WO2021062412A1 - Lung fibrosis model and methods of using the same - Google Patents

Lung fibrosis model and methods of using the same Download PDF

Info

Publication number
WO2021062412A1
WO2021062412A1 PCT/US2020/053164 US2020053164W WO2021062412A1 WO 2021062412 A1 WO2021062412 A1 WO 2021062412A1 US 2020053164 W US2020053164 W US 2020053164W WO 2021062412 A1 WO2021062412 A1 WO 2021062412A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
lung
pats
alveolar
cell
Prior art date
Application number
PCT/US2020/053164
Other languages
French (fr)
Inventor
Purushothama Rao TATA
Aleksandra TATA
Arvind KONKIMALLA
Yoshihiko Kobayashi
Original Assignee
Duke University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Duke University filed Critical Duke University
Priority to US17/763,991 priority Critical patent/US20220341915A1/en
Priority to CA3156163A priority patent/CA3156163A1/en
Priority to EP20868029.8A priority patent/EP4034142A4/en
Priority to JP2022519551A priority patent/JP2022550141A/en
Publication of WO2021062412A1 publication Critical patent/WO2021062412A1/en

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/42Respiratory system, e.g. lungs, bronchi or lung cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0688Cells from the lungs or the respiratory tract
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082Supracellular entities, e.g. tissue, organisms
    • G01N33/5088Supracellular entities, e.g. tissue, organisms of vertebrates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/135Platelet-derived growth factor [PDGF]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00Coculture with; Conditioned medium produced by
    • C12N2502/27Lung cells, respiratory tract cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00Use of cells in diagnostics
    • C12N2503/04Screening or testing on artificial tissues

