Method for differentiating epithelial cells or basal cells

Abstract

The present disclosure relates to a method for differentiating pluripotent stem cells or lung progenitor cells into epithelial cells or basal cells. The present disclosure also relates to epithelial cells or basal cells produced by such a method, organoids containing such epithelial cells or basal cells, and methods of using the same. The present disclosure also relates to a differentiation medium for use therewith.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 390,454, filed Jul. 19, 2022, which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates to methods of differentiating pluripotent stem cells or lung progenitor cells into epithelial cells or basal cells. The present disclosure also relates to epithelial cells or basal cells produced by such methods, organoids containing such epithelial cells or basal cells, and methods of using the same. The present disclosure also relates to differentiation media for such use.

Background Art

[0003] Pluripotent stem cells (PSCs) are undifferentiated or partially differentiated cells that can differentiate into various other cell types. Induced pluripotent stem cells (iPSCs) are a type of PSC derived from adult somatic cells that have been genetically reprogrammed to an ESC - like state by the expression of genes and factors important for maintaining the properties that define embryonic stem cells (ESCs). iPSCs have gained interest in the medical community in recent years because they address many of the obstacles associated with the use of embryonic stem cells, allowing for the generation of patient - specific PSCs, which can be genetically modified, differentiated into somatic lineages, and returned to the same patient as autologous transplants. Yamanaka et al., Cell Stem Cell. 1(1):39 - 49 (2007); Nishikawa et al., Nat. Rev. Mol. Cell Biol. 9:725 (2008). In addition to genetic disorders, iPSCs can be used for tissue regeneration and disease modeling. Kogut et al., Methods Mol. Biol. 1195:1 - 12 (2014).

Summary of the Invention

[0004] The present disclosure relates to methods of differentiating pluripotent stem cells (PSCs) or lung progenitor cells (LPCs) into epithelial cells or basal cells.

[0005] In some embodiments, the method comprises: (i) culturing LPCs in a base culture medium comprising fibroblast growth factor 2 (FGF2), fibroblast growth factor 10 (FGF10), dexamethasone, cyclic adenosine monophosphate (cAMP), an inhibitor of cyclic nucleotide phosphodiesterase, and a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor; (ii) separating cells of (i) that do not express or have low expression of nerve growth factor receptor (NGFR) to form basal progenitor cells; (iii) culturing the cells of (ii) in a base culture medium comprising FGF10, keratinocyte growth factor (KGF), a transforming growth factor β type I receptor kinase (ALK5) inhibitor, an inhibitor of phosphorylation of SMAD (suppressor of mothers against decapentaplegic), and a ROCK inhibitor, and (iv) separating cells of (iii) that have expression of NGFR to form basal cells.

[0006] In some embodiments, the method further comprises: (v) culturing the cells of (iv) that have expression of NGFR in a base culture medium comprising FGF10, KGF, an ALK5 inhibitor, an inhibitor of phosphorylation of SMAD, and a ROCK inhibitor. In some embodiments, the culturing of (v) is performed on a laminin-coated surface. In some embodiments, the laminin is laminin-521.

[0007] In some embodiments, the culturing of (i) is in a three-dimensional (3D) matrix. In some embodiments, the culturing of (ii) is in a 3D matrix. In some embodiments, the 3D matrix is Matrigel®.

[0008] In some embodiments, FGF2 is present in the culture medium at a concentration of about 250 ng / mL.

[0009] In some embodiments, FGF10 is present in the culture medium at a concentration of about 100 ng / mL. In some embodiments, FGF10 is present in the culture media of (ii) and (iv) at a concentration of about 20 ng / mL.

[0010] In some embodiments, dexamethasone is present in the culture medium at a concentration of about 50 nM.

[0011] In some embodiments, cAMP is present in the culture medium at a concentration of about 100 μM.

[0012] In some embodiments, the inhibitor of cyclic nucleotide phosphodiesterase is 3-isobutyl-1-methylxanthine (IBMX). In some embodiments, IBMX is present in the culture medium at a concentration of about 100 μM.

[0013] In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, Y-27632 is present in the culture medium at a concentration of 10 μM.

[0014] In some embodiments, KGF is present in the culture media of (ii) and (iv) at a concentration of about 20 ng / mL.

[0015] In some embodiments, the ALK5 inhibitor is A83-01. In some embodiments, A83-01 is present in the culture medium at a concentration of about 1 μM.

[0016] In some embodiments, the inhibitor of SMAD phosphorylation is DMH-1. In some embodiments, DMH-1 is present in the culture medium at a concentration of about 0.5 μM.

[0017] The present disclosure also provides epithelial cells produced by the differentiation methods disclosed herein, basal cells produced by the differentiation methods disclosed herein, organoids containing the epithelial cells disclosed herein, organoids containing the basal cells disclosed herein, and certain methods of using them.

