Pseudociliated airway epithelial cells, differentiation induction method and production method

Abstract

Provided is a method for producing a cell population that includes pseudostratified ciliated respiratory epithelial cells, the method including culturing, in a medium that flows in a particular direction, a cell population that includes at least one of respiratory epithelial precursor cells and respiratory epithelial tissue stem cells. Also provided is a cell population obtained by said production method. Provided is a method for inducing differentiation of respiratory epithelial precursor cells and respiratory epithelial tissue stem cells into pseudostratified ciliated respiratory epithelial cells, the method including culturing, in a medium that flows in a particular direction, a cell population that includes at least one of respiratory epithelial precursor cells and respiratory epithelial tissue stem cells. Also provided is a cell population that includes pseudostratified ciliated respiratory epithelial cells that have been differentiated from pluripotent stem cells, endodermal precursor cells, respiratory epithelium precursor cells, or respiratory epithelium tissue stem cells, and that demonstrates well-coordinated ciliary movement across cells.

Description

[Technical Field]

[0001] This patent application claims priority to Japanese Patent Application No. 2020-081522, the entire contents of which are incorporated herein by reference. The present disclosure relates to pseudostratified ciliated airway epithelial cells, a method for inducing differentiation, and a method for producing them. [Background technology]

[0002] Airway epithelial cells, which line the surface of the airways and bronchi, play a crucial role in removing foreign particles and pathogens through mucociliary clearance. Diseases associated with abnormalities in mucociliary clearance include chronic obstructive pulmonary disease, bronchial asthma, bronchiectasis, cystic fibrosis, and primary ciliary dyskinesia. Research into these diseases relies on primary airway epithelial cells obtained from patients or volunteers, but their supply is limited.

[0003] In recent years, methods for inducing differentiation of pluripotent stem cells such as iPS cells and ES cells into various functional cells have been developed. For example, Patent Document 1 and Non-Patent Document 1 disclose a method for inducing differentiation of iPS cells into airway epithelial cells by a process including air-liquid interface (ALI) culture. Furthermore, Non-Patent Document 2 discloses that primary cultured airway epithelial cells have been induced to differentiate into ciliated epithelial cells by ALI culture.

[0004] Meanwhile, microdevices called "organ chips" have been developed. Conventional two-dimensional or three-dimensional cell cultures are insufficient to reproduce the functions of living organisms. However, organ chips can provide tissue or organ models with higher-dimensional functions by reconstructing the tissue microenvironment essential for organ function. Non-Patent Document 3 reports an "airway chip" fabricated by ALI culture of primary cultured airway epithelial cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Application Publication No. 2016 / 148307 [Non-patent literature]

[0006] [Non-Patent Document 1] Konishi, S. et al. Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells. Stem Cell Reports 6, 18-25, doi:10.1016 / j.stemcr.2015.11.010 (2016). [Non-patent document 2] de Jong PM, et al. Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface. Am J Respir Cell Mol Biol. 1994 Mar;10(3):271-7. [Non-patent document 3] Benam, KH et al. Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro. Nat Methods 13, 151-157, doi:10.1038 / nmeth.3697 (2016). Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present disclosure is to provide a cell population comprising pseudostratified ciliated airway epithelial cells in which ciliary beats are coordinated among the cells in vitro or ex vivo. [Means for solving the problem]

[0008] The present inventors have found that culturing cells under fluid flow during differentiation into multistratified ciliated airway epithelial cells results in intercellular coordination of ciliary movement, which was not observed in conventional methods. Accordingly, the present invention provides the following aspects.

[0009] In one aspect, the present disclosure provides a method for producing a cell population comprising pseudostratified ciliated airway epithelial cells, comprising culturing a cell population comprising at least one of airway epithelial progenitor cells or airway epithelial tissue stem cells in a medium that is flowing in a certain direction.

[0010] In one aspect, the present disclosure provides a method for inducing differentiation of airway epithelial progenitor cells or airway epithelial tissue stem cells into pseudostratified ciliated airway epithelial cells, comprising culturing a cell population comprising at least one of airway epithelial progenitor cells or airway epithelial tissue stem cells in a medium that is flowing in a certain direction.

[0011] In one aspect, the present disclosure provides a cell population comprising pseudostratified ciliated airway epithelial cells in which ciliary movement is coordinated among cells, which are induced to differentiate from pluripotent stem cells, endodermal progenitor cells, airway epithelial progenitor cells, or airway epithelial tissue stem cells. [Effects of the Invention]

[0012] The present disclosure makes it possible to obtain, in vitro or ex vivo, a cell population containing pseudostratified ciliated airway epithelial cells in which ciliary beats are coordinated among the cells. [Brief explanation of the drawings]

[0013] [Figure 1] Schematic diagram of the stepwise culture method for inducing multistratified ciliated airway epithelial cells (MCACs) from human iPS cells (iPSCs) at the ALI or on an airway chip. [Figure 2] Schematic diagram of the airway chip. MCACs were cultured under fluid shear stress (FSS) induced by medium flow using a microperistaltic pump. VANGL1 is localized intracellularly at the cell interface opposite the direction of ciliary beating. [Figure 3] Figure 1 shows the subcellular localization of VANGL1 in iPS cell-derived airway cell sheets. OCLN was used as a marker for cell boundaries. Angular histograms of VANGL1 subcellular localization were calculated based on immunostaining of samples under each condition (n = 611, 189, and 545 cells from three independent ALIs, FSS(-), and FSS(+) airway chip cultures, respectively). [Figure 4] The direction of fluorescent microbead flow in iPS cell-derived airway cell sheets is shown. Angular histograms of fluorescent microbead flow were calculated based on the trajectories of samples under each condition (a total of 3–5 independent ALIs, FSS(-) airway chip cultures, and FSS(+) airway chip cultures; n (number of trajectories) = 1075, 516, and 4216, respectively). [Figure 5] The directional index (Io) of VANGL1 subcellular localization was calculated based on immunostaining of samples under each condition (mean ± standard error, n = 611, 189, and 545 cells for three independent ALIs, FSS(-), and FSS(+) airway tip cultures, respectively). One-way analysis of variance and Tukey's multiple comparison test were used. *p < 0.05. [Figure 6] Io values ​​calculated based on the fluorescent microbead trajectories of samples under each condition are shown (mean ± standard error, n = 1075, 516, and 4216 trajectories for three to five independent ALIs, FSS(-), and FSS(+) airway tip cultures, respectively). One-way analysis of variance and Tukey's multiple comparison test were used. *p < 0.05. [Figure 7] RT-qPCR results showing gene expression of ciliated airway epithelial cell markers (FOXJ1, DNAH5, and SNTN) under each condition (mean ± standard error, n = 4–5). One-way analysis of variance and Dunnett's multiple comparison test were used. DETAILED DESCRIPTION OF THE INVENTION

[0014] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0015] As used herein, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all values ​​between and including the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0016] In this disclosure, the term "cell" refers to a single cell, and may encompass multiple cells depending on the context. Multiple cells may also be referred to as a "cell population," which may be a cell population consisting of one type of cell or a cell population containing multiple types of cells depending on the context.

[0017] The disclosed method for producing a cell population containing pseudostratified ciliated airway epithelial cells, or the method for inducing differentiation into pseudostratified ciliated airway epithelial cells, comprises culturing a cell population containing airway epithelial progenitor cells or airway epithelial tissue stem cells in a medium that is flowing in a certain direction.

[0018] In the present disclosure, airway epithelial progenitor cells refer to cells that are positive for NKX2-1, SOX2, CPM, and CD47. Airway epithelial progenitor cells may further be positive for at least one of SOX9, TP63, SFTPC, SFTPB, or SCGB3A2. Preferably, airway epithelial progenitor cells are positive for NKX2-1, SOX2, CPM, and CD47, and negative for PAX8. Airway epithelial progenitor cells can differentiate into, for example, pseudostratified ciliated airway epithelial cells, airway mucus-producing cells, airway epithelial basal cells, neuroendocrine epithelial cells, myoepithelial cells and / or club cells derived from the airway submucosa.

[0019] In the present disclosure, airway epithelial tissue stem cells refer to tissue stem cells that can differentiate into ciliated epithelial cells, mucus-producing cells, goblet cells, chloride cells, brush cells, neuroendocrine epithelial cells, and airway submucosal gland cells, such as airway epithelial basal cells, club cells, or myoepithelial cells derived from the airway submucosal gland, preferably airway epithelial basal cells. Airway epithelial basal cells are positive for at least one of p63, KRT5, or NGFR. Club cells are positive for at least one of SCGB1A1 or SCGB3A2. Myoepithelial cells derived from airway submucosal glands are positive for at least one of KRT5, KRT14, or αSMA.

[0020] A cell population containing at least one of airway epithelial progenitor cells or airway epithelial tissue stem cells is, for example, a cell population in which 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more, preferably 80% or more, of the cells contained in the cell population are airway epithelial progenitor cells or airway epithelial tissue stem cells.

[0021] Airway epithelial progenitor cells or airway epithelial tissue stem cells may be isolated from a mixture of cells or tissue using a marker. A reagent with specific affinity for the cell surface marker may be used for isolation. Here, the reagent with specific affinity is an antibody, aptamer, peptide, or a compound that specifically recognizes the marker, and is preferably an antibody or a fragment thereof. The antibody may be a polyclonal or monoclonal antibody. Examples of antibody fragments include portions of antibodies (e.g., Fab fragments) or synthetic antibody fragments (e.g., single-chain Fv fragments, "ScFv").

[0022] To recognize or isolate cells expressing a cell surface marker, the affinity reagent may be bound or conjugated to a detectable substance, such as a fluorescent label, a radioactive label, a chemiluminescent label, an enzyme, biotin, or streptavidin, or to a substance that enables isolation and extraction, such as protein A, protein G, beads, or magnetic beads. The affinity reagent may also be indirectly labeled, for example, using a pre-labeled antibody (secondary antibody) that specifically binds to the antibody.

[0023] Examples of methods for isolating cells include methods in which an affinity reagent is attached to particles and then precipitated, methods in which cells are magnetically selected using magnetic beads (e.g., MACS), methods in which a cell sorter is used with fluorescent labels (e.g., FACS), and methods in which a carrier on which antibodies or the like are immobilized (e.g., a cell concentration column) is used.

