The epithelial lining of regeneratively quiescent lungs is composed of alveolar type 2 (AT2) progenitor and differentiated alveolar type 1 (AT1) cells. To replace aged AT1 cells, AT2 cells undergo self-renewal to maintain alveolar epithelial homeostasis1. The regenerative potential of AT2 cells could be activated for timely recovery from lung epithelial injury2-4, including lobectomy and infections5 , 6. A marked suppression in fibrinolytic activity in local respiratory illnesses (e.g., inhaled smoke and aspirated gastric juice) and pulmonary complications of systemic diseases (e.g., sepsis) has been reported clinically and in animal models7 , 8. Migration and differentiation of mesenchymal stem cells (MSCs) in inflamed tissues are regulated by dynamic fibrinolytic niche9-12. The proteolysis of extracellular matrix substrates by urokinase plasminogen activator (uPA) could be involved in the benefit of the fibrinolytic niche to the regeneration of skeletal muscles and fractured cartilage13-17. For example, uPA and plasmin, two critical components of the fibrinolytic niche, cleave epithelial sodium channels (ENaC)18 , 19. In addition, functionally multifaceted uPA regulates alveolar epithelial function20 and possesses an A6 motif with a high affinity to CD44 receptors21 , 22. CD44 + AT2 cells show a higher proliferative capacity in fibrotic lungs23. The fibrinolytic niche in alveolar epithelial homeostasis and regeneration mediated by AT2 cells, however, has not been studied systematically. Our results have tested the potential novel contribution of uPA-PAI1-A6-CD44-ENaC cascade to the fibrinolytic niche in regulating the fate of AT2 cells.
To examine the effects of fibrinolytic niche on AT2 cells in normal and injured lungs, we infected wt and Plau -/mice with the PR8 H1N1 type A influenza virus intranasally24 , 25. The severity of injured regions was classified as mild, moderate, and severe based on the lung injury score26. Randomly selected fields were analyzed with the ImageJ software to count proSPC + AT2 cells (green) and the total cells for wt (Fig. 1A) and Plau -/mice (Fig. 1B) .
Although AT1 cells were stained with anti-pdpn antibody, it was not possible to clearly distinguish from other non-epithelial cells, i.e., basal cells, leukocytes, endothelial cells, and fibroblasts due to considerable overlap. AT2 cells per field of wt lungs were approximately two-fold (12.6 ± 0.55%) that of Plau -/lung preparations (6.0 ± 0.35 %, n=26 from 8 different mice). Moreover, influenza-infected lungs displayed widespread alveolar collapse and increased thickness of the lung interstitial septa in a severity-dependent manner. AT2 cells per field were significantly reduced in both moderate and severe injury regions as compared to wt controls ( Fig. 1A-B) .
Similarly, this finding was observed in Plau -/mice ( Fig. 1B-C) . AT2 cells per field for Plau -/mice were markedly fewer than in wt animals ( Fig. 1C. n=9 mice, 5 fields per mice). This is consistent with a reduction in the yield of total AT2 cells (1.7 ´ 10 6 cells for Plau -/mice vs 2.6 ´ 10 6 cells for wt mice, Fig. 1D) . We compared CD44 + AT2 cells between healthy and ARDS patients (n = 3 patients per group). As shown by fluorescenceactivated cell sorting (FACS) data, CD44 + AT2 cells were reduced in ARDS patients (Fig. 1E-G) . These results suggest that Plau -/mice may mimic impaired fibrinolytic niches in the lung of ARDS patients.
To determine whether the Plau gene is required for re-alveolarization in vitro, we quantified spheroids formed by primary AT2 cells from both wt and Plau -/mice in parallel ( Fig. 2A) . Organoids were captured as 4´ DIC images from 4 to 12 days post seeding (Fig. S2) .
Organoids with a diameter > 50 µm (Fig. 2B) and colony forming efficiency (CFE) (Fig. 2C) were significantly decreased (n = 12, P < 0.01) in Plau -/cultures compared with wt controls. Apparently, the organoids with a diameter ranging from 50 -200 μm resulted in a reduction in colonies and CFE (Fig. 2D) . The suppression in organoid formation associated with Plau -/cells contributed to the reduction of total surface area, a clinical parameter for epithelial repair and development (Fig. 2E) . The large organoids were filled with culture medium, having a smaller lumen with thicker walls for Plau -/cultures over wt controls (Fig. 2F-G) . These data provide direct evidence for the regulation of AT2-mediated re-alveologenesis by uPA.
