key: cord-0253278-zyu4fze2
authors: Taghizadeh, Sara; Heiner, Monika; Wilhelm, Jochen; Herold, Susane; Chen, Chengshui; Zhang, JinSan; Bellusci, Saverio
title: Characterization in mice of the stromal niche maintaining AT2 stem cell self-renewal in homeostasis and disease
date: 2021-01-28
journal: bioRxiv
DOI: 10.1101/2021.01.28.428090
sha: 146d3fca335b7a0031e6b89a5f859e6c41934a64
doc_id: 253278
cord_uid: zyu4fze2

Resident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described. We propose that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1PosFgf10Pos) are equally important to maintain AT2 stem cell self-renewal. The alveolosphere model, based on the AT2-rMC co-culture in growth factor reduced Matrigel, was used to test the efficiency of different rMC subpopulations isolated by FACS from adult murine lung to sustain the self-renewal and differentiation of AT2 stem cells. We demonstrate that rMC-Sca1PosFgf10Pos cells are efficient to promote the self-renewal and differentiation of AT2 stem cells. Co-staining of adult lung for Fgf10 mRNA and Sftpc protein respectively, indicate that 28% of Fgf10Pos cells are located close to AT2 cells. Co-ISH for Fgf7 and Fgf10 indicate that these two populations do not significantly overlap. Gene arrays comparing rMC-Sca1PosAxin2Pos and rMC-Sca1PosFgf10Pos support that these two cell subsets express differential markers. In addition, rMC function is decreased in diabetic and obese ob/ob mutant compared to WT mice with a much stronger loss of function in males compared to females. In conclusion, rMC-Sca1PosFgf10Pos cells play important role in supporting AT2 stem cells self-renewal and differentiation. This result sheds a new light on the subpopulations of rMCs contributing to the AT2 stem cell niche in homeostasis and in the context of COVID-19 pathogenesis. Key message What is already known about the subject? Resident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described. What does this study add? Our study shows that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1PosFgf10Pos) is equally important to maintain AT2 stem cell self-renewal. rMC-Sca1PosFgf10Pos are LipidToxHigh and are located close to AT2s. In addition, rMC-Sca1PosFgf10Pos cells support AT2 stem cell self-renewal and differentiation thereby identifying these cells as bone fide functional lipofibroblasts (LIFs). We have previously reported that LIF can transdifferentiate into activated MYF in the context of bleomycin-induced fibrosis in mice [1] and that activated MYF isolated from the lungs of end stage idiopathic fibrosis human patients can respond to Metformin to undergo transdifferentiation back to the LIF phenotype [2]. We also show that the function of rMCs-Sca1Pos is negatively impacted by gender and obesity, which represent two major aggravating factors for COVID-19 pathogenesis, leading to either death or major complications after infection recovery such as lung fibrosis. How might this impact on clinical practice and future development? By establishing that rMC-Sca1PosFgf10Pos are different from the MANCs, our study opens the way for a new key mesenchymal cell population that should be targeted to either prevent or reverse fibrosis. In addition, as this population maintains the AT2 stem cells self-renewal and differentiation, such targeting will also allow to progressively recover the loss in respiratory function.

Lung fibrosis is characterized by an accumulation in the respiratory airways of mesenchymal cells, called activated myofibroblasts (MYF) which over time, lead to impaired lung function [3] . Through the use of single cell transcriptomic approach, the heterogeneity of the different resident mesenchymal (rMC) populations present in the lung is starting to emerge. A key stromal cell is represented by the lipofibroblasts (LIFs), which are rich in lipid-droplets and can be stained and isolated using the vital dye LipidTox (LT). They express Perilipin 2, platelet derived growth factor receptor alpha (Pdgfra) and are negative for Acta2 and most importantly are located close to alveolar type 2 cells (AT2) [4] . They are proposed to supply AT2s with the triglycerides needed for the elaboration of surfactant. Using a co-culture assay of Pdgfrα High mesenchymal cells and AT2s in growth factor reduced Matrigel, it has been proposed that the rMC-LIFs are essential for the maintenance of the self-renewal and differentiation of AT2 stem cells [5] .

