WO2016100956A1 - Cellules progénitrices pour la régénération du tissu pulmonaire - Google Patents

Cellules progénitrices pour la régénération du tissu pulmonaire Download PDF

Info

Publication number
WO2016100956A1
WO2016100956A1 PCT/US2015/066950 US2015066950W WO2016100956A1 WO 2016100956 A1 WO2016100956 A1 WO 2016100956A1 US 2015066950 W US2015066950 W US 2015066950W WO 2016100956 A1 WO2016100956 A1 WO 2016100956A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
lung
epithelial
progenitor cells
krt5
Prior art date
Application number
PCT/US2015/066950
Other languages
English (en)
Inventor
Harold A. Chapman
Andrew E. VAUGHAN
Original Assignee
The Regents Of The University Of California
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 The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO2016100956A1 publication Critical patent/WO2016100956A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/26—Cyanate or isocyanate esters; Thiocyanate or isothiocyanate esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0688—Cells from the lungs or the respiratory tract
    • C12N5/0689—Stem cells; Progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/42—Notch; Delta; Jagged; Serrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1402—Data analysis by thresholding or gating operations performed on the acquired signals or stored data
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1477—Multiparameters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1488—Methods for deciding

Definitions

  • the present disclosure is generally related to methods for isolating epithelial progenitor cells from mammalian lung tissue.
  • the present disclosure provides compositions comprising the isolated epithelial progenitor cells, and methods of use for repairing injured lung.
  • IPF Idiopathic pulmonary fibrosis
  • IPF prognosis is poor, with a median survival after diagnosis of only about 3 years (Blackwell et al., Am J Respir Crit Care Med, 189:214-222, 2014).
  • pharmacological interventions have been explored, many of these are no longer considered to be effective treatment options.
  • Lung transplantation in patients capable of undergoing major surgery reduces risk of death in the majority of recipients.
  • the availability of donor lungs is limited. Thus, there remains a need in the art for therapies to restore lung function.
  • the present disclosure is generally related to methods for isolating epithelial progenitor cells from mammalian lung tissue.
  • the present disclosure provides compositions comprising the isolated epithelial progenitor cells, and methods of use for repairing injured lung.
  • the present disclosure provides methods of isolating epithelial progenitor cells, comprising: (a) isolating primary epithelial cells from mammalian lung tissue by positive selection for expression of E-cadherin; and (b) isolating epithelial progenitor cells from the primary epithelial cells by positive selection for expression of integrin beta-4 (ITGB4), CD 14, and CD200.
  • the methods further comprise a step before (a) of obtaining the lung tissue from a mammalian subject.
  • the lung tissue is adult lung tissue.
  • the mammalian lung is human lung.
  • step (a) further comprises removing hematopoietic cells from the lung tissue by negative selection for expression of CD45, and one or both of CD16 and CD32.
  • the isolated progenitor cells are among a cell population that is substantially devoid of one or more of Krt5, CC10, SPC and FoxJl.
  • Some methods further comprise (c) culturing the epithelial progenitor cells in vitro under suitable conditions for obtaining an expanded population of epithelial progenitor cells.
  • the expanded population comprises is at least 2 fold (at least 2X, 3X, 4X, 5X, etc. more cells) greater or from about 2 to 20 fold greater than the starting population of isolated progenitor cells.
  • the present disclosure also provides methods comprising isolating a population of epithelial progenitor cells from a population of mammalian primary epithelial cells by positive selection for expression of integrin beta-4 (ITGB4), CD 14, and CD200, wherein the epithelial progenitor cells have the ability to differentiate into mature epithelial cells.
  • the selection is by flow cytometry or immunomagnetic separation.
  • the selection involves the use of a monoclonal antibody specific for ITGB4, a monoclonal antibody specific CD 14, and a monoclonal antibody specific for CD200.