In addition, the organoids were further visualized with cell-permeable green fluorescent calcein to measure the cystic fibrosis transmembrane conductance regulator (CFTR) and ENaC activity (Fig. S3) . The surface area was not altered in 30 minutes in the presence of cAMP-elevating forskolin, CFTRinh, and amiloride. One potential explanation is that these organoids do not develop tight junctions intercellularly as well as polarized monolayers.
Plau gene regulates polarization and bioelectric features of AT2 monolayers. The divergent bioelectric features in polarized AT2 monolayers, including transepithelial resistance (RT) and short-circuit currents (ISC) were measured. Plau -/monolayers showed a lower ISC level compared to wt monolayer and diminished significantly by replacing Na + -free bath solution to inhibit Na + ion transport ( Fig. 3A-B) , consistent with our previous studies in the airway epithelial cells27. Moreover, Plau -/-AT2 cells showed a reduced amiloridesensitive ISC level compared to wt cells (Fig. 3C-D) . However, the amiloride affinity was not altered significantly ( Fig. 3D) . In addition, a greater RTE value on day 5 was measured in wt monolayers compared to Plau -/monolayer cultures (Fig. 3E) . Thus, the ENaC activity seems to be regulated by the Plau gene.
Plau gene up-regulates the fate of AT2 cells. To quantify AT1 and AT2 cells in colonies, 3D images were obtained by stacking Z sections of each organoid (Fig. S4) . Organotypic cultures showed a reduction in the number of epithelial cells in Plau -/organoids as compared to wt cultures ( Fig. 4A-C) . Similarly, polarized monolayers exhibited a decline in both AT1 and AT2 cells in Plau -/cells, too ( Fig. 4D-E) . Further, this reduction in AT1, AT2, and total cells in Plau -/monolayers compared to wt cultures was confirmed by FACS (Fig. 4F) .
Also, cells counted in polarized monolayers showed a similar decline in both AT1 and AT2 cells (Fig. S5A-B) .
However, there was not a difference in the geometric volume and surface of monolayers between wt and Plau -/cultures ( Fig. S5C-E) , perhaps due to the analysis of a small portion (0.12%, 0.0004 cm 2 ) of entire monolayers (0.33 cm 2 ). These results suggest that Plau regulates the renewal and differentiation of AT2 cells.
Plau gene facilitates DNA synthesis. To analyze DNA synthesis accompanied by cell proliferation, AT2 cells with active DNA synthesis were assessed by the EdU incorporation assays in both monolayers and organoids.
Monolayer cultures of Plau -/-AT2 cells showed significantly lesser EdU + cells (Fig. 5A-B) . Further, organoids were analyzed with 3D stacking images to count EdU + cells through an entire spheroid (Fig. S6) . Plau -/colonies showed a significantly reduced percentage of EdU + cells compared with wt controls (Fig. 5C-D) . The difference in EdU + cells is in agreement with the diverse proliferative AT2 cells between wt and Plau -/cultures.
The CD44 receptor and the uPA signal pathway involve in alveologenesis. The A6 peptide is derived from the connecting domain of uPA (from aa136 to aa143). The A6 peptide may serve as a mediator for uPA to bind with CD44 receptors located at the plasma membrane of AT2 cells. If so, the application of A6 peptides may restore the dysfunctional fate of Plau deficient AT2 cells. A6 peptides but not scramble controls (sA6) significantly increased spheroids ( Fig. 6A-B) . Also, A6 peptide markedly augmented the surface area of Plau -/organoids per well (1.35 ± 0.13 mm 2 vs. 0.82 ± 0.05 mm 2 for sA6 group). In addition, blockade of CD44 receptors with a neutralizing antibody reduced wt organoids and corresponding surface area (Fig. S6C) . Furthermore, the CD44 antibody resulted in a significant decrease in the number of AT2 cells in addition to a slight reduction in AT1 cells without statistical significance in wt organoids (Fig. 6C, 6E-F) . In contrast, the number of both AT1 and AT2 cells was reduced in Plau -/organoids ( Fig. 6C-F) , which were partially restored by A6 peptides. These two sets of experiments demonstrate that the binding of A6 peptides to CD44 receptors mediates the regulation of AT2 fate by uPA.
