Devolución

BCs have been described as progenitors of the airway epithelium: Breuer et al. showed that BCs act as progenitors in the steady state in conducting airways, but are substituted by SCs as progenitor population for the maintenance of the epithelium in the smaller airways where BCs are rarer (Breuer et al., 1990). This is consistent with the studies performed by Reynolds in 2000 (Reynolds et al., 2000) showing that an ablation of SCs in mice causes a loss of MCCs in the terminal bronchioles but not in proximal airways. Different populations of cells affect therefore the fate of the epithelium, and these differences depend of regions of the airways that are considered.

BCs constitute a multipotent progenitor population that gives rise to other BCs, SCs and MCCs, playing an important role in the regeneration of the epithelium. Experiments of Hong and Gosh revealed a BC subset expressing KRT5 and KRT14 responsible of the regeneration of the epithelium after injury in mouse through naphthalene-induced ablation of the SC population (Kyung U Hong et al., 2004; Kyung U. Hong et al., 2004) or in increased proliferating cells due to remodelling events in cultured primary cells from cadaveric tracheobronchial tissue (Ghosh et al., 2013).

Edith Puchelle and Christelle Coraux tried identified the BC population as the responsible of the differentiation a regeneration of the epithelium, they FACS-sorted BCs and columnar cells separately and analysed the properties of these cell suspensions. They showed that only cells of the BC suspension, once seeded in plastic or in denuded rat tracheas, were able to attach, proliferate and differentiate. They also studied the telomerase activity of the two cells suspension and they only detected active telomerase in the BC suspension, which is a feature of stem cells (Rodolphe Hajj et al., 2007).

To characterize better the BC population and improve cell lineage tracing during epithelial homeostasis and regeneration, the group of Hogan genetically tagged KRT5+ cells in adult mouse under tamoxifen induction, and they reported that just after lineage induction only BCs were tagged. Over time, the percentage of tagged BCs declined, while the number of tagged SCs and MCCs increased (Rock et al., 2009). Since the number of tagged SCs was considerably bigger that the number of MCCs, two possible hypotheses were raised: (1) MCCs have a longer life span than SCs, so BCs give rise to more SCs than MCCs in a certain period of time; (2) BCs give rise to SCs that slowly transitioned to ciliated cells. The latter hypothesis indeed agrees well with the results of Plopper’s group (Evans et al., 2001) and with their own study (Rawlins et al., 2009) on the role of SCs during homeostasis and repair of lung airway. In the same way, they analysed the cellular composition and behaviour of BCs in postnatal growth and showed that BCs were able to self-renew and generate SC and MCCs.

Hogan’s group also performed experiments in mice after injury using sulfur dioxide (SO2)and analysed

the repair model with their KRT5 genetically tagged mouse line (Rock et al., 2009). SO2 caused an extensive

damage in the airways. During repair they analysed the BC population and they showed that this population was highly proliferative and gave rise to patches of cilia and SCs. In contrast with the behaviour showed during homeostasis, the process of regeneration after injury by BCs gave rise to more cilia than SC cells. This plasticity suggests that the fate of the progeny of BCs is really influenced by the tissue conditions.

Already in 1986 Evans et al. used a model of “mild” injury in rats that consisted in the exposure to NO2. This kind of injury led to the destruction of the more outer cell layer, meaning that only GCs and MCCs were ablated. After injury they studied the regeneration of the epithelium, focusing on the region of the bronchi with diameters between 1.47 and 1.89, a level where BCs are still present. They revealed that the SC

population was the responsible of the restoration of the ablated cell types after the injury and not the BCs (Evans et al., 1986).

Teixeira and colleagues used a statistical lineage tracing model based in the study on mitochondrial mutations in the epithelial cells as a marker to analyse clonal expansion. They showed that in the human upper airways, the model of homeostatic maintenance of the epithelium was consistent with a model where a unique, multipotent progenitor cell is loosed and replaced in a stochastic manner, and this progenitor cell would be a BC (Teixeira et al., 2013).