Definitions

  • the present disclosure provides the identification of a transitional cell state in alveolar regeneration, a model to ablate lung alveolar type-1 cells that leads to lung fibrosis and emphysema, a scalable, ex vivo lung fibrosis model that uses co-cultured lung fibroblasts and pre-alveolar type-1 transitional cell state (PATS) cells for the use of disease modeling, drug discovery, and/or drug screening, and methods of using same.
  • PATS pre-alveolar type-1 transitional cell state
  • AEC2s cuboidal surfactant-producing type-2 alveolar epithelial cells
  • AECls cuboidal surfactant-producing type-2 alveolar epithelial cells
  • Recent studies have identified a subset of AEC2s that are enriched for active Wnt signaling and have higher ‘sternness’ compared with Wnt-inactive AEC2s.
  • the present disclosure is based, in part, on the discovery by the inventors that a transitional cell state exists in alveolar regeneration, which lends itself to models for the use for understanding lung disease, drug discovery, and/or drug screening.
  • One aspect of the present disclosure provides a method of generating a lung injury organoid model, the method comprising: i) providing a co-culture of cells in a culture medium adapted for the culture of said cells, wherein the co-culture comprises pre-alveolar type-1 transitional cell state (PATS) cells and alveolar fibroblasts; ii) contacting the culture medium with one or more agents; and iii) analyzing the biological effect of the agent on at least one expression marker of the PATS cells and/or the alveolar fibroblasts relative to a control cell culture medium that has not been contacted by the agent.
  • PATS pre-alveolar type-1 transitional cell state
  • the PATS cells are isolated from diseased tissue.
  • the diseased tissue is chronic obstructive pulmonary disease (COPD) lung tissue, pulmonary fibrosis lung tissue, idiopathic pulmonary fibrosis lung tissue, emphysema lung tissue, lung cancer tissue, Sarcoidosis lung tissue, interstitial pneumonia lung tissue, sepsis lung tissue, lung tissue having viral and bacterial infections, acute respiratory distress syndrome lung tissue, or bronchopulmonary dysplasia lung tissue.
  • COPD chronic obstructive pulmonary disease
  • the PATS cells are generated by exposing lung epithelial cells to an injury-causing agent (e.g., bleomycin, diphtheria toxin (DT), tamoxifen, irradiation, a virus, a bacterium, or a fungus) in vivo or in vitro.
  • an injury-causing agent e.g., bleomycin, diphtheria toxin (DT), tamoxifen, irradiation, a virus, a bacterium, or a fungus
  • the alveolar fibroblasts are lipofibroblasts.
  • the alveolar fibroblasts have disease-state characteristics.
  • the disease-state characteristic comprises lipofibroblast cells that express ACTA2, the presence of myofibroblast cells, and/or cell changes from a seriate shape to a banded shape.
  • the culture medium comprises a platelet- derived growth factor receptor (PDGFR) ligand.
  • PDGFR platelet- derived growth factor receptor
  • the culture medium comprises an agent that is capable of maintaining or modulating PDGFR expression.
  • Another aspect of the disclosure provides a method of generating an ex vivo model for lung fibrosis, the method comprising: i) providing a culture of AEC1 cells that have undergone ablation in vivo in a culture medium adapted for the culture of said cells; ii) contacting the culture medium with one or more agents; and iii) analyzing the biological effect of the agent on at least one expression marker of the AEC1 cells relative to a control cell culture medium that has not been contacted by the agent.
  • the AEC1 cells have undergone at least one round of ablation. In other embodiments, the AEC1 cells have undergone ablation alone or in combination with other injury models. In some embodiments, the AEC1 cells have undergone repeated ablation, for example, over the course of about three days to about two weeks.
  • the AEC1 cells have undergone ablation by being exposed to bleomycin, diphtheria toxin (DT), asbestos, chemical toxic agents, tamoxifen, a virus, a bacterium, or a fungus.
  • the AEC1 cells express aSMA and/or ACTA2.
  • the AEC1 cells exhibit characteristics of a fibromyocyte cell.
  • the alveolar cells are selected from the group consisting of tracheal basal cells, bronchiolar secretory cells (also known as club cells or Clara cells), club variant cells, alveolar epithelial progenitor (AEP) cells, clara variant cells, distal lung progenitors, p63+ Krt5- airway cells, lineage negative epithelial progenitors, bronchioalveolar stem cells (BASCs), Sox9+ p63+ cells, neuroendocrine progenitor cells, distal airway stem cells, submucosal gland duct cell, induced pluripotent stem cell-derived lung stem cells and alveolar type 2 epithelial (AEC2) cells.
  • tracheal basal cells also known as club cells or Clara cells
  • AEP alveolar epithelial progenitor
  • AEP alveolar epithelial progenitor
  • Clara cells clara variant cells
  • distal lung progenitors p63+ Krt5
  • the alveolar cells are obtained from the lung of a patient suffering from chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, idiopathic pulmonary fibrosis, emphysema, lung cancer, Sarcoidosis, interstitial pneumonia, sepsis, a viral infection, a bacterial infection, a fungal infection, acute respiratory distress syndrome, and/or bronchopulmonary dysplasia.
  • COPD chronic obstructive pulmonary disease
  • pulmonary fibrosis pulmonary fibrosis
  • idiopathic pulmonary fibrosis emphysema
  • lung cancer emphysema
  • Sarcoidosis interstitial pneumonia
  • sepsis a viral infection
  • bacterial infection bacterial infection
  • a fungal infection a fungal infection
  • acute respiratory distress syndrome acute respiratory distress syndrome
  • bronchopulmonary dysplasia bronchopulmonary dysplasia
  • identifying pre-alveolar type-1 transitional cell state (PATS) cells or PATS-like cells further comprises contacting the alveolar cells with an agent (e.g., bleomycin, diphtheria toxin (DT), tamoxifen, a virus, a bacterium, or a fungus).
  • an agent e.g., bleomycin, diphtheria toxin (DT), tamoxifen, a virus, a bacterium, or a fungus.
  • Yet another aspect of the present disclosure provides a method of generating an in vivo tissue fibrosis model, the method comprising: i) introducing a genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling in a subject; and/or ii) administering an agent to a subject that can modulate PDGF-PDGFR signaling; and iii) analyzing cells from the subject to determine the biological effect of genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling and/or the biological effect of the agent that can modulate PDGF-PDGFR signaling relative to a control subject.
  • PDGFR PDGF ligands and/or PDGF receptor
  • the agent that can modulate PDGF-PDGFR signaling comprises a PDGFR inhibitor, a PDGFR agonist, a PDGF inhibitor, or a PDGF agonist.
  • Yet another aspect of the disclosure provides a method of generating an in vivo tissue fibrosis model, the method comprising: i) introducing a genetic loss of RUNX1, RUNX2, RUNX3, or CBFb in a subject; and/or ii) administering to the subject a RUNX1, RUNX2, RUNTX3, or CBFb inhibitor; and iii) analyzing cells from the subject to determine the biological effect of the genetic loss of RUNX1, RUNX2, RUNX3, or CBFp and/or the biological effect of the RUNX1, RUNX2, RUNX3, or CBFP inhibitor relative to a control subject.
  • the inhibitor is Ro 5-3335, AI-10-49, or a neutralizing antibody against RUNX1, RUNX2, RUNX3, and/or CBFp.
  • the in vivo model is a mouse model.
  • the in vivo model is a disease model for fibrosis in the lung, liver, kidney, skin, intestines, or bone marrow.
  • UMAP shows major cell populations including epithelial cells (5,163 cells), fibroblasts (5,686 cells), and some minor populations such as endothelial cells (66 cells) and macrophages (33 cells) in alveolar organoids (right panel).
  • FIG. 1C is an integrated UMAP showing cells derived from alveolar organoids (4,787 cells), in vivo homeostatic mouse lung (1,878 cells) and LPS-treated mouse lung (14,323 cells) (left). Expression of indicated genes in the integrated UMAP (right).
  • FIG. 1C is an integrated UMAP showing cells derived from alveolar organoids (4,787 cells), in vivo homeostatic mouse lung (1,878 cells) and LPS-treated mouse lung (14,323 cells) (left). Expression of indicated genes in the integrated UMAP (right).
  • FIG. IE are UMAP plots showing the expression of the indicated genes in epithelial populations in cultured alveolar organoids.
  • FIG. IE are UMAP plots showing the expression of the indicated genes in epithelial populations in cultured alveolar organoids.
  • FIG. 1H is a schematic of alveolar organoid culture using fibroblasts and AEC2s. Tmx, tamoxifen.
  • FIG. 2 is a schematic showing the experimental workflow of bleomycin-induced lung injury in Ctgf-GFP mice.
  • FIGS. 5A-5F shows that PATS cells originate from alveolar stem cells after lung injury in vivo.
  • FIG. 5A is a schematic showing an experimental workflow for the sequential administration of tamoxifen, followed by bleomycin injury and tissue collection for analysis using Sftpc-CreER ; R26R-tdTomato mice.
  • FIG. 5B show a time course of immunostaining for CLDN4, Sftpc- tdt and LGALS3 after bleomycin injury. Scale bars, 10 pm. Images are representative of three mice.
  • 5D is a graph showing cell length of CLDN4+, CLDN4+LGALS3+ and LGALS3+ cells.
  • FIG. 5E is a schematic showing the pneumonectomy injury model.
  • FIG. 5F is a schematic showing transition from AEC2 to AECl through different PATS subtypes.
  • FIGS. 6A-6H show lineage tracing revealed that PATS cells generate AECls.
  • FIG. 6C is a schematic showing the experimental workflow for the sequential administration of bleomycin (injury) or PBS (control), followed by tamoxifen (to label Krt 19-expressing cells) in Krtl9-CreER ; R26R- tdTomato mice.
  • FIG. 6D are images showing immunostaining for SFTPC, Krtl9-tdt and AGER.
  • the white arrows indicate AGER+Av7/9-tdt+ cells. Scale bars, 30 pm. Images are representative of three mice, repeated independently with similar results.
  • FIG. 6E are images showing co-staining for SFN, Krtl9-tdt and AGER. The white arrows indicate SFN+A77/9- tdt+ cells. Scale bars, 50 mih. Images are representative of three mice, repeated independently with similar results.
  • FIG. 6D are images showing immunostaining for SFTPC, Krtl9-tdt and AGER. The white arrows indicate AGER+Av7/9-tdt+ cells. Scale bars, 30 pm. Images are representative of three mice, repeated independently with similar results.
  • FIG. 6E are images showing co-staining for SFN, Krtl9-tdt and AGER. The white arrows indicate
  • FIGS. 8A-8I shows that PATS cells undergo stretch-induced DNA damage and senescence in vivo and ex vivo.
  • FIG. 8A are images showing staining for b-galactosidase activity in bleomycin-injured (right) and control (left) lungs. Scale bars, 100 pm.
  • FIG. 8A are images showing staining for b-galactosidase activity in bleomycin-injured (right) and control (left) lungs. Scale bars, 100 pm.
  • FIG. 8B are images showing immunostaining for CDKN1A, Sftpc- tdt and AGER in control (top) and bleomycin-injured (bottom) lungs.
  • the arrowheads indicate CDKN1A+ cells.
  • the white dotted lines indicate AECls and the dotted lines outline tdt+ cells. Scale bars 50 pm.
  • FIG. 8C are images showing immunostaining for gH2AC, Sftpc- tdt and AGER in control (top) and bleomycin-injured (bottom) lungs. Scale bars 50 pm.
  • FIG. 8C are images showing immunostaining for gH2AC, Sftpc- tdt and AGER in control (top) and bleomycin-injured (bottom) lungs. Scale bars 50 pm.
  • FIG. 8C are images showing immunostaining for gH2AC, Sftpc- tdt and AGER in control (top)
  • FIG. 8E are images showing staining for b-galactosidase activity in DT-treated and control lungs. Scale bars, 100 pm.
  • FIG. 8F are images showing immunostaining for CDKN1A, LGALS3 and AGER in control (top) and DT-treated lungs (bottom). The arrowheads indicate CDKN1A+ cells. Scale bars 20 pm.
  • FIG. 8E are images showing staining for b-galactosidase activity in DT-treated and control lungs. Scale bars, 100 pm.
  • FIG. 8F are images showing immunostaining for CDKN1A, LGALS3 and AGER in control (top) and
  • FIG. 8H is a schematic of 2D culture of AEC2s.
  • FIG. 81 are images showing immunostaining for Sftpc-G FP, gH2AC and AGER in 2D culture of AEC2s. The white arrows indicate cells with DNA-damage marker. Scale bars 20 pm Images are representative of three mice, repeated independently with similar results. DAPI stains nuclei. [0038] FIGS.
  • FIG. 9C is a schematic of alveolar organoid culture treated with Nutlin-3a.
  • FIG. 9D are images showing immunostaining for Ki67 (green) and AGER (grey) in control or Nutlin-3a treated alveolar organoids. Scale bar: 30 pm.
  • FIG. 9C is a schematic of alveolar organoid culture treated with Nutlin-3a.
  • FIG. 9D are images showing immunostaining for Ki67 (green) and AGER (grey) in control or Nutlin-3a treated alveolar organoids. Scale bar: 30 pm.
  • FIG. 9E is a schematic showing the experimental workflow for the sequential administration of tamoxifen to delete TP53 in AEC2s, followed by bleomycin injury (day 0) in Sftpc-CreER ; R26R-tdTomato ; 7/yGJfl/fl or control mice ( Sftpc-CreER ; R26R-tdTomato ; Trp53+/+ ).
  • FIG. 9F is a graph showing the proportion of the total Sftpc- tdt+ cells from for SFTPC, Sftpc- tdt and AGER, and CLDN4, Sftpc- tdt and LGALS3 in control and ///GJ-knockout mice that are AGER+.
  • FIGS. 10A-10E shows transcriptional control of PATS by TP53 signaling.
  • FIG. 10A is a graph showing the distribution of H3K4me3 peaks in PATS marker gene loci in PATS (upper line) and homeostatic AEC2s (lower line).
  • FIG. 10B is a schematic showing the experimental workflow for bleomycin-induced lung injury in Sftpc-CreER ; R26R- tdTomato; Ctgf-GFP mice, followed by PATS sorting and ChIP analysis.
  • FIG. IOC is ChIP enrichment for TRP53, H3K4me3 (active promoter) and H3K27ac (active enhancer) shown in IGV.
  • FIG. 11C shows Hematoxylin and Eosin staining on IPF lung tissue sections. Representative image depicting fibrotic (right side square box) and non-fibrotic (left side square box) regions in IPF lung. Scale bar indicates 200 pm.
  • FIG. 11D are images showing immunostaining for ACTA2, SFN and SFTPC in healthy human lungs (left), and non-fibrotic (middle) and severe-fibrotic (right) regions of lungs from patients with IPF.
  • FIG. 11F are images showing immunostaining for SFN, CLDN4 and AGER in a fibrotic region of an IPF lung.
  • FIG. 11H are images showing immunostaining for SFN, KRT17 and TP63 in a region of an IPF lung with severe fibrosis (arrowheads indicate SFN+KRT17+TP63+ cells).
  • FIG. 11K are violin plots showing expression of IPF -relevant genes in the indicated cell types/states in control
  • FIG. 12 is a schematic describing emergence of a novel transitional cell state in alveolar stem cell-mediated epithelial regeneration and its persistence in disease pathogenesis.
  • Alveolar stem cells replicate in response to damage and generate a novel transitional cell state which normally matures into functional alveolar type-1 epithelial cells.
  • the previously unappreciated transitional state is directly regulated by TP53 signaling, vulnerable to DNA damage and undergoes a transient senescent state. This state is enriched in human fibrotic lungs.
  • FIG. 13A is a schematic of AEC1 specific ablation model.
  • FIG. 13B are images showing immunostaining for SFN, ACTA2, LGALS3, and DAPI for cells that had undergone four rounds of AEC1 ablation and analyzed on day 3, day 6, day 10, and day 60.
  • FIG. 14A is a schematic of repeated AEC1 ablation.
  • FIG. 14B are images showing H&E and trichome after 22 days.
  • FIG. 14C are images showing immunostaining for LGALS3, ACTA2, and DAPI of ablated tissue after 22 days.
  • FIG. 14D is a schematic showing morphological changes with repeated AEC1 ablation.
  • FIG. 14E are images showing widespread fibrosis in single ablation of AEC1 in combination with pneumonectomy induced injury.
  • FIG. 15 is a schematic and coculture of PATS or AEC2s with lipofibroblasts. The data on the right demonstrates that coculture of PATS but not AEC2s induces the conversion of lipofibroblasts into myofibroblasts.
  • FIG. 16 is a violin plot showing that PDGFA is predominantly expressed in AECl.
  • PDGFA transcripts are also observed in the transition cells during AEC2s differentiation into AECl.
  • FIG. 17 are images showing immunostaining for aSMA, PDGFRA, and DAPI in control and PDGFRA-CreER/PDGFRA ⁇ ox mice. These data demonstrate that loss of PDGFRA in fibroblasts is sufficient to induce their conversion into myofibroblasts.
  • FIG. 18 is a violin plot showing that Runxl is upregulated in fibroblasts post- AECl ablation and following bleomycin injury.
  • FIG. 19 are images showing immunostaining for Runxl, aSMA, Pdgfa, and DAPI on the AECl ablation model, repeated AECl ablation model, and PDGFRA deletion. These data demonstrate that RUNX1 expression is increased in single and repeat AECl ablation mouse models as well as in targeted loss of PDGFRA in fibroblasts.
  • FIG. 20A are images showing immunostaining for GFP, aSMA, Pdgfa, and DAPI in RUNX1 deficient cells.
  • FIG. 20B are images showing immunostaining for GFP, aSMA, Pdgfa, and DAPI in RUNX1 deficient cells containing TGF-B. These data demonstrate that loss of RUNX1 blocks fibroblasts conversion into myofibroblasts.
  • Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article.
  • an element means at least one element and can include more than one element.
  • “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • disease includes, but is not limited to, any abnormal condition and/or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease, or by internal dysfunctions, such as cancer, cancer metastasis, and the like.
  • an effective amount or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • treatment refers to the clinical intervention made in response to a disease, disorder, or pathogen infection manifested by a patient or to which a patient may be susceptible.
  • the aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, disease causative agent (e.g., bacteria, viruses, a chemotherapeutic agent, radiation), or condition and/or the remission of the disease, disorder or condition.
  • a novel alveolar epithelial type-I cell also known as pneumocyte type- I
  • the inventors established a first genetic model that develops lung fibrosis.
  • the inventors have developed an ex vivo lung fibrosis models that uses co-culture of above described novel transitional cell state (PATS) and alveolar fibroblasts.
  • PATS novel transitional cell state
  • These novel in vivo and ex vivo fibrosis models are scalable and allows for disease modeling and drug screening. These features are often found in progressive lung disease and one can observe enrichment of cells in a PATS-like state in human fibrotic lungs, indicating persistence of the transitional state in fibrosis.
  • the studies provided herein thus implicate a transient state associated with senescence in normal epithelial tissue repair and its abnormal persistence in disease conditions.
  • organs refers to self-organized three-dimensional (3D) structures or entities that are derived from stem cells grown in culture. Organoids cultures can replicate the complexity of an organ or can express selected aspects of an organ, such as by producing only certain types of cells. Alternatively, at certain stages before differentiation, they can be comprised only of stem cells.
  • Stem cells are cells that have the ability to both replicate themselves (self-renew) and give rise to other cell types. When a stem cell divides, a daughter cell can remain a stem cell or become a more specialized type of cell, or give rise to other daughters that differentiate into one or more specialized cell types.
  • Two types of mammalian stem cells are: pluripotent embryonic stem cells that are derived from undifferentiated cells present in blastocyst or pre-implantation embryos, and adult stem cells that are found in adult tissues or organs.
  • Adult stem cells can maintain the normal turnover or regeneration of the tissue or organ and can repair and replenish cells in a tissue or organ after damage.
  • stem cell refers to an undifferentiated cell that is capable of proliferation and self-renewal and of giving rise to progenitor cells with the ability to generate one or more other cell types, or to precursors that can give rise to differentiated cells.
  • the daughter cells or progenitor or precursor cells that can give rise to differentiated cells In certain cases the daughter cells or progenitor or precursors cells can themselves proliferate and self-renew as well as produce progeny that subsequently differentiate into one or more mature cell types.
  • a progenitor cell refers to a cell that is similar to a stem cell in that it can either self-renew or differentiate into a differentiated cell type, but a progenitor cell is already more specialized or defined than a stem cell.
  • Stems cells of the present disclosure can be derived from any animal, including but not limited to, human, mouse, rat, rabbit, dog, pig, sheep, goat, and non-human primates.
  • the stem cells that can be used in the methods and models of the present disclosure can be normal (e.g., cells from healthy tissue of a subject) or abnormal cells (e.g., transformed cells, established cells, or cells derived from diseased tissue samples).
  • the cells used in the methods and models of the present disclosure can be derived from lung stem cells. Division of lung stem cells can promote renewal of the lung’s structure.
  • lung stem cells include, but are not limited to tracheal basal cells, bronchiolar secretory cells (also known as club cells or Clara cells), club variant cells, alveolar epithelial progenitor (AEP) cells, clara variant cells, distal lung progenitors, p63+ Krt5- airway cells, lineage negative epithelial progenitors, bronchioalveolar stem cells (BASCs), Sox9+ p63+ cells, neuroendocrine progenitor cells, distal airway stem cells, submucosal gland duct cell, induced pluripotent stem cell-derived lung stem cells and alveolar type 2 epithelial (referred to herein as AEC2 or AT2) cells.
  • tracheal basal cells also known as club cells or Clara cells
  • AEP alveolar epithelial progenitor
  • AEP alveolar epithelial progenitor
  • Clara cells clara variant cells
  • distal lung progenitors p63
  • the cells used in the methods and models of the present disclosure can be derived from basal stem cells from organs including, skin, mammary gland, esophagus, bladder, prostate, ovary, and salivary glands.
  • Another aspect of the present disclosure provides a model system to ablate lung alveolar type-I cells (also known as pneumocyte type-I) that leads lung fibrosis, emphysema (similar to the one observed in human combined pulmonary fibrosis and emphysema syndrome (CPFE)).
  • lung alveolar type-I cells also known as pneumocyte type-I
  • CPFE pulmonary fibrosis and emphysema syndrome
  • the model system comprises an animal model.
  • the animal comprises a mouse.
  • the animal comprises any animal that has lungs.
  • Another aspect of the present disclosure provides a method of generating a lung injury organoid model, the method comprising, consisting of, or consisting essentially of: i) providing a co-culture of cells in a culture medium adapted for the culture of said cells, wherein the co-culture comprises pre-alveolar type-1 transitional cell state (PATS) cells and alveolar fibroblasts; ii) contacting the culture medium with one or more agents; iii) analyzing the biological effect of the agent on at least one expression marker of the PATS cells and/or the alveolar fibroblasts relative to a control cell culture medium that has not been contacted by the agent.
  • PATS pre-alveolar type-1 transitional cell state
  • Alveolar fibroblasts are cells that are involved in the lung repair/fibrosis process in the lung. Alveolar fibroblasts can synthesize extracellular matrix and collagen and produce stroma in animal tissues. Examples of alveolar fibroblasts include but are not limited to, human fetal lung fibroblast cells (e.g., HFL1, MRC-5, 2BS, and WI38), Chinese hamster lung fibroblast (V-97) cell, C57BL/6 mouse primary lung fibroblasts, and CC-2512 cells.
  • human fetal lung fibroblast cells e.g., HFL1, MRC-5, 2BS, and WI38
  • V-97 Chinese hamster lung fibroblast
  • C57BL/6 mouse primary lung fibroblasts CC-2512 cells.
  • the alveolar fibroblasts are lipofibroblasts. In some embodiments, the alveolar fibroblasts have disease-state characteristics.
  • disease-state characteristics refers to phenotypic or genotypic properties of a cell that indicates the cell is a disease cell. These characteristics can be the expression of certain markers or morphological changes in the cell that indicate disease state.
  • disease-state characteristic of alveolar fibroblasts comprises lipofibroblast cells that express ACTA2, the presence of myofibroblast cells, and/or cell changes from a seriate shape to a banded shape.
  • the PATS cells are isolated from diseased tissue.
  • diseased tissue refers to the tissue of subject (e.g., human or animal model) that is abnormal compared to healthy tissue.
  • the diseased tissue can be obtained from the tissue of a riving or deceased subject.
  • Example of diseased tissue include, but are not limited to, chronic obstructive pulmonary disease (COPD) lung tissue, pulmonary fibrosis lung tissue, idiopathic pulmonary fibrosis lung tissue, emphysema lung tissue, lung cancer tissue, Sarcoidosis lung tissue, interstitial pneumonia lung tissue, sepsis lung tissue, ring tissue having viral and bacterial infections, acute respiratory distress syndrome lung tissue, and bronchopulmonary dysplasia lung tissue.
  • COPD chronic obstructive pulmonary disease
  • the PATS cells are isolated from pulmonary fibrosis lung tissue or idiopathic pulmonary fibrosis lung tissue.
  • Lung cancer cells can be isolated from a subject suffering from a lung cancer.
  • the lung cancer cells can be isolated from a primary lung tumor or a secondary lung tumor (e.g., a cancer that starts in another tissue and metastasizes to the lungs).
  • Examples of lung cancer cells include but are not limited to small cell lung cancer cells or non-small cell lung cancer cells, including but not limited to, small cell carcinoma, combined small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, pancoast tumor cells, neuroendocrine tumor, or lung carcinoid tumor cells.
  • lung cancer cell lines can also be used in the methods and models of the present disclosure.
  • Lung cancer cell lines that can be used in the methods and models of the present disclosure can be found on the ATCC website.
  • lung cancer cell lines include but are not limited to, EML4-ALK Fusion- A549 Isogenic cell line, NCI-H838 [H838], HCC827, SK-LU-1, HCC2935, HCC4006, NCI-H1819 [H1819], NCI-H676B [H676B], Hs 618.
  • HLF-a Hs 913T, GCT [Giant Cell Tumor]
  • SW 900 SW-900, SW900]
  • LL/2 LLC1
  • HBE135-E6E7 Tera-2
  • NCI-H292 H292]
  • sNF02.2 NCI-H1703 [H1703]
  • NCI- H2172 H2172
  • NCI-H2444 H2444
  • NCI-H2135 NCI- H2347 [H2347]
  • NCI-H810 H810]
  • NCI-H1993 H1993
  • NCI-H1792 NCI-H1792
  • the PATS cells are isolated from healthy tissue (e.g., normal lung tissue).
  • tissue refers to an aggregate of cells that can be of a particular kind together with their intercellular substance that form the structural material of the subject from which the tissue is obtained.
  • the PATS cells can be isolated and purified from the tissue.
  • the PATS cells can be generated by exposing lung epithelial cells (e.g., AEC2s or AECls) to an injury-causing agent in vivo or in vitro.
  • lung epithelial cells e.g., AEC2s or AECls
  • injury-causing agent refers to an agent that is capable of converting or transitioning a healthy cell to a diseased-cell state.
  • injury-causing agent refers to an agent that is capable of ablating a normal cell such that a disease-state cell arises.
  • the injury-causing agent can be, for example, a chemotherapy agent (e.g., bleomycin, tamoxifen, actinomycin-D, mitomycin, taxanes, and gemcitabine), an exotoxin (e.g., diphtheria toxin (DT)), a virus, a bacterium, a fungus, asbestos, chemical toxic agents, or irradiation.
  • a chemotherapy agent e.g., bleomycin, tamoxifen, actinomycin-D, mitomycin, taxanes, and gemcitabine
  • an exotoxin e.g., diphtheria toxin (DT)
  • DT diphtheria toxin
  • virus e.g., a virus, a bacterium, a fungus, asbestos, chemical toxic agents, or irradiation.
  • the injury-causing agent can be a pathogen (e.g., a bacterium, virus, or fungus) that is capable of infecting lung tissue of humans or any animal with lungs.
  • a pathogen e.g., a bacterium, virus, or fungus
  • Bacteria that can infect lungs include, but are not limited to Bordetella pertussis, Streptococcus pneumonia , Haemophilus influenza , Staphylococcusaureus, Moraxellacatarrhalis, Streptococcuspyogenes, Pseudomonas aeruginosa,
  • Fungus that can infect lungs include, but are not limited to, Aspergillosis.
  • the culture medium and/or subjects (e.g., mice) used in the methods and models of the disclosure comprise a platelet-derived growth factor receptor (e.g., platelet-derived growth factor receptor- A, platelet-derived growth factor receptor-B, or platelet-derived growth factor receptor-AB).
  • a platelet-derived growth factor receptor e.g., platelet-derived growth factor receptor- A, platelet-derived growth factor receptor-B, or platelet-derived growth factor receptor-AB.
  • the culture medium and/or subjects (e.g., mice) used in the methods and models of the disclosure comprises a platelet-derived growth factor receptor (PDGFR) ligand.
  • the PDGFR ligand is platelet-derived growth factor (PDGF).
  • PDGF is a growth factor that can regulate cell growth and division.