To corroborate these observations, we sorted and compared CD44 + and CD44 -AT2 cells between wt and Plau -/mice by FACS. CD44 receptors at the AT2 cell surface were recovered for 24 -36 h post enzymatic isolation.
Significantly more CD44 + AT2 cells were harvested from wt mice than those from Plau -/mice ( Fig. 7A-B) . We then cultured the organoids with these sorted CD44 + and CD44cells. Organoids developed by CD44 + , Plau -/-AT2 cells were significantly reduced (112 ± 10 organoids vs. 182 ± 12 organoids for CD44 + , Plau +/+ cells). In contrast, there was no significant difference in the number of organoids grown from CD44 -AT2 cells between wt and Plau -/groups (30 ± 1 organoids for wt and 33 ± 2 organoids for Plau -/line) ( Fig. 7C-E) . AT1 and AT2 cells in CD44 + Plau -/organoids were fewer (n=6 transwells in 3 independent experiments) than wt CD44 + organoids ( Fig. 7F-G) . However, a significant decrease was observed only in AT1 but not AT2 cells of CD44 -Plau -/organoids (Fig. 7H) . Hence, both in vivo and in vitro data suggest that the Plau gene regulates the fate of CD44 + AT2 progenitor cells in vivo and that alveologenesis in vitro is utmost predominately determined by CD44 + cells.
Fibrinolytic activity is required for re-alveolarization and tissue homeostasis. To further substantiate the role of the fibrinolytic niche in lung regeneration, we compared the proliferation and differentiation of AT2 cells between wt and Serpine1 transgenic (PAI-1 Tg ) mice. Elevated PAI-1 level is a hallmark of lung injury patients28-33. Genetically engineered humanized mice expressing a gain-of-function PAI-1 human gene mimicked the disrupted fibrinolytic niche in septic ARDS patients34 , 35. Lesser AT2 cells were harvested from PAI-1 Tg mice compared to wt (Fig. 8A) . These cells were then sorted for CD44 + and CD44cells (Fig. 8B) . A significant decrease in CD44 + AT2 cells was observed in PAI-1 Tg mice, which was similar to that in Plau -/mice (Fig. 8C) .
Moreover, CD44 + AT2 cells of PAI-1 Tg mice developed fewer organoids than wt controls (61 ± 9 organoids vs. 91 ± 7 organoids for wt controls, n=12) ( Fig. 8D-E) . Correspondingly, the total surface area of organoids of PAI-1 Tg AT2 cells was significantly reduced compared with wt cultures (Fig. 8F) . Further, duplication of PAI-1 Tg AT2 but not AT1 cells in organoids were down-regulated ( Fig. 8G-H) . On the other hand, the transepithelial resistance in wt AT2 monolayers was reduced with a maximal difference on day 5 than that in PAI-1 Tg cultures (Fig. 8I) .
Basal, amiloride-sensitive, and amiloride-resistant fractions of the short-circuit (ISC) currents in PAI-1 Tg monolayers were reduced (Fig. 8J) . However, the amiloride sensitivity remained unchanged by over-expression of the PAI-1 gene, as shown by apparent ki values (Fig. 8K) . Consistent with the results in Plau -/mice and cultures, disruption of the fibrinolytic niche by PAI-1 overexpression attenuates the rate of AT2 cells for renewal and differentiation to AT1 cells.
The primary objective of this study was to decipher the role of the fibrinolytic niche in the re-alveolarization mediated by progenitor AT2 cells and underlying mechanisms in healthy and injured lungs. We employed influenza-infected mice, Plau -/and Serpine1 Tg mouse strains, primary human AT2 cells from the human lungs of brain-dead patients with ARDS, and 3D organoids and polarized monolayers of AT2 cells to trace the cell fate in vivo and in vitro. The results demonstrate that the impaired fibrinolytic niche in influenza-infected lungs and genetically engineered mice modeling impaired fibrinolytic niche results in a significant decline in the proliferative AT2 and differentiated AT1 cells.