[0024] A cell population containing at least one of airway epithelial progenitor cells and airway epithelial tissue stem cells is prepared by, for example, adding approximately 1.0 × 10 5 pieces / cm 2 ~1.0×10 9 pieces / cm 2 , about 1.0×10 5 pieces / cm 2 ~1.0×10 8 pieces / cm 2 , about 1.0×10 6 pieces / cm 2 ~1.0×10 8 pieces / cm 2 , about 2.0×10 6 pieces / cm 2 ~5.0×10 7 pieces / cm 2 , about 5.0×10 6 pieces / cm 2 ~2.0×10 7 pieces / cm 2 , for example, about 1.0 × 10 7 pieces / cm 2 The cells may be dissociated and then seeded. Methods for dissociating the cells include, for example, mechanical separation, separation using a separation solution having protease activity and / or collagenase activity (e.g., Accutase (商標) , Accumax (商標) and trypsin, etc.) are preferred. A separation solution having protease activity and collagenase activity (particularly preferred is Accutase (商標) The cell population is allowed to adhere to the culture vessel and then cultured in a flowing medium.

[0025] The medium used in this step can be prepared using a medium used for culturing animal cells as a basal medium. Examples of basal media include Pneumacult (商標) -ALI Supplement and Pneumacult (商標) -Pneumacult with ALI Maintenance Supplement (商標) Examples of suitable media include ALI Basal Medium (STEMCELL Technologies), IMDM medium, Medium 199 medium, EMEM medium, αMEM medium, DMEM medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium, and mixtures thereof. The media may contain serum or may be serum-free. If necessary, the media may contain one or more serum substitutes, such as albumin, transferrin, KSR, N2 supplement, B27 supplement, fatty acids, insulin, ITS premix, collagen precursor, trace elements, 2-mercaptoethanol, monothioglycerol, lipids, amino acids, L-glutamine, Glutamax, etc. (商標) The composition may contain one or more substances such as non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. (商標) -ALI Supplement and Pneumacult (商標) -Pneumacult with ALI Maintenance Supplement (商標)- ALI Basal Medium may be used. A known airway differentiation induction medium (e.g., those described in Patent Document 1, Non-Patent Document 1, Miller AJ, et al., In Vitro Induction and In Vivo Engraftment of Lung Bud Tip Progenitor Cells Derived from Human Pluripotent Stem Cells. Stem Cell Reports. 2018 Jan 9;10(1):101-119, or Huang SX et al., The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells. Nat Protoc. 2015 Mar;10(3):413-25) may also be used.

[0026] The medium may contain at least one of hydrocortisone, heparin, a ROCK inhibitor, and a NOTCH signaling inhibitor. In some embodiments, the medium contains a NOTCH signaling inhibitor. In some embodiments, the medium contains hydrocortisone, heparin, a ROCK inhibitor, and a NOTCH signaling inhibitor.

[0027] The concentration of hydrocortisone in the medium is, for example, about 0.01 to 10 μM, about 0.1 to 5 μM, or about 0.5 to 2 μM, such as about 1.0 μM, but is not limited to these. The concentration of heparin in the medium is, for example, but not limited to, about 0.1 to 100 μg / ml, about 0.5 to 20 μg / ml, or about 1 to 10 μg / ml, such as about 4 μg / ml, or about 0.1 to 100 units / ml, about 0.5 to 20 units / ml, or about 1 to 10 units / ml, such as about 4 units / ml.

[0028] ROCK inhibitors are substances that suppress the function of Rho kinase (ROCK), and examples thereof include Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride) (see, e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil / HA1077 (see, e.g., Uenata et al., Nature 389: 990-994 (1997)), H-1152 (see, e.g., Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), and Wf-536 (see, e.g., Nakajima et al., Cancer Chemother. Pharmacol. 52(4): 319-324 (2003)) and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (eg, siRNA), dominant-negative mutants, and expression vectors thereof. Other small molecule compounds are also known as ROCK inhibitors, and such compounds or their derivatives can also be used (see, for example, U.S. Patent Application Publication Nos. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, 20030087919, and International Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796). One or more ROCK inhibitors can be used. For example, Y-27632 can be used. The concentration of Y-27632 is, for example, about 0.1 to 50 μM, about 1 to 25 μM, or about 5 to 15 μM, for example, about 10 μM, but is not limited to these.

[0029] The NOTCH signal inhibitor is a substance that inhibits the Notch signal, and examples thereof include DAPT (N-[2S-(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl ester-glycine), DBZ (N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenzo[b,d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide), Compound E (N-[(1S)-2-[[(3S)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide), FLI-06 (cyclohexyl 1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-4-(4-nitrophenyl)-5-oxo-3-quinolinecarboxylate), LY411575 (N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide) and the like. For example, DAPT can be used at a concentration of, for example, about 0.1 to 50 μM, about 1 to 25 μM, or about 5 to 15 μM, such as about 10 μM, but is not limited to these.

[0030] The size and shape of the culture vessel are not limited. For example, a culture vessel having a channel (flow path) can be used to allow the culture medium to flow in a certain direction. For example, cells can be attached to the bottom of the channel, and the culture medium can be delivered from one end of the channel. The channel can be straight or curved, and can be bent. It can also intersect with other channels. Two or more channels can be bundled, and they can be connected via a porous membrane (permeable membrane). For example, a culture vessel can be used that has upper and lower channels connected via a porous membrane. A culture vessel with a channel can be provided with structures such as an inlet for delivering culture medium, culture medium components, or cells into the channel, and a solution reservoir for receiving the delivered culture medium.Examples of such culture vessels include microfluidic devices known in the art (e.g., Benam, KH et al. Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro. Nat Methods 13, 151-157, doi:10.1038 / nmeth.3697 (2016); Bhatia, SN & Ingber, DE Microfluidic organs-on-chips. Nat. Biotechnol. 32, 760-772 (2014); Esch, EW, Bahinski, A. & Huh, D. Organs-on-chips at the frontiers of drug discovery. Nat. Rev. Drug Discov. 14, 248-260 (2015); Ingber, DE Reverse Engineering Human Pathophysiology with Organs-on-Chips. Cell 164, 1105-1109, doi:10.1016 / j.cell.2016.02.049 (2016); Huh, D. et al. Reconstituting organ-level lung functions on a chip. Science 328, 1662-1668, doi:10.1126 / science.1188302 (2010); Huh, D. et al. A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Sci. Transl. Med. 4, 159ra147 (2012)).

[0031] The material of the culture vessel is not particularly limited, and examples thereof include silicone resin (e.g., polymethylsiloxane (PDMS)), polymethyl methacrylate, polyurethane, polystyrene, SU-8, glass, and metal.

[0032] The channels of the culture vessel can be fabricated using a grooved layer. For example, a culture vessel with one channel can be fabricated by bonding a grooved layer to a flat layer. For example, a culture vessel with upper and lower microchannels connected via the porous membrane can be fabricated by bonding a porous membrane to a grooved layer and then bonding another grooved layer to the opposite side. The grooved layer can be fabricated by molding the material in a mold using standard soft lithography methods. The mold can be fabricated, for example, by coating a glass substrate with a negative photoresist, such as SU-8 photoresist, masking it to leave the desired shape of the channel, exposing it to light, and removing the unexposed resist.

[0033] The cross-sectional shape of the channel is not particularly limited, and may be, for example, a rectangle. The inner length, width, and height of the channel are not particularly limited, as long as they allow cell culture and the flow of culture medium. The width of the bottom of the channel is, for example, about 0.1 mm to 20 cm, about 0.1 mm to 12 cm, about 0.1 mm to 10 cm, about 0.1 mm to 5 cm, about 0.2 mm to 1 cm, about 0.5 mm to 5 mm, about 0.7 mm to 3 mm, for example, about 1 mm, and the height is, for example, about 30 μm to 30 mm, about 60 μm to 15 mm, about 150 μm to 3 mm, or about 200 μm to 1 mm, for example, about 300 μm. The cross-sectional area of ​​the channel is, for example, about 0.003 to 6000 mm. 2 , approximately 0.03 to 100 mm 2 , about 0.05~10mm 2 , about 0.1~5mm 2 or approximately 0.1 to 1 mm 2 , for example, about 0.3 mm 2 is.

[0034] The culture vessel may be coated. The coating agent may be a naturally derived or artificially synthesized extracellular matrix, such as Matrigel, collagen (such as rat tail collagen), gelatin, laminin, heparan sulfate proteoglycan, entactin, or Geltrex. (商標)(Life Technologies; containing laminin, collagen IV, entactin, and heparin sulfate proteoglycan), and combinations thereof. In one embodiment, the coating agent is laminin, particularly recombinant laminin-511 (e.g., iMatrix (商標) The inner wall of the container may be made hydrophilic, for example by oxygen plasma treatment using methods well known to those skilled in the art.

[0035] The method for flowing the culture medium in a certain direction is not particularly limited, and any method known in the art may be used. For example, the flow of the culture medium can be achieved using a liquid delivery device that delivers the culture medium. As the liquid delivery device, an infusion pump known in the art can be used, for example, a peristaltic infusion pump.

[0036] The flow rate of the medium is, for example, about 5 μm to 5 mm / s, about 10 μm to 4 mm / s, about 500 μm to 3 mm / s, about 300 μm to 1.5 mm / s, or 1 mm to 2 mm / s, e.g., about 1.4 mm / s. The flow rate can be measured, for example, by the following method: a liquid (e.g., medium, pure water, or ultrapure water) is pumped into a culture vessel having a channel using a liquid pumping device (such as a peristaltic infusion pump) for a certain period of time (e.g., 1 hour), and the volume of the liquid pumped can be measured to calculate the volumetric flow rate (μl / h) per hour pumped by the liquid pumping device. Furthermore, the flow rate (mm / s) within the channel can be calculated from the cross-sectional area of ​​the channel. For example, a volumetric flow rate of 1500 μl / h (0.42 mm 3 / s, channel width 1 mm, height 0.3 mm, volumetric flow rate is calculated by dividing the cross-sectional area (0.3 mm 2 ), the flow rate can be calculated to be approximately 1.4 mm / s.

