In vitro culture of aberrant basal-like cells from fibrotic lung tissue

Rationale In idiopathic pulmonary fibrosis (IPF) atypical epithelial cells are present in the alveolar compartment. Their origin and contribution to IPF pathogenesis is unknown. We recently cultured a distinct population of cells, which readily grew from fibrotic lung tissue, but only rarely from non-fibrotic tissue. Here we aimed to characterize these fibrosis-enriched cells and determine transcriptomic differences between cells derived from IPF and patients with other interstitial lung diseases (ILD). Methods Cells were cultured from peripheral lung tissue of ILD patients and analysed by bulk or single cell RNA sequencing (scRNA-seq), TaqMan-PCR, immunofluorescence (IF), immunoblotting or electron microscopy (EM). Results scRNA-seq demonstrated an overall homogeneity and epithelial origin of the cells. The majority of cells expressed basal cell markers (Cytokeratin (KRT) 5 and 17, TP63), of which a fraction co-expressed mesenchymal cell markers (VIM, FN1, CDH2), alveolar (SLC34A2, ABCA3, LPCAT1, EMP2, HOPX) and/or secretory epithelial cell markers (SCGB1A1, MUC4). Interestingly, most of the cells showed closest transcriptomic similarity to recently described aberrant basal-like cells. Cells derived from IPF versus other ILD patients revealed significant transcriptomic differences with an up-regulation of fibrosis-associated and a down-regulation of inflammatory pathways in IPF cells. Conclusion We here confirm the presence of aberrant basal-like cells in fibrotic lung tissue and, importantly, are the first to describe their in vitro characteristics and a way of culturing these cells in vitro. Cultured basal-like cells co-express epithelial and mesenchymal markers, suggesting a partial epithelial to mesenchymal transition (EMT). A subset of cells co-express alveolar, ciliated or secretory epithelial cell markers, possibly indicating differentiation towards these cell linages. Furthermore, cultured basal-like cells display a disease-specific transcriptome, possibly induced by their specific microenvironment. Our findings will contribute to a better understanding of the cells origin and their potential contribution to IPF pathogenesis.


Introduction
Idiopathic pulmonary fibrosis (IPF) is a rare, chronic, and irreversible interstitial lung disease characterized by a progressive destruction of the lung parenchyma and the respective loss of lung function [1]. The initial events leading to the development and progression of IPF are still not elucidated, however, evidence suggests that repeated 3 injury to the alveolar epithelial cells plays an important role [2]. In the healthy lung, the alveoli are lined with alveolar epithelial cells type 1 and 2 (AT-1 and 2). AT-2 cells maintain the alveolar epithelium by their ability to self-renew and they serve as progenitors for AT-1 cells [3]. The conducting airway is lined with p63+/KRT5+ basal cells and different secretory and ciliated cells. Basal cells have the ability to differentiate into all other types of airway epithelial cells, and therefore serve as progenitors to regenerate the tracheobronchial epithelium after injury [4]. During normal lung homeostasis there is no overlap of epithelial cell types of the alveolar and the conducting regions. Interestingly, severe lung injury in mice through H1N1 influenza infection or bleomycin resulted in an accumulation of p63+/KRT5+ basallike cells in the alveolar region [5,6], and it was suggested that these cells could contribute to alveolar epithelium regeneration [7,8]. However, the presence of basallike cells in bronchoalveolar lavage (BAL) fluid or in the alveolar region of lung tissue derived from IPF patients [9][10][11] was associated with increase mortality [11] and pathological bronchiolization and honeycomb formation [11,12]. Two recent studies performing single-cell RNA-seq analysis of cells derived from dissociated human fibrotic and control lung tissue described a novel fibrosis-enriched aberrant basal-like cell population, named "aberrant basaloid" by Adams et al. [13] and "KRT5-/KRT17+" by Habermann et al. [14]. These aberrant basal-like cells were shown to express some but not all canonical basal cell markers along with mesenchymal cell markers, senescence markers, and IPF-associated molecules [13,14]. Interestingly, these unique cells were shown to localize on the surface of IPF fibroblastic foci (FF).
Recently, we observed a fibrosis-enriched outgrowth of a distinct cell population from peripheral lung tissue of ILD patients in vitro: Similar to mesenchymal stem cells (MSC), these cells stained positive for CD44, CD90 and CD105 and had the capacity to differentiate into adipocytes, osteocytes and chondrocytes [15]. However, when cultured in an epithelial cell specific medium, these cells acquired cobblestone morphology typical of epithelial cells [15]. A potential for epithelial differentiation of mesenchymal progenitor cells was described earlier [16][17][18], but is controversial. On the other hand, epithelial cells were shown to acquire MSC-like features when cultured under specific conditions [19]. Therefore, we here aimed to characterize our fibrosis-specific cell population in greater detail. 4 Cell culture Fibrosis-enriched cells or fibroblasts were cultured from transbronchial or surgical lung biopsies derived from IPF or non-IPF (other interstitial lung diseases or nonfibrotic diseases) patients and from lung-explants derived from patients undergoing lung transplantation due to terminal fibrotic lung disease. Cells were cultured and conditioned medium collected as previously described [15,20]

TaqMan RT-PCR, Immunoblotting, Immunofluorescence & Electron microscopy
TaqMan® PCR, immunoblotting and IF stainings were performed as previously described [15,22]. Details for primer and antibodies are listed in supplement table 1.
For electron microscopy (EM) imaging, cells were fixed in 4% paraformaldehyde/0.1% glutaraldehyde/ 0.2M HEPES and embedded in epoxy resin (Epon®) and subjected to EM imaging as previously described [23]. Semi-thin sections were cut and stained with toluidine blue for light microscopic investigation.
Afterwards, ultrathin sections were used for transmission electron microscopy (Morgagni, FEI, Eindhoven, The Netherlands) to characterize the ultrastructure of isolated stem-like cells.