These results thus demonstrate the novel finding that that the fibrinolytic niche may be critical for realveologenesis. Previous studies suggest that Plau -/and PAI-1 Tg mice are more susceptible to lung fibrosis post bleomycin injury36 , 37. Clinically, impaired fibrinolytic niche, including elevated PAI-1 contents and suppressed fibrinolytic activity, are prognostic markers for ARDS patients38 , 39. Deficiency of plasminogen activators and plasminogen leads to dysfunctional repair of injured tissues (i.e., skin and muscle)40 , 41. In contrast, regeneration of injured muscle is improved by knocking out the Serpine1 gene142. The Plau gene regulates the proliferation of various cells, including renal epithelial cells43 , 44, umbilical vein endothelial cells45, vascular endothelial cells46, and keratinocytes47 , 48. Contrary to this, Serpine1 is a negative regulator of cell proliferation through the phosphatidylinositol3-kinase/Akt pathway in endothelial cells49 , 50. Our study uncovers a new signaling pathway of the fibrinolytic niche in the AT2 cell-mediated re-alveolarization. The uPA/A6/CD44 /ENaC cascade is involved in the regulation of self-renewal and differentiation of mouse and human AT2 cells, particularly the CD44 + subpopulation. This conclusion was further substantiated in Serpine1 Tg mice that have a disrupted fibrinolytic niche similar to the Plau -/mice, and both mouse lines are reasonable pre-clinical models of ARDS.
The proteolytic link between the fibrinolytic niche and ENaC proteins has been provided by other groups and us in vivo and in vitro27 , 51-53. The differences between each component of the fibrinolytic niche are described previously. For example, uPA but not tPA cleaves human gENaC subunits51. PAI-1 or antiplasmin alone does not affect ENaC activity. The slight diversity between the Plau -/and Serpine1 Tg models in this study could be due to their differential regulation of ENaC and other down-stream molecules.
CD44 is a crucial mediator for the regulation of re-alveologenesis by AT2 cells. We previously demonstrated that CD44 receptors govern cell survival and the progression of lung fibrosis via the Toll-like receptors and hyaluronan54. The connective domain of uPA has an eight L-amino acid sequence (Ac-KPSSPPEE-NH2), known as A6 peptide55. Binding of the A6 peptide with the link domain of CD44 receptors initiates the downstream signaling56. CD44 -/mice demonstrate an inflammatory phenotype characterized by increased inflammatory cell recruitment and are more susceptible to LPS induced injury. CD44 deficiency leads to impaired expression of negative regulators of TLR signaling in macrophages, an essential event in the prevention of LPS induced inflammatory responses57. Our study demonstrates a new mechanism that the CD44 receptors at the plasma membrane of AT2 cells are a key player for AT2 cell-mediated re-alveolarization. In addition to the reduction in AT2 cells in injured mouse and human lungs associated with aberrant fibrinolytic niche, both Plau -/and Serpine1 Tg AT2 cells cannot develop organoids equal to those of wt mice in vitro, mostly likely due to the lesser expression of CD44 receptors in AT2 cells as A6 peptide compensates for the loss of organoids. This is supported by our previously unreported observations that CD44 + AT2 cells have an enhanced capacity for selfrenewal and differentiation to AT1 cells. Of note, both Plau -/and Serpine1 Tg mice have lesser CD44 + AT2 cells so that the total yield of primary AT2 cells is significantly reduced compared with wt controls. The brain ENaC proteins encoded by the Scnn1a, 1b, 1d, and 1g are involved in adult neurogenesis by the transmembrane sodium signals for the deletion of Scnn1a or functional blockade by amiloride in neural stem cells strongly impairs their proliferation and differentiation58. ENaC has been functionally detected in both club cells and AT2 cells59. Whether ENaC is a member of the fibrinolytic niche for lung stem cell-mediated regeneration is unknown. We recently reported that overexpression of the Scnn1d, a pseudogene in mice, significantly improves the development of organoids in 3D cultures of primary mouse AT2 cells60. Together with abnormal ENaC activity in Plau -/and Serpine1 Tg AT2 cells, we conclude that the ENaC signal may be an essential regulator for the fate of AT2 cells in normal and injured lungs.
In summary, our study uncovers a novel role of the fibrinolytic niche in alveolarization via the uPA/A6/CD44 and the uPA/ENaC signaling pathways. This study provides new evidence for the possible prognostic relevance of the fibrinolytic niche for ARDS patients. Targeting the abnormality of the fibrinolytic niche could be a promising pharmaceutic strategy to accelerate reparative processes of injured alveolar epithelium in clinical disorders such as ARDS.