[0037] The shear stress applied to cells by flowing the medium in a certain direction is, for example, about 0.0001 to 0.1 Pa, about 0.0005 to 0.08 Pa, about 0.001 to 0.05 Pa, about 0.01 to 0.04 Pa, e.g., about 0.02 Pa. Methods for measuring shear stress are known, and it can be measured, for example, by the method described in Bacabac RG, et al. Dynamic shear stress in parallel-plate flow chambers. J. Biomech, 2005. For example, the shear stress can be calculated using the following formula: Shear stress (dyne / cm 2 )=6×μ(Viscosity:dyne / s / cm 2 ) × Q (volume flow rate: cm 3 / s) / h 2 (Channel height: cm) / w(Channel width: cm) For example, a volumetric flow rate of 1500 μl / h (0.00042 cm 3 / s), channel width 1mm (0.1cm), height 0.3mm (0.03cm), viscosity 0.0078 dyne / s / cm 2 (constant for DMEM with supplements at 37°C), the shear stress is 6 x 0.0078 (dyne / s / cm 2 )×0.00042(cm 3 / s) / (0.03(cm)) 2 / 0.1(cm)=0.2167dyne / cm 2 It can be calculated as follows.

[0038] The culture period may be long enough to induce differentiation into pseudostratified ciliated airway epithelial cells, but may be longer as long as the cells remain viable. For example, culture may be continued for 7, 8, 9, 10, 11, 12, 13, or 14 days or longer, or for 1, 2, 3, or 4 weeks or longer. The medium may be changed, for example, once every 1 to 7 days, e.g., once every 4, 3, or 2 days. The culture temperature may be about 30 to 40°C, about 35 to 39°C, or about 36 to 38°C, e.g., about 37°C. The CO2 concentration may be about 2 to 10%, about 2 to 7%, or about 2 to 5%, e.g., about 5%.

[0039] The method of the present disclosure may further include a step of air-liquid interface (ALI) culture. ALI culture refers to culturing cells while simultaneously exposing them to a medium and a gas phase. For example, cells are cultured with the bottom surface of the cells in contact with a liquid medium and the top surface exposed to air. For example, ALI culture can be performed in a culture vessel having upper and lower chambers separated by a porous membrane by adding medium only to the lower chamber and culturing the cells on the membrane. Examples of culture vessels suitable for ALI culture include cell culture inserts available from Corning, Thermo Fisher Scientific, Merck, etc. Preferably, the method of the present disclosure does not include a step of ALI culture.

[0040] The presence of multistratified ciliated airway epithelial cells obtained by the methods of the present disclosure can be confirmed, for example, morphologically by the presence of cells with multiple, dynamic cilia. Alternatively, it can be confirmed by the expression of a multistratified ciliated airway epithelial cell marker. Numerous multistratified ciliated airway epithelial cell markers are known, and any marker can be used. Examples include acetylated tubulin (Ac-Tub), FOXJ1, DNAH5, SNTN, GAS8, RSPH4A, DNAAF2, and CCDC39. The expression of a multistratified ciliated airway epithelial cell marker can be detected by conventional biochemical or immunochemical methods (e.g., enzyme-linked immunosorbent assay, immunohistochemical assay, etc.). Alternatively, the expression of a nucleic acid encoding a multistratified ciliated airway epithelial cell marker can be evaluated. In certain embodiments, about 30% or more, about 35% or more, about 40% or more, or about 45% or more of the cells contained in the cell population obtained by the methods of the present disclosure have multiple, dynamic cilia or express a multistratified ciliated airway epithelial cell marker.

[0041] As demonstrated by the examples of the present disclosure, cell populations containing multistratified ciliated airway epithelial cells obtained by the methods of the present disclosure are characterized by coordinated ciliary beating. "Coordinated ciliary beating" means that the direction of ciliary beating of each cell in the cell population is within a range that allows mucociliary transport.

[0042] For example, the degree of coordination of ciliary beating can be quantified as a directional index. In one embodiment, a method for quantifying the directional index comprises the following steps: (1) determining multiple unit vectors that indicate the direction of ciliary movement in a cell population; (2) calculating an average vector of the unit vectors obtained in (1); and (3) A step of calculating the length of the average vector obtained in (2) and using the length as a direction index.

[0043] In step (1), for example, a unit vector indicating the direction of ciliary beating of each cell in a cell population may be determined. In one embodiment, step (1) comprises the following steps: (a) immunostaining cells in a cell population with an anti-VANGL1 antibody; and (b) In multiple VANGL1-positive cells, the position of the center of the cell body and the position of the center of the immunostained VANGL1 localized area are determined, and a unit vector from the position of the center of the cell body to the position of the center of the VANGL1 localized area is calculated.

[0044] VANGL1 is a planar cell polarity marker expressed on the opposite side of a protruding structure called the basal foot in multistratified ciliated airway epithelial cells, and has been reported to be localized on the opposite side to the direction of ciliary beating (Vladar, EK, Nayak, JV, Milla, CE & Axelrod, JD Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation. JCI Insight 1, doi:10.1172 / jci.insight.88027 (2016)). Immunostaining can be performed by methods well known in the art. In step (b), a plurality of VANGL1-positive cells are randomly selected. For example, 40 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, or 300 or more VANGL1-positive cells are randomly selected. For example, approximately 5000 μm 2 All VANGL1-positive cells within a certain area (e.g., a microscope field of view) are selected. In each pseudostratified ciliated airway epithelial cell, VANGL1 is localized to a portion of the cell membrane, and the immunostained image has a crescent-like shape. The position of the apex of the crescent arc is taken as the center of the VANGL1-localized area. To determine the center of the cell body, immunostaining with antibodies against cell membrane proteins such as OCLN, ZO-1, or E-CAD can be performed. The shape of the cell can be considered as a polygon, and the position of its center of gravity can be taken as the center of the cell body.

[0045] If we imagine a line connecting the center of the cell body and the center of the VANGL1 localization area, and the angle between this line and a reference line (e.g., a line parallel to the horizontal axis of the microscope field of view or the flow of the medium) is θi (i = 1, ..., N), the unit vector pi from the center of the cell body to the center of the VANGL1 localization area can be expressed as pi = (cosθi, sinθi).

[0046] Alternatively, in step (1), a unit vector indicating the direction of movement of particles flowing due to ciliary movement may be obtained. For example, mucociliary transport may be visualized using microbeads. A method for visualizing mucociliary transport using microbeads is described, for example, in Non-Patent Document 1. In one embodiment, step (1) comprises the following steps: (a) adding microbeads to the culture medium of a cell population and recording the flux of the microbeads; and (b) A process of determining the start and end points of the flow of multiple microbeads and calculating the unit vector from the start point to the end point.

[0047] Microbeads may be any microbeads that can be transported and observed via mucociliary transport. The size of the microbeads may be, for example, about 0.1 to 10 μm, about 0.1 to 5 μm, or about 0.5 to 5 μm, e.g., about 0.5 μm in diameter. In the present disclosure, the diameter of the microbeads refers to the weighted average particle size measured by centrifugal sedimentation, and may be measured using a disc centrifuge such as the CPS Disc Centrifuge, Model DC24000 (CPS Instruments, Inc.). The density, concentration, and material of the microbeads may be any microbeads that can be observed. Such microbeads are commercially available, for example, from Polysciences, Inc. Methods for detecting microbeads are well known to those skilled in the art. When detecting microbeads using fluorescent labels, the type or wavelength of the fluorescent substance may be any microbead that can be observed. For example, the flow of the microbeads can be recorded using a fluorescence microscope and a CCD camera. Microbeads that can be detected in bright field may also be evaluated in bright field. In step (b), multiple microbeads are randomly selected. For example, 40 or more, 100 or more, 200 or more, 300 or more, 500 or more, 800 or more, or 1000 or more microbeads are randomly selected. 2 Select all microbeads contained within a certain area (e.g., a microscope field of view) or more.

[0048] If we imagine a straight line connecting the start and end points of the flow of the microbeads and define the angle between it and a reference line (e.g., the horizontal axis of the microscope field of view or a line parallel to the flow of the culture medium) as θi (i = 1, ..., N), the unit vector pi from the start to the end point of the flow of the microbeads can be expressed as pi = (cosθi, sinθi).

[0049] In step (2), the mean vector can be calculated by the following formula:

number

[0050] In the present disclosure, when the directional index is equal to or greater than a threshold, it may be determined that the ciliary beating is coordinated between cells. For example, the threshold value of the directional index for determining whether the ciliary beating is coordinated between cells may be 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or 0.80.

[0051] Alternatively, whether ciliary beating is coordinated can be determined by a method including the following steps (see Figure 3). (1) immunostaining cells in a cell population with an anti-VANGL1 antibody; (2) determining the position of the center of the cell body and the position of the center of the immunostained VANGL1 localized region in a plurality of VANGL1-positive cells, imagining a line from the position of the center of the cell body to the position of the center of the VANGL1 localized region, and calculating the angle θ between this line and a line parallel to the flow of the medium, with the direction of the flow of the medium being set to 0°; and (3) determining that ciliary movement is coordinated when the angle θ obtained in (2) is within the range of approximately 90° to 270° in approximately 80% or more of the cells; The steps of this method can be performed in accordance with the steps of the method for quantifying the directional index of VANGL1. In one embodiment, in step (3), ciliary beating is determined to be coordinated when the angle obtained in (2) is within the range of about 120° to 240° in about 80% or more of the cells.

[0052] Alternatively, whether ciliary beating is coordinated can be determined by a method including the following steps (see Figure 4). (1) adding microbeads to the culture medium of a cell population and recording the flow of the microbeads; (2) determining the start and end points of the flow of a plurality of microbeads, imagining a straight line from the start point to the end point, and calculating the angle θ between the straight line and a line parallel to the flow of the culture medium, with the direction of movement of the culture medium being 0°; and (3) A step of determining that ciliary movement is coordinated when the angle θ obtained in (2) is within the range of approximately -90° to 90° (i.e., approximately 270° to 360° and 0° to approximately 90°) in approximately 80% or more of the cells. The steps of this method can be carried out in accordance with the steps of the method for quantifying the directional index of microbeads. In one embodiment, in step (3), the ciliary beating is determined to be coordinated when the angle obtained in (2) is within the range of about -60° to 60° (i.e., about 300° to 360° and 0° to about 60°) for about 80% or more of the cells.

