A new system to induce alveolar and airway epithelial cells

Professor Shimpei Gotoh and Junior Affiliate Professor Kazuo Takayama teamed up in examine to assemble a novel in vitro tradition system for alveolar and airway epithelial cells, using a biomaterials engineering technique referred to as micro-patterning method, and utilizing the newly devised system to simulate viral infections by SARS-CoV-2 variants. The examine is published within the journal Stem Cell Reviews.

The respiratory epithelium lining the airway and alveoli, the place oxygen and carbon dioxide trade happens, consists primarily of two cell sorts, kind 1 and a couple of alveolar epithelial cells, known as AT1 and AT2, respectively. Whereas AT1 cells are skinny and flat, optimum for mediating gasoline trade, cuboidal AT2 cells secrete surfactant proteins and act as tissue stem cells.

With their talents of self-renewal and AT1 differentiation, AT2 cells are of explicit curiosity in regenerative medication to facilitate the restore of injured or diseased lungs. The Gotoh Laboratory has beforehand devised a protocol to make use of iPS cells as a priceless beginning materials to generate AT2 cells.

Nevertheless, the requirement for Matrigel-based 3D tradition limits tradition dimension and direct image-based evaluation. Moreover, cells in such programs sometimes orient themselves with their apical floor dealing with inward, thus unable to mannequin precisely in vivo tissue construction for mechanistic research.

In an try to create a greater means to distinguish and develop iPS cell-derived AT2 cells, non-adhesive 2.5-dimensional (D) cell tradition plates have been micro-patterned with tiny circles of cell-adhesive coating, 100 or 200 μm in diameter (for comparability, the diameter of human hair ranges from 17-181 μm).

First, to make sure that such micro-patterned plates are suitable with AT2 cell induction, iPS cell-derived lung progenitor cells that specific a green fluorescent protein upon differentiation into AT2 cells have been seeded and handled with a tradition medium particularly for alveolar epithelial cell induction.

In comparison with typical 2D monolayer cultures, the relative proportion of fluorescent cells and the expression of SFTPC, a gene encoding one among a number of surfactant proteins, have been considerably increased within the micro-patterned system than in typical cultures. Notably, NaPi2b protein was noticed on the sting of colonies fashioned on the micro-patterned cell-adhesive coating, indicative of an “apical-out” orientation for these AT2 cells.

Throughout this examine, the researchers noticed {that a} DNA-binding fluorescent dye (Hoechst-33342) stained peripheral dwell cells rather more quickly than these within the middle. By separating cells primarily based on their fluorescence depth, they discovered extra cells with inexperienced fluorescence and AT2 marker genes to be enriched within the edge (Hoechst-33342high) subpopulation in comparison with the middle (Hoechst-33342low) subpopulation, additional indicating AT2 cells to be on the periphery.

The analysis crew subsequent separated edge and middle cells and analyzed their gene expression profiles by bulk RNA sequencing (RNA-seq). Amongst practically 2,000 differentially expressed genes, a number of AT2 marker genes and endogenous WNT suppressor genes have been considerably upregulated within the peripheral subpopulation in comparison with undifferentiated lung progenitor cells, indicative of suppressed WNT signaling in AT2 cells on the periphery. In distinction, the gene expression profile prompt Notch signaling activation within the core subpopulation.

Subsequent, to show that this micro-patterned system can generate multiciliated airway epithelial cells, lung progenitor cells have been handled with a medium to advertise differentiation in the direction of mature airway epithelium. Consequently, gene expression, immunofluorescence, and electron microscopic analyses detected the upregulation of multiciliated cell markers genes (FOXJ1 and SNTN) and proteins (FOXJ1 and acetylated tubulin), in addition to transferring cilia in multiciliated cells.

Furthermore, the micro-patterned system was extra environment friendly at producing FOXJ1+ cells than typical 2D cultures. By bulk RNA-seq, the researchers discerned that genes associated to goblet and mucous cell lineages have been equally upregulated in each the sting and middle subpopulations in comparison with lung progenitor cells, with the middle subpopulation additionally displaying upregulation of a number of marker genes related to multiciliated cells, along with an enrichment of a number of gene ontology (GO) phrases with its differentially upregulated genes.

The researchers additionally analyzed the assorted cell sorts within the micro-patterned colonies by single-cell (sc)RNA-seq induced into each epithelial cell sorts. Alveolar epithelial cells have been primarily segregated into three clusters: AT2 cells characterised by excessive expression of AT2 marker genes, proliferating AT2 cells with low marker expression, and respiratory bronchiole-like cells.

Conversely, a multiciliated cluster expressing SNTN and FOXJ1 and a number of clusters representing comparatively immature ciliated epithelial cells (FOXJ1, DRC1, and CCNO) or immature secretory cells (SERPINA1 and MUC5B) have been recognized within the airway epithelial cells. Curiously, an impartial cluster fashioned by cells within the periphery displayed transcriptomic adjustments resembling cells beforehand described as intermediate cells on the differentiation trajectory from AT2 to AT1 cells.

The analysis crew subsequent contaminated the micro-patterned alveolar or airway epithelial cultures with 5 SARS-CoV-2 variants, B.1.1.214, B.1.617.2 (delta), and omicron variants BA.1, BA.2, and BA.5, to look at the feasibility of utilizing this new system to check infections by respiratory viruses.

Notably, whereas alveolar epithelial cells contaminated with the B.1.1.214 and B.1.617.2 variants readily launched viruses, infections by the omicron variants led to a lot decrease virus launch. B.1.1.214-, B.1.617.2-, and BA.5-infected airway epithelial cells adopted an identical pattern, equally releasing excessive quantities of virus three days post-infection (dpi), however considerably declined on 4 dpi after an infection by B.1.617.2 or BA.5. In distinction, airway epithelial cells contaminated by BA.1 and BA.2 launched negligible ranges of virus.

Equally, when viral gene expression was examined, B.1.617.2 displayed excessive viral tropism, as each epithelial cell sorts confirmed dramatic will increase in viral gene expression and protein ranges. Notably, B.1.1.214 and BA.1 confirmed weak and robust specificity for alveolar and airway epithelial cells, respectively, as viral gene expression and protein ranges within the specified epithelial cells have been increased than the opposite.

Lastly, the analysis crew examined gene expression profiles of alveolar and airway epithelial cells because of an infection by B.1.617.2 and BA.1. Whereas B.1.617.2-infected alveolar and airway epithelial cells and BA.1-infected airway epithelial cells all confirmed an interferon response, airway epithelial cells contaminated by B.1.617.2 underwent gene expression adjustments indicative of apoptosis (programmed cell dying), which was confirmed by impartial assays.

Utilizing this micro-patterned tradition system, the analysis crew efficiently created a way to tradition AT2 cells within the right orientation (with their apical floor dealing with the medium) that’s amendable to imaging.

The researchers additionally demonstrated the applicability of alveolar and airway epithelial cells induced within the micro-patterned system to disclose variant-specific viral options like viral tropism, a property that makes the system adaptable for illness modeling and drug discovery.