Methods, including statements of data availability and any associated accession codes and references, are available in the online version of this paper.
This work was supported by NIH grant R01 HL134828 and Donor West (California). Human AT2 cells isolated from 6 donors with FACS were slightly cultured for 24 -48 h to improve viability, and then ran FACS for sorting CD44 + population was sorted by FACS for healthy control (e) and ARDS patients (f).
(g) Average CD44 + cells (%). The data were analysed with one-way ANOVA followed by the Tukey post hoc test. n = 12 samples. * P < 0.05 vs healthy controls. Data in c and g are mean ± sem and analyzed with one-way ANOVA. ** P ≤ 0.01 and *** P ≤ 0.001 vs wt controls at the same severity. # P ≤ 0.05 and ## P ≤ 0.01 compared with controls for wt. & P ≤ 0.05 and &&& P ≤ 0.001 vs controls for Plau -/group. 
Major critical reagents are listed in the 
All mice purchased from Jackson Laboratory were maintained in a pathogen-free facility. A 12-h light/dark cycle and ad libitum supply for food and water were provided. Age, sex, and weight-matched (4-12 months) wild type (wt), Plau -/-, and Sepine1 Tg mice were sacrificed for experiments as approved by the Institute of Animal Care and Use Committee of the University of Texas Health Science Center at Tyler.
Wt and Plau -/mice were infected intranasally with a dose of 3,660 pfu influenza virus (type A/PR8/34 H1N1, Charles River, USA) diluted in 50 µl PBS per anesthetized animal. Control animals were delivered intranasally with the same amount of PBS. Both control and infected mice were euthanized 5 d post infection. The trachea was tied with a suture followed by opening the chest cavity and removing the lungs subsequently. The dissected lungs were fixed with 10% neutral buffered formalin v/v (Richard-Allan Scientific) for 72 h at room temperature.
Lung tissues were then dehydrated with increasing ethanol grades, embedded in paraffin, and sectioned at a thickness of 7 µm.
Lung sections from both influenza-infected mice and brain dead patients who had ARDS based on the Berlin criteria were deparaffinized and rehydrated in xylene and a series of ethanol at cumulating concentrations and in H2O for 5 min. To unmask antigens, tissue slides were incubated with 10 mM sodium citrate buffer (Thermo Supplementary file Scientific), pH 6.0, for 20 min at 95°C, cooled for another 20 min at room temperature, and then washed with PBS. After blocking for 1 h with 1% BSA and 4% normal goat serum, tissue sections were incubated with following antibodies: anti-proSP-C (1:500, EMD Millipore), anti-pdpn (1:300, ThermoFisher) for mouse lung tissues and 1:100 dilution for both antibodies (Abcam) for human lung slices. After 3 times washing with PBS, secondary antibodies were applied: goat anti-rabbit IgG AF488, goat anti-hamster AF568, and goat anti-mouse AF568. Tissue sections were stained with DAPI and sealed. Images were captured using a Zeiss LSM 510 confocal microscope. Images were captured containing at least 7 -8 individual 1μm optical Z-sections. Z-stacks were obtained from at least five randomly selected areas. All images were subsequently processed with ImageJ software. AT2 cells were counted using a cell counter plug-in for ImageJ software. Cells were counted in at least 5 different areas to obtain a total above 1,000 cells and then analyzed statistically.