[0053] Airway epithelial progenitor cells can be differentiated from pluripotent stem cells. Pluripotent stem cells are stem cells that have the pluripotency to differentiate into all cells present in the body and the ability to proliferate. Examples include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, and Muse cells (e.g., Muse cells derived from cultured fibroblasts or bone marrow stem cells). iPS cells are preferably used. Details of these pluripotent stem cells are described, for example, in International Patent Publication No. 2016 / 148307 (Patent Document 1), which is incorporated herein by reference. Methods for inducing differentiation of pluripotent stem cells into airway epithelial progenitor cells are known, and any method may be used. For example, the methods disclosed in Patent Document 1, Non-Patent Document 1, KB McCauley et al. Cell Stem Cell 20, 844-857.e846 (2017), or YW Chen et al. Nat Cell Biol 19, 542-549 (2017), De Carvalho et al. Development 146, pii: dev171652 may be used.

[0054] In one example, airway epithelial progenitor cells are used that have been induced to differentiate from pluripotent stem cells by a method comprising the steps of: (1) culturing pluripotent stem cells in a medium containing activin A and a GSK3β inhibitor; (2) culturing the cell population obtained in step (1) in a medium containing a BMP inhibitor and a TGFβ inhibitor; (3) culturing the cell population obtained in step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor; and (4) Culturing the cell population obtained in step (3) in a medium containing a GSK3β inhibitor, FGF10, and a ROCK inhibitor.

[0055] (1) culturing pluripotent stem cells in a medium containing activin A and a GSK3β inhibitor; The medium used in step (1) can be prepared using a medium used for culturing animal cells as the basal medium. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), and mixtures thereof. The medium may contain serum or may be serum-free. Optionally, the medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS used in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, ITS premix, collagen precursors, trace elements, 2-mercaptoethanol, monothioglycerol, etc. It may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, etc. For example, RPMI 1640 medium supplemented with B27 supplement and antibiotics may be used.

[0056] Activin A is a homodimer of two beta A chains. The amino acid sequence of activin A is 100% homologous to the proteins of humans, mice, rats, pigs, cows, and cats, so the species is not particularly limited. Preferably, it is an active form in which the N-terminal peptide is cleaved, and is a homodimer in which the Gly311-Ser426 fragment obtained by cleaving the N-terminal peptide of the inhibin βA chain (e.g., NCBI accession number: NP_002183) is disulfide-bonded. Such activin A can be purchased, for example, from Wako or R&D Systems.

[0057] The concentration of activin A in the medium is, for example, about 10 to 1000 ng / ml, about 20 to 500 ng / ml, about 50 to 200 ng / ml, or about 75 to 120 ng / ml, for example, about 100 ng / ml, but is not limited to these.

[0058] GSK3β inhibitors are defined as substances that inhibit the kinase activity of GSK3β protein (for example, the ability to phosphorylate β-catenin), and many of them are already known, including the indirubin derivative BIO (also known as GSK3β inhibitor IX; 6-bromoindirubin 3'-oxime), the maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), and the phenyl α-bromo Examples of such inhibitors include the monomethyl ketone compound GSK-3β inhibitor VII (4-dibromoacetophenone), the cell membrane-permeable phosphorylated peptide L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), and the highly selective CHIR99021 (6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile). These compounds are commercially available from, for example, Calbiochem and Biomol, and are readily available, but they can also be obtained from other sources or prepared by the user.

[0059] The GSK3β inhibitor preferably used is CHIR99021. The concentration of CHIR99021 in the medium is, for example, but not limited to, about 1 nM to 50 μM, about 100 nM to 10 μM, about 200 nM to 5 μM, or about 500 nM to 2 μM, e.g., about 1 μM.

[0060] In this step, a ROCK inhibitor may be added to the medium. The ROCK inhibitor is as described above, preferably Y-27632. The concentration of Y-27632 is, for example, but not limited to, about 0.1 to 50 μM, about 1 to 25 μM, or about 5 to 15 μM, e.g., about 10 μM.

[0061] In this step, an HDAC inhibitor may be further added to the medium. HDAC inhibitors are defined as substances that inhibit or inactivate the enzymatic activity of histone deacetylase (HDAC). Examples of HDAC inhibitors include small molecule inhibitors such as valproic acid (VPA) (Nat. Biotechnol., 26(7): 795-797 (2008)), trichostatin A, sodium butyrate (NaB), MC1293, and M344; and siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool). (登録商標) (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.), and DNA methyltransferase inhibitors (e.g., 5'-azacytidine) (Nat. Biotechnol., 26(7): 795-797 (2008)).

[0062] The HDAC inhibitor preferably used is sodium butyrate (NaB), whose concentration in the medium is, for example, but not limited to, about 1 μM to 5 mM, about 20 μM to 1 mM, about 50 μM to 500 μM, or about 125 to 250 μM.

[0063] In this step, cells may be cultured in a coated culture vessel. The coating agent is as described above, and preferably Geltrex (商標) (Life Technologies) or recombinant laminin-511 (e.g., iMatrix (商標) -511(nippi)). This step may include a step of isolating the pluripotent stem cells. The method for isolating the cells is as described above.

[0064] The culture period is not particularly limited and can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more days, preferably 6 days or more, and particularly preferably 6 days. When a ROCK inhibitor is added, for example, the addition is made from the day after the start of this process, and the cells are cultured in the presence of the ROCK inhibitor for 1 or 2 days, preferably 2 days. When an HDAC inhibitor is added, for example, the addition is made from the day after the start of this process, and the cells are cultured in the presence of the HDAC inhibitor for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more days, preferably 5 days or more, and particularly preferably 5 days. The medium may be changed, for example, once every 1 to 7 days, for example, once every 4, 3, or 2 days. The culture temperature can be about 30 to 40°C, about 35 to 39°C, or about 36 to 38°C, for example, about 37°C. The CO2 concentration can be about 2-10%, about 2-7%, or about 2-5%, for example about 5%.

[0065] (2) culturing the cell population obtained in step (1) in a medium containing a BMP inhibitor and a TGFβ inhibitor; The medium used in this step can be prepared in the same manner as in step (1). Preferably, a mixed medium of DMEM medium and Ham's F12 medium supplemented with Glutamax, B27 supplement, monothioglycerol, ascorbic acid, and antibiotics is used.

[0066] Examples of BMP inhibitors include protein inhibitors such as chordin, noggin, and follistatin, dorsomorphin (i.e., 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), its derivatives (PB Yu et al. (2007), Circulation, 116: II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4: 33-41; J. Hao et al. (2008), PLoS ONE, 3(8): e2904), and LDN-193189 (i.e., 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). Dorsomorphin and LDN-193189 are commercially available from Sigma-Aldrich and Stemgent, respectively.

[0067] Noggin is preferably used as a BMP inhibitor. The concentration of Noggin in the medium is, for example, but not limited to, about 1 ng / ml to 2 μg / ml, about 10 ng / ml to 1 μg / ml, or about 50 ng / ml to 200 ng / ml, for example, about 100 ng / ml.

[0068] TGFβ inhibitors are substances that inhibit the signal transduction that follows from the binding of TGFβ to its receptor to SMAD, and they inhibit the binding of TGFβ to its receptor, the ALK family, or inhibit the phosphorylation of SMAD by the ALK family. Examples of TGFβ inhibitors include Lefty-1 (NCBI accession numbers include NM_010094 for mouse and NM_020997 for human), SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide), SB202190 (RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, and LY580276 (Lilly Research Laboratories), A-83-01 (WO2009 / 146408), and derivatives thereof.

[0069] As a TGFβ inhibitor, SB431542 is preferably used. The concentration of SB431542 in the medium is, for example, but not limited to, about 1 to 500 μM, about 2 to 100 μM, about 5 to 50 μM, for example, about 10 μM.

[0070] In this step, the culture vessel may be coated, as in step (1). This step can be carried out by exchanging the medium of the cell population obtained in step (1) with the above-mentioned medium. Alternatively, it may be carried out by dissociating the cells and reseeding them in a culture vessel. When dissociating the cells, specific cells, for example, SOX17-, FOXA2-, and / or CXCR4-positive cells (i.e., definitive endoderm cells), may be selected. Preferably, this step is carried out by exchanging the medium. As in step (1), a ROCK inhibitor may be added to the medium.

[0071] The culture period is not particularly limited and may be 1, 2, 3, 4, 5, 6, 7, 8, or more days, preferably 4 days. Other culture conditions are the same as those in step (1).

[0072] (3) culturing the cell population obtained in step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor; The medium used in this step can be prepared in the same manner as in step (1). Preferably, a mixed medium of DMEM medium and Ham's F12 medium supplemented with Glutamax, B27 supplement, monothioglycerol, ascorbic acid, and antibiotics is used.

[0073] BMP4 is a protein encoded by a polynucleotide represented by NCBI accession number NM_001202, NM_130850, or NM_130851, and may be in an activated form following cleavage by a protease.

[0074] The concentration of BMP4 in the medium is, for example, but not limited to, about 10 ng / ml to 1 μg / ml, about 10 ng / ml to 100 ng / ml, or about 10 ng / ml to 50 ng / ml, such as 20 ng / ml.

[0075] An example of retinoic acid is all-trans retinoic acid (ATRA). However, artificially modified retinoic acid that retains the functions of natural retinoic acid may also be used, such as 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-benzoic acid (AM580) (Tamura K, et al., Cell Differ Dev. 32: 17-26 (1990)), 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-benzoic acid (TTNPB) (Strickland S, et al., Cancer Res. 43: 5268-5272). (1983)), retinol palmitate, retinol, retinal, 3-dehydroretinoic acid, 3-dehydroretinol, 3-dehydroretinal, or compounds described in Abe, E., et al., Proc. Natl. Acad. Sci. (USA) 78: 4990-4994 (1981); Schwartz, EL et al., Proc. Am. Assoc. Cancer Res. 24: 18 (1983); Tanenaga, K. et al., Cancer Res. 40: 914-919 (1980).

[0076] The concentration of retinoic acid in the medium is, for example, about 1 nM to 10 μM, about 10 nM to 1 μM, about 50 nM to 1 μM, about 50 nM to 100 nM, about 10 nM to 100 nM, e.g., about 50 nM, but is not limited to these.

[0077] The GSK3β inhibitor is as described in step (1), preferably CHIR99021. The concentration of CHIR99021 in the medium is, for example, but not limited to, about 1 nM to 50 μM, about 100 nM to 20 μM, about 500 nM to 10 μM, or about 1 μM to 5 μM, e.g., about 3 μM.