Fibroblast growth factor 10 (Fgf10) is a mesenchymal-specific gene expressed in the distal part of the embryonic lung during the early pseudoglandular stage [6] . Our lineage tracing analyses have shown that a subpopulation of Fgf10 Pos cells at embryonic day 12.5 serves as progenitor for rMC-LIFs at later stages of lung development. Additionally, in postnatal lungs, around 30% of rMC-LIF express the Fgf10 gene [7] . The relevance of these rMC-Fgf10 Pos LIF in maintaining the self-renewal and differentiation of AT2 stem cells remains to be demonstrated. Interestingly, another population of stromal cells, called MANC (Mesenchymal Alveolar Niche Cells), which are positive for Axin2, Pdgfra, Wnt2, Il6 and Fgf7 [8] , were also described to locate close to AT2 cells. How different are the rMC-Fgf10 pos LIFs to the MANC is still unclear.

Using lineage tracing, we previously reported the reversible transdifferentiation of the LIFs into activated MYF during fibrosis formation and resolution [1] . In addition, Metformin, a 5 first line antidiabetic drug has been reported to enhance the activated MYF to LIF transition both in vitro and in vivo [2] . Repetitive damages to AT2s have been proposed to be linked to the development of lung fibrosis. Interestingly, one of the major and long-term complication for patients who survived SARS-CoV-2 infection (leading to COVID-19 disease) is fibrosis [9, 10] . SARS-CoV-2 binds to its receptor angiotensin converting enzyme (ACE) mostly abundantly expressed in the AT2s. Massive damages to the AT2s likely unleashes the associated LIFs to become activated MYFs, thereby leading to fibrosis. Pertinent to this background, obesity/diabetes and gender have been proposed to be predictive factors for the severity of the disease [11, 12] .

In this paper, we used the in vitro alveolosphere model based on the AT2-rMC co-culture in growth factor reduced Matrigel to test the efficiency of different rMC subpopulations isolated by FACS from adult murine lung. We used antibodies against Cd45, Cd31, Epcam and Stem cell antigen 1 (Sca1) to refine the initial rMC population responsible for the maintenance of AT2 stem cells. Using a fluorescent substrate for ß-galactosidase activity, rMC-Sca1 Pos Fgf10 Pos as well as rMC-Sca1 Pos Axin2 Pos cells were sorted. Additional sorting was achieved using LT staining for cells containing high level of neutral lipids. After 2 weeks of co-culture, organoid size and colony forming efficiency were quantified. RNAscope with Fgf7-and Fgf10-labelled riboprobes, to label rMC-Axin2 Pos and rMC-Fgf10 Pos respectively, combined with Sftpc immunofluorescence on adult lungs was carried out. Gene array analyses were used to characterize sorted rMC-Sca1 Pos Fgf10 Pos vs. rMC-Sca1 Pos Axin2 Pos . We used males and females C57BL6 and corresponding ob/ob mutant mice to determine the impact of obesity and gender on the functional capacity of the corresponding rMCs-Sca1 Pos to sustain AT2 stem cells.

Our results indicate that an rMC-Sca1 Pos Fgf10 Pos /LIF Pos subpopulation is essential for the self-renewal of the AT2 stem cells. This subpopulation is likely different from the previously 6 described MANC. In addition, obesity and gender impact the capacity of rMCs to maintain the self-renewal of AT2 stem cells indicating that future therapeutic approaches should be focussed on restoring the function of the mesenchymal niche. 