  • compositions comprising a pharmaceutically acceptable excipient and the progenitor cells obtained by any method of the preceding paragraph.
  • the pharmaceutical composition is a suspension of the progenitor cells in a sterile, isotonic solution.
  • the present disclosure provides methods for treating an injured lung, comprising administering the pharmaceutical composition to a mammalian subject in need thereof so as to treat the injured lung.
  • the mammalian subject is a human patient.
  • the administration is intrapulmonary administration.
  • the intrapulmonary administration is by intranasal administration.
  • the mammalian subject has a chronic fibrosing lung injury, a smoking related lung injury or an acute/subacute lung injury.
  • the chronic fibrosing lung injury is idiopathic pulmonary fibrosis.
  • the acute/subacute injury is as a result of a viral infection or a bacterial infection.
  • the epithelial progenitor cells have the ability to give rise to one or both of SPC+ cells and KRT5+ cells.
  • Some methods further comprising administering a Notch signaling pathway inhibitor to the mammalian subject after the progenitor cells have engrafted in the injured lung.
  • the Notch signaling pathway inhibitor is a ⁇ -secretase inhibitor.
  • the Notch signaling pathway inhibitor is an antibody that neutralizes one of the group consisting of Notch 1, Notch 2, Notch 3, Notch4, and ligands thereof.
  • the Notch signaling pathway inhibitor is a compound selected from the group consisting of genistein, sulforaphane, quercetin, curcumin, and resveratrol.
  • FIG. 1A is a schematic depicting lineage analysis methodology.
  • FIG. IB- ID shows a quantification of lineage tracing, expressed as percentage of cells counted bearing the respective lineage tag (GFP / tdTomato).
  • FIG. 2A shows FACS segregation of epithelial (EpCam+) cells by ⁇ 4 expression and a CC10-CreERT2 lineage tag (GFP), demonstrating a ⁇ 4+ population distinct from club cells.
  • FIG. 2B shows the hierarchical clustering / heat map of RNA-seq transcriptomes from single CC10- ⁇ 4+ cells (o) and distal Krt5-CreERT2 traced cells ( ⁇ ) (columns). Listed genes (rows) were selected from >1200 differentially expressed genes identified by ANOVA.
  • FIG. 2C is a schematic depicting orthotopic cell transplantation methodology.
  • FIG. 2D-2E shows FACS isolation of ⁇ 4+ CD200+ CD14+ LNEPs.
  • FIG. 2F shows FACS isolation of Krt5-CreERT2- labeled LNEPs.
  • FIG. 3A shows quantification of Krt5+ colonies in influenza-injured mice after exposure to BALF in conjunction with DAPT.
  • FIG. 3B shows Krt5+ cell activation / expansion after DAPT treatment as measured by fraction of lung section area.
  • FIG. 3C shows a quantification of SPC expression in LNEPs in vitro.
  • FIG. 3D shows a quantification of SPC expression in Krt5-Cre traced cells following intranasal administration of DBZ.
  • FIG. 4 shows a quantification of Hessl+ cells at late time points post-influenza in SPC+ type II cells in hyperplastic foci and Krt5-CreERT2-traced (tdTomato+) cells in cystic structures.
  • FIG. 5 shows levels of Krt5 protein from whole lung mouse lysate after either influenza injury or bleomycin treatment for 11 or 17 days.
  • FIG. 6A shows cytospins of sorted CC10- ⁇ 4+ cells with multiciliated cells (green, acetylated tubulin+) and ⁇ 63+ cells (red).
  • FIG. 6B shows a PCA plot of cells sequenced in FIG. 2B. The CC10- ⁇ 4+ population is within the dotted outline.
  • FIG. 6C shows the gating on
  • FIG. 6D shows qRT-PCR analysis of mature lineage genes and genes of interest in all populations.
  • FIG. 7A shows that distinct epithelial cell types contribute to regeneration depending on the severity of parenchymal injury. Examples of each are referenced.
  • FIG. 7B shows how Notch signaling regulates the activation, expansion, and differentiation of LNEPs.
  • the present disclosure is generally related to methods for isolating epithelial progenitor cells from mammalian lung tissue.
  • the present disclosure provides compositions comprising the isolated epithelial progenitor cells, and methods of use for repairing injured lung.
  • tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ and injury-specific 1"4 .
  • Current paradigms in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers 5- " 8.
  • LNEPs rare lineage-negative epithelial stem/progenitor
  • LNEPs transplantation of LNEPs, isolated by a definitive surface profile identified through single cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population.