  • the PDGFR ligand is platelet-derived growth factor subunit A (PDGF-A), platelet-derived growth factor subunit B (PDGF-B), platelet-derived growth factor subunit C (PDGF-C), platelet-derived growth factor subunit D (PDGF-D), and/or platelet-derived growth factor subunit AB (PDGF-AB).
  • Human genes that encode proteins that belong to the PDGF family include FIGF, PDGF A, PDGFB, PDGFC, PDGFd, PGF, VEGF, VEGF41, VEGFB, and VEGFC.
  • the methods and models of the present disclosure comprise the use of PDGF signaling peptides, PDGF receptors, PDGF agonists, PDGFR agonists, PDGF antagonists, PDGFR antagonists, or any agent that is capable of modulating the PDGF-PDGFR signaling pathway.
  • the methods and models of the present disclosure comprise the use of a pharmacological blockade of PDGF ligands and/or PDGFR receptors.
  • Modulators of the PDGF-PDGFR signaling pathway include substrates, inhibitors, activators, neurotransmitters, agonists, antagonists, inverse agonists, inverse antagonists, partial agonists, and partial antagonists.
  • Modulators of the PDGF-PDGFR signaling pathway include, but are not limited to, PDGF isoform antagonists, PDGFR activation blockers, agents capable of increasing or decreasing the expression of PDGF and/or PDGFR in a cell, chemical compounds, such as endogenous metabolites, non-endogenous metabolites, synthetic chemical compounds, polypeptides, amino acid residues, nucleic acids, siRNA, and antibodies (e.g., neutralizing antibodies).
  • agonist refers to a modulator that binds to a receptor (e.g., PDGFR) or protein (e.g., PDGF) and activates the receptor or protein to produce a biological response.
  • a receptor e.g., PDGFR
  • protein e.g., PDGF
  • the term “antagonist” refers to a modulator or ligand that blocks, impedes, or dampens a biological response by binding to and blocking a receptor or protein rather than activating it.
  • inverse agonist refers to a modulator or ligand that binds to the same receptor as an agonist but induces a biological response opposite to that agonist.
  • the culture medium used in the methods and models comprises an agent that is capable of maintaining PDGFR (e.g., PDGFR-A, PDGFR-B, or PDGFR-AB) expression in a cell.
  • PDGFR e.g., PDGFR-A, PDGFR-B, or PDGFR-AB
  • the agent capable of maintaining PDGFR-A expression in a cell is Ro 5-3335 or AI-10-49.
  • the agent capable of maintaining PDGFRA expression in a cell is any molecule that can modulate RUNX (e.g., RUNX1, RUNX2, or RUNX3) or core binding factor (CBF) related family of proteins (e.g., CBF-b).
  • RUNX e.g., RUNX1, RUNX2, or RUNX3
  • CBF core binding factor
  • Molecules that modulate RUNX and/or CBF can be molecules that interfere with the protein-protein interaction between RUNX and CBF.
  • These modulators can be inhibitors of RUNX (e.g., RUNX1, RUNX2, or RUNX3) or inhibitors CBF-b.
  • RUNX or CBF modulators include, but are not limited to, Ro 5-3335, AI-10-49, CADD522, muramyl dipeptide, L-quebrachitol, L-ascorbic acid 2-phosphate, ascorbic acid 2-phosphate magnesium, 2-pyridyl benzimidazole AI-4-57, RUNX1/MTG8 fusion protein, and a neutralizing antibody against RUNX1, RUNX2, RUNX3, and/or CBFb, and/or derivatives thereof,
  • agent refers to a small molecule, protein, peptide, gene, compound, or other pharmaceutically active ingredient.
  • the agent can be used for the treatment, prevention, or mitigation of a disease (e.g., lung fibrosis).
  • PATS cell expression markers refer to gene transcripts that are expressed on PATS cells or PATS-like cells.
  • PATS markers are unique to the PATS cell state and are not typically expressed on related cell states (e.g., AEC2s or AECls).
  • PATS cell markers can comprise CLDN4, KRT19, SFN, LGALS3, SOX4, S100A2, PTGS2, KRT17, KRT8, CALSl, MMP7, PRSS2, IGFBP7, COL1A1, MDK, TAGLN, GDF15, TM4SF1 TP63, and/or CTSE.
  • ablation refers to the removal or destruction of a tissue or cell, or their function. Ablation can be caused by surgery, hormones, drugs, radiofrequency, heat, or other methods that cause destruction of the tissue or cell.
  • the alveolar cells e.g. AEC1 cells
  • the injury-causing agent is bleomycin, diphtheria toxin (DT), tamoxifen, a virus, a bacterium, or a fungus.
  • the ablated cells express aSMA and/or ACTA2. In some embodiments, the ablated cells exhibit characteristics of a fibromyocyte cell.
  • Yet another aspect of the present disclosure provides, a method of identifying pre- alveolar type-1 transitional cell state (PATS) cells, the method comprising: i) obtaining lung cells; and ii) screening the cells for one or more markers, wherein the presence of the one or more markers indicates the presence of PATS cells.
  • PATS pre- alveolar type-1 transitional cell state
  • the lung cells can be selected from the group consisting of tracheal basal cells, bronchiolar secretory cells (also known as club cells or Clara cells), club variant cells, alveolar epithelial progenitor (AEP) cells, clara variant cells, distal lung progenitors, p63+ Krt5- airway cells, lineage negative epithelial progenitors, bronchioalveolar stem cells (BASCs), Sox9+ p63+ cells, neuroendocrine progenitor cells, distal airway stem cells, submucosal gland duct cell, induced pluripotent stem cell-derived lung stem cells and alveolar type 2 epithelial (AEC2) cells.
  • bronchiolar secretory cells also known as club cells or Clara cells
  • AEP alveolar epithelial progenitor
  • AEP alveolar epithelial progenitor
  • Clara cells clara variant cells
  • distal lung progenitors p63+ Krt5
  • the lung cells can be selected from the group consisting of AEC2 cells, AEC1 cells, or alveolar macrophages.
  • the lung cells are obtained from the lung of a patient suffering from chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, idiopathic pulmonary fibrosis, emphysema, lung cancer, Sarcoidosis, interstitial pneumonia, sepsis, a viral infection, a bacterial infection, a fungal infection, acute respiratory distress syndrome, and/or bronchopulmonary dysplasia.
  • COPD chronic obstructive pulmonary disease
  • pulmonary fibrosis pulmonary fibrosis
  • idiopathic pulmonary fibrosis emphysema
  • lung cancer emphysema
  • Sarcoidosis interstitial pneumonia
  • sepsis a viral infection
  • bacterial infection bacterial infection
  • a fungal infection a fungal infection
  • acute respiratory distress syndrome acute respiratory distress syndrome
  • bronchopulmonary dysplasia bronchopulmonary dysplasia
  • the method of identifying pre-alveolar type-1 transitional cell state (PATS) cells further comprises contacting the alveolar cells with an injury-causing agent.
  • the agent is bleomycin, diphtheria toxin (DT), tamoxifen, a virus, a bacterium, or a fungus.
  • Another aspect of the present disclosure provides an ex vivo lung fibrosis model that uses co-culture of above described novel transitional cell state (PATS) and alveolar fibroblasts that is scalable and allows for disease modeling and drug screening.
  • PATS novel transitional cell state
  • Another aspect of the present disclosure provides a method of generating an in vivo model of generating fibrosis in lung and other tissues.
  • the in vivo models can be used, for example, to test the ability for therapeutic agent to treat or prevent disease.
  • the in vivo models of present disclosure can utilize a variety of animal subjects (e.g., mouse, rat, rabbit, monkey, or pig or any animal that is capable of modeling fibrosis).
  • An in vivo model refers to the use of a whole living animal to investigate the effect of an agent and to study the biological processes of disease (e.g., lung fibrosis).
  • Another aspect of the present disclosure provides a method of generating an in vivo model of generating fibrosis in lung and other tissues, the method comprising: i) introducing a genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling in a subject; and/or ii) administering an agent to a subject that can modulate (e.g., block, enhance, increase, or decrease) PDGF -PDGFR signaling; and iii) analyzing cells from the subject to determine the biological effect of the genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling and/or the biological effect of the agent that can modulate (e.g., block enhance, increase, or decrease) PDGF -PDGFR signaling relative to a normal control subject.
  • the agent that can block PDGF-PDGFR signaling comprises a PDGFR inhibitor, neutralizing antibodies against PDGF ligands and/or
  • Another aspect of the present disclosure provides a method of generating an in vivo model of generating fibrosis in lung and other tissues, the method comprising: i) introducing a genetic loss of RUNX1, RUNX2, RUNX3, or CBFP in a subject; and/or ii) administering an agent toa subject that can modulate (e.g., inhibit) RUNX1, RUNX2, RUNX3, or CBFP; and iii) analyzing cells from the subject to determine the biological effect of genetic loss of RUNX1, RUNX2, RUNX3, or CBFp and/or the biological effect of modulating RUNX1, RUNX2, RUNX3, or CBFP relative to a normal control subject.
  • the RUNX1, RUNX2, RUNX3, or CBFP modulator is a small molecule inhibitor are a neutralizing antibody against RUNX1, RUNX2, RUNX3, and/or CBFp.
  • the models of the present disclosure are fibrosis models of lung fibrosis, cardiac fibrosis, liver fibrosis, kidney fibrosis, intestinal fibrosis, skin fibrosis, and/or bone marrow fibrosis.
  • the models of the present disclosure are lung fibrosis models that can model the impact of lung fibrosis on other tissues in the body, such as cardiac, liver, kidney, intestinal, skin, and/or bone marrow tissues.
  • the cells of the models of the present disclosure are lung, heart, kidney, liver, intestine, skin, or bone marrow.
  • mice Both male and female mice aged between 8-16 weeks were used for experiments. All of the mice were C57BL/6 unless otherwise indicated. The following mice were used for experiments: Sftpctml(cre/ERT2)Blh ( Sftpc-CreER ), Krtl9tml(cre/ERT)Ggu/J ( Krtl9 - CreER ), Rosa26R-CAG-lsl-tdTomato (crossed with Sftpc-CreER ), B6.Cg- Gt(ROSA)26Sortm 14(CAG-tdTomato)Hze/J ( R26R-tdTomato ) (crossed with Krtl9-CreER ), I giSl ⁇ J ' PC Gl ⁇ P) Heat (Sftpc-GFP)51, B6.Cg-Agertm2.1(cre/ERT2) Blh/J ( Ager-CreER ), B6-Gt(ROSA
  • R26R-DTR Mki67tml.lCle/J ( Mki67 RFP ), Tg(Ctgf EGFP)FX156Gsat ( Ctgf-GFP) ⁇ 5 and TP53fl/fl (mixed background).
  • 3-5 doses of 2 mg tamoxifen (Sigma- Aldrich) per 20 g of body weight were given via oral gavage or intraperitoneal injection.
  • mice For bleomycin-induced lung injury, 2.5 U kg-1 bleomycin was administered intranasally 2 weeks after the tamoxifen injection and the mice were monitored daily. Mice that were administered PBS served as controls. The mice were killed at different times after bleomycin injury.
  • mice Two weeks before DT administration, Ager-CreER ; R26R-DTR mice received tamoxifen via intraperitoneal injection.
  • One dose of 3 pg DT (Millipore, 322326) was administered via intraperitoneal injection and the mice were killed 6 d later for tissue collection and analysis.
  • the cell pellet was resuspended in red-blood-cell lysis buffer (155 mM NH4C1, 12 mM NaHC03 and 0.1 mM EDTA), incubated for 2 min and then filtered through a 40-pm strainer.
  • the cell pellet was resuspended in DMEM/F12 + 2% BSA and stained with the following antibodies: EpCAM (eBioscience, G8.8), PDGFRa (BioLegend, APA5) and Lysotracker (Thermo Fisher, L7526) as described previously56. Sorting was performed using a BD FACS Vantage SE, SONY SH800S or Beckman Coulter MoFlo Astrios EQ system. [00142] Alveolar organoid culture.
  • the DNA polymerase for the pre- amplification step was replaced by Terra PCR direct polymerase (Takara, 639271).
  • the other processes were performed as described in the original droplet-based scRNA-seq protocol.
  • the libraries were sequenced using a HiSeq X system with 150-bp paired-end sequencing.
  • Cell clusters of AEC2s, AECls, transitional AEC2s and KRT5-KRT17+ cells were extracted and analysed. Markers for each cluster, obtained using the FindAllMarkers command in Seurat, were utilized for identifying specific signalling pathways and gene ontology through Enrichr59. Z-scores were calculated based on the combined score in Kyoto Encyclopedia of Genes and Genomes (KEGG) to compare enrichment of signalling and ontology across different cell clusters. The results were displayed in heatmap format, generated using the R package pheatmap vl.0.12. Scaled data in Seurat object were extracted and the mean values of the scaled score of the gene members in each pathway were calculated and shown in EIMAP as enrichment of signaling pathways.
  • KEGG Kyoto Encyclopedia of Genes and Genomes
  • the gene-member lists of the utilized pathways were obtained from KEGG pathways60 and AmiG06T
  • PATS CD31-CD45-CD140a-CD326+C7g/-GFP+
  • AEC2s CD31-CD4 5-CD326+Lysotracker+Mki67-RFP- cells
  • T7 adaptor ligation was performed for 2 h and the samples then were split to give approximately 7,000 cells per antibody for histone-modification ChIP-seq.
  • TP53 we used approximately 40,000 cells for each replicate sample.
  • the samples were incubated with histone H3 (H3; 1 pi; Active Motif, 39763), histone H3 lysine 36 trimethylation (H3K36me3; 1 m ⁇ ; Active Motif, 61101), histone H3 lysine 4 trimethylation (H3K4me3; 1 m ⁇ ; Abeam, ab8580), histone H3 lysine 27 acetylation (H3K27ac; 1 pi; Active Motif, 39133) or TP53 (5 m ⁇ ; Cell Signaling Technology, 2524T) antibodies overnight at 4 °C.
  • DNA was purified followed by T7-RNA polymerase mediated in vitro transcription at 37 °C for 3 h. Reverse transcription was performed as described in the original protocol, followed by library preparation using Terra direct PCR polymerase (TaKaRa, 639271). Two experimental replicates were performed for each cell population. The libraries were sequenced (at least 5 c 106 reads of 150-bp paired-ends per sample) using the Hiseq X or NovaSeq 6000 systems.
  • FASTQ files were generated using Bcl2fastq. Additional demultiplexing for Mint- ChlP FASTQ files were performed using Je63. Low-quality reads were trimmed out from the FASTQ files using trimmomatic v0.38. The reads were mapped on the mmlO genome reference using BWA65. The packages were run through a pipeline called MintChIP (https://github.com/jianhong/MintChIP). HOMER66 was used to generate bedGraph files to visualize peaks in Integrative Genomics Viewer (IGV).
  • MintChIP https://github.com/jianhong/MintChIP
  • Peak calling for H3K4me3 was performed using the function getDifferentialPeaksReplicates.pl -region -size 1000 -minDist 2000 -C 0 -L 50 of HOMER with normalization by H3. Motif analysis was performed using the fmdMotifsGenome.pl function of HOMER. A chart of called peaks for H3K4me3 was generated using deepTools68. Called peaks for each genomic locus from different cell populations were prepared in Affinity Designer.
  • IPF IPF lungs
  • diagnoses were determined according to ATS/ERS consensus criteria. All studies were approved by the local Institutional Review Boards (IRB; Vanderbilt IRB nos 060165 and 171657, and Western IRB no. 20181836).
  • the diagnosis of IPF was evaluated by a surgical pathology team. The specimens were washed thoroughly in PBS before inflation and immersion in 4% paraformaldehyde at 4 °C overnight. The specimens were subsequently washed in PBS until the appearance of blood was minimal, followed by incubation in 30% sucrose at 4 °C. The samples were then incubated with OCT compound at a 1:1 ratio for 1 h at 4 °C before embedding in OCT. Section with a thickness of 7-9 pm were used for histological analysis.
  • Lungs and alveolar organoids were prepared as described previously. Briefly, the tissues were fixed with 4% paraformaldehyde at 4 °C for 4 h (for lungs) or at room temperature for 30 min (for organoids), and then embedded in OCT compound or paraffin. Sectioned samples (10 pm) were utilized for staining following incubation at 95 °C for 10-15 min for antigen retrieval using 10 mM sodium citrate.
  • pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D Systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176, 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181-200, 1:500), CLDN4 (Invitrogen, 36- 4800, 1:200; Proteintech, 16165-1-AR, 1:500), GFP (Novus Biologicals, NB 100-1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500), SOX4 (Invitrogen, MA5-31424, 1:250), SFN (Invitrogen, PA5-95056, 1:250; Proteintech, 66251-1-Ig, 1:500; Abeam, ab77187, 1:200), ACTA2 (Sigma, C6198,
  • Proximity ligation in situ hybridization was performed as described previously69. Briefly, frozen mouse lung sections were fixed with 4.0% paraformaldehyde for 20 min, treated with 20 pg ml-1 proteinase K for 9 min at 37 °C and dehydrated with an up-series of ethanol. The sections were incubated with gene-specific oligos in hybridization buffer (1 M sodium trichloroacetate, 50 mM Tris pH 7.4, 5 mM EDTA and 0.2 mg ml-1 heparin) for 2 h at 37 °C. Common-bridge and circle probes were added to the sections and incubated for 1 h, followed by a T4 DNA ligase reaction for 2 h.
  • hybridization buffer (1 M sodium trichloroacetate, 50 mM Tris pH 7.4, 5 mM EDTA and 0.2 mg ml-1 heparin
  • Rolling-circle amplification was performed using phi29 polymerase (Lucigen, 30221) for 12 h at 37 °C. Fluorophore-conjugated detection probe was applied and incubated for 30 min at 37 °C, followed by mounting in medium containing 4,6-diamidino-2-phenylindole (DAPI).
  • Example 1 Single-cell transcriptomics reveals previously unknown alveolar epithelial cell states in ex vivo organoids
  • AEC2s proliferate and give rise to AECls in response to lung injury. Moreover, AEC2s spontaneously generate AECls in alveolar organoids. However, the molecular mechanisms and transitional cell states underlying the differentiation of AEC2 into AECl are poorly understood. To address these questions, we purified AEC2s and PDGFRa+ fibroblasts to set up alveolar organoids. Single-cell transcriptome analysis was performed on cells isolated from the day- 10 organoid culture (FIG. 1A). Uniform manifold approximation and projection (UMAP) identified two major clusters, consisting of Epcam+ epithelial cells and Vim+Pdgfra+ fibroblasts (FIG. IB).
  • UMAP Uniform manifold approximation and projection
  • Example 2 PATS cells emerge in vivo after alveolar injury.
  • Example 3 Lineage tracing reveals that PATS cells originate from AEC2s.
  • Tdt+ cells co-expressing AGER displayed a flat, thin and elongated morphology at the later times, confirming that AEC2s transition to AECls.
  • lineage-labeled cells co-expressing PATS markers including SFN, CLDN4 and LGALS3 — had distinct morphological features (rounded, ‘stretched’ and elongated) during the repair process (FIG. 5B).
  • Example 4 Lineage-tracing analysis reveals PATS cells generate AECls [00173] Multiple lines of data from the experiments presented earlier suggest that PATS cells are en route to AEC1. To test this, an algorithm tool called Velocytol8 was used, which allows the prediction of cell differentiation trajectories based on the ratios between spliced and unspliced messenger RNA. Strong RNA velocities (trajectory) originating from the PATS1 Ctgf+ population to AEC1 through the PATS2 Lgals3+ population was observed (FIG. 6A and FIG. 6B).
  • a CreER allele of Krtl9, a gene enriched in PATS, combined with R26R-tdTomato ( Krtl9-tdt ) was then used to carry out lineage tracing after bleomycin injury to test whether the same trajectory occurs in regenerating alveoli in vivo.
  • mice were injured with bleomycin and tamoxifen was administered 7 d later to label A7v/9-expressing cells and their progeny (FIG. 6C). Immunofluorescence for AEC2, AEC1 and PATS markers was then performed.
  • Pathway enrichment analysis revealed that TP53, TNF-NF-KB, ErbB, HIF1, Hippo-YAP, cell-cycle arrest, cytoskeletal dynamics, tight-junction and TGFP signaling are activated in PATS cells compared with other populations (FIG. 7A and FIG. 7B).
  • Substantial enrichment for genes representative of cellular senescence and DNA-damage-response pathways was unexpectedly found (FIG. 7C and FIG. 7B).
  • Example 6 PATS cells naturally exhibit DNA damage and senescence during alveolar regeneration in vivo.
  • Example 7 PATS cells are vulnerable to mechanical stretch-induced DNA damage [00179] Previous studies have shown that ionizing radiation, oxidative stress and the mechanical forces that occur during cell migration and stretching can all cause DNA damage. Pathway analysis revealed an enrichment for genes involved in cytoskeletal changes but not oxidative stress. Given that AEC2 differentiation into AECl requires extensive cell spreading and cytoskeletal dynamics it was hypothesized that PATS cells experience mechanical stretching that can lead to DNA damage. To test this, AEC2s were purified from SFTPC GFP mice and cultured them on a plastic surface (two-dimensional (2D) culture), conditions under which AEC2s spread and differentiate into AECls (FIG. 8Hb).
  • 2D two-dimensional
  • Example 8 Genetic loss of function and pharmacological activation of TP53 signaling dysregulates AEC2 to AEC1 differentiation via PATS during alveolar injury-repair [00180] These data suggest that activation of TP53 signaling is associated with PATS in injured lungs. Previous studies in other tissues have suggested that TP53 signaling promotes differentiation and suppresses stem cell self-renewal.
  • Example 9 TP53 binds to PATS-associated gene loci and controls their expression [00183]
  • Chrin immunoprecipitation (ChIP) analysis was performed for the histones H3K4me3, H3K36me3 and H3K27ac to identify active promoters, transcribing genes and enhancers, respectively.
  • PATS cells and AEC2s from bleomycin-treated Ctgf-GFP and control mice were isolated, respectively, and used for histone-marks analysis.
  • H3K4me3 enrichment was found in genes corresponding to cell-type-specific promoters ( Sftpc in AEC2 and Fnl in PATS).
  • H3K4me3 peaks overlapping with the transcriptional start sites and promoters of transcripts specific to PATS cells from bleomycin-damage-induced lungs compared with AEC2s from controls (FIG. 10A).
  • Predicted binding sites were found for TP53 in the Mdm2 enhancer and Fas promoter, two well-known direct targets of TP53. Together, these data implicate a direct role for TP53 in the transcriptional control of PATS-specific genes.
  • ChIP analysis was performed for TP53 in PATS cells to empirically test whether TP53 directly regulates transcriptional programs associated with PATS. Given that the scRNA-seq analysis revealed that TP53 signaling is highly enriched in the Ctgf+ subset of PATS, a Sftpc-CreER ; R26R-tdTomato ; Ctgf-GFP mouse model was generated to purify AEC2-lineage-derived PATS cells for ChIP analysis.
  • PATS cells (tdt+GFP+) were purified from the lungs of Sftpc-CreER ; R26R-tdTomato ⁇ Ctgf-GFP mice on day 8 post bleomycin administration and performed multiplexed indexed T7 ChIP (Mint-ChIP) analysis for TP53 (FIG. 10B).
  • Example 10 PATS-associated gene-expression signatures and signaling pathways are enriched in human fibrotic lungs
  • the human IPF-specific cluster was therefore named as ‘PATS- like’ cells (FIG. 11A and FIG. 11B).
  • Other transcripts that were highly enriched in the PATS-like population are CALD1, PRSS2, MMP7 and S100A2.
  • immunofluorescence analysis was perfomed on healthy human and fibrotic and non- fibrotic regions of lungs with IPF (FIG. 11C).
  • KRT17 has been recently shown to be expressed in basal cells of the normal lung and basaloid-like cells in IPF lungs (Habermann et al., 2019, doi.org/10.1101/753806; Adams et al., 2019, doi.org/10.1101/759902).
  • TP63 another marker for basal cells, specifically in the fibrotic, but not in non-fibrotic, regions of the IPF lungs was also found (FIG. 11H, FIG. 111). It was found that PATS-like markers are highly enriched in regions of severe fibrosis (FIG. 11D-FIG. Ill), coinciding with high levels of COL1A1 and accumulation of myofibroblasts (FIG. 7D).
  • CDKN1A , CDKN2A, and MDM2 Gene ontology and pathway analysis revealed an enrichment for components of p53 signaling ( CDKN1A , CDKN2A, and MDM2, as well as CCND1, CCND1, SNF, GADD45A, BID, BAX, PMAIP1, BBC3, BCL2L1, PERP, ZMAT3, IGFBP3, CD82, THBS1, SERPINB5, RRM2B, SESN3, STEAP3, TP73 ), DNA-damage checkpoint (.
  • p53 signaling CDKN1A , CDKN2A, and MDM2, as well as CCND1, CCND1, SNF, GADD45A, BID, BAX, PMAIP1, BBC3, BCL2L1, PERP, ZMAT3, IGFBP3, CD82, THBS1, SERPINB5, RRM2B, SESN3, STEAP3, TP73
  • DNA-damage checkpoint DNA-damage checkpoint
  • PATS-like cells that are specifically present in the fibrotic regions of IPF lungs. Similar to murine PATS cells, PATS- like cells in human lungs are characterized by enrichment for genes associated with cellular senescence, TP53 signaling and TGFp -regulated genes — all of which are known to be involved in fibrosis in multiple organs, including the lung (Zhang et al., 2015, Cell Death Dis. 6, el847; Lipson et al., 2012, Fibrogenesis Tissue Repair 5, S24). In contrast, some differences were found in gene-expression signatures between murine and human IPF- specific PATS-like cells.
  • TP63 TP63
  • KRT17 KRT17
  • COL1A1 RNA-velocity projections from human scRNA-seq data suggest that these KRT17+TP63+ cells originate from AEC2s. Immunofluorescence for KRT17 and TP63 further suggested that these cells are surrounded by PATS-like cells within the same alveoli.
  • the transitional cell state has clinical implications, notably in lung diseases associated with DNA damage (da Silva, et al., 2013, BMC Med. Genet. 14, 93; Sauler et al., 2018, Eur. Respir. J. 52, 1701994).
  • the PATS population is enriched for SOX4 (known to regulate cytoskeletal genes), which is induced following DNA damage and is critical in TP53 stabilization and function.
  • SOX4 known to regulate cytoskeletal genes
  • Example 11 A novel and tunable lung injury model recapitulates transient fibrosis in vivo
  • Pulmonary fibrosis is a complex disease, involving alterations in the epithelial, mesenchymal, and immune populations, all in turn resulting in dramatic extracellular matrix changes and tissue remodeling.
  • the initiation of fibrosis is poorly understood, and has been assumed to be a result of a combination of all of these individual factors. Yet, it is still unknown whether damage to a single cell type can result in fibrotic initiation.
  • FIG. 13A cell type specific epithelial ablation models were developed (FIG. 13A), and it was found that ablation of a single cell type, AECls, results in rapid activation of alpha smooth muscle actin (aSMA or ACTA2, a marker of tissue fibrosis) in resident Pdgfra+ lipofibroblasts, while maintaining expression of Pdgfra and the stellate shape characteristic of lipofibroblasts. As these cells do not exhibit the morphological changes normally seen in myofibroblasts, they are termed “fibromyocytes” (FIG. 13B).
  • fibromyocytes FIG. 13B
  • fibromyocytes are transient, and as epithelial homeostasis is restored, ACTA2 expression gradually decreases.
  • specific ablation of AEC1 cells resulted in an intermediate PATS state, with rapid activation by day 3, and peaking at day 6.
  • PATS and fibromyocytes co-occurred, with PATS directly overlying the fibromyocyte-converted mesenchyme. PATS did not exist in significant numbers by day 10, suggesting that epithelial tissues resolve rapidly following this cell-type specific injury.
  • Example 12 Persistence of PATS drives lipofibroblasts to myofibroblasts in vivo [00194] Considering that a single round of AEC1 ablation resulted in only transient, reversible fibrosis, fibromyocyte transition, and no myofibroblasts, it was thought that repeated AEC1 ablation (a surrogate for repeated alveolar injury) may further drive fibrosis (FIG. 14A). The preliminary histological analysis of mouse lungs after repeat AEC1 ablation indeed revealed increased ACTA2 expression in the alveoli, as well as morphological changes in ACTA2+ cells from a stellate to a “banded” morphology (FIG. 14B, FIG. 14C, and FIG. 14D).
  • Example 13 A scalable co-culture model recapitulates lung fibrosis ex vivo [00196]
  • PATS play a direct role in myofibroblast conversion.
  • culture conditions were established that would support the growth of both epithelial and lipofibroblasts and maintain their normal phenotype.
  • Lipofibroblasts were then co-cultured with AEC2 cells (derived from SFTPC-GFP mice) or with PATS (isolated from CTGF-GFP mice (GFP + /EpCAM + ) cells following bleomycin injury) for seven days and analyzed by immunostaining.
  • lipofibroblasts spontaneously expressed ACTA2 only in the presence of PATS but not with AEC2s.
  • Example 14 Lipofibroblast identity is maintained by tonic, AECl-derived PDGFA signaling
  • PDGFA expression increases as cells transition from AEC2s, through the PATS state, and finally to AECls, suggesting that PDGFA is an important signal for epithelial-mesenchymal homeostatic maintenance (FIG. 16).
  • PDGFA maintains lipofibroblast identity, and loss of AECl and resulting loss of PDGFA is a cue for lipo-to-myofibroblast initiation.
  • AEC2s differentiate into AECls, PDGFA expression is restored, and this subsequently returns fibromyocytes to their native lipofibroblast state.
  • Example 15 Disruption of PDGFA signaling is sufficient for initiation of myofibrogenesis
  • Runxl is a master transcriptional regulator of myofibrogenic fate [00200] In order to determine master regulators effecting lipofibroblast to myofibroblast transitions, transcription factors were interrogated that are preferentially upregulated in post injury conditions. Runxl, a transcription factor known to be linked to fibrotic progression in multiple organs, was found to be strongly upregulated post-AECl ablation and following bleomycin injury (FIG. 18). Importantly, Runxl expression increases across all models of fibrotic injury models described here, including AEC1 ablation, repeated AEC1 ablation, and PDGFRA deletion (FIG. 19).
  • Example 17 Genetic and pharmacological inhibition of RUNX1 blocks TGFb induced fibrogenesis

