Single-cell RNA sequencing reveals novel cell differentiation dynamics during human airway epithelium regeneration
Preprint posted on October 24, 2018 https://www.biorxiv.org/content/early/2018/10/24/451807?%3Fcollection
By single cell RNA sequencing airway epithelial cells during their differentiation from basal progenitor cells to mature epithelial cell types, the authors of this preprint throw light on the step-wise differentiation of the respiratory epithelium.Rob Hynds
The airways conduct air to the alveoli in order for gas exchange to occur. The epithelium that lines the airways is an important barrier to the outside world: it is challenged by inhaled particulates and pathogens and mediates mucociliary clearance by producing mucous and actively beating trapped matter out of the lungs to be swallowed. Recently, a number of single cell RNA sequencing (scRNAseq) studies have begun to reveal new insights into the composition of this epithelium in humans. Most notably, two papers published in Nature by the Regev and Rajogopal laboratories and the Klein and Jaffe laboratories revealed the presence of cystic fibrosis transmembrane conductance regulator (CFTR)-rich population of cells that were dubbed ‘ionocytes’ because of their similarity to ion transporter-rich ionocyte cells found in Xenopus larval skin and populations of mammalian kidney and inner ear cells. A novel epithelial population, which expresses transcripts associated with both airway and alveolar lineages, has also been discovered in idiopathic pulmonary fibrosis.
In their pre-print, Sandra Ruiz Garcia, Marie Deprez and colleagues in Pascal Barbry’s team at Université Côte d’Azur in France study the regeneration of the airway epithelium from cultured basal cells in the widely used air-liquid interface cell culture model which allows differentiation of basal cells to mature airway cell types.
In their first analysis, the authors find a picture which fits neatly with previous studies; two populations of basal cells [cycling (Ki67+) and non-cycling (Ki67-)], a suprabasal population, which represent an intermediate ‘mid-differentiation’ cell type, secretory cells (Scgb1A1+), goblet cells (MUC5AC+) and ciliated cells (FOXJ1+). Using sub-clustering, in which less discrimination between clusters is accepted, the authors are able to distinguish important intermediate states within each of these baseline populations. Non-cycling basal cells can be divided into two subsets, one of which expresses genes associated with cell motility. Pseudotime analysis suggests that these cells are the most differentiated basal cells, or pre-suprabasal cells. Our knowledge around basal cell motility in humans is limited but very motile cells can be selected in airway cell cultures and clonal relationships between spatially separated pre-malignant airway lesions make this an interesting area for future study. Three populations of suprabasal cells and three populations of secretory cell can also be distinguished from one another. Intriguingly, one of the secretory populations differentially expresses transcripts which make it a candidate immune regulatory population, including TNF, IFNG, IL1 and IL6. As the authors acknowledge, future work might investigate the spatial distribution of these cells in human airways and also establish how dynamic the secretory subpopulation lineage hierarchy is. Finally, the authors are able to distinguish mature multiciliated cells from those that are actively undergoing multiciliogenesis (labelled ‘deuterosomal cells’). These cells selectively express genes such as DEUP1, which is involved in the massive centriole amplification necessary for multiciliation, and reactivate some cell cycle associated genes.
Figure 5E from the pre-print summarises the lineage hierarchy of the airway epithelium.
In further analyses, the authors investigate the dynamic changes in keratin gene expression during airway epithelial differentiation and investigate the expression of key airway cell signalling pathway components in a cell type specific manner. Notch signalling has been widely described as a key mediator of airway epithelial cell fate choice with no or little Notch activation in basal cells, Notch activation during secretory differentiation and then repression of Notch to generate ciliated cells. The new analysis suggests several potential Notch repressors that become expressed at the deuterosomal stage and finds, consistent with a previous report, that suprabasal cells are the first to activate Notch signalling by NOTCH3 expression. Interestingly, the putative immune-regulatory secretory population selectively express Wnt5a and Wnt4 and basal cells express both Wnt10a and Wnt target genes, suggesting their behaviour might be regulated in an autocrine fashion. Future experimental work is required to confirm these effects and to investigate how the epithelial gene expression landscape is altered by non-epithelial cell types in vivo.
- Analysis of airway epithelial regeneration from basal progenitor cells in vitro adds to our understanding of differentiation intermediates.
- Airway basal cells differentiate via suprabasal cells to secretory lineages which can further differentiate towards multiciliated cells given the correct cues.
- A subpopulation of relatively differentiated basal cells is enriched in migration-associated transcripts.
- A subpopulation of secretory cells is enriched in immune-modulatory genes and might orchestrate mucosal immunity.
Travaglini, K.J. and Krasnow, M.A.(2018). Profile of an unknown airway cell. Nature, 560, 313-314.
Questions for the authors
Q1. How much difference is there in practice between a secretory cell and a goblet cell? Is it an oversimplification to describe them by Scgb1a1 and MUC5AC in human proximal airway samples where club cells are rarely seen? Put another way, is there a meaningful difference between a BEGM ALI culture (which has no Scgb1a1+ or MUC5AC+ cells but ‘secretory-like’ cells that don’t express these specific markers) and a Pneumacult ALI culture (which has both populations)?
Q2. In the pre-print, cultured cells are airway epithelial cells but your fresh samples are nasal. Do you think that scRNAseq studies of different airway locations will reveal meaningful differences in different parts of the respiratory tree as suggested by cell culture studies?
Q3. The deuterosomal population is found consistently across samples – do the authors know how transient a state it is? (i.e. how long does multiciliogenesis take in human airways?)
Q4. The authors see significant differences between late-stage ALI cultures and fresh biopsy tissue. Since ALI cultures are imperfectly differentiated with incomplete ciliation and some squamous differentiation, do the authors think that the intermediate cells found in ALI cultures are physiologically relevant differentiation intermediates?
Posted on: 10th November 2018 , updated on: 17th November 2018Read preprint
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