.
The culture was grown from cells of a living donor, which were placed in a matrigel – a gelatinous environment similar to the extracellular environment of cells in the body (matrix). Growth factors involved in lung development were also added to the matrigel.
Under these conditions, alveolar cells formed various three–dimensional structures, including cysts similar to alveoli, in which surfactants were found - substances that prevent the adhesion of alveoli in real lungs. The authors report that the culture remains stable even after six months, which allows it to be used in long-term studies.
Types of colonies of alveolar cells formed in the matrix. On the left – hematoxylin and eosin staining, on the right – immunofluorescence. Drawings from the article by Youk et. al.
To study the mechanism of development of coronavirus infection, the researchers infected cultured cells with a virus isolated from a patient from South Korea. Human bronchial cells were used for control, as well as Vero cells obtained from African green macaques.
Before infection, the researchers depolymerized the matrix and destroyed the three-dimensional structures formed by the cells to give the virus access to their apical surface. The results were analyzed on the first, second and third days after infection. After just one day, a significant titer of the virus was observed in the pseudoalveoli, which indicates exponential replication even on the first day.
Aggregates of viral particles and components were observed in the cells themselves. Massive vacuoles similar to those occurring in epithelial cells infected with the Zika virus were also found.
A group of alveolar cells two days after infection. The red asterisks show the space of pseudo-alveoli. White arrows indicate aggregates of viral particles, white dotted lines indicate cell membranes. Vc – pathological vacuoles, Nu– nuclei.
When analyzing the transcriptome (a set of rna that allows us to judge the level of gene expression), scientists drew attention to a significant increase in transcription of genes associated with immunity. These were interferon genes, as well as genes whose expression interferon stimulates. These genes are involved in the antiviral response: they prevent the entry and exit of viruses from cells, inhibit their replication and the formation of proteins.
The authors draw attention to the fact that the source of the immune response here were the alveolar cells themselves – after all, immune cells were absent in the model. In their opinion, this brings the model infection closer to the earliest stage of human disease: there are practically no immune cells in the lungs before the infection develops.
As part of the study, scientists also tried to find out how many viral particles, as a rule, infect a cell. Scientists write that there are two possible scenarios: in the first, one cell is infected by one viral particle, in the second, infection occurs by several particles at once.
To identify the type of virus that infected an individual cell, a "silent" (that is, not affecting the virality of the pathogen) mutation contained in a small part (about four percent) of the viruses used in the experiment was used. After infection, the number of cells containing only mutant RNA (which means infected only with a mutant virus) turned out to be twice as large as the number of those in which both wild and mutant type RNA was found (n=8 vs. 4).
Statistical analysis also showed that the probability of infecting a cell with an individual viral particle is more than twice as high. According to the authors, this may indicate the possibility of viral interference in the development of pulmonary coronavirus infection.
The researchers believe that the created model will be extremely useful for understanding the mechanisms of disease development. To improve it, they propose to introduce immune cells into the culture, as well as create variations of the model using lung cells taken from elderly and deceased patients from this disease.
According to the authors, the combination of the approach described in the article with other techniques, such as joint cultivation with immune cells and in vitro testing of antiviral drugs, will be promising.
Well, without any connection with coronavirus infection, the model can be used to study the biology of alveolar cells of the second type and lung diseases affecting the alveoli.
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