RNA sequencing
Bulk and scRNA-seq datasets were generated at the Genomics Facility Basel of the ETH Zurich, Basel, and data were analysed by the Bioinformatics Core Facility, Department of Biomedicine, University of Basel. The processed bulk and scRNA-seq data can be obtained from the gene expression omnibus database (GEO) under accession GSE145441. A detailed description of the analysis methods used for these datasets and other public scRNA-seq datasets can be found in the supplement document. 5

Fibrosis-enriched cells with distinct morphology
We here confirmed our previous findings [15], showing an enhanced outgrowth of cells with distinct morphology from fibrotic lung tissue: peripheral lung tissue pieces obtained from explants derived from all 15 IPF patients that were put in culture showed outgrowth of the cells. In contrast, peripheral lung tissue pieces obtained from surgical lung biopsies of non-fibrotic donors (e.g., emphysema) showed outgrowth of the cells for only 3 out of 62 donors (5%).
The morphology of these cultured fibrosis-enriched cells impeded a clear classification to any known plastic adherent lung cell type as they neither displayed the morphology of cubical epithelial cells nor that of spindle-shaped mesenchymal cells (Figure 1 A, B). They were long and flat cells showing a certain polarity.
Microvilli-like structures were observed on their surface and the cells were joined by desmosomes and tight junctions. Multi-lamellate bodies were occasionally seen.
Bundles of intermediate filaments were often found, oriented along the axis of the cell and connected to the plaques of the desmosomes. Occasionally, late endosomes and autophagosomes were detected in the cells (Figure 1 C).

Fibrosis-enriched cells express mesenchymal-and epithelial marker
We performed bulk RNA-seq on the cultured cells derived from eight different ILD patients (patients details see supplement table 2). We observed expression of mesenchymal stem cell markers, as described previously [15]: CD44 was highly expressed, whereas CD90 (THY1) and CD105 (ENG) were expressed at lower levels (Figure 2 A). Furthermore, other mesenchymal markers such as vimentin (VIM), N-cadherin (CDH2), or fibronectin (FN1) were also highly expressed. We

The transcriptome of fibrosis-enriched cells shows closest similarity to basal epithelial cells
In order to characterize more specifically the cell-type composition of cultured fibrosis-specific cells, we reanalysed a publicly available scRNA-seq dataset of the whole lung (GEO accession GSE122960) [24], where each of the main cell types of the lung could be identified based on the expression of established cell markers (Supplement figure 1). After aggregating cells into "pseudo-bulk" samples [25,26]

scRNA-seq analysis of fibrosis-enriched cells
The high expression of both epithelial and mesenchymal markers in our samples could be due to the joint presence of both mesenchymal and epithelial cells within the samples, or to co-expression of these markers by the same cells. In order to disentangle these two possibilities, we performed scRNA-seq using the 10X Genomics technology on cultured cells obtained from IPF patient 6 (Patients details in supplement table 2). After quality control and filtering, a total of 7,498 cells were further analyzed (Methods in the supplement document).
Based on their expression profile, cells were grouped into seven clusters, each encompassing between 2% and 32% of the cells, visualized on the tSNE reduced dimensionality projection (  [27]; the general epithelial marker KRT7 [28], mesenchymal markers FN1 and VIM; the transcription factors SOX4 and SOX9, IPF-related matrix metalloprotease (MMP)7 [29], and secretory cell markers MUC4 and SCGB1A1 can be seen in  (Supplement table 3).
Thus, our cultured basal-like cells displayed obvious similarities to recently described aberrant basal-like cells also co-expressing mesenchymal markers [13,14]. We compared our dataset to the two scRNA-seq datasets used in the studies describing these aberrant basal-like cells (GEO accessions GSE136831 and GSE135893). We

