Review 1: "Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

***************************************

Review:

The authors’ description of a long term culture conditions that selects for two important progenitor cell populations in the distal lung epithelium represent a significant advance in the study of lung epithelial stem cells. Through careful characterizations of the culture conditions and subsequent infection of lung epithelial organoids, Salahudeen et al. identify distal lung club cells as a novel target cell population of SARS-CoV-2. The general conclusions drawn in the study are strongly supported by the data.

The study is primarily aimed towards establishing culture conditions for long term in vitro growth of distal lung epithelial progenitors. This is a significant step forward in our ability to study distal lung epithelium, especially mature alveolar epithelial cells (AEC2s) which are the cell type often observed to be aberrantly regulated in several diseases. Majority of our understanding of lung pathologies come from our study of the murine lung. However, recent studies have shown that adult human AEC2s possess remarkably higher regenerative activity than their murine counterpart. In addition, the distal airways of the human lung are replete with cytokeratin 5+ (KRT5) basal cells, which are extremely rare in the similar airways of the mouse lung. The robust growth of the distal lung airway and alveolar progenitors can now allow the researcher to study their function and provide scalable opportunities for injury and therapeutic screenings.

Through single cell RNA sequencing of their organoids, the authors identify the distal basal cells as a heterogeneous cell population that comprises of a non-proliferating basal cells and a proliferating TNFRSF12Ahi subfraction, which represents the primary regenerative basal cell population in their in vitro system. In contrast, a similar heterogeneity within the adult AEC2s did not exist. However, this apparent lack of heterogeneity implies that any AEC2 can spontaneously become regenerative in vitro although the data does not support such implications and the authors do not speculate whether that may or may not be the case. A minor, but important, criticism of their description of methods is that the authors also do not give as colony forming efficiency or how many cells were plated in the initial stages, points that are critical for careful evaluation of a novel cell culture method.

Having established the cell culture conditions for prolonged growth of distal airway and alveolar progenitors, the authors dedicate the final series of experiments to establish the infectability of these organoids by A/Puerto Rico/8/1934 (H1N1) [PR8] virus (a laboratory adapted strain of flu) and SARS-CoV-2 virus. In this set of organoids, it is interesting to see that the polarity of the organoids, as the authors call “apical-out” – referring to reversing the polarity such that the apical side is facing outwards and not towards the lumen, is critical for infection of the cells. Consistent with the general idea of the cell types infected by SARS-CoV-2, the authors show that the ~10% AEC2s are infected by the virus. A key weakness in the study is further characterizations of the pathway and mechanisms why only 10% of the AEC2s are infected and whether that is physiologically relevant. The authors can easily address this using their prolonged cell culture conditions. On the other hand, the infection of airway organoids revealed a new cell type, club cells, which were the primary target of SARS-CoV-2. This observation stands in stark contrast to early studies showing SARS-CoV-2 infection of basal and ciliated cells obtained by nasal brushes, and therefore capturing cells primarily from the most proximal airways. The inherent differences between the same cell type obtained from proximal and distal airways should be probed further to understand the pathogenesis and progression of the disease. Findings from this study need to be replicated in other cell culture system to ensure that it is not an artifact of in vitro organoid system, which, although attractive, is limited by its simplicity in that it does not allow for interaction of multiple cell types and the microenvironment of a living lung. Finally, careful histological evaluation of the pathology of SARS-CoV-2 infected lungs and whether there is a difference between proximal and distal disease lung may hold a key in contextualizing findings from this study and whether there is indeed a relevance to different cell populations being the target of SARS-CoV-2 in different regions of the lung. This study opens up new avenues for research of SARS-CoV-2 and how it may infect different regions of the lungs.