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Pulmonology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Physiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)

Abstract

The present disclosure provides a newly-identified transitional cell state in alveolar regeneration, models to ablate lung alveolar type-1 cells that leads to lung fibrosis and emphysema, a scalable, an ex vivo lung fibrosis model that uses co-cultured lung fibroblasts and pre-alveolar type-1 transitional cell state (PATS) for the use of disease modeling and drug screening, and methods of using same.

Description

LUNG FIBROSIS MODEL AND METHODS OF USING SAME
CROSS REFERENCE TO RELATED APPLICATIONS [001] This application claims priority to U.S. Provisional Patent Application Serial Number 62/975,294, filed February 12, 2020, U.S. Provisional Patent Application Serial Number 62/906,241, filed September 26, 2019, the contents of each of which are hereby incorporated by reference in their entirety.
FEDERAL FUNDING LEGEND
[002] This invention was made with government support under National Institutes of Health, National Heart, Lung, and Blood Institute Grant Nos. R00HL127181; R01HL153375. The Federal Government has certain rights to this invention.
BACKGROUND
[003] Field
[004] The present disclosure provides the identification of a transitional cell state in alveolar regeneration, a model to ablate lung alveolar type-1 cells that leads to lung fibrosis and emphysema, a scalable, ex vivo lung fibrosis model that uses co-cultured lung fibroblasts and pre-alveolar type-1 transitional cell state (PATS) cells for the use of disease modeling, drug discovery, and/or drug screening, and methods of using same.
Description of the Related Art
[005] Adult stem cells undergo dynamic changes in response to tissue damage. These changes include resurgence from a quiescent or poised state, onset of proliferation, activation of new gene expression and a return to homeostasis. In many cases, repair also involves changes in epithelial cell shape, for example, through transient stretching and expansion to cover areas of damage or denudation. Studies of stem cell regeneration usually focus on understanding how cells select new differentiation programs in response to signals from the niche. However, much less is known about the significance of changes in parameters such as cell shape and spreading and whether they involve the transient and tightly controlled expression of genes usually associated with pathological states including DNA repair and senescence.
[006] In the lung, the maintenance of the alveolar epithelium at homeostasis and its regeneration after injury are fuelled by cuboidal surfactant-producing type-2 alveolar epithelial cells (AEC2s), which can self-renew and differentiate into very large thin type-1 alveolar epithelial cells (AECls), specialized for gas exchange. Recent studies have identified a subset of AEC2s that are enriched for active Wnt signaling and have higher ‘sternness’ compared with Wnt-inactive AEC2s. Such differences in alveolar progenitor cell subsets have been attributed to differences in microenvironmental signals; in this case, to the vicinity of PDGFRa-expressing fibroblasts, which produce ligands to activate Wnt signaling in AEC2s. Recent studies have also implicated other signaling pathways — including BMP, Notch, TGFP, YAP and NF-KB — in the proliferation and differentiation of AEC2s, both at steady state and in response to alveolar injury. However, the precise mechanisms by which the cuboidal AEC2s orchestrate their dramatic changes in cell shape, structure and mechanical properties as they convert into thin flat AECls, remain elusive. In addition, the cellular mechanisms that drive AEC2s to express genes associated with cell senescence, a feature commonly observed in most progressive pulmonary diseases, remain unknown.
[007] Here, using organoid cultures and single-cell transcriptome studies, previously unknown, distinct cell states encompassing the transition between AEC2s and AECls were uncovered. Moreover, murine lineage tracing, coupled with injury-repair models, has revealed the existence of similar transition states in vivo. The study reveals signaling pathways that control these transition states. As described herein, these transitional states exhibit DNA-damage responses and express senescence-related genes en route to AECl. Mechanistically, the use of genetic loss of function, pharmacological gain of function and genomic binding assays revealed a direct transcriptional control of PATS by TP53 signaling. Importantly, these transitional states correlate with abnormal epithelial cells associated with defective fibrotic foci in human lungs with progressive pulmonary fibrosis.
BRIEF SUMMARY OF THE DISCLOSURE [008] The present disclosure is based, in part, on the discovery by the inventors that a transitional cell state exists in alveolar regeneration, which lends itself to models for the use for understanding lung disease, drug discovery, and/or drug screening.
[009] One aspect of the present disclosure provides a method of generating a lung injury organoid model, the method comprising: i) providing a co-culture of cells in a culture medium adapted for the culture of said cells, wherein the co-culture comprises pre-alveolar type-1 transitional cell state (PATS) cells and alveolar fibroblasts; ii) contacting the culture medium with one or more agents; and iii) analyzing the biological effect of the agent on at least one expression marker of the PATS cells and/or the alveolar fibroblasts relative to a control cell culture medium that has not been contacted by the agent.
[0010] In some embodiments of the disclosure, the PATS cells are isolated from diseased tissue. In some embodiments of the disclosure, the diseased tissue is chronic obstructive pulmonary disease (COPD) lung tissue, pulmonary fibrosis lung tissue, idiopathic pulmonary fibrosis lung tissue, emphysema lung tissue, lung cancer tissue, Sarcoidosis lung tissue, interstitial pneumonia lung tissue, sepsis lung tissue, lung tissue having viral and bacterial infections, acute respiratory distress syndrome lung tissue, or bronchopulmonary dysplasia lung tissue.
[0011] In some embodiments of the disclosure, the PATS cells are generated by exposing lung epithelial cells to an injury-causing agent (e.g., bleomycin, diphtheria toxin (DT), tamoxifen, irradiation, a virus, a bacterium, or a fungus) in vivo or in vitro.
[0012] In some embodiments of the disclosure, the alveolar fibroblasts are lipofibroblasts. [0013] In other embodiments of the disclosure, the alveolar fibroblasts have disease-state characteristics. In some embodiments of the disclosure, the disease-state characteristic comprises lipofibroblast cells that express ACTA2, the presence of myofibroblast cells, and/or cell changes from a seriate shape to a banded shape.
[0014] In some embodiments of the disclosure, the culture medium comprises a platelet- derived growth factor receptor (PDGFR) ligand.
[0015] In some embodiments, the culture medium comprises an agent that is capable of maintaining or modulating PDGFR expression.
[0016] Another aspect of the disclosure provides a method of generating an ex vivo model for lung fibrosis, the method comprising: i) providing a culture of AEC1 cells that have undergone ablation in vivo in a culture medium adapted for the culture of said cells; ii) contacting the culture medium with one or more agents; and iii) analyzing the biological effect of the agent on at least one expression marker of the AEC1 cells relative to a control cell culture medium that has not been contacted by the agent.
[0017] In some embodiments, the AEC1 cells have undergone at least one round of ablation. In other embodiments, the AEC1 cells have undergone ablation alone or in combination with other injury models. In some embodiments, the AEC1 cells have undergone repeated ablation, for example, over the course of about three days to about two weeks.
[0018] In some embodiments of the disclosure, the AEC1 cells have undergone ablation by being exposed to bleomycin, diphtheria toxin (DT), asbestos, chemical toxic agents, tamoxifen, a virus, a bacterium, or a fungus. [0019] In some embodiments, the AEC1 cells express aSMA and/or ACTA2. In some embodiments, the AEC1 cells exhibit characteristics of a fibromyocyte cell.
[0020] Yet another aspect of the disclosure provides a method of identifying pre-alveolar type-1 transitional cell state (PATS) cells or PATS-like cells, the method comprising: i) obtaining alveolar cells; and ii) screening the cells for one or more markers, wherein the presence of the one or more markers indicates the presence of PATS cells or PATS-like cells. [0021] In some embodiments of the disclosure, the alveolar cells are selected from the group consisting of tracheal basal cells, bronchiolar secretory cells (also known as club cells or Clara cells), club variant cells, alveolar epithelial progenitor (AEP) cells, clara variant cells, distal lung progenitors, p63+ Krt5- airway cells, lineage negative epithelial progenitors, bronchioalveolar stem cells (BASCs), Sox9+ p63+ cells, neuroendocrine progenitor cells, distal airway stem cells, submucosal gland duct cell, induced pluripotent stem cell-derived lung stem cells and alveolar type 2 epithelial (AEC2) cells.
[0022] In some embodiments of the disclosure, the alveolar cells are obtained from the lung of a patient suffering from chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, idiopathic pulmonary fibrosis, emphysema, lung cancer, Sarcoidosis, interstitial pneumonia, sepsis, a viral infection, a bacterial infection, a fungal infection, acute respiratory distress syndrome, and/or bronchopulmonary dysplasia.
[0023] In some embodiments of the disclosure of identifying pre-alveolar type-1 transitional cell state (PATS) cells or PATS-like cells further comprises contacting the alveolar cells with an agent (e.g., bleomycin, diphtheria toxin (DT), tamoxifen, a virus, a bacterium, or a fungus).
[0024] Yet another aspect of the present disclosure provides a method of generating an in vivo tissue fibrosis model, the method comprising: i) introducing a genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling in a subject; and/or ii) administering an agent to a subject that can modulate PDGF-PDGFR signaling; and iii) analyzing cells from the subject to determine the biological effect of genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling and/or the biological effect of the agent that can modulate PDGF-PDGFR signaling relative to a control subject.
[0025] In some embodiments, the agent that can modulate PDGF-PDGFR signaling comprises a PDGFR inhibitor, a PDGFR agonist, a PDGF inhibitor, or a PDGF agonist.
[0026] Yet another aspect of the disclosure provides a method of generating an in vivo tissue fibrosis model, the method comprising: i) introducing a genetic loss of RUNX1, RUNX2, RUNX3, or CBFb in a subject; and/or ii) administering to the subject a RUNX1, RUNX2, RUNTX3, or CBFb inhibitor; and iii) analyzing cells from the subject to determine the biological effect of the genetic loss of RUNX1, RUNX2, RUNX3, or CBFp and/or the biological effect of the RUNX1, RUNX2, RUNX3, or CBFP inhibitor relative to a control subject.
[0027] In some embodiments, the inhibitor is Ro 5-3335, AI-10-49, or a neutralizing antibody against RUNX1, RUNX2, RUNX3, and/or CBFp.
[0028] In some embodiments, the in vivo model is a mouse model.
[0029] In some embodiments, the in vivo model is a disease model for fibrosis in the lung, liver, kidney, skin, intestines, or bone marrow.
BRIEF DESCRIPTION OF THE DRAWINGS [0030] FIGS. 1A-1H Previously unknown alveolar epithelial cell states in ex vivo organoids revealed by scRNA-seq. FIG. 1A is a schematic of the alveolar organoid culture utilized for scRNA-seq. FIG. IB show a Pearson correlation plot visualizes the number of genes per cell (nGene) and unique molecular identifier (nUMI) in total cells derived from alveolar organoids (left panel, 10,948 cells). UMAP shows major cell populations including epithelial cells (5,163 cells), fibroblasts (5,686 cells), and some minor populations such as endothelial cells (66 cells) and macrophages (33 cells) in alveolar organoids (right panel). FIG. 1C is an integrated UMAP showing cells derived from alveolar organoids (4,787 cells), in vivo homeostatic mouse lung (1,878 cells) and LPS-treated mouse lung (14,323 cells) (left). Expression of indicated genes in the integrated UMAP (right). FIG. ID is a UMAP visualization of the epithelial populations in cultured alveolar organoids ( n = 4,573 cells): AEC2s ( n = 3,303 cells); AEC2-proliferating, proliferating AEC2s ( n = 696 cells); AECls ( n = 262 cells) and new cell states — Lgals3+ (n = 184 cells) and Ctgf+ cells ( n = 128 cells). FIG. IE are UMAP plots showing the expression of the indicated genes in epithelial populations in cultured alveolar organoids. FIG. IF are UMAP plots showing the expression of indicated genes in AEC2, AEC2-proliferating, AECl, and novel alveolar epithelial cell state in alveolar organoid scRNA-seq dataset (4,573 cells) and LPS treated or control lungs (13,204 cells). FIG. 1G is a volcano plot showing specific genes enriched in the Ctgf+ ( n = 128 cells) and Lgals3+ (n = 184 cells) transitional cell states. A Wilcoxon rank-sum test was used for the statistical analysis. Data points indicate transcripts that fall below the set threshold for fold change (Log2) and P value (LoglO). FIG. 1H is a schematic of alveolar organoid culture using fibroblasts and AEC2s. Tmx, tamoxifen. [0031] FIG. 2 is a schematic showing the experimental workflow of bleomycin-induced lung injury in Ctgf-GFP mice.
[0032] FIG. 3 are violin plots show the expression of Krt8 and Sfii in epithelial cell populations derived from alveolar organoid (top, n = 4,573 cells) and LPS -injured lung scRNA-seq datasets (bottom, n = 13,204 cells). Violin plots indicate distribution of the cells. [0033] FIGS. 4A-4B shows that PATS cells emerge transiently from alveolar stem cells after lung injury in vivo. FIG. 4A is a schematic showing the experimental design to ablate AECls in the Ager-CreER; R26R-DTR mouse model. The mice were administered tamoxifen, followed by DT, and tissues were collected on day 6. FIG. 4B is a graph showing proportion of elongated LGALS3+ cells in control and AECl-ablated lungs. ***P < 0.0008, two-tailed unpaired Student’s /-test; n = 3 mice.
[0034] FIGS. 5A-5F shows that PATS cells originate from alveolar stem cells after lung injury in vivo. FIG. 5A is a schematic showing an experimental workflow for the sequential administration of tamoxifen, followed by bleomycin injury and tissue collection for analysis using Sftpc-CreER ; R26R-tdTomato mice. FIG. 5B show a time course of immunostaining for CLDN4, Sftpc- tdt and LGALS3 after bleomycin injury. Scale bars, 10 pm. Images are representative of three mice. FIG. 5C is a graph showing quantificationt of CLDN4+, CLDN4+LGALS3+ and LGALS3+ cells at different times after injury n = 3 mice. FIG. 5D is a graph showing cell length of CLDN4+, CLDN4+LGALS3+ and LGALS3+ cells. *P = 0.049 (CLDN4+ versus CLDN4+LGALS3+) and 0.0235 (CLDN4+ versus LGALS3+), two- tailed unpaired Student’s /-test; n = 30 cells from three mice. FIG. 5E is a schematic showing the pneumonectomy injury model. FIG. 5F is a schematic showing transition from AEC2 to AECl through different PATS subtypes.
[0035] FIGS. 6A-6H show lineage tracing revealed that PATS cells generate AECls. FIG. 6A is a UMAP of the epithelial populations in cultured alveolar organoids (// = 4,573 cells). The arrow indicates the select cell populations in the oval that are shown in FIG. 6B. FIG. 6B are UMAP plots showing the expression of the indicated genes in the selected populations (oval in FIG. 6A; n = 574 cells). FIG. 6C is a schematic showing the experimental workflow for the sequential administration of bleomycin (injury) or PBS (control), followed by tamoxifen (to label Krt 19-expressing cells) in Krtl9-CreER ; R26R- tdTomato mice. FIG. 6D are images showing immunostaining for SFTPC, Krtl9-tdt and AGER. The white arrows indicate AGER+Av7/9-tdt+ cells. Scale bars, 30 pm. Images are representative of three mice, repeated independently with similar results. FIG. 6E are images showing co-staining for SFN, Krtl9-tdt and AGER. The white arrows indicate SFN+A77/9- tdt+ cells. Scale bars, 50 mih. Images are representative of three mice, repeated independently with similar results. FIG. 6F is a graph showing quantification of AGER+A /9-tdt+ cells in total Krtl9-tdt+ cells p = 0.0318 (one-tailed, Mann- Whitney) n = 3 mice. FIG. 6G are images showing immunostaining for CLDN4, Krtl9-tdt and LGALS3. The arrows indicate CLDN4+Xr/79-tdt+ cells. Scale bars, 50 pm. Images are representative of three mice, repeated independently with similar results. The arrowheads indicate a LG A LS 3 +Krt 19-tdt+ cell. FIG. 6H is a graph showing quantification of KRT8+Xr/79-tdt+ cells in total Kill 9 -tdt+ cells. » = 0.0318, (one-tailed, Mann-Whitney). n = 3 mice.
[0036] FIGS. 7A-7C show gene-expression signatures and signalling pathways enriched in PATS. FIG. 7A is a graph showing KEGG analysis reveals pathways enriched in PATS. The web-based tool EnrichrScale was used to determine the log2-tranformed combined score values. FIG. 7B is a heatmap that shows expression of known target genes of indicated signalling pathways in AEC2, proliferating AEC2 (AEC2pro), PATS and AEC1 in alveolar organoids (n = 4,573 cells) and LPS-treated murine lung (n = 13,204 cells). Scale indicates z- score. FIG. 7C are violin plots for senescence (top) and DNA-damage-response pathways (bottom) in the indicated cell populations from the LPS-injury model in vivo (// = 13,039 cells). The black dots represent cells. The violin bodies indicate the distribution of the cells. [0037] FIGS. 8A-8I shows that PATS cells undergo stretch-induced DNA damage and senescence in vivo and ex vivo. FIG. 8A are images showing staining for b-galactosidase activity in bleomycin-injured (right) and control (left) lungs. Scale bars, 100 pm. FIG. 8B are images showing immunostaining for CDKN1A, Sftpc- tdt and AGER in control (top) and bleomycin-injured (bottom) lungs. The arrowheads indicate CDKN1A+ cells. The white dotted lines indicate AECls and the dotted lines outline tdt+ cells. Scale bars 50 pm. FIG. 8C are images showing immunostaining for gH2AC, Sftpc- tdt and AGER in control (top) and bleomycin-injured (bottom) lungs. Scale bars 50 pm. FIG. 8D is a graph showing the proportion of the total yH2AX+Sftpc-tdt+ cells in control and bleomycin-injured mice on day 10 that are LGALS3+. *P < 0.0318, one-tailed Mann-Whitney test; n = 3 mice. FIG. 8E are images showing staining for b-galactosidase activity in DT-treated and control lungs. Scale bars, 100 pm. FIG. 8F are images showing immunostaining for CDKN1A, LGALS3 and AGER in control (top) and DT-treated lungs (bottom). The arrowheads indicate CDKN1A+ cells. Scale bars 20 pm. FIG. 8G is a graph showing the proportion of the total gH2AC+ cells in control and DT-treated lungs that are LGALS3+. *P = 0.0318, one-tailed Mann-Whitney test; n = 3 mice. FIG. 8H is a schematic of 2D culture of AEC2s. FIG. 81 are images showing immunostaining for Sftpc-G FP, gH2AC and AGER in 2D culture of AEC2s. The white arrows indicate cells with DNA-damage marker. Scale bars 20 pm Images are representative of three mice, repeated independently with similar results. DAPI stains nuclei. [0038] FIGS. 9A-9H show genetic and pharmacological modulation and genomic binding assays reveal transcriptional control of PATS by TP53 signalling. FIG. 9A is a schematic showing an experimental workflow for the sequential administration of tamoxifen followed by PBS or bleomycin (day 0) administration, Nutlin-3a or DMSO treatment (days 8-18) and tissue collection (day 20) for analysis using Sftpc-CreER ; R26R-tdTomato mice. FIG. 9B is a graph showing the proportion of the total Sftpc- tdt+ cells mice treated with PBS + Nutlin-3a, bleomycin + DMSO, and bleomycin + Nutlin-3a that are AGER+. *P = 0.0201, two-tailed unpaired Student’s /-test; n = 3 mice. FIG. 9C is a schematic of alveolar organoid culture treated with Nutlin-3a. FIG. 9D are images showing immunostaining for Ki67 (green) and AGER (grey) in control or Nutlin-3a treated alveolar organoids. Scale bar: 30 pm. FIG. 9E is a schematic showing the experimental workflow for the sequential administration of tamoxifen to delete TP53 in AEC2s, followed by bleomycin injury (day 0) in Sftpc-CreER ; R26R-tdTomato ; 7/yGJfl/fl or control mice ( Sftpc-CreER ; R26R-tdTomato ; Trp53+/+ ). FIG. 9F is a graph showing the proportion of the total Sftpc- tdt+ cells from for SFTPC, Sftpc- tdt and AGER, and CLDN4, Sftpc- tdt and LGALS3 in control and ///GJ-knockout mice that are AGER+. ****p < 0.0001, two-tailed unpaired Student’s /-test; n = 3 mice. FIG. 9G is a graph showing the proportion of CLDN4+, CLDN4+LGALS3+ and LGALS3+ cells (bars from left to right, respectively) in bleomycin-treated lungs. **P = 0.048 (CLDN4) and 0.0013 (LGALS3), two-tailed unpaired Student’s /-test; n = 3 mice. FIG. 9H is a graph showing the proportion of the total Sftpc- tdt+ cells from Sftpc- tdt and SFN in control and ///Gd-knockout mice treated with bleomycin that are p21+. ***R < 0.0001, two-tailed unpaired Student’s /- test; n = 3 mice.
[0039] FIGS. 10A-10E shows transcriptional control of PATS by TP53 signaling. FIG. 10A is a graph showing the distribution of H3K4me3 peaks in PATS marker gene loci in PATS (upper line) and homeostatic AEC2s (lower line). FIG. 10B is a schematic showing the experimental workflow for bleomycin-induced lung injury in Sftpc-CreER ; R26R- tdTomato; Ctgf-GFP mice, followed by PATS sorting and ChIP analysis. FIG. IOC is ChIP enrichment for TRP53, H3K4me3 (active promoter) and H3K27ac (active enhancer) shown in IGV. FIG. 10D IGV tracks does not show enrichment for TP53 binding in genomic loci corresponding to known targets of AEC1 gene loci. FIG. 10E IGV tracks does not show enrichment for TP53 binding in genomic loci corresponding to known targets of AEC2 gene loci. [0040] FIGS. 11A-11M show enrichment of PATS-like states in IPF suggests persistence of this state in pathological milieu. FIG. 11A is a UMAP that shows scRNA-seq data from AECs in healthy and IPF lungs ( n = 11,725 cells). FIG. 11B are UMAP plots that indicate the expression of genes in scRNA-seq data for healthy and IPF lungs. FIG. 11C shows Hematoxylin and Eosin staining on IPF lung tissue sections. Representative image depicting fibrotic (right side square box) and non-fibrotic (left side square box) regions in IPF lung. Scale bar indicates 200 pm. FIG. 11D are images showing immunostaining for ACTA2, SFN and SFTPC in healthy human lungs (left), and non-fibrotic (middle) and severe-fibrotic (right) regions of lungs from patients with IPF. FIG. 11E is a graph showing the proportion of SFTPC+, SFTPC+SFN+ and SFN+ cells in healthy lungs, and non-fibrotic and fibrotic regions of IPF lungs; n = 3 human samples. FIG. 11F are images showing immunostaining for SFN, CLDN4 and AGER in a fibrotic region of an IPF lung. FIG. 11G is a graph showing the proportion of the total SFN+ cells in non-fibrotic and severe-fibrotic regions of IPF lungs that are CLDN4+. *P = 0.0218, one-tailed Mann-Whitney test; n = 3 human samples. FIG. 11H are images showing immunostaining for SFN, KRT17 and TP63 in a region of an IPF lung with severe fibrosis (arrowheads indicate SFN+KRT17+TP63+ cells). FIG. Ill is a graph showing the proportion of total SFN+ cells that are KRT17+ or KRT17+TP63+ in non-fibrotic regions compared with regions of severe fibrosis in IPF lungs. *P = 0.0318, one-tailed Mann-Whitney test; n = 3 human samples. FIG. 11J are UMAP plots from scRNA-seq data show enrichment of candidate signaling pathways in healthy and IPF lungs {n = 11,725 cells). FIG. 11K are violin plots showing expression of IPF -relevant genes in the indicated cell types/states in control and IPF lungs {n = 11,725 cells). The violin plots indicate the cell distributions in healthy and IPF lungs. FIG. 11L is a graph showing the proportion of total SFN+ cells that are CDKN1A+. *P = 0.0318, one-tailed Mann-Whitney test; n = 3 human samples. FIG. 11M is a graph showing the proportion of total SFN+ cells that are gH2AC+. *P = 0.0218, one-tailed Mann-Whitney test; n = 3 healthy and 4 IPF human samples. Insets: single-channel images of the region in a white box.
[0041] FIG. 12 is a schematic describing emergence of a novel transitional cell state in alveolar stem cell-mediated epithelial regeneration and its persistence in disease pathogenesis. Alveolar stem cells replicate in response to damage and generate a novel transitional cell state which normally matures into functional alveolar type-1 epithelial cells. The previously unappreciated transitional state is directly regulated by TP53 signaling, vulnerable to DNA damage and undergoes a transient senescent state. This state is enriched in human fibrotic lungs. [0042] FIG. 13A is a schematic of AEC1 specific ablation model. FIG. 13B are images showing immunostaining for SFN, ACTA2, LGALS3, and DAPI for cells that had undergone four rounds of AEC1 ablation and analyzed on day 3, day 6, day 10, and day 60.
[0043] FIG. 14A is a schematic of repeated AEC1 ablation. FIG. 14B are images showing H&E and trichome after 22 days. FIG. 14C are images showing immunostaining for LGALS3, ACTA2, and DAPI of ablated tissue after 22 days. FIG. 14D is a schematic showing morphological changes with repeated AEC1 ablation. FIG. 14E are images showing widespread fibrosis in single ablation of AEC1 in combination with pneumonectomy induced injury.
[0044] FIG. 15 is a schematic and coculture of PATS or AEC2s with lipofibroblasts. The data on the right demonstrates that coculture of PATS but not AEC2s induces the conversion of lipofibroblasts into myofibroblasts.
[0045] FIG. 16 is a violin plot showing that PDGFA is predominantly expressed in AECl. In addition, PDGFA transcripts are also observed in the transition cells during AEC2s differentiation into AECl.
[0046] FIG. 17 are images showing immunostaining for aSMA, PDGFRA, and DAPI in control and PDGFRA-CreER/PDGFRA ^ox mice. These data demonstrate that loss of PDGFRA in fibroblasts is sufficient to induce their conversion into myofibroblasts.
[0047] FIG. 18 is a violin plot showing that Runxl is upregulated in fibroblasts post- AECl ablation and following bleomycin injury.
[0048] FIG. 19 are images showing immunostaining for Runxl, aSMA, Pdgfa, and DAPI on the AECl ablation model, repeated AECl ablation model, and PDGFRA deletion. These data demonstrate that RUNX1 expression is increased in single and repeat AECl ablation mouse models as well as in targeted loss of PDGFRA in fibroblasts.
[0049] FIG. 20A are images showing immunostaining for GFP, aSMA, Pdgfa, and DAPI in RUNX1 deficient cells. FIG. 20B are images showing immunostaining for GFP, aSMA, Pdgfa, and DAPI in RUNX1 deficient cells containing TGF-B. These data demonstrate that loss of RUNX1 blocks fibroblasts conversion into myofibroblasts.
PET ATT, ED DESCRIPTION
[0050] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
[0051] Definitions
[0052] Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
[0053] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
[0054] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).
[0055] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. Thus, the term "consisting essentially of as used herein should not be interpreted as equivalent to “comprising.”