Fluorescein di (b-D-galactopyranoside) (aka FDG) (Thermo Fischer Scientific #F1930) was used to isolate by FACS, cells expressing ß-galactosidase from Fgf10 LacZ and Axin2 LacZ reporter lines. Lungs were collected from 6-8 weeks old Fgf10 LacZ and Axin2 LacZ reporter mice. According to manufacturer's instruction, single-cell suspension and FDG working solution were prewarmed and the cells were resuspended with chloroquine followed by loading by FDG. After incubation for 20 minutes, FDG loading is stopped by adding ice cold staining medium containing propidium iodide and chloroquine. Cells are then placed on ice 8 and incubated with antibodies against Cd45, Cd31, Sca1, and Epcam (for details see Lung dissociation and Fluorescence-Activated Cell Sorting) before sorting using the FACSAria™ III (BD Bioscience) cell sorter. Cells were sorted through a flow chamber with a 100-μm nozzle tip under 25 psi sheath fluid pressure. Cells were collected in sorting media (advanced DMEM:F12 (gibco#12634-010) plus 10% FBS and 1% P/S).

5000 Lyso Pos Tom Pos cells (AT2s from adult Sftpc CreERT2/+ ; tdTomato flox/+ lungs) and 50000 rMCs cells were resuspended in 100 µl culture medium (sorting media plus 1% ITS (gibco # 41400-045)) and mixed 1:1 with 100 µl growth factor-reduced phenol Red-free Matrigel (Corning #356231). Cells were seeded in individual 24-well 0.4 µm Transwell inserts (Falcon, SARSTEDT). After incubation at 37°C for 15 minutes, 500 µl of culture was placed in the lower chamber and the plate was placed at 37°C in 5% CO 2 /air. The culture medium was changed every other day. ROCK inhibitor (10 µM, Y27632 STEMCELL#72304) was included in the culture medium for the first two days of culture. Organoids were counted and measured at day 14. Colony-forming efficiency (CFE) is calculated as the ratio between the numbers of spheres observed over the initial number (5000) of AT2 cells. At day 14, organoids were processed for whole-mount immunofluorescence staining.

Co-staining: RNA in situ hybridization assay and IF 9

See supplementary methods. GEO accession: GSE162859

All results are mean ± SEM. All error bars on graphs represent SEM. Statistical tests are 2tailed t tests. P ≤ 0.05 was considered statistically significant.

Patient and public involvement in research: n/a. This is a basic research project. 1 0

The capacity of Cd45 Neg Cd31 Neg Epcam Neg resident mesenchymal cells (rMCs) to functionally support the self-renewal and differentiation of alveolar epithelial type 2

The aim of this study is to refine the adult lung resident mesenchymal cell population, called thereafter rMC and defined by flow cytometry as Cd45 Neg Cd31 Neg Epcam Neg cells. This cell population is capable of sustaining AT2 stem cell renewal using the previously described alveolosphere in vitro assay [5] . In this approach, the use of antibodies against Cd45 and Cd31 allowed us to remove, from the target subpopulation, the hematopoietic and endothelial cells, respectively.

In the context of a previous milestone study generating organoids in Matrigel arising from a co-culture of Epcam High Cd24 Low epithelial cells (previously called epithelial stem/progenitor cells or EpiSPC) with rMC, it was demonstrated that rMC expressing the stem cell antigen 1 (Sca1) were capable of sustaining the self-renewal and differentiation of these cells [13, 14] .

However, in this study, the impact of the different subpopulations of rMCs on AT2 stem cell self-renewal and differentiation was not analysed.

For this purpose, we have sorted an enriched population of mature AT2 cells from the Sftpc CreERT2/+ ; Tomato flox/+ lungs. We started first by isolating Cd31 Neg Cd45 Neg Epcam Pos cells and then applied two successive and stringent sorting approaches: first using LysoTracker, a fluorescent dye that labels acidic compartments found abundantly in the lamellar bodies of AT2 cells and second, using the tomato reporter expressed specifically in AT2 cells (Fig.   1A ). In addition, we sorted also rMCs-Sca1 Pos as well as rMCs-Sca1 Neg (Cd45 Neg Cd31 Neg Epcam Neg Sca1 Neg ). In order to have sufficient cells to carry out our assay, we pooled the lungs of four adult (6-8 weeks old) C57BL/6 mice. This allowed us to carry 1 1 out 6 independent co-cultures. rMCs-Sca1 Pos represent around 25% of the total rMC population (Fig. 1A) . When co-cultured with sorted mature AT2s, rMCs-Sca1 Pos led to the formation of organoids (Fig. 1B) with a colony forming efficiency of 5% ( Fig. 1C ) which is in line with previously published alveolosphere experiments. Interestingly, rMCs-Sca1 Neg fail to sustain alveolosphere formation (Fig. 1B) . The difference in organoid size and CFE is highly significant between the two rMC subpopulations (Fig. 1C , P < 0.01).