Basal-like cells do not undergo a full EMT in vitro and do not induce a-SMA expression in cultured fibroblasts
Co-expression of epithelial and mesenchymal markers in basal-like cells suggests that the cells underwent a partial EMT. In order to determine whether the cells become fully mesenchymal after a prolonged culture period, we observed cultured basal-like cells over a period of ten days. Interestingly, after ten days of culture the cells did not undergo dramatic morphological changes, and still highly expressed the epithelial cell markers E-cadherin and Nkx2. (Figure 7 A, B). Our previous observation of a disease-enriched outgrowth of a distinct cell population from peripheral lung tissue derived from IPF and other ILD patients [15] was confirmed in this study. Based on the expression of the mesenchymal stem cell markers CD44, CD90 and CD105, their differentiation potential and the lack of the current advanced transcriptomic tools we earlier named these cells mesenchymal stem cells [15]. Using RNA-seq in this study, we confirmed the expression of mesenchymal cell markers, but however surprisingly, observed closest transcriptional similarity of these cells to epithelial cells. Epithelial origin of the cells was corroborated by EM images, showing polarity to some extent, microvilli-like structures on the cell surface and occasionally the presence of multi-lamellate bodies in the cells. Furthermore, the cells were joined by desmosomes and tight junctions.
In line with our findings, epithelial cells with similar characteristics reported here were detected in the alveolar compartment of fibrotic lung tissue and were suggested to be bronchial basal cell-derived [34,35]. The disease-specific presence of atypical epithelial cells in the peripheral IPF lung was observed before: DNP63 [12] , KRT5 [10], or DNP63 and KRT5/6 [11] expressing cells as well as recently described KRT17+/KRT5-aberrant basal-like cells [13,14] or KRT8+ alveolar epithelial progenitor cells [27,36] were detected in the alveolar compartment of lung tissue derived from IPF patients, but were absent in tissue from control donors. In addition, KRT5/6+ and KRT5+ cells were present in BAL of IPF patients, but were only rarely observed in BAL from control donors [11]. 11 scRNA-seq data demonstrated an overall homogeneity and epithelial origin of the cultured cell population. Importantly, comparison of our dataset to those describing the aberrant basal-like cell population [13,14] [13,14] and to our cultured basallike cells, expressing KRT17 along with varying levels of KRT5, seemingly differentiating towards a more mature airway basal-like cell in vitro. Airway basal cells have the capacity to differentiate into secretory or ciliated epithelial cells [4], 12 which likely explains the presence of secretory or ciliated epithelial cell markers in the most differentiated subset of our cultured cells. [13,14] and in the here described cultured cells points to a partial EMT within these cells.

The high expression of mesenchymal markers in aberrant basal-like cells in vivo
Interestingly, others have demonstrated that epithelial cells that underwent a partial EMT acquire MSC-like properties [19], which may explain our previously observed similarities to MSCs [15]. More importantly, aberrant basaloid cells localize on the surface of fibroblastic foci (FF) in IPF lung tissue [13,14]. Earlier studies suggested After determining an overall homogeneity of the cultured cell population using scRNA seq, we went back to analyse our bulk RNA-seq samples. Interestingly, we revealed a significant difference in the transcriptome of cultured basal-like cells derived from IPF compared to patients with other ILDs. IPF derived basal-like cells were enriched with fibrosis-associated genes mediating EMT, myogenesis or angiogenesis, whereas pathways involved in inflammation, which is known to play a minor role in IPF pathogenesis [2], were down-regulated in IPF cells. This suggests epigenetic changes of the cells, potentially induced by the disease-specific microenvironment that persists in vitro. Basal-like cells accumulating within the alveolar region in mice after severe lung injury were suggested to help regenerating the alveolar epithelium by their ability to differentiate into alveolar epithelial cells [5,6]. Furthermore, we previously observed anti-fibrotic effects of basal-like cell-derived conditioned medium (CM) in vitro: IPF fibroblast proliferation was inhibited and epithelial wound repair increased after treatment with basal-like cell-CM [15]. However, in IPF the presence of basal-like cells in the alveolar region was associated with pathological bronchiolization [12]. Furthermore, enrichment of genes that are highly expressed in 13 airway basal cells in BAL from IPF patients was associated with higher mortality [11].
Interestingly, it was shown that bronchial epithelial stem cells are able to contribute to pathological bronchiolization and alveolar regeneration, depending on the activation of specific signaling pathways [8]. Therefore, it can be speculated that the fibrotic microenvironment directs basal-like cells to contribute to pathological bronchiolization rather than alveolar epithelial regeneration. Studying the differentiation potential of fibrosis-specific basal-like cells exposed to different profibrotic factors/microenvironments would therefore be of uttermost interest in future studies.
It is important to mention that our results are limited by the low number of patients included in the study. Furthermore, we had to exclude two outlier samples from our bulk RNA-seq analysis, both coming from our only female patients, leaving the possibility that the reported differential expression patterns are only male-specific. An increase of the patient numbers in future studies would corroborate our findings.
Regarding the scRNA-seq analysis of basal-like cells, it would have been interesting to compare our results to basal-like cells grown from non-fibrotic tissue. However, we clearly showed in this and our previous study [15] that under standardized cell culture conditions the basal-like cell population readily grows from fibrotic, but only rarely from non-fibrotic tissue, making it difficult to provide a control group of cultured non-fibrotic basal-like cells.
To the best of our knowledge, this is the first study describing the culture and in vitro characteristics of aberrant basal-like cells from peripheral lung tissue of IPF patients.
Culturing these cells in vitro opens up the opportunity to study their role, function and origin in the IPF lung. Understanding the cells origin, differentiation potential, and how they are influenced by pro-fibrotic factors/different microenvironments may lead to the development of novel therapeutic targets in IPF.