Tuesday , January 26 2021

Synthetic lung organoids provide a high-performance platform for identifying new SARS-CoV-2 cell targets

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that caused the 2019 coronavirus disease pandemic (COVID-19), is still spreading and mutating. Scientists are working with therapists to control the virus. An interesting new study published on bioRxiv * the preprint server describes a new platform that allows a high-performance workflow to create matching human lung buds in the tens of thousands. This allows researchers to study SARS-CoV-2 infection and therapies in lung tissue that reflect key aspects of human lung development.

The need for lung models

SARS-CoV-2 infection can occur through the nose or the airways of the lungs and alveoli. The virus appears to target the lungs, particularly with acute lung injury, and pneumonia-related complications are the most common complications of severe COVID-19. The virus attacks the eyelashes and pneumonia type II lungs through the angiotensin-converting enzyme 2 (ACE2) receptor and other enzymes that act as cofactors upon entry of the virus.

However, the way the virus damages lung tissue is still unclear. Currently, most research on SARS-CoV-2 is conducted in human primary lung tissue. However, these in vitro systems are unpredictable and cells vary widely depending on the genetic and phenotypic profile of the tissue donor. These differences are important in their impact on virus replication and clinical impact.

An alternative to this is the use of differentiated human pluripotent stem cells (hPSCs) in lung respiratory and alveolar cells, while other researchers have developed hPSC lung cells from organoids to identify and test small molecules that inhibit SARS-CoV-2. However, these models do not show tissue organization in developing and adult lung tissue resulting from the embryonic signaling pathways that guide lung development. In addition, lung cells need 1-3 months to differentiate from hPSC.

Synthetic human lung buds

The aim of this study was to create a platform for the creation of synthetic embryonic lung tissues. These are formed from hPSCs and use their ability to self-organize when grown in a specific geometry on micro chip samples. With the help of special activators, thousands of lung stem cells arranged in colonies were created on a single microchip.

Ustvarjanje samoorganiziranih epitelijskih popkov na omejenih geometrijah.  A) Protokol za ustvarjanje sintetičnih pljučnih brstov na omejenih geometrijah v mikro vzorcih.  BD) Generacija SOX17 + endodermnih celic (B), FOXA2 + sprednjih endodermnih celic (C) in NKX2.1 + multipotentnih pljučnih prednikov (D) na koncu dokončne faze indukcije endoderma (DE), črevesja in pljučnega progenitorja.  E) Delež NKX2.1 + pljučnih prednikov pri modulaciji z BMP4, KGF ali BMP4 / KGF 7 dni.  FG) Pogled od zgoraj in s strani 3D epitelijskih brstov, ki vsebujejo NKX2.1 + multipotentne pljučne prednike v kolonijah 225 in 500 μm.  I) Kolonije mikropattern, ki vsebujejo NKX2.1 + epitelijski brsti pri naraščajočih odmerkih KGF.  J) Delež NKX2.1 + progenitornih celic pri naraščajočih odmerkih KGF.  K) Kvantifikacija območja epitelijskega pljučnega popka v kolonijah mikro vzorcev pri različnih odmerkih KGF.  (** p <0,01, *** p <.001, **** p <0,0001, Dunnettov večkratni primerjalni test; merilna lestvica, 50 μm)

Creating self-organized epithelial buds on bounded geometries. A) Protocol for the creation of synthetic lung buds on limited geometries in micro samples. BD) Generation of SOX17 + endoderm cells (B), FOXA2 + anterior endoderm cells (C) and NKX2.1 + multipotent lung ancestors (D) at the end of the final phase of endoderm (DE) induction, intestinal and pulmonary progenitor. E) Proportion of NKX2.1 + pulmonary ancestors in modulation with BMP4, KGF or BMP4 / KGF 7 days. FG) Top and side view of 3D epithelial buds containing NKX2.1 + multipotent lung ancestors in colonies 225 and 500 μm. I) Colonies of micropatterns containing NKX2.1 + epithelial buds at increasing doses of KGF. J) Proportion of NKX2.1 + progenitor cells at increasing doses of KGF. K) Quantification of the epithelial lung umbilical region in colonies of micro samples at different doses of KGF. (** p <0.01, *** p <.001, **** p <0.0001, Dunnett multiple comparison test; scale, 50 μm)

They found that these synthetic human lung buds not only show early parameters of differentiation, but were organized into human lung buds. They were also able to discover some key steps in early differentiation and discover new relationships between alveolar cells.

The virus was found to preferentially infect these lung buds, both eyelashes and type II alveolar cells. The latter are more susceptible because they have higher levels of ACE2 and TMPRSS2, as well as furin expression, all of which are essential for SARS-CoV-2 infection. They also found that alveolar stem cell divisions are also more vulnerable to the virus.

The researchers measured the infection within 48 and 96 hours of onset and found that the infected cells eventually increased in all cell types, suggesting that viral spread occurs within synthetic human lung buds. Apoptosis has also been found to occur in alveolar and airway cells. Thus, hundreds of synthetic human lung buds could be grown with a single microchip to monitor susceptibility to infections, identify susceptible cell types, assess virus transmission between cells, visualize cytopathic effects on hundreds of synthetic lung buds, and thus follow the entire life cycle of the virus. .

They then performed a review by therapists who demonstrated in vitro efficacy in binding assays and in cell lines. Neutralizing antibodies in convalescent human plasma were tested to prevent infection at various dilutions. They found that the results were comparable to those obtained with cell lines. Again, they found that neutralizing antibodies can also inhibit the spread of the virus through the organoid even after infection. This supports the potential of these drugs as therapeutics that act as biological inhibitors of SARS-CoV-2 infection and intercellular transmission.

What are the consequences?

Scientists have not only shown that lung stem cells can self-organize if they are grown in limited geometries, but that these organoids repeat the same path that fetal lung ancestors follow during lung development. This could be very useful in lung disease and regenerative medicine research involving lung tissue models by providing rapid and unrestricted access to synthetic genetically matched human organoid lung tissue.

With this platform, we can also quantitatively examine differences in disease susceptibility between different cells and differences in viral impact on different cell types. In fact, this model allowed scientists to identify some important cell types that are otherwise difficult to study. It may also lead to a better understanding of why SARS-CoV-2 is more pathogenic than other coronaviruses.

In addition, these findings suggest new cell types and pathways of cell development in fetal life, which could help to understand how human lungs differ. This could also shed light on the origins of lung cancer and other diseases.

We expect our synthetic human lung model to be transformative to elucidate the molecular basis of respiratory infections and lung diseases for which there is currently no cure.. This study also highlights the potential of using synthetic lungs to identify new drugs that block infection with SARS-CoV-2, endemic coronaviruses, and other respiratory viruses.. “

* Important notice

bioRxiv publishes prior scientific reports that have not been peer reviewed and should not be considered definitive, guide clinical practice / health-related behavior, or treat it as established information.

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