  • LNEPs require Notch signaling to activate the ⁇ 63/ ⁇ 5+ program whereas subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signaling post-injury led to
  • the present disclosure provides methods of isolating mammalian epithelial progenitor cells, comprising: isolating epithelial progenitor cells from primary epithelial cells by positive selection for expression of integrin beta-4 (ITGB4), CD 14, and CD200. In some embodiments, the methods further comprise the step of isolating primary epithelial cells from mammalian lung tissue by positive selection for expression of E-cadherin.
  • E-cadherin As used herein, the terms "epithelial cadherin,” “E-cadherin,” “CDHE”, “ECAD,” “cadherin-1,” “CDH1,” “uvomorulin,” “UVO,” and “CD324" refer to human protein set forth as UniProtKB - P12830 (CADH1_HUMAN), and mammalian counterparts thereof.
  • the term E- cadherin and the like also encompass variants that are at least 95% identical to the amino acid sequence of the extracellular domain of the exemplary human protein.
  • positive selection for expression of E-cadherin is accomplished using a
  • the terms "integrin beta-4," “ITGB4,” and “CD 104" refer to a human protein as set forth as UniProtKB - P16144 (ITB4_HUMAN) and mammalian counterparts thereof.
  • ITGB4 and the like also encompass variants that are at least 95% identical to the amino acid sequence of the extracellular domain of the exemplary human protein.
  • positive selection for expression of ITGB4 is accomplished using a monoclonal antibody that binds to the extracellular domain of ITGB4, which extends from positions 28-710 of the 1822 residue exemplary human protein.
  • CD 14 myeloid cell-specific leucine-rich glycoprotein
  • CD 14 and the like also encompass variants that are at least 95% identical to the amino acid sequence of the extracellular domain of the exemplary human protein.
  • positive selection for expression of CD 14 is accomplished using a monoclonal antibody that binds to the extracellular domain of CD 14, which extends from positions 20-345 of the 375 residue exemplary human protein.
  • OX-2 membrane glycoprotein refers to a human protein set forth as UniProtKB - P41217 (OX2G_HUMAN) and mammalian counterparts thereof.
  • the term CD200 and the like also encompass variants that are at least 95% identical to the amino acid sequence of the extracellular domain of the exemplary human protein.
  • positive selection for expression of CD200 is accomplished using a monoclonal antibody that binds to the extracellular domain of CD200, which extends from positions 31-232 of the 278 residue exemplary human protein.
  • treating or “treatment” of a disease refer to executing a protocol, which may include administering one or more pharmaceutical compositions to an individual (human or other mammal), in an effort to alleviate signs or symptoms of the disease.
  • treating does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a palliative effect on the individual.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival of an individual not receiving treatment.
  • “Palliating" a disease or disorder means that the extent and/or undesirable clinical manifestations of the disease or disorder are lessened and/or time course of progression of the disease or disorder is slowed, as compared to the expected untreated outcome.
  • Notch signalling pathway inhibitor refers to compounds that reduce the activity or expression of Notch receptors. Notch signalling pathway inhibitors that reduce activity may bind to Notch receptors (e.g., Notch 1, Notch2, Notch3 or Notch 4) or their ligands (e.g., Jaggedl/2, DLL1, DLL3, etc.). These inhibitors may include neutralizing antibodies, fragments or derivatives thereof, as well as decoys, gamma- secretase inhibitors, blocking peptides and natural compounds (see, e.g., Espinoza and Miele, Pharmacol Ther, 139:95-110, 2013).
  • isolated and purifying refer to cells or compounds that are removed from their natural environment, such that they are at least 75%, 90%, 95%, 98% or 99% free from other components with which they are naturally associated.
  • a substantially pure population of epithelial progenitors cells expressing ITGB4, CD15 and CD200 may include up to 1%, 2%, 5%, 10% or 25% by number of cells of other phenotypes (e.g., cells devoid of one or more of ITGB4, CD 15 and CD200).
  • removing and “depleting” as used herein in reference to cells or compounds refer to cells or compounds that are removed from a composition containing the cells or compounds, such that the composition is at least 75%, 90%, 95%, 98% or 99% free of the cells or compounds.