[0056] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
[0057] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. [0058] The term “disease” as used herein includes, but is not limited to, any abnormal condition and/or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease, or by internal dysfunctions, such as cancer, cancer metastasis, and the like.
[0059] The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
[0060] As used herein, “treatment” or “treating” refers to the clinical intervention made in response to a disease, disorder, or pathogen infection manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, disease causative agent (e.g., bacteria, viruses, a chemotherapeutic agent, radiation), or condition and/or the remission of the disease, disorder or condition.
[0061] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
[0062] PATS
[0063] Stem cells undergo dynamic changes in response to injury to regenerate lost cells. However, the identity of transitional states and the mechanisms that drive their trajectories remain understudied. Using lung organoids, multiple in vivo repair models, single-cell transcriptomics and lineage tracing, the inventors have found that alveolar type-2 epithelial cells undergoing differentiation into type-1 cells acquire pre-alveolar type-1 transitional cell state (PATS) en route to terminal maturation. The inventors have further found that transitional cells undergo extensive stretching during differentiation, making them vulnerable to DNA damage. The inventors have determined that cells in the PATS show an enrichment of TP53, TGFp, DNA-damage-response signaling and cellular senescence. As described in the examples, gain and loss of function as well as genomic binding assays revealed a direct transcriptional control of PATS by TP53 signaling. The inventors have also found that accumulation of PATS-like cells in human fibrotic lungs was observed, suggesting persistence of the transitional state in fibrosis. The results described in the example section herein thus implicate a transient state associated with senescence in normal epithelial tissue repair and its abnormal persistence in disease conditions.
[0064] Using a novel alveolar epithelial type-I cell (also known as pneumocyte type- I) ablation model, the inventors established a first genetic model that develops lung fibrosis. In addition, the inventors have developed an ex vivo lung fibrosis models that uses co-culture of above described novel transitional cell state (PATS) and alveolar fibroblasts. These novel in vivo and ex vivo fibrosis models are scalable and allows for disease modeling and drug screening. These features are often found in progressive lung disease and one can observe enrichment of cells in a PATS-like state in human fibrotic lungs, indicating persistence of the transitional state in fibrosis. The studies provided herein thus implicate a transient state associated with senescence in normal epithelial tissue repair and its abnormal persistence in disease conditions.
[0065] As used herein, the term “organoid” refers to self-organized three-dimensional (3D) structures or entities that are derived from stem cells grown in culture. Organoids cultures can replicate the complexity of an organ or can express selected aspects of an organ, such as by producing only certain types of cells. Alternatively, at certain stages before differentiation, they can be comprised only of stem cells.
[0066] Stem cells are cells that have the ability to both replicate themselves (self-renew) and give rise to other cell types. When a stem cell divides, a daughter cell can remain a stem cell or become a more specialized type of cell, or give rise to other daughters that differentiate into one or more specialized cell types. Two types of mammalian stem cells are: pluripotent embryonic stem cells that are derived from undifferentiated cells present in blastocyst or pre-implantation embryos, and adult stem cells that are found in adult tissues or organs. Adult stem cells can maintain the normal turnover or regeneration of the tissue or organ and can repair and replenish cells in a tissue or organ after damage.
[0067] As used herein, the term “stem cell” refers to an undifferentiated cell that is capable of proliferation and self-renewal and of giving rise to progenitor cells with the ability to generate one or more other cell types, or to precursors that can give rise to differentiated cells. In certain vases the daughter cells or progenitor or precursor cells that can give rise to differentiated cells. In certain cases the daughter cells or progenitor or precursors cells can themselves proliferate and self-renew as well as produce progeny that subsequently differentiate into one or more mature cell types.
[0068] A progenitor cell refers to a cell that is similar to a stem cell in that it can either self-renew or differentiate into a differentiated cell type, but a progenitor cell is already more specialized or defined than a stem cell.
[0069] Stems cells of the present disclosure can be derived from any animal, including but not limited to, human, mouse, rat, rabbit, dog, pig, sheep, goat, and non-human primates. [0070] The stem cells that can be used in the methods and models of the present disclosure can be normal (e.g., cells from healthy tissue of a subject) or abnormal cells (e.g., transformed cells, established cells, or cells derived from diseased tissue samples). [0071] In some embodiments, the cells used in the methods and models of the present disclosure can be derived from lung stem cells. Division of lung stem cells can promote renewal of the lung’s structure. Examples of lung stem cells include, but are not limited to tracheal basal cells, bronchiolar secretory cells (also known as club cells or Clara cells), club variant cells, alveolar epithelial progenitor (AEP) cells, clara variant cells, distal lung progenitors, p63+ Krt5- airway cells, lineage negative epithelial progenitors, bronchioalveolar stem cells (BASCs), Sox9+ p63+ cells, neuroendocrine progenitor cells, distal airway stem cells, submucosal gland duct cell, induced pluripotent stem cell-derived lung stem cells and alveolar type 2 epithelial (referred to herein as AEC2 or AT2) cells.
[0072] In some embodiments, the cells used in the methods and models of the present disclosure can be derived from basal stem cells from organs including, skin, mammary gland, esophagus, bladder, prostate, ovary, and salivary glands.
[0073] Methods and Models
[0074] Accordingly, one aspect of the present disclosure provides the identification of a newly-identified transitional cell state (PATS) in alveolar regeneration. This transitional cell state induces lung fibroblasts conversion into pathological fibroblasts (myofibroblasts). Further, this transitional cell state is also found in human fibrotic (e.g., idiopathic lung fibrosis and interstitial lung diseases) lungs.
[0075] Another aspect of the present disclosure provides a model system to ablate lung alveolar type-I cells (also known as pneumocyte type-I) that leads lung fibrosis, emphysema (similar to the one observed in human combined pulmonary fibrosis and emphysema syndrome (CPFE)).
[0076] In some embodiments, the model system comprises an animal model. In one embodiment, the animal comprises a mouse. In some embodiments, the animal comprises any animal that has lungs.
[0077] Another aspect of the present disclosure provides a method of generating a lung injury organoid model, the method comprising, consisting of, or consisting essentially of: i) providing a co-culture of cells in a culture medium adapted for the culture of said cells, wherein the co-culture comprises pre-alveolar type-1 transitional cell state (PATS) cells and alveolar fibroblasts; ii) contacting the culture medium with one or more agents; iii) analyzing the biological effect of the agent on at least one expression marker of the PATS cells and/or the alveolar fibroblasts relative to a control cell culture medium that has not been contacted by the agent. [0078] Alveolar fibroblasts are cells that are involved in the lung repair/fibrosis process in the lung. Alveolar fibroblasts can synthesize extracellular matrix and collagen and produce stroma in animal tissues. Examples of alveolar fibroblasts include but are not limited to, human fetal lung fibroblast cells (e.g., HFL1, MRC-5, 2BS, and WI38), Chinese hamster lung fibroblast (V-97) cell, C57BL/6 mouse primary lung fibroblasts, and CC-2512 cells.
[0079] In some embodiments, the alveolar fibroblasts are lipofibroblasts. In some embodiments, the alveolar fibroblasts have disease-state characteristics.
[0080] The term “disease-state characteristics” as used herein refers to phenotypic or genotypic properties of a cell that indicates the cell is a disease cell. These characteristics can be the expression of certain markers or morphological changes in the cell that indicate disease state. In some embodiments, disease-state characteristic of alveolar fibroblasts comprises lipofibroblast cells that express ACTA2, the presence of myofibroblast cells, and/or cell changes from a seriate shape to a banded shape.
[0081] In some embodiments, the PATS cells are isolated from diseased tissue.
[0082] The term “diseased tissue” refers to the tissue of subject (e.g., human or animal model) that is abnormal compared to healthy tissue. The diseased tissue can be obtained from the tissue of a riving or deceased subject. Example of diseased tissue include, but are not limited to, chronic obstructive pulmonary disease (COPD) lung tissue, pulmonary fibrosis lung tissue, idiopathic pulmonary fibrosis lung tissue, emphysema lung tissue, lung cancer tissue, Sarcoidosis lung tissue, interstitial pneumonia lung tissue, sepsis lung tissue, ring tissue having viral and bacterial infections, acute respiratory distress syndrome lung tissue, and bronchopulmonary dysplasia lung tissue. In some embodiments, the PATS cells are isolated from pulmonary fibrosis lung tissue or idiopathic pulmonary fibrosis lung tissue. [0083] Lung cancer cells can be isolated from a subject suffering from a lung cancer. The lung cancer cells can be isolated from a primary lung tumor or a secondary lung tumor (e.g., a cancer that starts in another tissue and metastasizes to the lungs). Examples of lung cancer cells include but are not limited to small cell lung cancer cells or non-small cell lung cancer cells, including but not limited to, small cell carcinoma, combined small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, pancoast tumor cells, neuroendocrine tumor, or lung carcinoid tumor cells. Established lung cancer cell lines can also be used in the methods and models of the present disclosure. Lung cancer cell lines that can be used in the methods and models of the present disclosure can be found on the ATCC website. Examples of lung cancer cell lines include but are not limited to, EML4-ALK Fusion- A549 Isogenic cell line, NCI-H838 [H838], HCC827, SK-LU-1, HCC2935, HCC4006, NCI-H1819 [H1819], NCI-H676B [H676B], Hs 618. T, HBE4-E6/E7 [NBE4- E6/E7], NCI-H1666 [H1666, H1666], NCI-H23 [H23], NCI-H1435 [H1435], NCI-H1563 [H1563], 703D4, and NCI-H1688 [H1688], NCI-H187 [H187], NCI-H661 [H661], NCI- H460 [H460], NCI-H1299, NCI-H1155 [HI 155], DMS 114, NCI-H69 [H69], DMS 79, DMS 53, SW 1271 [SW1271, SW1271], SHP-77, NCI-H209 [H209], NCI-H146 [H146], NCI- H345 [H345], NCI-H1341 [H1341], DMS 153, NCI-H82 [H82], NCI-H1048 [H1048], NCI- H128 [H128], NCI-H446 [H446], NCI-H128 [H128], NCI-H510A [H510A, NCI-H510], H69AR. HLF-a, Hs 913T, GCT [Giant Cell Tumor], SW 900 [SW-900, SW900], LL/2 (LLC1), HBE135-E6E7, Tera-2, NCI-H292 [H292], sNF02.2, NCI-H1703 [H1703], NCI- H2172 [H2172], NCI-H2444 [H2444], NCI-H2110 [H2110], NCI-H2135 [H2135], NCI- H2347 [H2347], NCI-H810 [H810], NCI-H1993 [H1993], and NCI-H1792 [H1792]
[0084] In some embodiments, the PATS cells are isolated from healthy tissue (e.g., normal lung tissue).
[0085] The term “tissue” as used herein refers to an aggregate of cells that can be of a particular kind together with their intercellular substance that form the structural material of the subject from which the tissue is obtained. In some embodiments of the disclosure, the PATS cells can be isolated and purified from the tissue.
[0086] In some embodiments of the disclosure, the PATS cells can be generated by exposing lung epithelial cells (e.g., AEC2s or AECls) to an injury-causing agent in vivo or in vitro.
[0087] The term “injury-causing agent” as used herein refers to an agent that is capable of converting or transitioning a healthy cell to a diseased-cell state. The term “injury-causing agent” as used herein also refers to an agent that is capable of ablating a normal cell such that a disease-state cell arises.
[0088] The injury-causing agent can be, for example, a chemotherapy agent (e.g., bleomycin, tamoxifen, actinomycin-D, mitomycin, taxanes, and gemcitabine), an exotoxin (e.g., diphtheria toxin (DT)), a virus, a bacterium, a fungus, asbestos, chemical toxic agents, or irradiation.
[0089] In some embodiments, the injury-causing agent can be a pathogen (e.g., a bacterium, virus, or fungus) that is capable of infecting lung tissue of humans or any animal with lungs.
[0090] Bacteria that can infect lungs include, but are not limited to Bordetella pertussis, Streptococcus pneumonia , Haemophilus influenza , Staphylococcusaureus, Moraxellacatarrhalis, Streptococcuspyogenes, Pseudomonas aeruginosa,
Neisseriameningitidis, Klebsiellapneumoniae .
[0091] Viruses that can infect lungs include, but are not limited to, 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU 1 (beta coronavirus), MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome, or MERS), SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS), or SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or COVID-19), an influenza-A virus (e.g., H1N1, H7N9, low pathogenic avian flu, high pathogenic avian flu, or H5N1), an influenza-B virus, respiratory syncytial virus (RSV), or an enterovirus (e.g. enterovirus 71). In some embodiments, the virus is SARS-CoV-2.
[0092] Fungus that can infect lungs include, but are not limited to, Aspergillosis.
[0093] In some embodiments, the culture medium and/or subjects (e.g., mice) used in the methods and models of the disclosure comprise a platelet-derived growth factor receptor (e.g., platelet-derived growth factor receptor- A, platelet-derived growth factor receptor-B, or platelet-derived growth factor receptor-AB).
[0094] In some embodiments, the culture medium and/or subjects (e.g., mice) used in the methods and models of the disclosure comprises a platelet-derived growth factor receptor (PDGFR) ligand. In some embodiments, the PDGFR ligand is platelet-derived growth factor (PDGF). PDGF is a growth factor that can regulate cell growth and division. In some embodiments, the PDGFR ligand is platelet-derived growth factor subunit A (PDGF-A), platelet-derived growth factor subunit B (PDGF-B), platelet-derived growth factor subunit C (PDGF-C), platelet-derived growth factor subunit D (PDGF-D), and/or platelet-derived growth factor subunit AB (PDGF-AB). Human genes that encode proteins that belong to the PDGF family include FIGF, PDGF A, PDGFB, PDGFC, PDGFd, PGF, VEGF, VEGF41, VEGFB, and VEGFC.
[0095] In some embodiments, the methods and models of the present disclosure comprise the use of PDGF signaling peptides, PDGF receptors, PDGF agonists, PDGFR agonists, PDGF antagonists, PDGFR antagonists, or any agent that is capable of modulating the PDGF-PDGFR signaling pathway. In other embodiments, the methods and models of the present disclosure comprise the use of a pharmacological blockade of PDGF ligands and/or PDGFR receptors.
[0096] Modulators of the PDGF-PDGFR signaling pathway include substrates, inhibitors, activators, neurotransmitters, agonists, antagonists, inverse agonists, inverse antagonists, partial agonists, and partial antagonists. Modulators of the PDGF-PDGFR signaling pathway include, but are not limited to, PDGF isoform antagonists, PDGFR activation blockers, agents capable of increasing or decreasing the expression of PDGF and/or PDGFR in a cell, chemical compounds, such as endogenous metabolites, non-endogenous metabolites, synthetic chemical compounds, polypeptides, amino acid residues, nucleic acids, siRNA, and antibodies (e.g., neutralizing antibodies).
[0097] Examples of modulators of the PDGF-PDGFR signaling pathway include but are not limited to, fibronectin peptides, Ras-GAP, tyrosine phosphatases (e.g., PTP1B, TC-PTP, PTPRJ/DEP-1), SHP-2, integrins, the low density lipoprotein receptor-related protein, hyaluronan receptor CD44, sorafenib, nilotinib, pazopanib, regorafenib, AG1295 and AG1296, celecoxib, etoricoxib and DFU, cyclooxygenase-2 (COX-2) inhibitors (coxibs), pazopanib HC1, pazopanib, radotinib, AZD2932, toceranib phosphate, TAK-593, crenolanib, toceranib, SU 14813, SU 14813 maleate, orantinib, SU 4312, Sunitinib Malate, Linifanib, KG 5, N-Desethyl Sunitinib, CP-673451, VEGFR2 Kinase Inhibitor II, VEGFR2 Kinase Inhibitor II, AC710 Mesylate, AC710, DMPQ dihydrochloride, ripretinib, lenvatinib, nintedanib, regorafenib, sunitinib, pnatibib, sunitnib malate, TG 100572, ilorasertib, imatinib, axitinib, imatinib mesylate, pazopanib, crenolanib, SU 5402, avapritinib, ripretinib, cediranib, CP 673451, dovitinib, TAK 593, toceranib, PDGFRa kinase inhibitor- 1, SU16f, agonistic human monoclonal autoantibodies targeting PDGFRa, and non-agonistic human monoclonal autoantibodies targeting PDGFRa, and or derivatives thereof.
[0098] As used herein, the term “agonist” refers to a modulator that binds to a receptor (e.g., PDGFR) or protein (e.g., PDGF) and activates the receptor or protein to produce a biological response.
[0099] As used herein, the term “antagonist” refers to a modulator or ligand that blocks, impedes, or dampens a biological response by binding to and blocking a receptor or protein rather than activating it.
[00100] As used herein, the term “inverse agonist” refers to a modulator or ligand that binds to the same receptor as an agonist but induces a biological response opposite to that agonist.
[00101] As use herein, the term “partial antagonist” refers to a modulator or ligand that can bind to a receptor but does not completely block the receptor’s effects, but rather decreases the maximum potential of the receptor.
[00102] In some embodiments, the culture medium used in the methods and models comprises an agent that is capable of maintaining PDGFR (e.g., PDGFR-A, PDGFR-B, or PDGFR-AB) expression in a cell. In some embodiments, the agent capable of maintaining PDGFR-A expression in a cell is Ro 5-3335 or AI-10-49.
[00103] In some embodiments, the agent capable of maintaining PDGFRA expression in a cell is any molecule that can modulate RUNX (e.g., RUNX1, RUNX2, or RUNX3) or core binding factor (CBF) related family of proteins (e.g., CBF-b).
[00104] Examples of molecules that regulate RUNX or CBF related proteins can be RUNX and/or CBF substrates, inhibitors, activators, neurotransmitters, agonists, antagonists, inverse agonists, inverse antagonists, partial agonists, and partial antagonists.
[00105] Molecules that modulate RUNX and/or CBF can be molecules that interfere with the protein-protein interaction between RUNX and CBF. These modulators can be inhibitors of RUNX (e.g., RUNX1, RUNX2, or RUNX3) or inhibitors CBF-b. Examples of RUNX or CBF modulators include, but are not limited to, Ro 5-3335, AI-10-49, CADD522, muramyl dipeptide, L-quebrachitol, L-ascorbic acid 2-phosphate, ascorbic acid 2-phosphate magnesium, 2-pyridyl benzimidazole AI-4-57, RUNX1/MTG8 fusion protein, and a neutralizing antibody against RUNX1, RUNX2, RUNX3, and/or CBFb, and/or derivatives thereof,
[00106] An “agent” as used herein refers to a small molecule, protein, peptide, gene, compound, or other pharmaceutically active ingredient. In some embodiments, the agent can be used for the treatment, prevention, or mitigation of a disease (e.g., lung fibrosis).
[00107] PATS cell expression markers refer to gene transcripts that are expressed on PATS cells or PATS-like cells. In some embodiments, PATS markers are unique to the PATS cell state and are not typically expressed on related cell states (e.g., AEC2s or AECls). PATS cell markers can comprise CLDN4, KRT19, SFN, LGALS3, SOX4, S100A2, PTGS2, KRT17, KRT8, CALSl, MMP7, PRSS2, IGFBP7, COL1A1, MDK, TAGLN, GDF15, TM4SF1 TP63, and/or CTSE.
[00108] In some embodiments, the PATS markers can comprise a marker signature of two or more markers that are co-expressed. In some embodiments, the PATS marker signature is CLDN4, LGALS3, and LGALS3. In some embodiments, the PATS marker signature is CLDN4, KRT19, and SFN. In some embodiments, the PATS marker signature is CALDl, PRSS2, MMP7, and S100A2.
[00109] In some embodiments, the PATS marker signature is TP63, KRT17 and COL1A1. [00110] In some embodiments, the PATS marker signature is AREG, TGFB1, TGFB2 and TEMPI. [00111] Alveolar fibroblasts expression markers refer to gene transcripts that are expressed on alveolar fibroblasts cells. Alveolar fibroblasts can comprise Pdgfra.
[00112] Another aspect of the present disclosure provides a method of generating an ex vivo model for lung fibrosis, the method comprising: i) providing a culture of alveolar cells (e.g. AEC1 cells) that have undergone ablation in vivo in a culture medium adapted for the culture of said cells; ii) contacting the culture medium with one or more agents; iii) analyzing the biological effect of the agent on at least one expression marker the alveolar cells (e.g. AEC1 cells) relative to a control cell culture medium that has not been contacted by the agent.
[00113] The term “ablation” as used herein refers to the removal or destruction of a tissue or cell, or their function. Ablation can be caused by surgery, hormones, drugs, radiofrequency, heat, or other methods that cause destruction of the tissue or cell.
[00114] In some embodiments, the alveolar cells (e.g. AEC1 cells) have undergone at least one round of ablation (e.g., at least one, two, three, four, five, six, seven, eight, nine, or ten rounds of ablation). In some embodiments, the alveolar cells (e.g. AEC1 cells) have undergone between one and five rounds of ablation. In some embodiments, the cells have undergone ablation alone or in combination with other injury models described herein.
[00115] In some embodiments, the alveolar cells (e.g. AEC1 cells) have undergone repeated ablation. In some embodiments, the repeated ablation can occur over the course of about three days to about two weeks. In some embodiments, the repeated ablation can occur over the course of about three days to about one year.
[00116] In some embodiments, the alveolar cells (e.g. AEC1 cells) have undergone ablation by being exposed to an injury-causing agent. In some embodiments, the injury- causing agent is bleomycin, diphtheria toxin (DT), tamoxifen, a virus, a bacterium, or a fungus.
[00117] In some embodiments, the ablated cells express aSMA and/or ACTA2. In some embodiments, the ablated cells exhibit characteristics of a fibromyocyte cell.
[00118] Yet another aspect of the present disclosure provides, a method of identifying pre- alveolar type-1 transitional cell state (PATS) cells, the method comprising: i) obtaining lung cells; and ii) screening the cells for one or more markers, wherein the presence of the one or more markers indicates the presence of PATS cells.
[00119] In some embodiments, the lung cells can be selected from the group consisting of tracheal basal cells, bronchiolar secretory cells (also known as club cells or Clara cells), club variant cells, alveolar epithelial progenitor (AEP) cells, clara variant cells, distal lung progenitors, p63+ Krt5- airway cells, lineage negative epithelial progenitors, bronchioalveolar stem cells (BASCs), Sox9+ p63+ cells, neuroendocrine progenitor cells, distal airway stem cells, submucosal gland duct cell, induced pluripotent stem cell-derived lung stem cells and alveolar type 2 epithelial (AEC2) cells.
[00120] In some embodiments, the lung cells can be selected from the group consisting of AEC2 cells, AEC1 cells, or alveolar macrophages.
[00121] In some embodiments, the lung cells are obtained from the lung of a patient suffering from chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, idiopathic pulmonary fibrosis, emphysema, lung cancer, Sarcoidosis, interstitial pneumonia, sepsis, a viral infection, a bacterial infection, a fungal infection, acute respiratory distress syndrome, and/or bronchopulmonary dysplasia.
[00122] In some embodiments, the method of identifying pre-alveolar type-1 transitional cell state (PATS) cells further comprises contacting the alveolar cells with an injury-causing agent. In some embodiments, the agent is bleomycin, diphtheria toxin (DT), tamoxifen, a virus, a bacterium, or a fungus.
[00123] Another aspect of the present disclosure provides an ex vivo lung fibrosis model that uses co-culture of above described novel transitional cell state (PATS) and alveolar fibroblasts that is scalable and allows for disease modeling and drug screening.
[00124] Another aspect of the present disclosure provides a method of generating an in vivo model of generating fibrosis in lung and other tissues. Like the in vitro and ex vivo models of the present disclosure, the in vivo models can be used, for example, to test the ability for therapeutic agent to treat or prevent disease. The in vivo models of present disclosure can utilize a variety of animal subjects (e.g., mouse, rat, rabbit, monkey, or pig or any animal that is capable of modeling fibrosis).
[00125] An in vivo model refers to the use of a whole living animal to investigate the effect of an agent and to study the biological processes of disease (e.g., lung fibrosis).
[00126] Another aspect of the present disclosure provides a method of generating an in vivo model of generating fibrosis in lung and other tissues, the method comprising: i) introducing a genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling in a subject; and/or ii) administering an agent to a subject that can modulate (e.g., block, enhance, increase, or decrease) PDGF -PDGFR signaling; and iii) analyzing cells from the subject to determine the biological effect of the genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling and/or the biological effect of the agent that can modulate (e.g., block enhance, increase, or decrease) PDGF -PDGFR signaling relative to a normal control subject. [00127] In some embodiments, the agent that can block PDGF-PDGFR signaling comprises a PDGFR inhibitor, neutralizing antibodies against PDGF ligands and/or PDGF receptor (PDGFR).
[00128] Another aspect of the present disclosure provides a method of generating an in vivo model of generating fibrosis in lung and other tissues, the method comprising: i) introducing a genetic loss of RUNX1, RUNX2, RUNX3, or CBFP in a subject; and/or ii) administering an agent toa subject that can modulate (e.g., inhibit) RUNX1, RUNX2, RUNX3, or CBFP; and iii) analyzing cells from the subject to determine the biological effect of genetic loss of RUNX1, RUNX2, RUNX3, or CBFp and/or the biological effect of modulating RUNX1, RUNX2, RUNX3, or CBFP relative to a normal control subject.
[00129] In some embodiments, the RUNX1, RUNX2, RUNX3, or CBFP modulator is a small molecule inhibitor are a neutralizing antibody against RUNX1, RUNX2, RUNX3, and/or CBFp.