Mouse AT2 cells were isolated from wt, Plau -/-, and Sepine1 Tg strains of C57BL/6 animals (Jackson Laboratory, USA) as previously reported with modifications1. Briefly, mice were euthanized and exsanguinated, followed by perfusing lungs with 10 -20 mL DPBS until pink lungs turned to white. The trachea was cannulated with a 20G catheter to instill 1.5 -2.0 mL dispase followed by 0.5 mL of 1% low melting point agarose. The lungs were dissected and incubated in 50 U/mL dispase solution for 45 min at room temperature. The lungs were gently teased in DMEM/F-12 + 0.01% DNase I and incubated for 10 min at room temperature. Cells were passed through a serial filtration (100, 40, 30, and 10 µm cell strainers) and centrifuged at 300 × g for 10 min at 4 o C. Cells were resuspended in 10 mL medium (DMEM/F-12 + 10% FBS + P/S) supplemented with biotinylated antibodies, rat anti-mouse CD16/32 (0.65 µg/million cells), rat anti-mouse CD45 (1.5 µg/million cells), and rat anti-mouse Ter119 (5 µg) and incubated for 30 min at 37 o C on an incubator shaker at 60 rpm. Resuspended cells were then incubated with pre-washed streptavidin-coated magnetic particles for 30 min at room temperature to remove undesired cells. Selected cells were then resuspended in 10 mL medium (DMEM/F-12 + 10% FBS + P/S) and incubated for 30 min at 37 o C in a sterile Petri dish to allow residual fibroblasts to adhere to the bottom of the dish.
Suspended cells were transferred in plates pre-coated with mouse IgG for 2 h in a 5% CO2 incubator to remove Supplementary file macrophages. Unattached cells were collected and centrifuged at 300 × g for 10 min at 4 o C. Cell pellets were then resuspended in a complete mouse medium (CMM: DMEM/F-12 supplemented with 2 mM L-glutamine, 0.25% bovine serum albumin, 10 mM HEPES, 0.1 mM non-essential amino acids, 0.05% ITS, 100 µg/mL primocin, and 10% newborn calf serum). The viability of harvested AT2 cells was assessed by the trypan blue exclusion assay followed by cell counting for the yield.
The purity of isolated AT2 cells was confirmed by the Papanicolaou stain, immunofluorescent staining with anti-proSP-C antibody, and fluorescence-activated cell sorting (FACS) with anti-EpCAM antibody. 1) PAP stain:
freshly isolated AT2 cells suspended in DPBS + 10% FBS (2 ´ 10 5 cells/mL) were centrifuged at 600 rpm for 4 min by a Shandon CytoCentrifuge. Slides were air-dried overnight and stained using a modified PAP stain method2. Briefly, slides were stained with hematoxylin for 3.5 min. After rinsing in dH2O, slides were incubated in lithium carbonate for 2 min and rinsed with dH2O again. Slides were dehydrated in serial ethanol dilutions, then in xylene: ethanol (1:1) for 30 s, and finally in 100% xylene for 60 s. Slides were mounted with the Permount mounting medium. Randomly selected images were captured from 6 independent experiments with an Olympus BX41 microscope (40 ´). AT2 cells with large nuclei and blue colored granules spread in the cytoplasm were counted and calculated for purity (%). 2) Immunofluorescent stain: cytospanned freshly isolated AT2 cells and cells cultured on coverslips for 48 h were incubated with rabbit anti-proSP-C (1:500) overnight at 4 o C. Secondary antibody, either Alexa Flour 488 or 568-conjugated anti-rabbit IgG, was added to recognize proSP-C antibody. (Fig. S1A-C) . Cell viability was approximately 94%. Further, FACS showed that approximately 95% of cells were EpCAM positive (Fig. S1D-E) .
Human epithelial AT2 cells were isolated from six human lungs not used for transplantation by the Northern California Transplant Donor Network as previously described3 , 4. Three of the lungs were normal and three of the lungs met ARDS criteria before they were harvested. After cold preservation at 4°C, the right middle lobe was selected for cell isolation if no apparent signs of consolidation or hemorrhage by gross inspection were seen. The cell suspension was further incubated on Petri dishes coated with human IgG antibodies (Sigma) overnight at 37°C to remove the remaining macrophages and fibroblasts. Cell viability was assessed by the trypan blue exclusion method. AT2 cell purity was evaluated by Papanicolaou (Pap) staining.
Freshly isolated cells were seeded on either collagen IV (for mouse AT2, 10 μg/cm 2 ) or collagen I (for human AT2, 10 μg/cm 2 ) coated plates for 24 -36 h to revive CD44 expression diminished by digestive enzymes. Both unattached and attached (trypsinized) cells were collected and blocked with 1% BSA, 4% normal goat serum in PBS. Cells were stained with AF488-EpCAM (BioLegend), APC anti-human CD44 (BioLegend), and their respective isotypes. Cells were sorted using a Beckman Coulter MoFlo high-speed cell sorter. Unstained, isotype Supplementary file 6 and single-color controls were performed. The gates for CD44 and EpCAM were set based on the results of isotype, and single-color controls were run in parallel. The results were analyzed using FlowJo 10.1 software.