  • removing hematopoietic cells from a cell suspension derived from lung tissue is intended to result in a cell suspension that includes only up to 1%, 2%, 5%, 10% or 25% by number of hematopoietic cells (e.g., cells expressing CD45 and one or both of CD 16 and CD32).
  • the term "expanded" as used herein in reference to a population of cells refers to a population of cells that has been increased by at least two-fold in number in comparison to the starting population of cells (e.g., epithelial cell population prior to culture in vitro or growth in vivo). In some embodiments, the expansion is by from 2-fold to 100-fold, preferably 5-fold to 50-fold, more preferably from 10-fold to 25-fold.
  • a polynucleotide includes one or more polynucleotides.
  • references to "about" a value or parameter describes variations of that value or parameter.
  • the term about when used in reference to 1 x 10 6 epithelial progenitor cells encompasses 0.9 x 10 6 to 1.1 x 10 6 epithelial progenitor cells.
  • BALF bronchioalveolar lavage fluid
  • IPF idiopathic pulmonary fibrosis
  • LNEP lineage-negative epithelial stem/progenitor
  • LNEP rare lineage-negative epithelial stem/progenitor
  • mice are previously described.
  • Trt5 tmL1(cre/ERT2)Blh CC10-CreERT2 (Scgblal tml(cre ERT)Blh ), FoxJl-CreERT2 (Tg (Foxjl" cre ERT2)iBih ⁇ and Cp _ eGF p (Tg(Cp-EGFP)25Gaia) mice are previously described.
  • Tg(UBC-GFP)30Scha 27 were used for donor cells. For all experiments, 6-8 week old animals of both sexes were used in equal proportions. Investigators were not blinded to mouse identity. All studies were approved by UCSF IACUC, protocol AN088356-03.
  • mice were administered three doses (Krt5-CreERT2) or five doses (SPC-CreERT2 and CC10-CreERT2) of 0.25 mg / g body weight tamoxifen in 50 ⁇ corn oil. A chase period >21 days was employed to insure the absence of residual tamoxifen prior to injury.
  • Infective viral particles were assayed by inoculation of either stock virus or homogenate (in 1 ml PBS) of left lung, spleen, and brain onto 96 well plates of confluent MDCK cells. After one hour, samples were decanted and replaced with serum- free media containing TPCK trypsin at 100 ⁇ g/ml.
  • the cells were fixed in 100% methanol, and then underwent indirect immunocytochemistry using Millipore mouse anti-influenza A (MAB 8257) at 1.25 ⁇ g/ml, followed by Vector® 102 biotinylated horse anti-mouse, and the biotin/avidin system (PK-4002) with diaminobenzidine as a chromogen. Samples were processed in triplicate over dilutions, and foci were counted in wells that yielded 30-100 discrete foci.
  • Millipore mouse anti-influenza A MAB 8257
  • Vector® 102 biotinylated horse anti-mouse and the biotin/avidin system (PK-4002) with diaminobenzidine as a chromogen.
  • mice 1.7 U / kg body weight bleomycin was administered intra-tracheally. Mice were weighed twice a week. For lineage tracing Krt5+ cells post-bleomycin, a single dose of 0.125 mg / g body weight tamoxifen was administered at day 17 post-bleomycin. [0038] Treatment of animals with ⁇ -secretase inhibitors. For DAPT administration, mice received 50 mg / kg body weight DAPT in 20 ⁇ DMSO per intraperitoneal injection, for the indicated periods.
  • Notchl (1: 1000; Abeam, ab8925), mouse anti-acetylated tubulin (1:500, Sigma, 6-1 IB- 1).
  • recipient mice were administered 50 mg / kg body weight Edu (Santa Cruz) in PBS daily. Edu was detected with Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Invitrogen).
  • BALF Bronchioalveolar lavage fluid
  • LNEPs maintained as above were dissociated and re-plated directly into SAGM baseline media with added DAPT or GSI-X (Calbiochem) at 40 or 20 ⁇ concentrations (unless otherwise indicated). For SPC induction experiments, IBMX was added when indicated. LNEPs were cultured for 7-10 days and then analyzed by immunofluorescent staining. [0051] Immunofluorescence analysis of cultured cells. Cells grown on matrigel were fixed
  • Krt5-CreERT2 / tdTomato mice were administered 280 FFU PR8 (as above) and received a single 0.25 mg/kg dose of tamoxifen 24 hours prior to sacrifice at the indicated time points. Injured mice were euthanized and perfused and lavaged with PBS. Lungs were instilled with 2% low-melting point agarose and -300 um slices were prepared on a vibratome. Lung slices were maintained in SAGM + 10 ng/ml KGF during imaging with the addition of 500 nM hydroxytamoxifen (Sigma) in order to induce recombination in all Krt5- expressing cells. Slices were imaged continuously for 12 hours in a 37° C chamber on an inverted stage with a Leica SP5 confocal microscope. Images obtained were deconvoluted with Bitplane Imaris for presentation.