[0078] In this step, the culture vessel may be coated, as in step (1). This step can be carried out by exchanging the medium of the cell population obtained in step (2) with the above-mentioned medium. Alternatively, it may be carried out by dissociating the cells and reseeding them in a culture vessel. When dissociating the cells, specific cells, for example, SOX2 and / or FOXA2 positive cells (i.e., anterior foregut endoderm cells), may be selected. Preferably, this step is carried out by exchanging the medium. As in step (1), a ROCK inhibitor may be added to the medium.

[0079] The culture period is not particularly limited and may be 1, 2, 3, 4, 5, 6, 7, 8, or more days, preferably 4 days or more, more preferably 4 days. Other culture conditions are the same as those in step (1).

[0080] (4) culturing the cell population obtained in step (3) in a medium containing a GSK3β inhibitor, FGF10, and a ROCK inhibitor; The medium used in this step can be prepared in the same manner as in step (1). Preferably, a mixed medium of DMEM medium and Ham's F12 medium supplemented with Glutamax, B27 supplement, L-ascorbic acid, monothioglycerol, and antibiotics is used.

[0081] The GSK3β inhibitor is as described in step (1), preferably CHIR99021. The concentration of CHIR99021 in the medium is, for example, but not limited to, about 1 nM to 50 μM, about 100 nM to 20 μM, about 500 nM to 10 μM, or about 1 μM to 5 μM, e.g., about 3 μM.

[0082] FGF10 is a protein encoded by a polynucleotide designated NCBI accession number NM_004465, and may be in an activated form following cleavage by a protease. FGF10 is available from, for example, Life Technologies or Wako.

[0083] The concentration of FGF10 in the medium is, for example, about 1 ng / ml to 1 μg / ml, about 10 ng / ml to 500 ng / ml, or about 50 ng / ml to 200 ng / ml, such as about 100 ng / ml, but is not limited to these.

[0084] The ROCK inhibitor is as described in step (1), preferably Y-27632. The concentration of Y-27632 is, for example, but not limited to, about 0.1 to 50 μM, about 1 to 25 μM, or about 5 to 15 μM, such as about 10 μM.

[0085] In this step, the culture vessel may be coated, as in step (1). This step can be carried out by replacing the medium of the cell population obtained in the previous step with the medium described above, or by separating the cells and reseeding them in a culture vessel.

[0086] When the cells are dissociated and reseeded into a culture vessel, a cell population containing NKX2-1-positive cells (i.e., ventral anterior foregut endoderm cells) may be isolated from the cell population obtained in step (3). For example, a cell population containing about 50%, about 60%, about 70%, about 80%, or about 90% or more of the cells may be NKX2-1-positive cells. NKX2-1-positive cells are known to be CPM-positive, CD47-positive, and CD26-negative, and can be isolated using at least one of these cell surface markers (see Non-Patent Document 1 and Hawkins F, et al. Prospective isolation of NKX2-1-expressing human lung progenitors derived from pluripotent stem cells. J Clin Invest. 2017 Jun 1;127(6):2277-2294).

[0087] In one embodiment, three-dimensional culture is performed in this step. Three-dimensional culture refers to culturing cells in a suspended state as clumps (spheroids). Three-dimensional culture can be performed using, for example, cell culture inserts provided by BD.

[0088] In the three-dimensional culture, cells may be co-cultured with other cell types. Examples of such cells include human lung fibroblasts and human fetal lung fibroblasts. Such cells are available from, for example, the American Type Culture Collection (ATCC) and DV biologics. The target cells and other cell types may be mixed at a ratio of, for example, 1:10 to 1:500. Preferably, co-culture is not performed. The cell density in the medium is, for example, about 2.0 × 10 4 ~2.0×10 8 pieces / cm 3 , about 2.0×10 5 ~2.0×10 7 pieces / cm 3 , preferably about 2.0 x 10 6 pieces / cm 3 is.

[0089] In three-dimensional culture, the above-mentioned medium may be supplemented with an extracellular matrix. The ratio of medium to extracellular matrix is, for example, 1:0.25 to 10, preferably 1:1. Here, the extracellular matrix is ​​a supramolecular structure present outside the cells, and may be naturally derived or artificial (recombinant or peptide hydrogel). Examples include collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin, or fragments thereof. These extracellular matrices may be used in combination, for example, Corning Matrigel (商標) The artificial material may be a preparation from cells such as laminin fragments or Corning PuraMatrix. (商標) is exemplified.

[0090] The culture period is not particularly limited. In this step, there may be a period of culture in the absence of a ROCK inhibitor. For example, in the absence of a ROCK inhibitor, the cells are cultured for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more, preferably 14 days or more, more preferably 14 days, and then a ROCK inhibitor is added and the cells are cultured for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more, preferably 14 days or more, more preferably 14 days. Alternatively, the cells may be cultured in the presence of a ROCK inhibitor from the beginning of the process for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days, preferably for 14 days or more, more preferably for 14 days. Other culture conditions were the same as in step (1).

[0091] Airway epithelial progenitor cells may be differentiated from endodermal progenitor cells. Endodermal progenitor cells refer to cells that emerge during the differentiation process from pluripotent stem cells to airway epithelial progenitor cells. Endodermal progenitor cells at any differentiation stage can be used, and may be naturally derived or artificially obtained by differentiation induction. For example, endodermal progenitor cells may be cells isolated from embryos using endodermal progenitor cell markers. Alternatively, they may be cells obtained by any step of the above-mentioned method for inducing differentiation of airway epithelial progenitor cells from pluripotent stem cells. Examples of endodermal progenitor cells include definitive endoderm cells (SOX17 and / or FOXA2 positive), anterior foregut endoderm cells (SOX2, SOX17, FOXA2, and / or CXCR4 positive), and ventral anterior foregut endoderm cells (NKX2-1 positive). Endodermal precursor cells can be induced to differentiate into airway epithelial precursor cells by referring to the above-mentioned method for inducing differentiation of pluripotent stem cells into airway epithelial precursor cells.

[0092] Airway epithelial tissue stem cells can be derived from living organisms. Airway epithelial tissue stem cells can be obtained by extracting airway epithelial tissue from lung tissue and culturing it in an airway epithelial cell medium (see, for example, Ando K, et al. Isolation of individual cellular components from lung tissues of patients with lymphangioleiomyomatosis. Am J Physiol Lung Cell Mol Physiol. 2016 May 15;310(10):L899-908). Airway epithelial cell media are known, and include, for example, a medium used for culturing animal cells supplemented with bovine pituitary extract, epidermal growth factor, insulin, hydrocortisone, epinephrine, triiodothyronine, transferrin, and retinoic acid. Airway epithelial cell media are commercially available, for example, as Airway Epithelial Cell Medium (PromoCell GmbH). Cells may be isolated from airway epithelial tissue, and airway epithelial basal cells, club cells, and / or myoepithelial cells derived from the airway submucosa may be selected using markers and cultured in airway epithelial cell medium. Commercially available airway epithelial tissue stem cells may also be used, such as Lonza's normal human bronchial epithelial cells (NHBE).

[0093] The airway epithelial tissue stem cells may be differentiated from pluripotent stem cells or endodermal progenitor cells. Methods for inducing differentiation of pluripotent stem cells or endodermal progenitor cells into airway epithelial tissue stem cells are known, and any method may be used. For example, the methods disclosed in Miller AJ, et al. (supra), Huang SX et al. (supra), and https: / / www.biorxiv.org / content / 10.1101 / 2020.02.21.959395v1 may be used. In one example, airway epithelial basal cells can be obtained by inducing differentiation of NKX2-1-positive cells from pluripotent stem cells or endodermal progenitor cells by steps (1) to (3) of the method for inducing differentiation of airway epithelial progenitor cells from pluripotent stem cells described above, optionally isolating the NKX2-1-positive cells and culturing them in a medium containing FGF2, FGF10, and a ROCK inhibitor, and optionally isolating NGFR-positive cells and culturing them in a medium containing a TGF-β inhibitor and a BMP inhibitor.

[0094] In one aspect, a cell population comprising pseudostratified ciliated airway epithelial cells obtained by any of the above methods is provided.

[0095] In one embodiment, a cell population is provided that contains multistratified ciliated airway epithelial cells induced to differentiate from pluripotent stem cells, endodermal progenitor cells, airway epithelial progenitor cells, or airway epithelial tissue stem cells, and that exhibit coordinated ciliary beating among the cells. In this embodiment, differentiation is artificially performed in vitro or ex vivo, and the presence of multistratified ciliated airway epithelial cells in the cell population can be confirmed, for example, by the presence of NKX2-1-positive, FOXJ1-positive, and Ac-Tub-positive cells. Coordinated ciliary beating among the cells can be confirmed by at least one of the above-mentioned methods using VANGL1 or microbeads. For example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more, preferably 80% or more, of the cells in the cell population are multistratified ciliated airway epithelial cells.

[0096] The cell density of the cell population containing pseudostratified ciliated airway epithelial cells is, for example, about 1.0 × 10 4pieces / mm 2 ~1.0×10 5 pieces / mm 2 , about 1.0×10 4 pieces / mm 2 ~5.0×10 4 pieces / mm 2 or about 1.5 x 10 4 pieces / mm 2 ~2.5×10 4 pieces / mm 2 The above cell population is, for example, about 1.0 × 10 4 pieces~1.0×10 10 pieces, approximately 2.5×10 4 pieces~1.2×10 9 pieces, approximately 1.0 pieces x 10 5 pieces~1.0×10 8 In one embodiment, the cell population is in the form of a cell sheet, i.e., layered. The cell sheet may contain one or more cell layers, and the number of cell layers may vary in some areas, and preferably contains one cell layer. The size of the cell sheet may be about 5 mm to 12 cm, about 10 mm to 10 cm, or about 20 mm to 5 cm, e.g., about 20 mm, in length, and about 0.5 mm to 10 cm, about 0.6 mm to 5 cm, about 0.7 mm to 1 cm, about 0.8 mm to 5 mm, or about 0.9 mm to 2 mm, e.g., about 1 mm, in width. The area of ​​the cell sheet is 2.5 mm 2 ~120cm 2 , about 5mm 2 ~10cm 2 , about 10mm 2 ~100mm 2 , for example, about 20 mm 2 The cell population may be provided in a medium or solution suitable for its survival or preservation. Such medium or solution is known to those skilled in the art and can be easily prepared or purchased. The cell population may be provided together with a culture vessel. The cell population may or may not be frozen.

[0097] In one aspect, a composition comprising any of the above cell populations is provided, which may be used, for example, as a pharmaceutical.