[00130] In some embodiments, the models of the present disclosure are fibrosis models of lung fibrosis, cardiac fibrosis, liver fibrosis, kidney fibrosis, intestinal fibrosis, skin fibrosis, and/or bone marrow fibrosis. In some embodiments, the models of the present disclosure are lung fibrosis models that can model the impact of lung fibrosis on other tissues in the body, such as cardiac, liver, kidney, intestinal, skin, and/or bone marrow tissues.
[00131] In some embodiments, the cells of the models of the present disclosure are lung, heart, kidney, liver, intestine, skin, or bone marrow.
[00132] The following Examples are provided by way of illustration and not by way of limitation.
EXAMPLES
[00133] Materials and Methods [00134] Mice.
[00135] Both male and female mice aged between 8-16 weeks were used for experiments. All of the mice were C57BL/6 unless otherwise indicated. The following mice were used for experiments: Sftpctml(cre/ERT2)Blh ( Sftpc-CreER ), Krtl9tml(cre/ERT)Ggu/J ( Krtl9 - CreER ), Rosa26R-CAG-lsl-tdTomato (crossed with Sftpc-CreER ), B6.Cg- Gt(ROSA)26Sortm 14(CAG-tdTomato)Hze/J ( R26R-tdTomato ) (crossed with Krtl9-CreER ), I giSl· J'PC Gl· P) Heat (Sftpc-GFP)51, B6.Cg-Agertm2.1(cre/ERT2) Blh/J ( Ager-CreER ), B6-Gt(ROSA ) 26Sortm 1 (HBEGF)Awai/J (. R26R-DTR ), Mki67tml.lCle/J ( Mki67 RFP ), Tg(Ctgf EGFP)FX156Gsat ( Ctgf-GFP)\5 and TP53fl/fl (mixed background). For lineage tracing with Sftpc-CreER ; R26R-tdTomato mice, 3-5 doses of 2 mg tamoxifen (Sigma- Aldrich) per 20 g of body weight were given via oral gavage or intraperitoneal injection. For lineage tracing using Krtl9-CreER ; R26R-tdTomato , one dose of 1 mg tamoxifen per 20 g body weight was given via intraperitoneal injection 7 d after bleomycin injury or PBS administration. The animal experiments were approved by the Duke University Institutional Animal Care and Use Committee in accordance with US National Institutes of Health guidelines.
[00136] Bleomycin injury.
[00137] For bleomycin-induced lung injury, 2.5 U kg-1 bleomycin was administered intranasally 2 weeks after the tamoxifen injection and the mice were monitored daily. Mice that were administered PBS served as controls. The mice were killed at different times after bleomycin injury.
[00138] Administration of DT
[00139] Two weeks before DT administration, Ager-CreER ; R26R-DTR mice received tamoxifen via intraperitoneal injection. One dose of 3 pg DT (Millipore, 322326) was administered via intraperitoneal injection and the mice were killed 6 d later for tissue collection and analysis.
[00140] Mouse lung dissociation and FA CS.
[00141] Lung dissociation and FACS were performed as described previouslyll. Briefly, the lungs were intratracheally inflated with 1 ml enzyme solution (dispase, 5 U ml-1, Corning, 354235; DNase I, 0.33 U ml-1; and collagenase type I, 450 U ml-1, Gibco, 17100- 017) in DMEM/F12 medium. The separated lung lobes were diced and incubated with 3 ml enzyme solution for 25 min at 37 °C with rotation. The reaction was quenched with an equal amount of medium containing 10% fetal bovine serum (FBS) and filtered through a 100-pm strainer. The cell pellet was resuspended in red-blood-cell lysis buffer (155 mM NH4C1, 12 mM NaHC03 and 0.1 mM EDTA), incubated for 2 min and then filtered through a 40-pm strainer. The cell pellet was resuspended in DMEM/F12 + 2% BSA and stained with the following antibodies: EpCAM (eBioscience, G8.8), PDGFRa (BioLegend, APA5) and Lysotracker (Thermo Fisher, L7526) as described previously56. Sorting was performed using a BD FACS Vantage SE, SONY SH800S or Beckman Coulter MoFlo Astrios EQ system. [00142] Alveolar organoid culture.
[00143] Alveolar organoid culture was performed as described previously (Barkauskas et ak, 2013, J. Clin. Invest. 123:1118-1123). Briefly, lineage-labeled AEC2s (1-3 x 103) from Sftpc-GFP or Sftpc-CreER ; R26R-tdTomato mice treated with Tmx were sorted by FACS, and PDGFRa+ (5 c 104) fibroblasts were resuspended in MTEC/Plus and mixed with an equal amount of growth-factor-reduced Matrigel (Coming, 354230). The medium was changed every other day.
[00144] Nutlin-3a treatment.
[00145] For in vivo studies, Sftpc-CreER ; R26R-tdTomato mice were injected with one dose of tamoxifen and rested for 2 weeks, followed by bleomycin or PBS administration. Nutlin-3a (Selleckchem, S8059) or DMSO (control) were administered by intraperitoneal injection 8 d after injury at a concentration of 20 mg kg-1 d-1 for ten consecutive days and samples were collected 20 d after the bleomycin administration. For ex vivo studies, alveolar organoids were cultured for 7 d, followed by Nutlin-3a (2 mM) treatment for 8 d before being harvested.
[00146] Droplet-based scRNA-seq.
[00147] Organoids embedded in Matrigel were incubated with Accutase solution (Sigma, A6964) at 37 °C for 20 min, followed by incubation with 0.25% trypsin-EDTA at 37 °C for 10 min. The trypsin was inactivated using DMEM/F12 Ham medium supplemented with 10% FBS and the cells were then resuspended in PBS supplemented with 0.01% BSA. After filtration through a 40-pm strainer, the cells were ran through microfluidic channels at 3,000 pi h-1 at a concentration of 100 cells pi h-1 together with mRNA-capture beads at 3,000 mΐ h-1 and droplet-generation oil at 13,000 mΐ h-1. The DNA polymerase for the pre- amplification step (one cycle of 95 °C for 3 min; 15-17 cycles of 98 °C for 15 s, 65 °C for 30 s and 68 °C for 4 min; and one cycle of 72 °C for 10 min) was replaced by Terra PCR direct polymerase (Takara, 639271). The other processes were performed as described in the original droplet-based scRNA-seq protocol. The libraries were sequenced using a HiSeq X system with 150-bp paired-end sequencing.
[00148] Computational analysis of scRNA-seq.
[00149] Analysis of the scRNA-seq of alveolar organoids was performed by processing FASTQ files using dropSeqPipe v0.3 (https://hoohm.github.io/dropSeqPipe) and mapped on the GRCm38 genome reference with annotation version 91. Unique molecular identifier (UMI) counts were then further analysed using the R package Seurat v3.1.1 (ref. 58). The UMI count matrix of murine lungs treated with LPS (GSE130148)14 was obtained from Gene Expression Omnibus (GEO). The UMI counts were normalized using SCTransform. The cell barcodes for the clusters of interest were extracted and utilized for the velocyto run command in velocyto. py vO.17.15 (ref. 18) as well as to generate RNA velocity plots using velocyto. R v0.6 in combination with the R package SeuratWrappers vO.l.O (https://github.com/satijalab/seurat-wrappers). Twenty -five nearest neighbours in slope calculation smoothing was used for the RunVelocity command. After excluding duplets, specific cell clusters were isolated based on enrichment for Sftpc , Sftpal , Sftpa2 , Sftpb, Lamp3 , Abca3, Hopx , Ager, Akap5, Epcam , Cdhl , A7v7, AvV#, Krtl8 , Krtl9 , Scgblal and Scgb3al as well as negative expressions of 17/??, Acta2, Pdgfra and Pdgfrb in UMAP plots. The Rds files for control and idiopathic pulmonary fibrosis (IPF) lungs were obtained from GEO (GSE135893)29. Cell clusters of AEC2s, AECls, transitional AEC2s and KRT5-KRT17+ cells were extracted and analysed. Markers for each cluster, obtained using the FindAllMarkers command in Seurat, were utilized for identifying specific signalling pathways and gene ontology through Enrichr59. Z-scores were calculated based on the combined score in Kyoto Encyclopedia of Genes and Genomes (KEGG) to compare enrichment of signalling and ontology across different cell clusters. The results were displayed in heatmap format, generated using the R package pheatmap vl.0.12. Scaled data in Seurat object were extracted and the mean values of the scaled score of the gene members in each pathway were calculated and shown in EIMAP as enrichment of signaling pathways. The gene-member lists of the utilized pathways were obtained from KEGG pathways60 and AmiG06T The log2 fold change and P values for each gene, extracted using the FindMarkers command in Seurat with the Wilcoxon rank-sum test, were shown in a volcano plot using the R package Enhanced Volcano vT3.1 (https://github.com/ kevinblighe/EnhancedVolcano) to show specific markers for Ctgf+ cells.
[00150] Mint-ChIP -seq.
[00151] For the ChIP analysis of histone marks, PATS (CD31-CD45-CD140a-CD326+C7g/-GFP+) cells were sorted from Ctgf-GFP mice on day 12 post bleomycin-induced lung injury. AEC2s (CD31-CD4 5-CD326+Lysotracker+Mki67-RFP- cells) were sorted from Mki67 RbP homeostatic mice. MM67 mice were used to eliminate any cells that were undergoing cell division by negatively gating out RFP+ cells. The Mint-ChIP protocol was described previously62. For TP53 ChIP sequencing (ChIP-seq), PATS (CD31 -CD45- V///?6-tdTomato+Ctgf-GFP+ cells) were sorted from Sftpc-CreER ; R26R-tdTomato\ Ctgf-GFP mice on day 8 post bleomycin administration. In this case, we used a modified Mint-ChIP (version 3) protocol as described previously (https://tinyurl.com/udqksct). Following cell lysis, the chromatin was digested with 300 units of MNase (New England Biolabs, M0247S) at 37 °C for 10 min. T7 adaptor ligation was performed for 2 h and the samples then were split to give approximately 7,000 cells per antibody for histone-modification ChIP-seq. For TP53, we used approximately 40,000 cells for each replicate sample. The samples were incubated with histone H3 (H3; 1 pi; Active Motif, 39763), histone H3 lysine 36 trimethylation (H3K36me3; 1 mΐ; Active Motif, 61101), histone H3 lysine 4 trimethylation (H3K4me3; 1 mΐ; Abeam, ab8580), histone H3 lysine 27 acetylation (H3K27ac; 1 pi; Active Motif, 39133) or TP53 (5 mΐ; Cell Signaling Technology, 2524T) antibodies overnight at 4 °C. DNA was purified followed by T7-RNA polymerase mediated in vitro transcription at 37 °C for 3 h. Reverse transcription was performed as described in the original protocol, followed by library preparation using Terra direct PCR polymerase (TaKaRa, 639271). Two experimental replicates were performed for each cell population. The libraries were sequenced (at least 5 c 106 reads of 150-bp paired-ends per sample) using the Hiseq X or NovaSeq 6000 systems.
[00152] Computational analyses of Mint-ChIP.
[00153] FASTQ files were generated using Bcl2fastq. Additional demultiplexing for Mint- ChlP FASTQ files were performed using Je63. Low-quality reads were trimmed out from the FASTQ files using trimmomatic v0.38. The reads were mapped on the mmlO genome reference using BWA65. The packages were run through a pipeline called MintChIP (https://github.com/jianhong/MintChIP). HOMER66 was used to generate bedGraph files to visualize peaks in Integrative Genomics Viewer (IGV). Peak calling for H3K4me3 was performed using the function getDifferentialPeaksReplicates.pl -region -size 1000 -minDist 2000 -C 0 -L 50 of HOMER with normalization by H3. Motif analysis was performed using the fmdMotifsGenome.pl function of HOMER. A chart of called peaks for H3K4me3 was generated using deepTools68. Called peaks for each genomic locus from different cell populations were prepared in Affinity Designer.
[00154] Human lung tissue.
[00155] Excised sub-transplant-quality human lung tissues from donors without pre existing chronic lung diseases and explanted fibrotic human lungs were procured through the BioRepository and Precision Pathology Center at Duke University in accordance with institutional procedures (Duke University Pro00082379 — ‘Human Lung Stem Cells’; exempt research as described in 45 CFR 46.102(f), 21 CFR 56.102(e) and 21 CFR 812.3(p), which satisfies the Privacy Rule as described in 45CFR164.514). Samples of the IPF tissue used for scRNA-seq were obtained from lungs removed at the time of lung transplantation at two lung transplant centres (VUMC and NTI). Non-fibrotic control tissue samples were obtained from lungs declined for organ donation. For IPF lungs, diagnoses were determined according to ATS/ERS consensus criteria. All studies were approved by the local Institutional Review Boards (IRB; Vanderbilt IRB nos 060165 and 171657, and Western IRB no. 20181836). The diagnosis of IPF was evaluated by a surgical pathology team. The specimens were washed thoroughly in PBS before inflation and immersion in 4% paraformaldehyde at 4 °C overnight. The specimens were subsequently washed in PBS until the appearance of blood was minimal, followed by incubation in 30% sucrose at 4 °C. The samples were then incubated with OCT compound at a 1:1 ratio for 1 h at 4 °C before embedding in OCT. Section with a thickness of 7-9 pm were used for histological analysis.
[00156] Immunostaining.
[00157] Lungs and alveolar organoids were prepared as described previously. Briefly, the tissues were fixed with 4% paraformaldehyde at 4 °C for 4 h (for lungs) or at room temperature for 30 min (for organoids), and then embedded in OCT compound or paraffin. Sectioned samples (10 pm) were utilized for staining following incubation at 95 °C for 10-15 min for antigen retrieval using 10 mM sodium citrate. Primary antibodies were as follows: pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D Systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176, 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181-200, 1:500), CLDN4 (Invitrogen, 36- 4800, 1:200; Proteintech, 16165-1-AR, 1:500), GFP (Novus Biologicals, NB 100-1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500), SOX4 (Invitrogen, MA5-31424, 1:250), SFN (Invitrogen, PA5-95056, 1:250; Proteintech, 66251-1-Ig, 1:500; Abeam, ab77187, 1:200), ACTA2 (Sigma, C6198, 1:500), g-H2AC (R&D, 4418-APC, 1:500; Novus Biologicals, NB100-74435, 1:250), CDKN1A (Sigma, ZRB1141, 1:200; BD Biosciences, 556430, 1:200), COL1A1 (Proteintech, 67288-Ig, 1:1,000), YAP (Cell Signaling Technology, 4912S, 1:250).
[00158] X-gal and haematoxylin-and-eosin staining.
[00159] Paraformaldehyde-fixed frozen sections were incubated overnight with X-gal staining buffer containing 1 mg ml-1 X-gal (Thermo, R0941), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgC12, 0.01% sodium deocycholate and 0.02% NP-40 at 37 °C. The sections were washed three times in PBS and mounted. For haematoxylin-and-eosin staining, 10-pm paraffin sections were submerged in Histo-clear and a series of ethanol. Mayer’s haematoxylin was used to stain the nuclei, followed by staining using 1% eosin Y.
[00160] Proximity ligation in situ hybridization.
[00161] Proximity ligation in situ hybridization was performed as described previously69. Briefly, frozen mouse lung sections were fixed with 4.0% paraformaldehyde for 20 min, treated with 20 pg ml-1 proteinase K for 9 min at 37 °C and dehydrated with an up-series of ethanol. The sections were incubated with gene-specific oligos in hybridization buffer (1 M sodium trichloroacetate, 50 mM Tris pH 7.4, 5 mM EDTA and 0.2 mg ml-1 heparin) for 2 h at 37 °C. Common-bridge and circle probes were added to the sections and incubated for 1 h, followed by a T4 DNA ligase reaction for 2 h. Rolling-circle amplification was performed using phi29 polymerase (Lucigen, 30221) for 12 h at 37 °C. Fluorophore-conjugated detection probe was applied and incubated for 30 min at 37 °C, followed by mounting in medium containing 4,6-diamidino-2-phenylindole (DAPI).
[00162] Image acquisition, processing and quantification.
[00163] Images were captured using an Olympus FV3000 confocal microscope with a x20, x40 or x60 objective, a Zeiss wide-field fluorescence microscope (X-gal staining) and a Zeiss Axio Imager wide-field fluorescence microscope (haematoxylin and eosin). The cells were manually counted based on immunohistochemistry markers and DAPI. For the determination of the average intersects per linear distance, a mean linear intercept analysis was conducted as previously described over the single-channel immunofluorescence stain of interestll. Images were processed using the Olympus CellSens application or ImageJ and figures were prepared using Affinity Designer.
[00164] Statistics and reproducibility.
[00165] Experiments were performed on at least three biological replicates (except organoid scRNA-seq and ChIP-seq). The sample size was not pre-determined. Data are presented as the mean with the s.e.m. to indicate the variation within each experiment. Statistical analyses were performed in GraphPad Prism. Two-tailed and unpaired Student’s t- tests were used for comparisons between two experimental conditions. The Mann-Whitney one-tailed test was used for comparisons between two conditions that showed non-normal distributions.
Example 1: Single-cell transcriptomics reveals previously unknown alveolar epithelial cell states in ex vivo organoids
[00166] Recent studies have shown that AEC2s proliferate and give rise to AECls in response to lung injury. Moreover, AEC2s spontaneously generate AECls in alveolar organoids. However, the molecular mechanisms and transitional cell states underlying the differentiation of AEC2 into AECl are poorly understood. To address these questions, we purified AEC2s and PDGFRa+ fibroblasts to set up alveolar organoids. Single-cell transcriptome analysis was performed on cells isolated from the day- 10 organoid culture (FIG. 1A). Uniform manifold approximation and projection (UMAP) identified two major clusters, consisting of Epcam+ epithelial cells and Vim+Pdgfra+ fibroblasts (FIG. IB). Next, we further deconvoluted and visualized epithelial cell populations and compared them with a publicly available single-cell RNA sequencing (scRNA-seq) dataset of lipopolysaccharide (LPS)-injured alveolar epithelial cellsl4. Organoid-derived epithelial cells overlapped with their in vivo counterparts, revealing transcriptional and cell-state similarities (FIG. 1C). We observed multiple sub-clusters among the organoid-derived epithelial cells: cells expressing Sftpc (a marker for AEC2), cells expressing Ager (a marker for AEC1) and Sftpc+Mki67+ proliferating AEC2s (FIG. ID).
[00167] In addition, we identified a population of alveolar epithelial cells expressing Cldn4 , Krtl9 and Sfn (FIG. ID, FIG. IE, and FIG. IF,). The marker genes unique to this cluster showed two distinct patterns when visualized in UMAP and volcano plots (Fig. lb-d). One subset (Ctgf+ cells) was enriched for Ctgf, Clu , Sox4 and Acini, whereas the other ( Lgals3+ cells) was enriched for Lgals3, Csrpl , S100al4 and Cldnl8. Additional transcripts enriched in the Lgals3+ sub-cluster including Ager, Emp2 and Hopx (markers of AECls) suggest resemblances and a potential lineage hierarchy between Lgals3+ cells and AECls (FIG. 1G). These data suggest that the Cldn4+Krtl9+Sfti+ population is an intermediate between AEC2 and AEC1. This population was termed “pre-alveolar type-1 transitional cell state” (PATS). We performed immunofluorescence for PATS markers on alveolar organoids to validate the single-cell data (FIG. 1H). Images were representative of 30 organoids from three mice. This confirmed the presence of cells expressing CLDN4, LGALS3 and SOX4 in alveolospheres. Together, these data identified unique cell states during alveolar epithelial stem cell differentiation in organoid cultures.
Example 2: PATS cells emerge in vivo after alveolar injury.
[00168] It was then investigated whether PATS cells can be observed in vivo in homeostatic and regenerating alveolar tissues. To test this, we rendered scRNA-seq data from LPS-treated and control mouse lungs in UMAP plots and found a population that is unique to LPS injury. UMAP plots showed the expression of genes in AEC2 (. Lamp and Sftpb), AEC2- proliferating (Mki67 and Top2d), AEC1 ( Ager and AkapS), and novel alveolar epithelial cell state in an alveolar organoid scRNA-seq dataset (4,573 cells) and LPS treated or control lungs (13,204 cells). Importantly, this population is enriched for genes expressed in PATS, including Cldn4, Sox4, Lgals3, S100al4, and Fnl.
[00169] To further characterize PATS, a Ctgf-green fluorescent protein ( GFP ) transgenic mouse line was utilized (Hall-Glennet ah, 2012, PLoS ONE 7:e30562). Mice were exposed to to bleomycin, a drug that causes transient fibrosis, and collected the lungs of the mice on day 12 post injury (FIG. 2). Immunofluorescence for GFP in uninjured Ctgf-GFP mice revealed GFP signal specifically in fibroblasts and not in alveolar epithelial cells. In contrast, in bleomycin-injured lungs, GFP expression was found in epithelial cells co-labeled with PATS markers including CLDN4, LGALS3 and SFN. A small fraction of GFP+ cells co-expressing low levels of AEC2 marker was observed, probably due to perdurance of SFTPC protein as AEC2s transition to the PATS.
[00170] The expression of KRT8 in bleomycin-injured lungs was tested. A significant increase in overlap between KRT8- and Ctgf-GFP-expressing cells was found by immunostaining for Ctgf-G FP, KRT8, and SFTPC in control lung and bleomycin treated lungs on day-12. These data further corroborate with elevated levels of Krt8 expression in the scRNA-seq data from organoids and LPS injury (FIG. 3). Together, these data reveal that PATS cells emerge in alveoli after bleomycin injury in vivo.
[00171] It was then tested whether PATS cells are specific to bleomycin injury or appear in other injury models. To test this, Ager-CreER; R26R-DTR mice were administered tamoxifen, followed by diphtheria toxin (DT), which resulted in selective ablation of AECls. We collected samples on days 6 and 28 after DT injection and performed immunostaining (FIG. 4A). Interestingly, cells expressing CLDN4, SFN, KRT19 and LGALS3 with elongated morphology in AEC1 -ablated lungs on day 6 but not in the controls was observed (FIG. 4B). Low levels of SFTPC expression in the elongated cells that co-expressed PATS markers was also observed by immunostaining, probably due to perdurance of SFTPC during the transition of AEC2 to PATS. Note no PATS markers were observed by immunostaining in AEC1 -ablated lungs on day 28, suggesting that PATS cells had matured into fully differentiated AECls. These data suggest that the emergence of the PATS is a general mechanism in alveolar regeneration.
Example 3: Lineage tracing reveals that PATS cells originate from AEC2s.
[00172] The data presented earlier from organoids and injury models suggested that PATS cells originate from AEC2s. To empirically test this hypothesis, we used a Sftpc-CreER ; R26R-tdTomato (hereafter refered to as Sftpc-tdT+) mouse model, in which the administration of tamoxifen permanently induces tdTomato expression specifically in AEC2s and their descendants (FIG. 5A). On day 10 post bleomycin administration, lineage-labeled (tdTomato+, tdt+) cells co-expressing LGALS3, SFN and CLDN4 in damaged alveolar regions but not in control lungs was observed. Tdt+ cells co-expressing AGER displayed a flat, thin and elongated morphology at the later times, confirming that AEC2s transition to AECls. Interestingly, lineage-labeled cells co-expressing PATS markers — including SFN, CLDN4 and LGALS3 — had distinct morphological features (rounded, ‘stretched’ and elongated) during the repair process (FIG. 5B). To quantify this, we scored the lineage positive cells according CLDN4+ and/or LGALS3+ expression as well as their shape (FIG. 5C). Three distinct patterns of marker expression was observed, CLDN4+, CLDN4+LGALS3+ and LGALS3+, and found that these correlated with round, stretched and elongated morphologies, respectively (FIG. 5C and FIG. 5D). Moreover, we observed a substantial enrichment of CLDN4+ cells on day 8, CLDN4+LGALS3+ on day 10 and LGALS3+ on day 12 following bleomycin-induced injury (Fig. 21). It was noticed CLDN4+ and CLDN4+LGALS3+ cell states in the scRNA-seq dataset but not a distinct LGALS3+ population. Similar PATS marker expression was detected in AEC2-lineage-labelled mice during pneumonectomy-induced ‘physiological’ regeneration (FIG. 5E). The discrepancy between the expression of LGALS3 in scRNA-seq versus immunostaining can be due to perdurance of LGALS3 protein in the terminal AEC1. Together, this analysis revealed that the differentiation of AEC2 into AEC1 goes through two distinct states (FIG. 5F).
Example 4: Lineage-tracing analysis reveals PATS cells generate AECls [00173] Multiple lines of data from the experiments presented earlier suggest that PATS cells are en route to AEC1. To test this, an algorithm tool called Velocytol8 was used, which allows the prediction of cell differentiation trajectories based on the ratios between spliced and unspliced messenger RNA. Strong RNA velocities (trajectory) originating from the PATS1 Ctgf+ population to AEC1 through the PATS2 Lgals3+ population was observed (FIG. 6A and FIG. 6B). A CreER allele of Krtl9, a gene enriched in PATS, combined with R26R-tdTomato ( Krtl9-tdt ) was then used to carry out lineage tracing after bleomycin injury to test whether the same trajectory occurs in regenerating alveoli in vivo. First, mice were injured with bleomycin and tamoxifen was administered 7 d later to label A7v/9-expressing cells and their progeny (FIG. 6C). Immunofluorescence for AEC2, AEC1 and PATS markers was then performed.
[00174] Numerous Krtl9-t&t+ cells co-expressing AGER, a marker for AECl, were found on day 12 post bleomycin administration (FIG. 6D, FIG. 6E, FIG. 6F). In uninjured lungs, we observed tdt expression in some LGALS3+ macrophages and rarely in AECls (FIG. 6G). We did not find Krtl9-t&t+ cells co-expressing SFTPC in uninjured regions of bleomycin- treated mice, indicating that Krtl9 expression is specifically activated in response to injury in the damaged regions and the conditions we tested are well suited for labelling and tracing PATS. Co-immunostaining revealed stretched tdTomato labelled cells co-expressing PATS markers including SFN, CLDN4 and LGALS3 in bleomycin-treated lungs but not in the controls (FIGS. 6D-6G, and FIG. 6H). Thus, the combined RNA-velocity and lineage tracing analyses reveal that the newly identified PATS traverse between AEC2 and AEC1.
Example 5: Conserved transcriptional programs and pathways in PATS.
[00175] Differentiation of AEC2s to AECls is associated with a dramatic change in the cell shape and structure, from cuboidal to flat and thin. Such a transition is typically accompanied by many changes in the expression of signaling and structural proteins. It was therefore thought that these changes occur in PATS cells. To determine whether these changes occur in PATS cells, scRNA-seq data from organoid cultures (4,573 cells) and LPS- induced injury models (13, 204 cells) were analyzed. Numerous genes were conserved among PATS cells, including: genes in the TGFP signaling pathway ( Cdkn2b , Mapk3 , Myc, Nbll , Ppp2rla , Rbxl, Rhoa , Skpla , Tgfbl, Tgifl and Thbsl ), genes in the p53 signaling pathway {Bax, Casp8 , Ccndl , Ccnd2 , Ceng I , Cdk4 , Cdknla , Cycs, Ei24, Gadd45a, Gadd45b , Sesn3, Sfii , Shisa5, Thbsl and Trp53 ), genes involved in cellular senescence (Calm I , Calm2 , Capn2 , Ccndl , Ccnd2 , Cdk4 , Cdknla , Cdkn2b , Eif4ebpl , Etsl, Gadd45b , H 2-1)1, H2-K1 , h2-Q4 , H2-Q6, H2-Q7 , H2-T22 , H2-T23 , Arav, Map2k2 , Mapkl 3, Mapk3 , Myc, Ppid , Ppplca , Ppp3ca , Rbbp4 , Rheb , A v, Sqstml , Slc25a4 , Tgfb2, Trp53 , Vdacl , Vdac2 , Vdac3 and Zfp36ll ), tight junction signaling genes ( Actb , Actgl , Acini, Actn4 , Actr3, Ccndl , Cdc42 , Cdk4, Cldn3, Cldn4, Cldn7 , Cldrnl8 , clln, Ezr , Fllr , HspaA, Itgbl , ./»//, Ms//, Myh9 , My 16, Myl9 , Myll2a , Myll2b , Mynl2a , Nedd4 , Ocln , Ppp2rla , Rad, Rap I a, Rhoa , Slc9a3rl , Tuba I a, Tubalb , Tubalc and Vasp), and DNA-damage checkpoint genes (Ccndl, Ccngl, Cdknla, Ddx39b, ler3, Prpfl9, Rpl26, Rps27l, Sox4, Syf2 and Trp53).