[0018] The present disclosure also provides certain differentiation media. In some embodiments, the differentiation media provided herein comprises a basal culture medium, FGF10, KGF, an ALK5 inhibitor, an inhibitor of SMAD phosphorylation, and a ROCK inhibitor. In some embodiments, the differentiation media provided herein comprises a basal culture medium, about 20 ng / mL of FGF10, about 20 ng / mL of KGF, about 1 μM of A83-01, about 0.5 μM of DMH-1, and about 10 μM of Y-27632.

[0019] Some embodiments of the invention are described herein with reference to the accompanying drawings, which are for illustrative purposes only. Although the drawings are referred to specifically herein, it should be emphasized that the details shown are for illustrative purposes and for the purpose of explicitly discussing embodiments of the invention.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2-1

Figure 2-2

Figure 3-1

Figure 3-2

Figure 4-1

Figure 4-2

Figure 4-3

Figure 4-4

Figure 4-5

Figure 5-1

Figure 5-2

Figure 5-3

Figure 5-4

Figure 5-5

Figure 6

Mode for Carrying Out the Invention

[0021] I. General Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless the context requires otherwise, the singular terms shall include the plural and the plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0022] Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, but suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and the claims.

[0023] To further define the present disclosure, the following terms and definitions are provided.

[0024] The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. The term "a" (or "an") and the terms "one or more" and "at least one" may be used interchangeably herein. In certain embodiments, the term "a" or "an" means "single." In other embodiments, the term "a" or "an" includes "two or more" or "plural."

[0025] As used herein, the term "about" is used to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower limits of the indicated value. In general, the term "about" is used herein to modify a value up and down (higher or lower) by 10 percent to include numerical values above and below the indicated value.

[0026] Throughout this disclosure, various aspects of the invention are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as the individual numerical values within that range. For example, a range description such as 1 - 6 should be considered to have specifically disclosed sub-ranges such as 1 - 3, 1 - 4, 1 - 5, 2 - 4, 2 - 6, 3 - 6, etc., as well as the individual numbers within that range, for example 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. The recited numerical ranges include the numbers defining the range and each integer within the defined range.

[0027] Units, prefixes, and symbols are expressed in their forms recognized in the International System of Units (SI). Numerical ranges include the numbers defining the range. When a range of values is stated, each integer value between the values stated as the upper and lower limits of the range, and each fraction thereof, are specifically disclosed, together with each sub-range between such values. The upper and lower limits of any range can be included in the range independently or excluded therefrom, and each range where either limit is included, or neither limit is included, or both limits are included, is also encompassed by the scope of the present disclosure. Accordingly, ranges described herein are understood to be a shorthand for all values within the range including the recited endpoints. For example, the range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0028] When values are explicitly recited, values of amounts or quantities that are substantially equivalent to the recited values are also understood to be within the scope of the present disclosure. When combinations are disclosed, each sub-combination of the elements of the combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, when different elements or groups of elements are disclosed individually, the combination is also disclosed. When any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure where each alternative is excluded, either alone or in any combination with other alternatives, are also disclosed herein; a plurality of elements of a disclosure can have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0029] As used herein, the term "and / or" is construed as a specific disclosure for each of two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" as used in expressions such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0030] It is understood that when an aspect is described herein in the expression "comprising", other similar aspects described in the words "consisting of" and / or "consisting essentially of" are also provided.

[0031] II. Differentiation Methods The present disclosure relates to methods for differentiating pluripotent stem cells (PSCs) or lung progenitor cells (LPCs) into epithelial cells or basal cells. Such methods improve, for example, the viability, yield, and / or differentiation characteristics of epithelial cells or basal cells.

[0032] As used herein, the terms "differentiation" and "differentiate" refer to the process of inducing or reprogramming young or immature cells (e.g., pluripotent stem cells) into more mature or specialized cells (e.g., epithelial cells or basal cells). Generally, the differentiation of pluripotent stem cells can be carried out by changing the cell culture conditions, such as by changing the stimulant in the culture medium or the physical state of the cells.

[0033] As used herein, the terms "pluripotent stem cell" and "PSC" refer to young or immature cells that can give rise to more mature or specialized cells (e.g., epithelial or basal cells).

[0034] In some embodiments, PSCs include, but are not limited to, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic germ cells, adult stem cells, or combinations thereof. In some embodiments, the PSCs are of human origin. In some embodiments, the PSCs are of animal origin. In some embodiments, the animal is a sheep, a pig, or a primate.

[0035] As used herein, the terms "induced pluripotent stem cell" and "iPSC" refer to cells that are induced or reprogrammed from differentiated adult, neonatal, or fetal cells to produce pluripotent stem cells.