Collectively, these results suggest that there are at least two populations of progenitor cells: one BC population that maintains the homeostasis at long-term and self-renews, and SCs that acts as facultative progenitors.

The statistical model of cell lineage developed by Texeira and colleagues revealed that the model of proliferation of the BC population led to three different outcomes for the daughter cells, where the division of the progenitor cell could give rise to two progenitor cells or to two cells that were committed to differentiation (symmetrical division), and could give rise to an asymmetric division generating one progenitor cell and one cell prepared to differentiate, this both kinds of division were also described later on by the group of Rawlins (Teixeira et al., 2013; Watson et al., 2015).

Boers’s group in 1998 described the heterogeneity of the BC population describing the presence of BCs expressing KRT5 and KRT14 and other called “paraBCs” expressing KRT1γ. They did a measurement of the number of BCs expressing KRT5 and KRT14 and paraBCs expressing KRT13 in healthy human lungs and they quantified also the proportion of these cells contributing to the proliferative fraction of the epithelium in proximal and distal airways. In the proximal fraction of the airways there were 31% of BCs, 51% of them were proliferating and 7% of paraBCs with 33% proliferating. In distal airways they did not observe any paraBC and only a 6% of BCs with a 30% of proliferating BCs (James E Boers, Ambergen and Thunnissen, 1998). The group of Rawlins performed single cell RT-qPCR over 67 isolated single cells and described the presence of a BC population expressing Krt5 but also the luminal keratin marker Krt8 (Watson et al., 2015). This KRT5+/KRT8+ population was also described by the group of Cardoso suggesting that these paraBCs were the progenitors of the differentiated luminal cells (Mori et al., 2015).

Very recently a study of in vitro regeneration of airway epithelium in mouse, described the presence of a KRT4+/KRT13+ population that could also correspond to supra-basal cells. During mouse regeneration of airway epithelium in vivo, the authors describe the presence of this population in “blocks” what they called “hillocks”, they suggested that this population can act as an alternative way from BCs to SCs (Montoro et al., 2018). Also very recenty a single cell transcriptome analysis of Mouse Tracheal Epithelial Cells (MTECs) showed a discrete cluster of cells expressing Krt4 and Ktr13 (Plasschaert et al., 2018). In our study of in vitro and in vivo HAECs differentiation by single cell RNAseq, our data showed a transitioning BC population precursor of the SC population (Ruiz Garcia et al., 2018).

The groups of Wa Xian and Frank McKeon described the presence of clusters of Trp63+ and Krt5+ cells in distal airways after H1N1 influenza virus infection. They termed them “Distal Airway Stem Cell” (DASCP63/Ktr5). These authors describe how these cells are able to prolifreate and disperse to injured areas where they proliferate and differentiate into cells expressing genes linked to alveolar function. They demonstrate that DASC contribute to lung regeneration giving rise to multiple epithelial lineages including cell types of the distal lung (Kumar et al., 2011; Zuo et al., 2014).

The groups of John Ngai and Russell B. Fletcher used a murine model of injury to assess the role of the BCs during the recovery of the airway epithelium and the derivatives of these BCs, to do so they used single cell techniques and clonal lineage tracing of BCs. They confirmed the BC quiescence at the homeostatic level and that after injury, BCs show multipotency and divide in a symmetric or asymmetric fashion early in the regeneration. To study the cell fate time course of the BC after injury, they used single cell transcriptomic analysis of lineage traced cells at time before injury and after injury in an early and late state. They show a lineage trajectory where they describe the presence of different populations of BCs in the regenerating epithelium, these BC populations are transitioning states with different transcriptome level of expression of basal markers such as P63, Krt5 and Krt14. Moreover, they see clear differences between the BCs in homeostasis and the BCs after injury, after injury the BCs shift to express genes involved in wound-response transcriptional program such as Krt6a and Krt16 and members of the SPRR gene family (Gadye et al., 2017). However, our analysis of single cell transcriptome of HAECs showed the expression of these markers in the BC population in a condition of homeostasis in vitro and in vivo without injury (Ruiz Garcia et al., 2018).