[00176] Pathway enrichment analysis revealed that TP53, TNF-NF-KB, ErbB, HIF1, Hippo-YAP, cell-cycle arrest, cytoskeletal dynamics, tight-junction and TGFP signaling are activated in PATS cells compared with other populations (FIG. 7A and FIG. 7B). Substantial enrichment for genes representative of cellular senescence and DNA-damage-response pathways was unexpectedly found (FIG. 7C and FIG. 7B).
Example 6: PATS cells naturally exhibit DNA damage and senescence during alveolar regeneration in vivo.
[00177] These data indicated that DNA-damage- and senescence-associated genes are highly enriched in PATS. To validate these findings, the lungs of bleomycin- and PBS- treated mice were assayed after 10 days for b-galactosidase activity — which serves as a biomarker of senescent cells — and gH2AC — a marker of DNA damage. Interestingly, numerous b-galactosidase-active cells were detected, based on 5-bromo-4-chloro-3-indolyl^- d-galactopyranoside (X-gal)-derived blue-colour deposition, in the bleomycin-injured but not control alveoli (FIG. 8A). This was further supported by the expression of the senescence marker CDKN1A (p21), specifically in bleomycin-treated lungs (FIG. 8B). CDKN1A expression was observed in stretched tdt+ cells but not in AGER+ cells, indicating that senescence is not observed in AECls (FIG. 8B). Similarly, an accumulation of gH2AC puncta was also detected in the nuclei of stretched Sftpc- tdt+ cells but not in AGER+ cells in bleomycin-treated lungs (FIG. 8C) Further analysis revealed a significant number of gH2 A X+Sflpc-tdt+ cells co-expressing LGALS3 in bleomycin-injured lungs but not in controls, indicating that PATS cells undergo DNA damage during alveolar regeneration (FIG. 8D).
[00178] Bleomycin is known to induce DNA damage in cells. However, both scRNA-seq analysis and immunostaining revealed a strong enrichment of DNA-damage-repair signaling in PATS cells. To avoid any potential effects from bleomycin on senescence and DNA damage, marker analysis was performed on lungs from the AECl -specific ablation model (FIG. 4A). Numerous X-gal+ cells detected and expression of CDKN1A or gH2AC in LGALS3+ cells in AECl -ablated lungs but not in control alveoli was observed (FIG. 8E, FIG. 8F, and FIG. 8G)
Example 7: PATS cells are vulnerable to mechanical stretch-induced DNA damage [00179] Previous studies have shown that ionizing radiation, oxidative stress and the mechanical forces that occur during cell migration and stretching can all cause DNA damage. Pathway analysis revealed an enrichment for genes involved in cytoskeletal changes but not oxidative stress. Given that AEC2 differentiation into AECl requires extensive cell spreading and cytoskeletal dynamics it was hypothesized that PATS cells experience mechanical stretching that can lead to DNA damage. To test this, AEC2s were purified from SFTPC GFP mice and cultured them on a plastic surface (two-dimensional (2D) culture), conditions under which AEC2s spread and differentiate into AECls (FIG. 8Hb). Most of the AEC2s stretched and either lost (GFP-) or downregulated (GFPlo) GFP expression within 5 d following plating. These cells increased their surface area through stretching and began to express AECl markers. Interestingly, AGER+ cells were also positive for the DNA-damage marker gH2AC (FIG. 8H and FIG. 81). Note that no DNA-damage markers were observed in cells on day 2 after plating (FIG. 81). Together, these data show that cuboidal AEC2s that differentiate into large and thin AECls naturally experience DNA-damage repair and undergo transient senescence.
Example 8: Genetic loss of function and pharmacological activation of TP53 signaling dysregulates AEC2 to AEC1 differentiation via PATS during alveolar injury-repair [00180] These data suggest that activation of TP53 signaling is associated with PATS in injured lungs. Previous studies in other tissues have suggested that TP53 signaling promotes differentiation and suppresses stem cell self-renewal. To determine whether enhanced activation of TP53 signaling increases AEC2 differentiation into AEC1 involving PATS after injury, bleomycin-induced injury in Sftpc-CreER ; R26-tdTomato mice was performed, followed by Nutlin-3a (TP53 activator) or dimethylsulfoxide (DMSO; control) administration starting on day 8 after injury and tissue collection on day 20 (FIG. 9A). Immunostaining for AGER revealed that Nultin-3a treatment led to significantly higher differentiation of Sftpc- tdt+ cells into AECls compared with DMSO treatment (FIG. 9B). There were no AGER-bS//pc-tdt+ cells in uninjured lungs that received Nutlin-3a, observed suggesting that Nutlin-3a-induced TP53 activation alone is not sufficient to induce differentiation of AEC2s into AECls (FIG. 9B). In addition, the effect of Nutlin-3a on the AEC2s in alveolar organoids was tested. The organoids were treated with Nutlin-3a or DMSO starting on day 7 until harvest on day 15 (FIG. 9C). The organoids treated with Nutlin-3a showed an increase in the number and intensity of AGER+ cells as well as a decrease in the number of Ki67+ proliferating cells compared with the controls (FIG. 9D). These data further support that ectopic activation of TP53 signaling enhances AEC2 differentiation in vivo and ex vivo. [00181] The data indicated that activation of TP53 signaling enhances AEC2 differentiation. To test whether TP53 is necessary for differentiation of AEC2s into AECls, the Sftpc-CreER ; R 26-tdTomato ; 7// Jfl/fl mouse model was utilized, in which tamoxifen administration allows the deletion of TP53 and expression of tdTomato specifically in AEC2s. Bleomycin injury was performed, followed by tissue collection on day 12 (FIG. 9E). Sftpc-CreER ; R26-tdTomato ; Trp53+/+ mice were used as controls. Immunostaining revealed numerous stretched lineage-labeled cells co-expressing AGER in the bleomycin-treated control mice (FIG. 9F). In contrast, tdt+ cells in TP 53 -deficient lungs showed no AGER expression after bleomycin treatment and had an abnormal morphology (FIG. 9F). Note that the TP53-deficient AEC2s looked normal in the regions that did not have bleomycin-induced injury. Immunostaining and quantification revealed a significant increase in the number of C L D N4+5/// 6-tdt+ cells and fewer LG A LS 3 +Sftpc-iAi+ cells in the lungs of TP53-mutant mice compared with controls following injury (FIG. 9G).
[00182] Expression analysis revealed a significant decrease in the levels of CDKN1A (a known target of TP53) in ZP53fl/fl lungs compared with controls (FIG. 9H). Immunostaining for cell-death marker revealed no difference between TP53 mutant and controls, indicating that the loss of TP53 did not affect cell death. Together, these data revealed that TP53 is essential for the differentiation of AEC2s into AECls, and TP53- deficient AEC2s are stuck in PATS and unable to progress to AEC1.
Example 9: TP53 binds to PATS-associated gene loci and controls their expression [00183] To identify the transcription-factor modules that are potentially active in PATS cells, chromatin immunoprecipitation (ChIP) analysis was performed for the histones H3K4me3, H3K36me3 and H3K27ac to identify active promoters, transcribing genes and enhancers, respectively. PATS cells and AEC2s from bleomycin-treated Ctgf-GFP and control mice were isolated, respectively, and used for histone-marks analysis. H3K4me3 enrichment was found in genes corresponding to cell-type-specific promoters ( Sftpc in AEC2 and Fnl in PATS). Further analysis revealed enrichment of numerous H3K4me3 peaks overlapping with the transcriptional start sites and promoters of transcripts specific to PATS cells from bleomycin-damage-induced lungs compared with AEC2s from controls (FIG. 10A). Motif analysis of the H3K4me3 peaks predicted enrichment of binding sites for transcription factors, including TP53, ETS1, NF1, ATF3 and SOX4 — all of which have been implicated in PATS-enriched pathways. Enrichment for H3K4me3, H3K27ac and H3K36me3 in known TP53 target gene loci (Fas and Mdm2) in PATS compared to AEC2s. Predicted binding sites were found for TP53 in the Mdm2 enhancer and Fas promoter, two well-known direct targets of TP53. Together, these data implicate a direct role for TP53 in the transcriptional control of PATS-specific genes.
[00184] ChIP analysis was performed for TP53 in PATS cells to empirically test whether TP53 directly regulates transcriptional programs associated with PATS. Given that the scRNA-seq analysis revealed that TP53 signaling is highly enriched in the Ctgf+ subset of PATS, a Sftpc-CreER ; R26R-tdTomato ; Ctgf-GFP mouse model was generated to purify AEC2-lineage-derived PATS cells for ChIP analysis. PATS cells (tdt+GFP+) were purified from the lungs of Sftpc-CreER ; R26R-tdTomato\ Ctgf-GFP mice on day 8 post bleomycin administration and performed multiplexed indexed T7 ChIP (Mint-ChIP) analysis for TP53 (FIG. 10B). Data visualization on Integrative Genome Viewer (IGV) tracks revealed strong enrichment of TP53 on previously described targets including Mdm2, Trp53 and Hasp l . More importantly, strong enrichment of TP53 was observed on many (22 of 28) PATS target genes that we analyzed — including Fnl , Sfii , Actnl , Nuprl , My 112a, Calml , Ctgf, Cdknla and Krtl9 (FIG. IOC). Note TP53 enrichment was not observed on AEC1 or AEC2 gene loci (FIG. 10D and FIG. 10E). In addition, overlap was also found between TP53-enrichment regions and H3K4me3 or H3K27ac marks, consistent with TP53 binding on the promoters and enhancers of the PATS -associated genes (FIG. IOC). Together, these data revealed that TP53 directly binds to PATS-enriched gene promoters and enhancers.
Example 10: PATS-associated gene-expression signatures and signaling pathways are enriched in human fibrotic lungs
[00185] Recent studies suggested that the alveolar epithelial cells that line fibrotic foci in idiopathic pulmonary fibrosis (IPF) show features of senescence, growth arrest and differentiation blockade. To test whether alveolar progenitors stall their differentiation process in a PATS-like state in response to a non-permissive pathological microenvironment, a recently reported scRNA-seq dataset from the lungs of human patients with IPF29 was analyzed. First, all non-epithelial and airway cells were excluded from further analysis. Numerous AEC2s and AECls in both healthy and IPF lungs were observed, with apparent overlap in UMAP plots (FIG. 11 A). In contrast, a distinct cell cluster that was highly enriched in IPF samples and did not overlap with either AECls or AEC2s was found (FIG. 11B). These cells were previously annotated by their marker gene expression as KRT5-KRT17+. Notably, analysis of differential gene expression revealed a striking resemblance of transcripts — including Sfii , Sox4 and Fnl , and also S100A2, PTGS2, KRT17 , KRT8, CALS1 , CLDN4, MMP7 , PRSS2, IGFBP7 , COL1A1 , MDK, TAGLN , GDF15, TM4SF1 , and CTSE — between the IPF-enriched cell cluster and PATS cells from the murine organoids and injury model. The human IPF-specific cluster was therefore named as ‘PATS- like’ cells (FIG. 11A and FIG. 11B). Other transcripts that were highly enriched in the PATS-like population are CALD1, PRSS2, MMP7 and S100A2. To validate the scRNA-seq data, immunofluorescence analysis was perfomed on healthy human and fibrotic and non- fibrotic regions of lungs with IPF (FIG. 11C). Co-immunofluorescence analysis of PATS- like cells (SFN, CLDN4 and KRT17), AEC2s (SFTPC and HTP-280), AECls (AGER) and myofibroblasts (ACTA2) revealed the expression of PATS-like markers specifically in the fibrotic regions of IPF lungs but not in the healthy controls and regions of IPF lungs with a healthy appearance (FIG. 11D, FIG. HE, FIG. 11F, FIG. 11G, FIG. 11H, FIG. Ill) Interestingly, KRT17 has been recently shown to be expressed in basal cells of the normal lung and basaloid-like cells in IPF lungs (Habermann et al., 2019, doi.org/10.1101/753806; Adams et al., 2019, doi.org/10.1101/759902). A similar expression pattern of TP63, another marker for basal cells, specifically in the fibrotic, but not in non-fibrotic, regions of the IPF lungs was also found (FIG. 11H, FIG. 111). It was found that PATS-like markers are highly enriched in regions of severe fibrosis (FIG. 11D-FIG. Ill), coinciding with high levels of COL1A1 and accumulation of myofibroblasts (FIG. 7D). Quantification of PATS markers (SFN, CLDN4, KRT17 and TP63) further supported these findings (FIG. HE, FIG. 11G, and FIG. 111). Moreover, it was observed that PATS cells acquired an elongated morphology in IPF lungs as opposed to the cuboidal AEC2s in healthy lungs (FIG. 11D, FIG. 11F, and FIG. 11G). Note that AGER expression was absent in regions in IPF lungs with many PATS- like cells (FIG. 11F). Gene ontology and pathway analysis revealed an enrichment for components of p53 signaling ( CDKN1A , CDKN2A, and MDM2, as well as CCND1, CCND1, SNF, GADD45A, BID, BAX, PMAIP1, BBC3, BCL2L1, PERP, ZMAT3, IGFBP3, CD82, THBS1, SERPINB5, RRM2B, SESN3, STEAP3, TP73 ), DNA-damage checkpoint (. RPS27L and PLK3, as well as BRSK1, HMGA2, PEA15, PLK2, PRKDC, SOX4, TRIAP1 ) and cellular senescence ( TGFB2 and HIPK2 , as well as CDKN2B, CXCL8, EIF4EBP1, ESI, NFATC1, SERPINE1, TGFB 1 ,ZFP 36L 7); FIG. 11J). Moreover, the PATS-like cluster was enriched for components of focal adhesion, tight junction and regulation of the actin cytoskeleton, indicating a remarkable resemblance between PATS-like cells in IPF and those in organoids and regenerating alveoli. A significant enrichment for AREG, TGFB1 , TGFB 2 and TIMP1 — known regulators of fibrosis — were found in PATS-like cells (FIG. 11K). Next, markers of cell senescence (b-galactosidase activity and CDKN1A) and DNA-damage response (gH2AC) were analyzed in IPF and healthy lungs. The data revealed specific expression of these markers in the PATS-like population in IPF but not in healthy lungs (FIG. 11L and FIG. 11M). Together, the analysis revealed similarities between PATS cells from regenerating tissues and PATS-like cells that are specific to pathological fibrotic lungs. [00186] Discussion
[00187] This study uncovered a previously unknown transitional state (PATS) that traverses between AEC2 and AEC1 and has a specific gene expression signature. Fate mapping studies demonstrate a clear lineage relationship from AEC2 to PATS to AECl. Therefore, with the addition of these transitional cell states, this work revises the hierarchy of alveolar epithelial cells. Specific gene-expression signatures indicate that PATS are not merely undergoing a gradual loss of AEC2 characteristics but represent a unique transitional population (FIG. 12). These cells are enriched for pathways — including TP53, NF-kB, YAP, TGFp and HIF1 — previously shown to be important for lung regeneration (LaCanna, et al., 2019, J. Clin. Invest. 129, 2107-2122; Riemondy et al., 2019, JCI Insight 5, el23637; Cheng et al., 2007, J. Immunol. 178, 6504-6513; McConnell et al., 2016, Cell Rep. 17, 2173-2182). Enrichment for transcripts associated with cell-cycle arrest, senescence and SOX4 were observed, a known regulator of epithelial-mesenchymal transition and cell adhesion in other tissues33. Through genetic and pharmacological modulation, these data demonstrate that TP53 signaling is necessary and sufficient to promote AEC2 differentiation into AEC1 involving PATS in regenerating tissues. Analysis by ChIP revealed a direct control of PATS genes by TP53. This study provides direct evidence for binding of TP53 to numerous cytoskeletal genes. Moreover, TP53 binding was observed on genes that are implicated in DNA-damage pathways (Nuprl and Sox4).
[00188] The above analysis of human lungs has identified PATS-like cells that are specifically present in the fibrotic regions of IPF lungs. Similar to murine PATS cells, PATS- like cells in human lungs are characterized by enrichment for genes associated with cellular senescence, TP53 signaling and TGFp -regulated genes — all of which are known to be involved in fibrosis in multiple organs, including the lung (Zhang et al., 2015, Cell Death Dis. 6, el847; Lipson et al., 2012, Fibrogenesis Tissue Repair 5, S24). In contrast, some differences were found in gene-expression signatures between murine and human IPF- specific PATS-like cells. These include TP63, KRT17 and COL1A1, which are found only in human PATS-like cells. The RNA-velocity projections from human scRNA-seq data suggest that these KRT17+TP63+ cells originate from AEC2s. Immunofluorescence for KRT17 and TP63 further suggested that these cells are surrounded by PATS-like cells within the same alveoli.
[00189] The findings showed that PATS cells undergo extensive stretching during AEC2 differentiation into thin and large AECls, which makes them vulnerable to DNA damage, a feature associated with most degenerative lung diseases — notably pulmonary fibrosis and cancers (Kropski et al., 2013, Dis. Models Mechanisms 6, 9-17; da Silva, et al., 2013, BMC Med. Genet. 14, 93). Previous studies revealed that cell stretching causes DNA damage when they migrate or squeeze through narrow spaces (McGregor et al., 2016, Curr. Opin. Cell Biol. 40, 32-40). The injury models and 2D culture indeed suggest that AEC2s undergo extensive stretching during differentiation into AECls. Therefore, the transitional cell state has clinical implications, notably in lung diseases associated with DNA damage (da Silva, et al., 2013, BMC Med. Genet. 14, 93; Sauler et al., 2018, Eur. Respir. J. 52, 1701994). Interestingly, the PATS population is enriched for SOX4 (known to regulate cytoskeletal genes), which is induced following DNA damage and is critical in TP53 stabilization and function. Together, these data support a model in which cells evolved co-transcriptional programs to combat DNA damage, which can occur when cells undergo stretching. In addition, genome-wide studies have identified mutations in DNA-damage-repair components — such as A7Y ’(’-family genes, Z/G7, TERC, PARP and RTEL1 — with emphysema and pulmonary fibrosis40. Thus, the newly identified transitional state implicates cell-shape changes and associated vulnerabilities accompanying alveolar stem cell differentiation in the lung pathogenesis. [00190] Senescence is often seen as an age-associated pathological state in which cells acquire an abnormal and irreversible state. As described herein, it was found that alveolar stem cell differentiation involves a transitional state that exhibits cardinal features of senescence in normal tissue regeneration. Previous studies have indeed found senescent cells in developing limb bud tissues (Storer et ah, 2013, Cell, 115, 1119-1130). Therefore, senescence may not necessarily occur exclusively in aged tissues but can be a reversible transient state accompanying tissue regeneration. This study thus redefines senescence as a state that can occur as part of normal tissue-maintenance programs and can be derailed in human diseases.
[00191] In conclusion, using alveolar organoid and in vivo injury-repair models, a pre- AEC1 transitional state in lung regeneration has been identified. This unique state is associated with cellular senescence and enrichment for defective alveolar regeneration pathways (FIG. 12). These results strongly suggest that prolonged senescence and stress- mediated pathways in transitional cell states can lead to diseases such as fibrosis.
Example 11: A novel and tunable lung injury model recapitulates transient fibrosis in vivo
[00192] Pulmonary fibrosis is a complex disease, involving alterations in the epithelial, mesenchymal, and immune populations, all in turn resulting in dramatic extracellular matrix changes and tissue remodeling. As a result of these complex interactions, the initiation of fibrosis is poorly understood, and has been assumed to be a result of a combination of all of these individual factors. Yet, it is still unknown whether damage to a single cell type can result in fibrotic initiation.
[00193] To address this, cell type specific epithelial ablation models were developed (FIG. 13A), and it was found that ablation of a single cell type, AECls, results in rapid activation of alpha smooth muscle actin (aSMA or ACTA2, a marker of tissue fibrosis) in resident Pdgfra+ lipofibroblasts, while maintaining expression of Pdgfra and the stellate shape characteristic of lipofibroblasts. As these cells do not exhibit the morphological changes normally seen in myofibroblasts, they are termed “fibromyocytes” (FIG. 13B). Notably, following a single round of AEC1 ablation, fibromyocytes are transient, and as epithelial homeostasis is restored, ACTA2 expression gradually decreases. Moreover, specific ablation of AEC1 cells resulted in an intermediate PATS state, with rapid activation by day 3, and peaking at day 6. Intriguingly, PATS and fibromyocytes co-occurred, with PATS directly overlying the fibromyocyte-converted mesenchyme. PATS did not exist in significant numbers by day 10, suggesting that epithelial tissues resolve rapidly following this cell-type specific injury.
Example 12: Persistence of PATS drives lipofibroblasts to myofibroblasts in vivo [00194] Considering that a single round of AEC1 ablation resulted in only transient, reversible fibrosis, fibromyocyte transition, and no myofibroblasts, it was thought that repeated AEC1 ablation (a surrogate for repeated alveolar injury) may further drive fibrosis (FIG. 14A). The preliminary histological analysis of mouse lungs after repeat AEC1 ablation indeed revealed increased ACTA2 expression in the alveoli, as well as morphological changes in ACTA2+ cells from a stellate to a “banded” morphology (FIG. 14B, FIG. 14C, and FIG. 14D). Additionally, repeated ablation enabled identification of fibrotic foci and Masson’s tri chrome analysis demonstrated an increase in ECM production in these lungs. [00195] While repeat ablation increases the amount of time PATS are present, as a result of a recurrent need for differentiation and therefore PATS accumulation, it was also hypothesized that a single, severely damaging injury would similarly result in increased PATS and fibrosis. To demonstrate this, two distinct injury models were combined, pneumonectomy and AEC1 ablation. Importantly, the two injuries were timed such that PATS would accumulate at the same time in these two injuries. Under these conditions, severe fibrosis and myofibrogenesis was extended from the periphery of the lung to throughout the lung, as indicated by aSMA staining (FIG. 14E). Notably, in this dual injury model, thick myofibroblast bands, fibrotic foci, as well as residual PATS presence was observed in these myofibroblast-dominated regions.
Example 13: A scalable co-culture model recapitulates lung fibrosis ex vivo [00196] Collectively, the in vivo data suggested that PATS play a direct role in myofibroblast conversion. To definitively demonstrate this inter-cellular communication, culture conditions were established that would support the growth of both epithelial and lipofibroblasts and maintain their normal phenotype. Lipofibroblasts were then co-cultured with AEC2 cells (derived from SFTPC-GFP mice) or with PATS (isolated from CTGF-GFP mice (GFP+/EpCAM+) cells following bleomycin injury) for seven days and analyzed by immunostaining. Under these conditions, lipofibroblasts spontaneously expressed ACTA2 only in the presence of PATS but not with AEC2s. These data further suggest that PATS produce pro-fibrotic factors and induce the conversion of lipofibroblasts into myofibroblasts (FIG. 15)
Example 14: Lipofibroblast identity is maintained by tonic, AECl-derived PDGFA signaling
[00197] However, while the data from these three injury models suggested that the presence of PATS drives further fibrogenesis, in single AEC1 ablation, it was observed that aSMA expression in native mesenchyme precedes PATS appearance. This suggested that loss of an AEC1 in isolation is sufficient to induce a lipofibroblast to myofibroblast transition, and it was hypothesized that AECls thus maintain lipofibroblast identity via intercellular communication.
[00198] To further probe the interactions between AEC1 and fibroblasts, epithelial- mesenchymal ligand-receptor expression data was examined from homeostatic single-cell data using Cellphone DB. Notably, of the two epithelial cells found in homeostasis, AECls, as compared to AEC2s, were predicted to have more numerous and stronger communication interactions with lipofibroblasts. Interestingly, it was found that AECls directly secrete the PDGFA ligand, for which the cognate receptor, PDGFRA, is expressed solely by lipofibroblasts in the lung mesenchyme. Importantly, PDGFA expression increases as cells transition from AEC2s, through the PATS state, and finally to AECls, suggesting that PDGFA is an important signal for epithelial-mesenchymal homeostatic maintenance (FIG. 16). Thus, it was hypothesized that PDGFA maintains lipofibroblast identity, and loss of AECl and resulting loss of PDGFA is a cue for lipo-to-myofibroblast initiation. As AEC2s differentiate into AECls, PDGFA expression is restored, and this subsequently returns fibromyocytes to their native lipofibroblast state.
Example 15: Disruption of PDGFA signaling is sufficient for initiation of myofibrogenesis
[00199] To test whether loss of the PDGFA signaling axis is sufficient for myofibroblast conversion, a cell-type specific conditional knockout model of PDGFRA, PDGFRA- CreER/PDGFR P0X was generated. This mouse line only has a single functional allele of PDGFRA, and upon tamoxifen administration, results in complete loss of PDGFRA expression exclusively in PDGFRA+ cells. Notably, after just two days following PDGFRA deletion, these mice began to express aSMA in the lung mesenchyme, indicative of rapid myofibroblast conversion upon loss of the PDGFRA signaling axis (FIG. 17). Based on these data, it was hypothesized that PDGFA is necessary and sufficient for maintenance of lipofibroblasts.
Example 16: Runxl is a master transcriptional regulator of myofibrogenic fate [00200] In order to determine master regulators effecting lipofibroblast to myofibroblast transitions, transcription factors were interrogated that are preferentially upregulated in post injury conditions. Runxl, a transcription factor known to be linked to fibrotic progression in multiple organs, was found to be strongly upregulated post-AECl ablation and following bleomycin injury (FIG. 18). Importantly, Runxl expression increases across all models of fibrotic injury models described here, including AEC1 ablation, repeated AEC1 ablation, and PDGFRA deletion (FIG. 19).
Example 17: Genetic and pharmacological inhibition of RUNX1 blocks TGFb induced fibrogenesis
[00201] Using the culture system, the direct role of Runxl in myofibrogenesis was determined. Wild-type lipofibroblasts treated with TGF-B in culture express aSMA and downregulate PDGFRA. Lipofibroblasts from Runxl flox/flox mice were cultured and administered Adeno-Cre-GFP virus, resulting in efficient Runxl deletion in culture (FIG. 20A). Following Runxl deletion, myofibrogenesis was induced using TGF-B. However, unlike wild-type fibroblasts, Runxl deleted fibroblasts did not express aSMA, and maintained PDGFRA expression, indicating that Runxl is necessary for myofibrogenesis (FIG. 20B). In addition to genetic deletion, these experiments were repeated with Runxl pharmacologic inhibition, with the drug Ro 5-3335. Lipofibroblasts were cultured in the presence of Ro 5-3335 and TGF-B for 48 hours. As with Runxl deletion, Ro 5-3335 treated lipofibroblasts maintained PDGFRA expression and their stellate shape, with virtually no aSMA expression and myofibrotic conversion.
[00202] One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.
[00203] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise.
[00204] The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.