[0098] For example, the present application provides the following embodiments. [1] A method for producing a cell population containing pseudostratified ciliated airway epithelial cells, comprising culturing a cell population containing at least one of airway epithelial progenitor cells or airway epithelial tissue stem cells in a medium that is flowing in a certain direction. [2] A method for inducing differentiation of airway epithelial progenitor cells or airway epithelial tissue stem cells into pseudostratified ciliated airway epithelial cells, comprising culturing a cell population containing at least one of airway epithelial progenitor cells or airway epithelial tissue stem cells in a medium that is flowing in a certain direction. [3] The method according to item 1, wherein a cell population containing airway epithelial progenitor cells is cultured. [4] The method according to item 2, comprising culturing a cell population containing airway epithelial progenitor cells and inducing differentiation of the airway epithelial progenitor cells into pseudostratified ciliated airway epithelial cells. [5] The method according to item 3 or 4, wherein a cell population containing airway epithelial progenitor cells induced to differentiate from pluripotent stem cells or endodermal progenitor cells is cultured. [6] The method according to item 5, further comprising inducing differentiation of airway epithelial progenitor cells from pluripotent stem cells or endodermal progenitor cells. [7] The method according to any one of items 3 to 6, wherein a cell population containing airway epithelial progenitor cells induced to differentiate from pluripotent stem cells is cultured. [8] The method according to item 7, further comprising inducing differentiation of airway epithelial progenitor cells from pluripotent stem cells. [9] The method according to item 7 or 8, wherein the pluripotent stem cells are ES cells or iPS cells.

[10] The method according to any one of items 7 to 9, wherein the pluripotent stem cells are iPS cells.

[0099]

[11] The method according to any one of items 3 to 10, comprising culturing a cell population containing airway epithelial progenitor cells induced to differentiate by a method comprising the following steps: (1) culturing pluripotent stem cells in a medium containing activin A and a GSK3β inhibitor; (2) culturing the cell population obtained in step (1) in a medium containing a BMP inhibitor and a TGFβ inhibitor; (3) culturing the cell population obtained in step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor; and (4) Culturing the cell population obtained in step (3) in a medium containing a GSK3β inhibitor, FGF10, and a ROCK inhibitor.

[12] The method according to item 11, further comprising inducing differentiation of airway epithelial progenitor cells by a method comprising the steps of: (1) culturing pluripotent stem cells in a medium containing activin A and a GSK3β inhibitor; (2) culturing the cell population obtained in step (1) in a medium containing a BMP inhibitor and a TGFβ inhibitor; (3) culturing the cell population obtained in step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor; and (4) Culturing the cell population obtained in step (3) in a medium containing a GSK3β inhibitor, FGF10, and a ROCK inhibitor.

[13] The method according to paragraph 11 or 12, wherein step (4) further comprises isolating a cell population containing NKX2-1-positive cells from the cell population obtained in step (3).

[14] The method according to any one of items 11 to 13, wherein in step (4), the cell population is three-dimensionally cultured.

[15] The method according to paragraph 1, wherein a cell population containing airway epithelial tissue stem cells is cultured.

[16] The method according to item 2, wherein a cell population containing airway epithelial tissue stem cells is cultured and the airway epithelial tissue stem cells are induced to differentiate into pseudostratified ciliated airway epithelial cells.

[17] The method according to item 15 or 16, wherein a cell population containing airway epithelial tissue stem cells induced to differentiate from pluripotent stem cells or endodermal progenitor cells, or a cell population containing airway epithelial tissue stem cells collected from a living body, is cultured.

[18] The method according to any one of items 15 to 17, wherein a cell population containing airway epithelial tissue stem cells induced to differentiate from pluripotent stem cells or endodermal progenitor cells is cultured.

[19] The method according to any one of items 15 to 17, wherein a cell population containing airway epithelial tissue stem cells collected from a living body is cultured.

[20] The method according to any one of items 15 to 19, wherein the airway epithelial tissue stem cells are airway epithelial basal cells, club cells, or airway submucosal-derived myoepithelial cells.

[21] The method according to any one of items 15 to 20, wherein the airway epithelial tissue stem cells are airway epithelial basal cells.

[0100]

[22] The method according to any one of items 1 to 21, which does not include an ALI culture step.

[23] The method according to any one of items 1 to 22, wherein the flow rate of the medium is about 5 μm / s to 5 mm / s.

[24] The method of claim 23, wherein the flow rate is about 1.4 mm / s.

[25] The method according to any one of items 1 to 24, wherein the shear stress caused by the flow of the culture medium is approximately 0.0001 to 0.1 Pa.

[26] The method of claim 25, wherein the shear stress is about 0.02 Pa.

[27] The method according to any one of items 1 to 26, wherein the cell population is cultured in a microchannel.

[28] A method according to any one of items 1 to 27, comprising seeding a cell population containing at least one of airway epithelial progenitor or airway epithelial tissue stem cells on a permeable membrane of a microfluidic device having upper and lower microchannels connected via a permeable membrane, and flowing the culture medium in the upper microchannel in a certain direction.

[29] The method of paragraph 27 or 28, wherein the width of the microchannel is about 0.5 to 5 mm.

[30] The method according to any one of items 27 to 29, wherein the width of the microchannel is about 1 mm.

[31] The cross-sectional area of ​​the microchannel is approximately 0.1–5 mm 2 31. The method according to any one of items 27 to 30, wherein

[32] The cross-sectional area of ​​the microchannel is approximately 0.3 mm 2 32. The method according to any one of items 27 to 31, wherein

[33] The method according to any one of items 1 to 32, wherein the medium flowing in a certain direction contains at least one of hydrocortisone, heparin, a ROCK inhibitor, and a NOTCH signal inhibitor.

[34] The method according to any one of items 1 to 33, wherein the medium flowing in a certain direction contains a NOTCH signal inhibitor.

[35] The method according to any one of items 1 to 34, wherein the medium flowing in a certain direction contains hydrocortisone, heparin, a ROCK inhibitor, and a NOTCH signal inhibitor.

[0101]

[36] The method according to any one of items 1 to 35, for producing a cell population containing multistratified ciliated airway epithelial cells in which ciliary movement is coordinated among the cells.

[37] The method according to any one of items 1 to 36, which produces a cell population containing pseudostratified ciliated airway epithelial cells having a directionality index equal to or greater than a threshold value, as quantified by a method comprising the following steps: (1) determining multiple unit vectors that indicate the direction of ciliary movement in a cell population; (2) calculating an average vector of the unit vectors obtained in (1); and (3) A step of calculating the length of the average vector obtained in (2) and using the length as a direction index.

[38] The method according to paragraph 37, wherein step (1) comprises the following steps: (a) immunostaining cells in a cell population with an anti-VANGL1 antibody; and (b) In multiple VANGL1-positive cells, the position of the center of the cell body and the position of the center of the immunostained VANGL1 localized area are determined, and a unit vector from the position of the center of the cell body to the position of the center of the VANGL1 localized area is calculated.

[39] The method according to paragraph 37, wherein step (1) comprises the following steps: (a) adding microbeads to the culture medium of a cell population and recording the flux of the microbeads; and (b) A process of determining the start and end points of the flow of multiple microbeads and calculating the unit vector from the start point to the end point.

[40] The method according to any one of items 37 to 39, wherein 40 or more unit vectors are determined in step (1).

[41] The method according to any one of paragraphs 37 to 40, wherein the threshold is 0.40.

[42] The method according to any one of paragraphs 37 to 40, wherein the threshold is 0.45.

[43] The method according to any one of paragraphs 37 to 40, wherein the threshold is 0.50.

[44] The method according to any one of paragraphs 37 to 40, wherein the threshold is 0.55.

[45] The method according to any one of paragraphs 37 to 40, wherein the threshold is 0.60.

[46] The method according to any one of paragraphs 37 to 40, wherein the threshold is 0.65.

[47] The method according to any one of paragraphs 37 to 40, wherein the threshold is 0.70.

[48] ​​The method according to any one of paragraphs 37 to 40, wherein the threshold is 0.75.

[49] The method according to any one of paragraphs 37 to 40, wherein the threshold is 0.80.

[0102]

[50] A method for quantifying intercellular coordination of ciliary beating in a cell population as a directional index, comprising the following steps: (1) determining multiple unit vectors that indicate the direction of ciliary movement in a cell population; (2) calculating an average vector of the unit vectors obtained in (1); and (3) A step of calculating the length of the average vector obtained in (2) and using the length as a direction index.

[51] The method according to paragraph 50, wherein step (1) comprises the steps of: (a) immunostaining cells in a cell population with an anti-VANGL1 antibody; and (b) In multiple VANGL1-positive cells, the position of the center of the cell body and the position of the center of the immunostained VANGL1 localized area are determined, and a unit vector from the position of the center of the cell body to the position of the center of the VANGL1 localized area is calculated.

[52] The method according to paragraph 50, wherein step (1) comprises the steps of: (a) adding microbeads to the culture medium of a cell population and recording the flux of the microbeads; and (b) A process of determining the start and end points of the flow of multiple microbeads and calculating the unit vector from the start point to the end point.

[53] The method according to any one of items 50 to 52, wherein 40 or more unit vectors are determined in step (1).

[0103]

[54] A cell population containing pseudostratified ciliated airway epithelial cells, obtained by the method according to any one of items 1 to 49.

[55] A cell population that is induced to differentiate from pluripotent stem cells, endodermal progenitor cells, airway epithelial progenitor cells, or airway epithelial tissue stem cells, and contains pseudostratified ciliated airway epithelial cells in which ciliary movement is coordinated among cells.

[56] The cell population described in item 55, wherein the pseudostratified ciliated airway epithelial cells are differentiated by culturing pluripotent stem cells, endodermal progenitor cells, airway epithelial progenitor cells, or airway epithelial tissue stem cells in a medium flowing in a certain direction.

[57] The cell population described in item 55 or 56, wherein the pseudostratified ciliated airway epithelial cells are differentiated by culturing airway epithelial progenitor cells or airway epithelial tissue stem cells in a medium flowing in a certain direction.

[58] The cell population according to any one of items 55 to 57, comprising pseudostratified ciliated airway epithelial cells induced to differentiate from pluripotent stem cells, endodermal progenitor cells, or airway epithelial progenitor cells.

[59] The cell population according to any one of items 55 to 58, comprising pseudostratified ciliated airway epithelial cells induced to differentiate from pluripotent stem cells.

