Airway Basal Cells Show Regionally Distinct Potential to Undergo Metaplastic Differentiation

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Basal cells are multipotent stem cells of a variety of organs, and in the lung are known as crucial components of the airway epithelium. However, it remains unclear how diverse basal cells are and whether distinct subpopulations respond differently to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the murine trachea. Among these we found two major subpopulations comprising the most stem-like progenitor compartment, but with distinct signatures and ability to self-renew and differentiate. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to initiate an aberrant program of differentiation in response to environmental perturbations in primary cultures and in injury mouse models in vivo . We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of cartilage-deficient mouse mutants. Lastly, we show that key features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide critical insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.

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  1. Evaluation Summary:

    Whether airway basal cells exhibit extensive cell state heterogeneity and whether this is relevant for their function has been unclear. This study provides important evidence that such heterogeneity exists and may dictate airway basal cell function in a spatially restricted manner.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    In the manuscript "Airway Basal Cells Show Regionally Distinct Potential to Undergo Metaplastic Differentiation" by Yizhou, Yang et al., the authors take an unbiased approach to interrogate basal cell heterogeneity in the trachea. Their single-cell RNA-seq data suggests that several sub-populations of basal cells exist. Follow-up studies support the conclusion that two major basal cell populations exist corresponding to the dorsal and ventral trachea. Strikingly, their functional data also supports that the microenvironment of the dorsal or ventral trachea, being surrounded by smooth muscle or cartilage respectively, and that loss of cartilage leads to aberrant patterning of BC1 and BC2. Overall, this is an interesting study with reasonable conclusions that are supported by the data, and, the data is clear and of high quality. One point that requires further discussion pertains to the KRT13 expression following injury, and whether calling KRT13 activation "aberrant" is appropriate if it is simply a part of the natural repair process.

  3. Reviewer #2 (Public Review):

    The manuscript by Zhou/Cardoso and colleagues addresses a topic of recent interest to the lung epithelial and stem cell research communities, namely the potential molecular, functional, and regional heterogeneity of airway basal stem cells, and the relevance of this heterogeneity for precancerous changes such as squamous metaplasia.

    There is much delineated in the scRNA-seq and immunostaining surveys provided that will be informative for the research community, including transcriptomic and protein staining markers that distinguish dorsal from ventral basal cells (BC-2 and BC-1 respectively; markers= Cd44, Cav1, Krt17, Isl1, Tgm2). Functional differences between these basal cell subtypes are important and relevant to biological behaviors as quantified here in assays that measure: colony forming efficiency, behavior in differentiation studies in vitro, differences in tracheal epithelial repopulation potential in vivo after 2 types of airway injuries (naphthalene and polidocanol), differences in squamous differentiation potential, and differential responses to blocking RA signaling. The work is strong and the conclusions and interpretations are supported by the data shown. However, it would be important to better quantify or discuss some of the differences in basal cell profiles or behaviors, and to consider alternative explanations to the hypothesis that these differences are due to biological signaling niches rather than just differences in structural properties of the dorsal vs ventral trachea.

  4. Reviewer #3 (Public Review):

    In this manuscript, Zhou et al describe basal cell heterogeneity in the mouse trachea. They describe how dorsally vs ventrally located tracheal basal cells which are supported by different stromal cell populations show differential potential to undergo squamous metaplastic differentiation. Furthermore, they suggest that the differences in these basal cells might be epigenetically programmed as they are maintained after these basal cells have been isolated and cultured in vitro. However, it is not clear whether dorsal vs ventral supporting stromal cell populations made it into the culture medium.