  • RNA-seq Single Cell RNA-seq.
  • Distal lung epithelial cells were isolated and FACS sorted as described above from CC10-CreERT2 / mTmG mice.
  • tdTomato+ cells were sorted from tamoxifen-treated Krt5-CreERT2 / tdTomato mice. Sorted single cells were captured on a medium-sized (10-17 ⁇ cell diameter) microfluidic RNA-seq chip (Fluidigm) using the Fluidigm CI system. All downstream steps (lysis, cDNA synthesis / amplification, library preparation, sequencing, and raw data processing) were performed exactly as previously described. 12 FPKM files for each cell were analyzed using Fluidigm Singular® software running in R.
  • Influenza infection challenges pulmonary regenerative capacity due to the widespread ablation of epithelial cells in substantial areas of lung .
  • a robust expansion of regenerative Krt5+ cells in the lung parenchyma following influenza infection has been observed in mice , which we confirmed.
  • These cells also appear variably after bleomycin injury, where -1/3 of the Krt5+ cells resolved into type II pneumocytes by 50 days post- injury.
  • Krt5-CreERT2 lineage label (FIG. ID), raising the possibility that tracheal basal cells might migrate distally during injury.
  • GFP+ trace
  • Abundant Krt5+ cells arose after infection but none were fluorescent.
  • Upper-airway basal cells therefore do not contribute to this phenomenon and instead implicate a lineage-negative epithelial progenitor(s) (LNEPs) as the major source of ANp63+/Krt5+ cells.
  • LNEPs lineage-negative epithelial progenitor
  • Cytospins of CCIO- ⁇ 4+ cells revealed primary cilia on ⁇ 63+ cells and additional cells without discernible ⁇ 63, suggesting the LNEP profile extends to a larger fraction of ⁇ 63 low/neg cells.
  • the presence of primary cilia has been linked to a stem / progenitor phenotype 13 ' 14 .
  • LNEPs To assess the potential of LNEPs in vivo, we devised a transplantation assay by which ⁇ 10 5 fluorescent CC10- 4+ cells were delivered orthotopically into influenza-injured mice (FIG. 2C). Seeded LNEPs developed into multicellular structures in two patterns seemingly dependent on location: areas of type II cells virtually indistinguishable from surrounding endogenous type II cells and engraftments expressing both Krt5 and CCIO near endogenous Krt5+/CC10+ structures. ⁇ 4- type II cells engrafted infrequently in small clusters ( ⁇ 8 cells), and expressed only alveolar markers such as SPC. CC10+ cells could engraft but exhibited scant differentiation, even losing CCIO expression.
  • LNEPs as well as the viability of orthotopic cell transplantation as a functional tool.
  • Notch 1 ICD and the canonical Notch target gene Hesl were evident in the nucleus of parenchymal Krt5+ cells post- influenza. Notch activity was further validated using a Notch reporter mouse (Cp-eGFP). Krt5+ cells arising in distal airways expressed GFP in Notch reporter mice 7 days after influenza infection.
  • Persistent cysts bear a strong resemblance to "micro-honeycombing" in lungs of Idiopathic Pulmonary Fibrosis (IPF) patients. These lungs (n 10) showed almost all cystic epithelia were comprised of Krt5+ cells surrounded by either additional metaplastic Krt5+ cells or pseudostratified epithelium with ectopic but otherwise typical basal cells 21. Distinct foci of hyperplastic SPC+ cells were also present. Notch activity correlated with Krt5+ cysts but was absent in most hyperplastic SPC+ cells (FIG. 4) and in normal alveolar regions.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Pulmonology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dermatology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Emergency Medicine (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne d'une manière générale des procédés permettant d'isoler des cellules progénitrices épithéliales à partir de tissu pulmonaire de mammifères. La présente invention concerne en outre des compositions comprenant les cellules progénitrices épithéliales isolées, et des méthodes d'utilisation desdites cellules pour la réparation de lésions pulmonaires.
PCT/US2015/066950 2014-12-19 2015-12-19 Cellules progénitrices pour la régénération du tissu pulmonaire WO2016100956A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462094883P 2014-12-19 2014-12-19
US62/094,883 2014-12-19