[60] The cell population according to any one of items 55 to 57, comprising pseudostratified ciliated airway epithelial cells induced to differentiate from airway epithelial tissue stem cells.

[0104]

[61] The cell population described in any one of items 54 to 60, wherein the directional index quantified by the method described in any one of items 50 to 53 is equal to or greater than a threshold value.

[62] The cell population described in paragraph 61, wherein the threshold is 0.40.

[63] The cell population described in paragraph 61, wherein the threshold is 0.45.

[64] The cell population described in paragraph 61, wherein the threshold is 0.50.

[65] The cell population described in paragraph 61, wherein the threshold is 0.55.

[66] The cell population described in paragraph 61, wherein the threshold is 0.60.

[67] The cell population of paragraph 61, wherein the threshold is 0.65.

[68] The cell population described in paragraph 61, wherein the threshold is 0.70.

[69] The cell population described in paragraph 61, wherein the threshold is 0.75.

[70] The cell population described in paragraph 61, wherein the threshold is 0.80.

[71] The cell population according to any one of items 54 to 70, which is in the form of a cell sheet.

[72] The cell population according to any one of items 54 to 71, provided together with a culture vessel.

[73] A composition comprising the cell population described in any one of items 54 to 72.

[74] A pharmaceutical composition comprising the cell population described in any one of items 54 to 72.

[0105] All documents cited herein are hereby incorporated by reference. All of the above descriptions are non-limiting and can be modified without departing from the scope of the present invention as defined in the appended claims. Furthermore, all of the following examples are non-limiting and are provided solely to illustrate the present invention. [Example]

[0106] Materials and Methods Fabrication of microfluidic devices A microfluidic device was fabricated with two microchannel layers separated by a semipermeable membrane. The microchannel layers were fabricated using polydimethylsiloxane (PDMS) by standard soft lithography techniques. PDMS prepolymer (Sylgard 184; Dow Corning) with a substrate to curing agent ratio of 10:1 was cast in a mold with a SU-8 2150 (MicroChem) pattern formed on a silicon wafer. The cross-sectional dimensions of the microchannels were 1 mm wide and 300 μm high. Two microchannel layers were bonded to a semipermeable PET membrane (#353090; Falcon) with 0.4 mm pores using a thin layer of liquid PDMS prepolymer as adhesive (Wu, H., Huang, B. & Zare, RN. Construction of microfluidic chips using polydimethylsiloxane for adhesive bonding. Lab Chip 5, 1393–1398, doi:10.1039 / b510494g (2005)). Specifically, the PDMS prepolymer was spin-coated (4000 rpm, 60 s) onto a glass slide. Then, both the top and bottom microchannel layers were placed on the glass slide, and a thin layer of PDMS prepolymer was attached to the embossed PDMS surface of the microchannel layer. The PET membrane was placed on top of the bottom microchannel layer and sandwiched between the top and bottom microchannel layers. The assembly was left at room temperature for 1 day to remove air bubbles, and then placed in an oven at 60 °C overnight to cure the PDMS (adhesive).

[0107] Differentiation of induced pluripotent stem cells (iPSCs) into multistratified ciliated airway epithelial cells (MCACs) Human iPS cells (201B7) were cultured in Essential 8 medium (Thermo Fisher Scientific) until differentiation began. After iPS cell generation, differentiation was performed within 20 passages. Mycoplasma contamination was detected using the MycoAlert mycoplasma detection kit (Lonza).

[0108] Human iPS cells were differentiated into functional airway epithelial cells by following the method described in Konishi, S. et al. Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells. Stem Cell Reports 6, 18-25, doi:10.1016 / j.stemcr.2015.11.010 (2016). Figure 1 shows an outline of the culture method, and Tables 1-1 and 1-2 show the composition of the culture medium used. Briefly, 5.0 × 10 4 pieces / cm 2 ~2.0×10 5 pieces / cm 2 Undifferentiated human iPS cells were seeded onto Geltrex (Gibco) or laminin-511-coated plates and differentiated into definitive endoderm cells in RPMI medium (Nacalai Tesque) containing a saturating dose of activin A (100 ng / ml) (Peprotech or Oriental Yeast), 1 μM CHIR99021 (Axon Medchem), 2% B27 supplement (Thermo Fisher Scientific), and 50 U / ml penicillin-streptomycin (Life Technologies). The medium was changed every two days. Y-27632 (LC Laboratories) was added on day 0, and sodium butyrate was added on days 1, 2, and 4. From days 6 to 10, the resulting definitive endoderm cells were cultured in anteriorization medium, and on day 10, they were switched to ventralization medium containing BMP4 (20 ng / ml; HumanZyme), CHIR99021 (3.0 μM), and ATRA (0.05 μM; Sigma-Aldrich). On day 14, Y-27632 was added to the medium, and the cells were detached with Accutase and transfected with NKX2-1. +Ventral anterior foregut endoderm cells (VAFEC) were immunostained using 0.2 μl of mouse anti-human CPM antibody (Abcam or Wako) per million cells in 20 μl of 1% BSA / PBS as the primary antibody. Cells were washed with 1% BSA / PBS and immunostained using anti-mouse IgG or IgG1-microbeads (Miltenyi Biotec) as the secondary antibody and LS columns (Miltenyi Biotec). + VAFEC was isolated. CPM isolated from VAFEC + cells 4.0x10 5 pieces / cm 2 The cells were resuspended in 112 μl of Step 4 medium and an equal volume of Matrigel (Corning). The total volume (224 μl) was carefully pipetted into a 12-well cell culture insert (#353180; Corning), and 1 ml of Step 4 medium was added to the lower chamber. The cells were cultured for 14 days with medium changes every other day to obtain airway epithelial progenitor cell spheroids.

[0109] In the 3D-ALI protocol, the medium in the lower chamber was switched to Step 5 medium on day 28 and changed every other day. Airway epithelial cell spheroids in 3D Matrigel blocks were isolated on day 42 and plated onto Geltrex-coated 12-well cell culture inserts at 7.5 × 10 cells in a total volume of 0.5 ml. 5 pieces / cm 2 The cells were replated at a density of 1000 μg / ml. Step 5 medium (1 ml) was added to the lower chamber, and the medium was changed every other day for 14 days. On day 43, the medium in the upper chamber was reduced to 0.125 ml to initiate ALI culture, which was continued until day 56.

[0110] A schematic diagram of microfluidic cell culture in the airway chip is shown in Figure 2. The microfluidic device was oxidized in oxygen plasma (Covance MP, Femto Science) for 5 minutes to make the surface hydrophilic. PBS was then immediately introduced into the microchannel, and the device was treated with UV light for 30 minutes in a safety cabinet (MHE-131AJ; SANYO). Prior to cell culture, the semipermeable membrane of the device was coated with recombinant laminin-511 (5.0 μg / cm). 2 The airway epithelial progenitor cell spheroids in Matrigel were dissociated on day 28 and plated on the laminin-coated membrane in the upper channel of the device at 1.0 × 10 cells per well. 7 Cells were seeded at a density of 50-100 μl / ml, and 100 μl of Step 5 medium was added to the lower channel. After 4 hours, 200 μl of Step 5 medium was added to the upper channel. After a further 8 hours, fluid flow was initiated using a microperistaltic pump (RP-TXP5F or RP-HXP5F; Aquatech Co. Ltd.) at a volumetric flow rate of 1500 μl / h (flow rate 1.39 mm / s, shear stress 0.022 Pa). The microfluidic device was placed in an incubator set at 5% CO2 and 37°C, and the Step 5 medium in both channels was changed every other day until day 42. The same culture was performed at different volumetric flow rates of 10.14 μl / h, 60 μl / h, and 360 μl / h.

[0111] [Table 1-1] [Table 1-2]

[0112] Immunofluorescence (IF) staining 2D cell culture samples were fixed with 100% methanol at -20°C for 7 minutes. The samples were permeabilized with 0.2% TritonX-100 / PBS for 15 minutes, immersed in a blocking solution containing 5% normal donkey serum (EMD-Millipore) and 1% BSA in PBS for 30 minutes, and immunostained with primary and secondary antibodies for 30 minutes each as previously described (Gotoh, S. et al. Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells. Stem Cell Reports 3, 394-403, doi:10.1016 / j.stemcr.2014.07.005 (2014)). All antibodies used in this study are listed in Table 2. Immunofluorescence images were obtained using a TCS SP8 confocal microscope (Leica Microsystems).

[0113] [Table 2]

[0114] Quantitative analysis of mucociliary transport in MCACs sheets Each sample was placed on a glass slide and immersed in 100 μl of Step 5 medium. For analysis of the MCACs sheet in the airway chip, the microfluidic device was disassembled with a disposable scalpel (Feather No. 11), and the cell culture membrane was removed and placed on a glass slide and immersed in 100 μl of Step 5 medium. The sample was placed on a thermoplate (37°C; Tokai Hit), and fluorescent microbeads (500-fold diluted Fluoresbrite) were applied to the MCACs sheet. (登録商標)YG Microspheres, 0.5 μm; Polysciences (17152) were placed on the periphery of the periphery. The flow of fluorescent microbeads was recorded at 36–42 fps using an Orca-ER CCD camera (Hamamatsu) connected to an upright fluorescence microscope (BX53; Olympus) equipped with a 60x water-immersion objective. A total of 64 frames were acquired per take. Fluorescent microbeads were traced using TrackMate (an ImageJ / Fiji software program plugin). To improve detection of the fluorescent microbeads, a Gaussian filter was applied to the original sequential images of the fluorescent microbeads, and the processed images were subtracted from the original sequential images to remove the diffraction rings of the fluorescent microbeads. After extracting the time series data of the fluorescent microbead positions, the trajectories of the fluorescent microbeads due to mucociliary transport were extracted using a previously reported method (Konishi, S. et al., op. cit.). More than 150 trajectories of the extracted fluorescent microbeads were analyzed in each experiment.

[0115] The time series data of the fluorescent microbead positions were used to evaluate the direction of each fluorescent microbead trajectory. The angle histograms of the fluorescent microbead trajectories in the cell sheets cultured under each condition were analyzed using a Matlab software program (Mathworks). We also analyzed the degree of unidirectionality of the fluorescent microbead trajectories using the orientation index used to evaluate the unidirectionality of basal bodies and foot pairs in mouse pseudostratified ciliated airway epithelial cells in a previous study (Herawati, E. et al. Multiciliated cell basal bodies align in stereotypical patterns coordinated by the apical cytoskeleton. J Cell Biol 214, 571–586, doi:10.1083 / jcb.201601023 (2016)).