To better characterize rMCs-Sca1 Pos lung cells and in particular whether this population may include the lipofibroblasts (LIFs), we used LipidTOX (LT), an efficient fluorescent stain for neutral lipid. As LIFs are abundant in neutral lipid droplets, LT has been previously used to quantify LIF population during lung development [4] . differentiation. RT-qPCR was also carried out to quantify the expression of Fgf10 and Pdfra, two well-known markers enriched in LIFs (Fig. 2D) . Our results indicate a trend towards an 1 2 increase in the expression of Fgf10 and Pdgfra in rMCs-Sca1 Pos LT High compared to rMCs-Sca1 Pos LT Neg (n=3 independent mice) (Fig. 2D ). These results are in line with the previous observation that Fgf10 and Pdgfra are also expressed in cells other than LIFs [4, 15] . Overall, our data therefore support the previous conclusion that it is the LIF subpopulation of the rMCs that can preferentially support AT2 stem cell survival and differentiation.

We To better characterize these two rMC subpopulations, we used specific reporter lines;

Fgf10 LacZ and Axin2 LacZ to monitor the distribution of LipidTOX staining in these two sublineages. By using FACSAria III cell sorter, we analysed 100,000 events, each sample contained harvested lung from one mouse -with the same age range (6-8 weeks old).

Cd45 Neg Cd31 Neg Epcam Neg Sca1 Pos sorted cells were processed for further analysis (Fig. 4A ). of Fgf10 Pos and 98% of rMC-Sca1 Pos Axin2 Pos cells were also LT High cells (Fig 4B and C, respectively). Based on LT staining, we also report that most of the rMCs-Sca1 Pos Fgf10 Neg as well as rMCs-Sca1 Pos Axin2 Neg subpopulations contain a high percentile of LT Low/High suggesting again a functional heterogeneity at the level of the LIFs (in regards to the maintenance of AT2 stem cell self-renewal) based on whether they express or not Fgf10 or

Axin2. 1 4 In order to better define at the transcriptomic level the difference between rMCs-Sca1 Pos Fgf10 Pos and rMCs-Sca1 Pos Axin2 Pos , we performed gene array analysis using the Agilent platform. Fig 4D shows 

To investigate the relative interaction between Fgf10 expressing cells and AT2 cells, we combined the in-situ hybridization technique for detecting Fgf10 mRNA expression with immunofluorescence staining for pro-SPC in adult wild type lungs. We found that 28% ± 1% (n=3) of total Fgf10 expressing cells are located close to pro-SPC expressing cells (Fig. 5A ).

This number is very similar to the one we reported before in the newborn lung using the S2 ) indicating that the signal observed in wild type lung is specific (Fig. 5B) . We found that around only 2.0% ± 0.2% (n=3) of Fgf7 expressing cells are in vicinity of Sftpc Pos cells (Fig.   5B ). Finally, we performed a co-staining for Fgf7 and Fgf10 mRNA using different fluorescent labelled probes. Our results indicate that 15.7% ± 3.0% (n=3) of total cells are formation [1] . Obesity/diabetes and gender have been proposed to be aggravating preexisting conditions predicting the severity of the disease [11] .

To explore the impact of obesity/diabetes and gender on the functionality of the rMCs- vs. 9%, respectively). We also functionally tested the rMCs-Sca1 Pos from these different mice by co-culturing them with sorted AT2 cells using the alveolosphere assay. When the rMCs-Sca1 Pos are isolated from Ob/Ob male mice presenting the two risk factors, obesity and male ( Fig. 6B) , we observed a complete absence of organoid formation (n=2). However, when rMCs-Sca1 Pos are isolated from female Ob/Ob mice presenting only obesity as a risk factor, a significant number of organoids are present (3% CFE, n=2). This CFE is, however, lower than the one observed in non-obese C57BL6 female wild type mice (5% CFE, n=2)

indicating that obesity alone is already impacting the functionality of the rMCs-Sca1 Pos .