Publications (1)

Publication Number Publication Date
WO2016100956A1 true WO2016100956A1 (fr) 2016-06-23

Family

ID=56127754

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/066950 WO2016100956A1 (fr) 2014-12-19 2015-12-19 Cellules progénitrices pour la régénération du tissu pulmonaire

Country Status (1)

Country Link
WO (1) WO2016100956A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007149447A2 (fr) * 2006-06-16 2007-12-27 The Board Of Trustees Of The Leland Stanford Junior University Cellules progénitrices pulmonaires, essais et leurs utilisations
WO2010085842A1 (fr) * 2009-01-27 2010-08-05 Australian Stem Cell Centre Limited Cellules progénitrices épithéliales de poumon, leurs utilisations et leurs procédés de fabrication
WO2013106677A1 (fr) * 2012-01-13 2013-07-18 The General Hospital Corporation Cellules progénitrices de poumon humain isolé et leurs utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007149447A2 (fr) * 2006-06-16 2007-12-27 The Board Of Trustees Of The Leland Stanford Junior University Cellules progénitrices pulmonaires, essais et leurs utilisations
WO2010085842A1 (fr) * 2009-01-27 2010-08-05 Australian Stem Cell Centre Limited Cellules progénitrices épithéliales de poumon, leurs utilisations et leurs procédés de fabrication
WO2013106677A1 (fr) * 2012-01-13 2013-07-18 The General Hospital Corporation Cellules progénitrices de poumon humain isolé et leurs utilisations

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DRISCOLL, BARBARA ET AL.: "Isolation and characterization of distal lung progenitor cells", AUTHOR MANUSCRIPT, METHODS IN MOLECULAR BIOLOGY, vol. 879, 2012, pages 109 - 122 *
SCHNIEDERMANN, JUDITH ET AL.: "Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels", BIOMED CENTRAL (BMC) CELL BIOLOGY, vol. 11, no. 50, 2010, pages 1 - 13 *
VAUGHAN, ANDREW E. ET AL.: "Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury", NATURE, vol. 517, no. 7536, 29 January 2015 (2015-01-29), pages 621 - 625 *

Similar Documents

Publication Publication Date Title
Vaughan et al. Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury
Hsieh et al. Galectin-3 regulates hepatic progenitor cell expansion during liver injury
Cheong et al. Identification of cell surface markers glypican-4 and CD200 that differentiate human corneal endothelium from stromal fibroblasts
US11624056B2 (en) Method for producing cartilage cells induced to be differentiated from stem cells
US20240000823A1 (en) Compositions and methods of treating fanconi anemia
US20230203450A1 (en) Method of producing organoid derived from lung epithelial cell or lung cancer cell
Lemaître et al. Cyclosporine A drives a Th17-and Th2-mediated posttransplant obliterative airway disease
CN108139405B (zh) Eb1作为药物应答的生物标记物的用途
WO2020150290A2 (fr) Procédés et compositions pour restaurer les taux de stmn2
US20170202910A1 (en) Differentiation marker and differentiation control of eye cell
CN109152799B (zh) 胰腺干细胞及其用途
Sachs et al. ADAM10-mediated ectodomain shedding is an essential driver of podocyte damage
WO2016104627A1 (fr) Fluide de surnageant de culture de cellules dérivé de tissu pulmonaire
Delaney et al. Human pluripotent stem cell modeling of tuberous sclerosis complex reveals lineage-specific therapeutic vulnerabilities
Koennecke et al. Neuronal differentiation capability of nasal polyps of chronic rhinosinusitis
WO2016100956A1 (fr) Cellules progénitrices pour la régénération du tissu pulmonaire
US20240003878A1 (en) Enriched bioactive renal cell populations, characteristics and uses thereof
KR101219624B1 (ko) 조직 손상의 치료 또는 조직 재생 촉진용 약학조성물
EP4182442A1 (fr) Méthodes permettant une infection d'organoïdes pulmonaires distaux humains par le sars-cov-2 et par d'autres agents pathogènes et leur différenciation
Yang et al. Human liver stem cells alleviate Con-A induced liver injury by regulating the balance of Treg/Th17 cells
WO2024063109A1 (fr) Dispositif d'élimination de cellules sénescentes
WO2021015218A1 (fr) Prévention ou traitement d'une fibrose ciblant le facteur associé à la transcription
EP3231434A1 (fr) Procédé de traitement de la maladie de parkinson
WO2022196747A1 (fr) Thérapie de l'insuffisance cardiaque et de ses maladies concomitantes, agent thérapeutique et méthode de diagnostic
Becknell et al. Keratin 5 basal cells are temporally regulated developmental and tissue repair progenitors in bladder urothelium

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: 15871260

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15871260

Country of ref document: EP

Kind code of ref document: A1