[0116] The directional index was calculated as follows: A set of vectors connecting the start and end points of each fluorescent microbead trajectory was extracted. To avoid overestimation of mucociliary transport, trajectories shorter than four frames were excluded. The angle between each vector and the horizontal axis was defined as θi (i = 1, …, N), where N represents the total number of trajectories detected by TrackMate (an ImageJ / Fiji software program plugin). The unit vector in two dimensions pi was defined as pi = (cosθi, sinθi). Based on this definition, a single average vector p was calculated using the following equation:

number

[0117] Quantitative analysis of subcellular localization of VANGL1 on MCACs To analyze the subcellular localization of VANGL1 on MCACs in cell sheets, cell sheets were simultaneously immunostained for VANGL1, OCLN, and Ac-TUB. VANGL1 is a protein previously reported to be central to planar cell polarity (PCP) in airway epithelial cells, including MCACs, and is known to be localized subcellularly opposite the direction of cilia beating (Vladar, EK, et al., supra). OCLN and Ac-TUB are markers of cell boundaries and cilia, respectively. Immunostained images were obtained using a confocal microscope (TCS SP8; Leica Microsystems) equipped with a 63x oil immersion objective. The recorded 3D images were converted to 2D images by maximum intensity projection in the z-direction. As a result, we were able to identify multistratified ciliated airway epithelial cells in all cell sheets, and we were able to observe the boundaries of multistratified ciliated airway epithelial cells and the subcellular localization of VANGL1. VANGL1 was significantly expressed in mature MCACs and localized at some of the cell boundaries. Next, MCACs were detected by the presence of multiple cilia stained with Ac-TUB. Using the ImageJ / Fiji software program, the positions of the center of the cell body and the tip of VANGL1 were extracted for all MCACs in the cell sheet under each condition. The angle between the horizontal axis and the line connecting the apex of the VANGL1 crescent to the center of the cell body was calculated, and a directional index was calculated, similar to the quantitative analysis of mucociliary transport. For each iPS cell line, more than 35 cells were analyzed per experiment.

[0118] Quantitative reverse transcription PCR (qRT-PCR) analysis RNA was obtained using the PureLink RNA mini kit (Invitrogen) or, for cells cultured in microfluidic devices, NucleoSpin (登録商標)Total RNA was extracted using RNA Plus XS (Takara Bio). cDNA was synthesized from 80 ng of total RNA per sample using SuperScript III reverse transcriptase (Thermo Fisher Scientific). Each cDNA was amplified using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) and quantified using QuantStudio 3 (Applied Biosystems). PCR was performed in duplicate for each sample. Expression of each gene was normalized to that of β-actin and compared with that of a fetal trachea sample (29 weeks gestation; #R1244160-10, Lot. B402231; Agilent Technologies) used as a control RNA. The primers used for qRT-PCR are listed in Table 3.

[0119] [Table 3]

[0120] statistical analysis Error bars indicate the standard error of the mean (SEM). Quantitative data were collected from three or more independent experiments. The statistical tests used are indicated in the legends of each figure. All statistical analyses were performed with the Prism 7 software program (GraphPad).

[0121] result Unidirectional mucociliary transport by MCACs after fluid shear stress (FSS) incubation We applied FSS to MCACs using a microfluidic device to investigate whether coordinated ciliary movement occurs between cells (Figure 2). Airway epithelial progenitor cell spheroids induced to differentiate from iPS cells were dissociated from the 3D Matrigel block on day 28 and reseeded in the microfluidic device (Figure 1). For airway chip cultures with FSS loading, FSS flow was initiated at a volumetric flow rate of approximately 1500 μl / h 12 hours after reseeding, and the cultures were continued until day 42. For airway chip cultures without FSS loading, the cultures were similar except that FSS flow was not applied. For ALI cultures, airway epithelial cell spheroids induced to differentiate from iPS cells were isolated on day 42 and cultured under ALI conditions until day 56. The resulting cell sheets were used for experiments. When the cell sheet was immunostained with anti-FOXJ1 antibody, a marker for MCACs, approximately 40% to 95% of the cells contained in the cell sheet obtained by airway chip culture loaded with FSS were FOXJ1 positive, indicating that they were induced to differentiate into MCACs with high efficiency.

[0122] Each cell sheet was simultaneously immunostained for VANGL1, OCLN, and Ac-TUB. Individual MCACs were identified by the presence of multiple cilia stained with Ac-TUB. Using ImageJ / Fiji software, the center of the cell body and the apex of the VANGL1-expressing crescent were extracted and analyzed for all MCACs in the cell sheet under each condition. In MCAC sheets cultured with FSS, VANGL1 was localized subcellularly in a direction roughly perpendicular to the FSS flow. In contrast, VANGL1 was not localized in a coordinated manner in MCAC sheets cultured with ALI or without FSS. Angle histograms of VANGL1 expression in MCAC sheets cultured under each condition are shown in Figure 3.

[0123] Mucociliary flow was observed for each cell sheet. The cell sheet was placed on a microscope slide, and the bead flow was observed on a horizontal stage. Fluorescent microbeads placed in MCACs of ALI cultures and airway chip cultures without FSS flowed in almost random directions, whereas in MCACs of airway chip cultures with FSS, the fluorescent microbeads flowed in the same direction as the FSS. Angle histograms of fluorescent microbead flow under each condition are shown in Figure 4.

[0124] Using IF staining of samples under each condition and the directional index (Io) based on the trajectory of fluorescent microbead flow, we quantified the intracellular localization of VANGL1 and the direction of fluorescent microbead flow under each condition. The results are shown in the table below and Figures 5 and 6. Both indices for MCACs in FSS-loaded airway chip cultures showed a more aligned polarity in the direction of FSS than for MCACs in ALI cultures and those in airway chip cultures without FSS loading. These results suggest that pre-loading MCACs in airway chips with FSS aligns the cellular polarity of MCACs, achieving coordinated ciliary beating between cells. [Table 4]

[0125] The expression of ciliated airway epithelial cell markers (FOXJ1, DNAH5, and SNTN) was evaluated in samples from each condition (Figure 7). FOXJ1: No difference in expression was observed between ALI and FSS(-) cultures (p>0.9999), but a difference in expression was observed between FSS(-) and FSS(+) cultures (p=0.0400). SNTN: No difference in expression was observed between ALI and FSS(-) cultures (p=0.2866), but a difference in expression was observed between FSS(-) and FSS(+) cultures (p=0.0427). DNAH5: No difference in expression was observed between ALI and FSS(-) cultures (p>0.9999), but a difference in expression was observed between FSS(-) and FSS(+) cultures (p=0.0427). Specifically, MCACs cultured in airway chips loaded with FSS induced the expression of ciliated airway epithelial cell markers more strongly than MCACs cultured at the ALI or without FSS. This result suggests that loading with FSS during differentiation into MCACs promotes differentiation. Coordinated ciliary beating was observed among cells in the resulting cell sheets, even when the volumetric flow rate of FSS loading was 10.14 μl / h, 60 μl / h, or 360 μl / h. [Industrial Applicability]

[0126] The present disclosure provides a cell population containing multistratified ciliated airway epithelial cells with intercellular coordination of ciliary beating. Such cell populations are expected to be useful for, for example, regenerative medicine and evaluation of drug efficacy or toxicity.

Claims

1. A method for producing a cell population containing pseudostratified ciliated airway epithelial cells, which comprises culturing a cell population containing at least one of airway epithelial progenitor cells or airway epithelial tissue stem cells in a medium that is flowing in a certain direction, and which does not include an ALI culture step.

2. The production method according to claim 1, which comprises culturing a cell population comprising airway epithelial progenitor cells induced to differentiate from pluripotent stem cells or endodermal progenitor cells.

3. The production method according to claim 1 or 2, wherein a cell population containing airway epithelial progenitor cells induced to differentiate is cultured by a method comprising the steps of: (1) culturing pluripotent stem cells in a medium containing activin A and a GSK3β inhibitor; (2) culturing the cell population obtained in step (1) in a medium containing a BMP inhibitor and a TGFβ inhibitor; (3) culturing the cell population obtained in step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor; and (4) Culturing the cell population obtained in step (3) in a medium containing a GSK3β inhibitor, FGF10, and a ROCK inhibitor.

4. The production method according to claim 1, which comprises culturing a cell population containing airway epithelial tissue stem cells induced to differentiate from pluripotent stem cells or endodermal progenitor cells, or a cell population containing airway epithelial tissue stem cells collected from a living body.

5. The method according to any one of claims 1 to 4, wherein the flow rate of the culture medium is 5 µm / s to 5 mm / s.

6. The method according to any one of claims 1 to 5, wherein the shear stress caused by the flow of the medium is 0.0001 to 0.1 Pa.

7. The method according to any one of claims 1 to 6, which produces a cell population containing pseudostratified ciliated airway epithelial cells in which ciliary movement is coordinated among the cells.

8. The production method according to any one of claims 1 to 7, wherein a cell population containing pseudostratified ciliated airway epithelial cells having a direction index equal to or greater than a threshold value as quantified by a method comprising the steps of: (1) determining a plurality of unit vectors that indicate the direction of ciliary beating in a cell population; (2) calculating an average vector of the unit vectors obtained in (1); and (3) A step of calculating the length of the average vector obtained in (2) and setting the length as a direction index.

9. The method of claim 8, wherein step (1) comprises the following steps: (a) immunostaining cells in the cell population with an anti-VANGL1 antibody; and (b) A process of determining the position of the center of the cell body and the position of the center of the immunostained VANGL1 localized area in multiple VANGL1-positive cells, and calculating the unit vector from the position of the center of the cell body to the position of the center of the VANGL1 localized area.

10. The method of claim 8, wherein step (1) comprises the following steps: (a) adding microbeads to the culture medium of a cell population and recording the flux of the microbeads; and (b) A step of determining the start and end points of the flow of a plurality of microbeads and calculating a unit vector from the start point to the end point.

11. A method for inducing differentiation of airway epithelial progenitor cells or airway epithelial tissue stem cells into pseudostratified ciliated airway epithelial cells, comprising culturing a cell population containing at least one of airway epithelial progenitor cells or airway epithelial tissue stem cells in a medium that is flowing in a certain direction, and not including an ALI culture step.

Images