Interestingly, non-obese C57BL6 male wild type mice display also a reduced CFE compared to non-obese C57BL6 female wild type mice (2% vs. 5%, respectively, n=2) indicating that the gender alone is also impacting the functionality of the rMCs-Sca1 Pos . We also observed that age, another risk factor for COVID-19, was also having the same effect than obesity and gender on the capacity of the rMCs-Sca1 Pos to maintain the self-renewal of the AT2 stem cells (data not shown).

A major problem arising from key publications using flow cytometry as a primary experimental approach is the lack of independent studies reporting the reproducibility of the results. This is due in part to the type of flow sorter used, the sophisticated flow cytometry protocols, including the gating conditions used and the lack of comparison with other endogenous population at the time of the sort as internal controls. This is particularly crucial in the context of the lung mesenchyme which is still a huge black box both in human and in mice in spite of recent scRNA seq studies [17] .In our study, we adopted an unbiased and reproducible FACS strategy to sort both epithelial and mesenchymal populations. We used Sca1 as a resident mesenchymal stem cell marker to refine the rMC population. In previous studies, Cd45 Neg Cd31 Neg Sca1 Pos cells (please note that Epcam was not used in that study)

were detectable in the postnatal lung coincidently with the transition from the saccular to the alveolar stage of lung development. This population also co-expressed Cd34, Thy1 (Cd90), Are the rMC-Sca1 Pos Fgf10 Pos more relevant than the rMC-Sca1 Pos Axin2 Pos for the repair process after injury?

Given the fact that both subpopulations appear to sustain the self-renewal and differentiation of AT2 stem cells in vitro, a natural question is therefore whether they have redundant functions or whether one population appears to be more crucial than the other. From the angle of Fgf signalling and based on the consequence of Fgf7 versus Fgf10 gene inactivation in mice, we can conclude that rMC-Sca1 Pos Fgf10 Pos are likely more important than rMC-Sca1 Pos Axin2 Pos . Fgf10 inactivation leads to lung agenesis [19] while Fgf7 null mice are viable and display no obvious lung phenotype [20] . Changes in endogenous Fgf10 expression has been correlated with disease progression and/or repair after lung injury both in mice and humans [21] [22] [23] . Evidence for such a role for Fgf7 in the embryonic or adult lung in human or mice are still lacking in spite of the fact that Fgf7 was discovered 7 years before Fgf10 [24, 25] . Another important question is whether it matters if the most efficient mesenchymal niche cells express Fgf7 versus Fgf10 as they are both ligands are acting through Fgfr2b.

Indeed, it does matter as Fgf7 and Fgf10, although belonging to the same Fgf subfamily of paracrine growth factors elicit different biological activities on isolated lung epithelium grown in Matrigel in vitro [6] . 

In the context of lung fibrosis, the accumulation of activated MYF producing extracellular matrix components, modify the lung structure and negatively impact gas exchange. The LIF to MYF reversible differentiation switch appears to be a key process in fibrosis formation and resolution. Moreover, this transition was also shown in vitro in response to hyperoxia, as a key event in bronchopulmonary dysplasia (BPD) [27] . AT2 cells express angiotensinconverting enzyme II (ACE), a main receptor for SARS-CoV-2 [28] . Interestingly, SARS- 

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We would like to thank Kerstin Goth and Jessica Nesswetha for the maintenance and management of the mouse colonies required for this study. 

The authors have declared that no conflict of interest exists.

ST has performed all experiments and MH has performed FACS analysis and sorting for samples. JW carried out the bioinformatics analysis. ST, JZ, SB, conceived the project and wrote the manuscript. All authors contributed to the article and approved the submitted version.