Transwell inserts (Costar 3470: 0.4 μm pore size, 0.33 cm 2 area; Corning Costar, USA) were pre-coated with mouse laminin 1 at 10 μg/cm 2 (for mouse AT2 cells; Trevigen, USA) for 4 -6 h at 37 o C or with rat tail collagen I at 10 μg/cm 2 (for human AT2 cells; Trevigen, USA) for 1 h at 37 o C. Freshly isolated AT2 cells were seeded at 
Anti-pdpn and anti-sftpc antibodies were used to detect AT1 and AT2 cells, respectively. Fluorescence conjugated secondary antibodies, goat anti-hamster AF568 and goat anti-rabbit IgG AF488 were used. Fluorescent images were projected with a Zeiss LSM 510 confocal microscope and stacked with a Fiji plug-in for ImageJ. Monolayers and organoids were scanned for Z sections with optimal depth from top to bottom. Images were stacked for pdpn and sftpc signal separately to count the number of positive cells precisely with a cell counter plug-in of ImageJ.
8 Alternatively, cells were collected from organoids and monolayers and sorted by FACS. Each slide was scanned for at least 6 different fields (n = 3 animals/experiments). For 3D organoids, all Z sections were stacked from top to bottom and saved as .avi files.
EdU assay for DNA synthesis AT2 cells with active DNA synthesis in organoids and monolayers were detected with a Click-iT TM EdU assay kit. Organoids and monolayers from both wt and Plau -/groups were stained with the Click-iT TM EdU Alexa flour 488 following the manufacturer's instructions. Images were captured and analyzed for the percentage of EdU + cells in different experimental groups. Ten randomly selected images across the monolayer from 3 independent experiments were captured and counted for total cells and EdU + portion. For organotypic cultures, all 3 organoid types were scanned (n = 5 colonies for each type). The z sections were stacked separately for DAPI (blue) and
EdU (green) to produce a 3D structure of organoids for counting total and EdU + cells, respectively, using a cell counter plug-in for the ImageJ. The percentage of EdU + cells was calculated for each group, and the difference among groups was compared statistically.
A6 and scrambled A6 (sA6) peptides were synthesized by Genscript. Stock solutions (2 mM) were prepared by dissolving peptides in water according to the manufacturer's instruction. For 3D matrigel cultures, sorted Plau -/-AT2 cells were preincubated with either A6 or sA6 peptide (1 µM) for 30 min at room temperature. Wt AT2 cells were treated with 30 ng/mL CD44-blocking antibody for 30 min at room temperature. The same concentrations of A6, sA6 peptides, and CD44-blocking antibody were added to both the matrigel/medium mix and the culture medium placed under the transwell inserts. The culture medium was changed every 48 h.
For analysis of AT2 cell proliferation and differentiation, AT2 organoids from different experimental groups were isolated from matrigel with dispase (10 U/mL) and dissociated in 0.25% trypsin-EDTA to get a single-cell suspension. Cells were then stained with antibodies AF488 conjugated EpCAM, APC conjugated ICAM, and APC conjugated PDPN. Gates for both colors were set by unstained cells and isotype controls for each antibody.
Supplementary file 9 We have used a strategy to use double color staining to enhance the separation of AT1 and AT2 cells in the cell suspension of organoids. Cells were analyzed by FACSCaliber TM (BD, USA), and the results were analyzed using FlowJow 10.1 software.
Data were presented as mean ± s.e.m. No animals were excluded. Normality tests were performed to determine whether the data were parametric or not. If the data were normally distributed and the variance between groups was not significantly different, mean differences in measured variables between the experimental and control group were assessed with the Student's two-tailed t-tests or one-way ANOVA followed by the Tukey's or Bonferroni's post hoc test. Otherwise, the Mann-Whitney U test was applied for analyzing non-parametric results.
Two-way ANOVA followed by Sidak's multiple-comparison test was used for multiple comparisons. Meanwhile, the actual power of the sample size was analyzed. Mean differences were considered statistically significant at the levels of P < 0.05, P < 0.01 and P < 0.001. Origin Pro 2018 was used for statistical analysis and plotting. 