Claims

Claims:
1. A method of generating a lung injury organoid model, the method comprising: i) providing a co-culture of cells in a culture medium adapted for the culture of said cells, wherein the co-culture comprises pre-alveolar type-1 transitional cell state (PATS) cells and alveolar fibroblasts; ii) contacting the culture medium with one or more agents; and iii) analyzing the biological effect of the agent on at least one expression marker of the PATS cells and/or the alveolar fibroblasts relative to a control cell culture medium that has not been contacted by the agent.
2. The method of claim 1, wherein the PATS cells are isolated from diseased tissue.
3. The method of claim 2, wherein the diseased tissue is chronic obstructive pulmonary disease (COPD) lung tissue, pulmonary fibrosis lung tissue, idiopathic pulmonary fibrosis lung tissue, emphysema lung tissue, lung cancer tissue, Sarcoidosis lung tissue, interstitial pneumonia lung tissue, sepsis lung tissue, lung tissue having viral and bacterial infections, acute respiratory distress syndrome lung tissue, and bronchopulmonary dysplasia lung tissue.
4. The method of claim 1, wherein the PATS cells are generated by exposing lung epithelial cells to an injury-causing agent in vivo or in vitro.
5. The method of claim 4, wherein the injury causing agent is bleomycin, diphtheria toxin (DT), tamoxifen, irradiation, a virus, a bacterium, or a fungus.
6. The method of claim 1, wherein the alveolar fibroblasts are lipofibroblasts.
7. The method of claim 1, wherein the alveolar fibroblasts have disease-state characteristics.
8. The method of claim 7, wherein the disease-state characteristic comprises lipofibroblast cells that express ACTA2, the presence of myofibroblast cells, and/or cell changes from a stellate shape to a banded shape.
9. The method of claim 1, wherein the culture medium comprises a platelet-derived growth factor receptor (PDGFR) ligand.
10. The method of claim 9, wherein the PDGFR ligand is platelet-derived growth factor subunit A (PDGFA), platelet-derived growth factor subunit B (PDGFB), platelet-derived growth factor subunit C (PDGFC), and/or platelet-derived growth factor subunit D (PDGFD).
11. The method of claim 1, wherein the culture medium comprises an agent that is capable of maintaining PDGFRA expression.
12. The method of claim 11, wherein the agent is Ro 5-3335, AI-10-49 or other molecules that regulate RUNX or core binding factor (CBF) proteins.
13. A method of generating an ex vivo model for lung fibrosis, the method comprising: i) providing a culture of AEC1 cells that have undergone ablation in vivo in a culture medium adapted for the culture of said cells; ii) contacting the culture medium with one or more agents; and iii) analyzing the biological effect of the agent on at least one expression marker the AEC1 cells relative to a control cell culture medium that has not been contacted by the agent.
14. The method of claim 13, wherein the AEC1 cells have undergone at least one round of ablation.
15. The method of claim 13, wherein the AEC1 cells have undergone ablation alone or in combination with other injury models.
16. The method of claim 13, wherein the AEC1 cells have undergone repeated ablation.
17. The method of claim 16, wherein the repeated ablation occurred over the course of about three days to about two weeks.
18. The method of claim 13, wherein the AEC1 cells have undergone ablation by being exposed to bleomycin, diphtheria toxin (DT), asbestos, chemical toxic agents, tamoxifen, a virus, a bacterium, or a fungus.
19. The method of claim 13, wherein the AEC1 cells express aSMA and/or ACTA2.
20. The method of claim 13, wherein the AEC1 cells exhibit characteristics of a fibromyocyte cell.
21. A method of identifying pre-alveolar type-1 transitional cell state (PATS) cells or PATS-like cells, the method comprising: i) obtaining alveolar cells; and ii) screening the cells for one or more markers, wherein the presence of the one or more markers indicates the presence of PATS cells or PATS-like cells.
22. The method of claim 21, wherein the alveolar cells are selected from the group consisting of tracheal basal cells, bronchiolar secretory cells (also known as club cells or Clara cells), club variant cells, alveolar epithelial progenitor (AEP) cells, clara variant cells, distal lung progenitors, p63+ Krt5- airway cells, lineage negative epithelial progenitors, bronchioalveolar stem cells (BASCs), Sox9+ p63+ cells, neuroendocrine progenitor cells, distal airway stem cells, submucosal gland duct cell, induced pluripotent stem cell-derived lung stem cells and alveolar type 2 epithelial (AEC2) cells.
23. The method of claim 21, wherein the alveolar cells are obtained from the lung of a patient suffering from chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, idiopathic pulmonary fibrosis, emphysema, lung cancer, Sarcoidosis, interstitial pneumonia, sepsis, a viral infection, a bacterial infection, a fungal infection, acute respiratory distress syndrome, and/or bronchopulmonary dysplasia.
24. The method of claim 21, wherein the method further comprises contacting the alveolar cells with an agent.
25. The method of claim 23, wherein the agent is bleomycin, diphtheria toxin (DT), tamoxifen, a virus, a bacterium, or a fungus.
26. The method of claim 21, wherein the one or more markers comprises CLDN4, KRT19, SFN, LGALS3, SOX4, S100A2, PTGS2, KRT17, KRT8, CALS1, MMP7, PRSS2, IGFBP7, COL1A1, MDK, TAGLN, GDF15, TM4SF1 TP63, and/or CTSE.
27. The method of claim 21, wherein the one or more markers is CLDN4, LGALS3, and LGALS3.
28. The method of claim 21, wherein the one or more markers is CLDN4, KRT19, and SFN.
29. The method of claim 21, wherein the one or more markers is CALDl, PRSS2, MMP7, and S100A2.
30. A method of generating an in vivo tissue fibrosis model, the method comprising: i) introducing a genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling in a subject; and/or ii) administering an agent to a subject that can modulate PDGF-PDGFR signaling; and iii) analyzing cells from the subject to determine the biological effect of genetic loss of PDGF ligands and/or PDGF receptor (PDGFR) signaling and/or the biological effect of the agent that can modulate PDGF-PDGFR signaling relative to a control subject.
31. The method of claim 30, wherein the agent that can modulate PDGF-PDGFR signaling comprises a PDGFR inhibitor, a PDGFR agonist, a PDGF inhibitor, or a PDGF agonist.
32. The method of claim 30, wherein the agent that can modulate PDGF-PDGFR signaling comprises a neutralizing antibody against PDGF or PDGFR.
33. A method of generating an in vivo tissue fibrosis model, the method comprising: i) introducing a genetic loss of RUNX1, RUNX2, RUNX3, or CBFb in a subject; and/or ii) administering to the subject a RUNX1, RUNX2, RUNX3, or CBFb inhibitor; and iii) analyzing cells from the subject to determine the biological effect of the genetic loss of RUNX1, RUNX2, RUNX3, or CBFp and/or the biological effect of the RUNX1, RUNX2, RUNX3, or CBFP inhibitor relative to a control subject.
34. The method of claim 33, wherein the inhibitor is Ro 5-3335, AI-10-49, or a neutralizing antibody against RUNX1, RUNX2, RUNX3, and/or CBFp.
35. The method of claims 30 or 33, wherein the subject is a mouse.
36. The method of claims 30 or 33, wherein the cells are from the subject’s lung, heart, kidney, liver, intestine, skin, or bone marrow.
37. The method of claims 30 or 33, wherein the model is a disease model for fibrosis in the lung, liver, kidney, skin, intestines, or bone marrow.
PCT/US2020/053164 2019-09-26 2020-09-28 Lung fibrosis model and methods of using the same WO2021062412A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US17/763,991 US20220341915A1 (en) 2019-09-26 2020-09-28 Lung fibrosis model and methods of using the same
CA3156163A CA3156163A1 (en) 2019-09-26 2020-09-28 Lung fibrosis model and methods of using same
EP20868029.8A EP4034142A4 (en) 2019-09-26 2020-09-28 Lung fibrosis model and methods of using the same
JP2022519551A JP2022550141A (en) 2019-09-26 2020-09-28 Pulmonary Fibrosis Model and Methods of Using The Same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201962906241P 2019-09-26 2019-09-26
US62/906,241 2019-09-26
US202062975294P 2020-02-12 2020-02-12
US62/975,294 2020-02-12

Publications (1)

Publication Number Publication Date
WO2021062412A1 true WO2021062412A1 (en) 2021-04-01

Family

ID=75166322

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2020/053164 WO2021062412A1 (en) 2019-09-26 2020-09-28 Lung fibrosis model and methods of using the same

Country Status (5)

Country Link
US (1) US20220341915A1 (en)
EP (1) EP4034142A4 (en)
JP (1) JP2022550141A (en)
CA (1) CA3156163A1 (en)
WO (1) WO2021062412A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021216378A1 (en) * 2020-04-23 2021-10-28 The Schepens Eye Research Institute, Inc. Methods and materials for treatment of fibrosis
WO2023073738A1 (en) * 2021-10-29 2023-05-04 Universita' Degli Studi Di Trento Genetic construct for tracking and/or ablating quiescent cells
US11739326B2 (en) 2017-11-14 2023-08-29 Massachusetts Eye And Ear Infirmary RUNX1 inhibition for treatment of proliferative vitreoretinopathy and conditions associated with epithelial to mesenchymal transition

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115772492B (en) * 2023-02-13 2023-05-02 淇嘉科技(天津)有限公司 Method for constructing pulmonary fibrosis disease model in vitro by using bleomycin

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BARKAUSKAS CHRISTINA E, CHUNG MEI-I; FIORET BRYAN; GAO XIA; KATSURA HIROAKI; HOGAN BRIGID L M: "Lung organoids: current uses and future promise Development", LUNG ORGANOIDS: CURRENT USES AND FUTURE PROMISE DEVELOPMENT, vol. 144, no. 6, March 2017 (2017-03-01), pages 986 - 997, XP055576557, DOI: 10.1242/dev.140103 *
BARKAUSKAS CHRISTINA E., CRONCE MICHAEL J., RACKLEY CRAIG R., BOWIE EMILY J., KEENE DOUGLAS R., STRIPP BARRY R., RANDELL SCOTT H.,: "Type 2 alveolar cells are stem cells in adult lung.", J CLIN INVEST., vol. 123, no. 7, July 2013 (2013-07-01), pages 3025 - 3036, XP055806798, DOI: 10.1172/JCI68782 *
MCCAULEY KATHERINE B., ALYSANDRATOS KONSTANTINOS-DIONYSIOS, JACOB ANJALI, HAWKINS FINN, CABALLERO IGNACIO S., VEDAIE MARALL, YANG : "Single-Cell Transcriptomic Profiling of Pluripotent Stem Cell-Derived SCGB3A2+ Airway Epithelium", STEM CELL REPORTS., vol. 10, no. 5, 8 May 2018 (2018-05-08), pages 1579 - 1595, XP055806799, DOI: 10.1016/j.stemcr.2018.03.013 *
SHIMIZU SHINO, GABAZZA ESTEBAN C., HAYASHI TATSUYA, IDO MASARU, ADACHI YUKIHIKO, SUZUKI KOJI: "Thrombin stimulates the expression of PDGF in lung epithelial cells", AM J PHYSIOL LUNG CELL MOL PHYSIOL, vol. 279, no. 3, September 2000 (2000-09-01), pages L503 - 510, XP055806800, DOI: 10.1152/ajplung.2000.279.3.L503 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11739326B2 (en) 2017-11-14 2023-08-29 Massachusetts Eye And Ear Infirmary RUNX1 inhibition for treatment of proliferative vitreoretinopathy and conditions associated with epithelial to mesenchymal transition
WO2021216378A1 (en) * 2020-04-23 2021-10-28 The Schepens Eye Research Institute, Inc. Methods and materials for treatment of fibrosis
WO2023073738A1 (en) * 2021-10-29 2023-05-04 Universita' Degli Studi Di Trento Genetic construct for tracking and/or ablating quiescent cells

Also Published As

Publication number Publication date
CA3156163A1 (en) 2021-04-01
EP4034142A4 (en) 2023-11-15
US20220341915A1 (en) 2022-10-27
JP2022550141A (en) 2022-11-30
EP4034142A1 (en) 2022-08-03

Similar Documents

Publication Publication Date Title
Kobayashi et al. Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis
Niethamer et al. Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury
Lynch et al. Submucosal gland myoepithelial cells are reserve stem cells that can regenerate mouse tracheal epithelium
US20220341915A1 (en) Lung fibrosis model and methods of using the same
Paris et al. STAT3–BDNF–TrkB signalling promotes alveolar epithelial regeneration after lung injury
Pentinmikko et al. Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium
Zepp et al. Distinct mesenchymal lineages and niches promote epithelial self-renewal and myofibrogenesis in the lung
Cassandras et al. Gli1+ mesenchymal stromal cells form a pathological niche to promote airway progenitor metaplasia in the fibrotic lung
Tasdemir et al. BRD4 connects enhancer remodeling to senescence immune surveillance
Choi et al. Release of Notch activity coordinated by IL-1β signalling confers differentiation plasticity of airway progenitors via Fosl2 during alveolar regeneration
Yu et al. The m6A readers YTHDF1 and YTHDF2 synergistically control cerebellar parallel fiber growth by regulating local translation of the key Wnt5a signaling components in axons
Biermann et al. Epigenetic priming of human pluripotent stem cell-derived cardiac progenitor cells accelerates cardiomyocyte maturation
Ji et al. Proteomic profiling of secretome and adherent plasma membranes from distinct mammary epithelial cell subpopulations
Moghieb et al. Time-resolved proteome profiling of normal lung development
Chen et al. Hepatic progenitor cells contribute to the progression of 2-acetylaminofluorene/carbon tetrachloride-induced cirrhosis via the non-canonical Wnt pathway
Faber et al. Functional effects of SDF-1α on a CD44+ CXCR4+ squamous cell carcinoma cell line as a model for interactions in the cancer stem cell niche
Yeo et al. Chromatin accessibility dynamics of neurogenic niche cells reveal defects in neural stem cell adhesion and migration during aging
Zheng et al. PRMT5 deficiency enforces the transcriptional and epigenetic programs of Klrg1+ CD8+ terminal effector T cells and promotes cancer development
Manieri et al. Role of PDGFRA+ cells and a CD55+ PDGFRALo fraction in the gastric mesenchymal niche
Zhou et al. STRA6 is essential for induction of vascular smooth muscle lineages in human embryonic cardiac outflow tract development
Sun et al. CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of subpopulation of AEC2s
US20210322454A1 (en) Hippo regulation of cardiac vascularity, fibrosis, and inflammation
Myszczyszyn et al. Mice with renal-specific alterations of stem cell-associated signaling develop symptoms of chronic kidney disease but surprisingly no tumors
Reinecke et al. Aberrant activation of wound healing programs within the metastatic niche facilitates lung colonization by osteosarcoma cells
Gao et al. Associative analysis of multi-omics data indicates that acetylation modification is widely involved in cigarette smoke-induced chronic obstructive pulmonary disease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20868029

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3156163

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2022519551

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020868029

Country of ref document: EP

Effective date: 20220426