[0036] As used herein, the term "lung progenitor cell" or "LPC" refers to a pluripotent cell that can differentiate into several cell types of the respiratory system, including but not limited to type I and type II alveolar cells, alveolar lining cells, smooth muscle cells, alveolar epithelial cells, endothelial cells, and erythrocytes. LPCs are specialized to the lung lineage and retain the ability to self-renew and can be identified by cell surface markers or intracellular proteins including, but not limited to, transcription termination factor 1 (TTF1), GATA-binding protein 6 (GATA6), Est1, Nkx2.1, surfactant protein C (SP-C), forkhead box A1 (FOXA1), FOXA2, SRY-box transcription factor 2 (SOX2), SOX9, CD49f, cytokeratin 8 (CK8), epithelial cell adhesion molecule (EpCAM), and p63. In some embodiments, the LPCs according to the present disclosure do not express certain markers such as markers of endothelial cells (e.g., CD144, CD31), markers of hematopoietic cells (e.g., CD43, CD45, CD235a, or CD41a), and / or markers of pluripotent stem cells (e.g., TRA1-60), or show negative or low expression of certain markers.

[0037] As used herein, the term "epithelial cell" or "epithelium" refers to cells or groups of cells that line hollow organs, as well as cells that make up glands and the outer surface of the body. Epithelial cells can include squamous epithelial cells, columnar epithelial cells, adenomatous epithelial cells, or transitional epithelial cells. Epithelial cells may be arranged in a single layer or in multiple layers, depending on the organ and location. As used herein, epithelial cells include mature epithelial cells, epithelial progenitor cells, and epithelial precursor cells.

[0038] In some embodiments, the cells of the differentiation methods provided herein express, or have expression of, one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures) typically associated with epithelial cells such as, for example, T1α, EpCAM, CD49f, and CD104. In some embodiments, the cells express, or have expression of, one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures) typically associated with basal epithelial cells such as, for example, integrin subunit α6 (ITGA6), integrin subunit β4 (ITGB4), keratin 14 (KRT14), KRT15, KRT5, and p63. In some embodiments, the cells express, or have expression of, one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures) typically associated with differentiated epithelial cells such as, for example, KRT4, KRT6, and KRT8. In some embodiments, the cells express, or have expression of, one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures) typically associated with airway epithelial cells such as, for example, HEY2 (Hes Related Family BHLH Transcription Factor With YRPW Motif 2), nerve growth factor receptor (NGFR), and bone morphogenetic protein 7 (BMP7).In some embodiments, the cell expresses or has expression of one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures), such as, for example, cyclin-dependent kinase inhibitor 2B (CDKN2B), CITED2 (Cbp / p300 Interacting Transactivator With Glu / Asp Rich Carboxy-Terminal Domain 2), CREG1 (Cellular Repressor Of E1A Stimulated Genes 1), DNA binding inhibitor 1 (ID1), mitogen-activated protein kinase 6 (MAP2K6), insulin-like growth factor binding protein 3 (IGFBP3), and IGFBP5.

[0039] In some embodiments, the cells of the differentiation methods provided herein do not express or express at low levels one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures) typically associated with epithelial stem cells, such as nerve growth factor receptor (NGFR), leucine rich repeat containing G protein-coupled receptor 5 (LGR5), or CD24. In some embodiments, the cells do not express or express at low levels one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures) typically associated with pluripotent stem cells, such as Lin-28 homolog A (LIN28A), NANOG, POU5F1 / OCT4, and SRY-related HMG-box2 (SOX2). In some embodiments, the cells do not express or express at low levels one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures) typically associated with terminally differentiated epithelial cells, such as cystic fibrosis transmembrane conductance regulator (CFTR), forkhead box J1 (FOXJ1), IVL, keratin 1 (KRT1), KRT10, KRT20, LOR, mucin 1 (MUC1), MUC5AC, secretoglobin family 1A member 1 (SCGB1A1), surfactant protein B (SFTPB), and SFTPD.

[0040] In some embodiments, the cells of the differentiation methods provided herein do not express or express at low levels NGFR and are basal progenitor cells. In some embodiments, the cells of the differentiation methods provided herein do not express or express at low levels NGFR and are epithelial cells or basal cells.

[0041] As used herein, "basal cell" or "BC" is a progenitor epithelial cell present in the epithelium of the airway, from the trachea and decreasing in number to the respiratory bronchioles. These are responsible for normal cell replacement and epithelial remodeling during lung injury. Basal cells can regenerate the airway epithelium by functioning as precursors to essential specialized epithelial cell types, including secretory cells (SC) and multiciliated cells (MCC). Basal cells are generally the cells in the deepest layer of stratified and pseudostratified epithelia.

[0042] In some embodiments, basal cells according to the present disclosure have one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures), including but not limited to CD49f, EpCAM, NGFR, NKX2-1, p63, and CK5. In some embodiments, basal cells according to the present disclosure do not express or show reduced expression of certain markers, such as markers of endothelial cells (e.g., CD144 and / or CD31), markers of hematopoietic cells (e.g., CD43, CD45, CD235a and / or CD41a), or markers of pluripotent stem cells (e.g., TRA1-60).

[0043] Methods of generating LPCs from PSCs (e.g., iPSCs) are known and are further described herein and, for example, in Jacob et al., Nature Protocols, 14:3303-3332, 2019; Hawkins et al., Cell Stem Cell, 28:79-95, 2021.

[0044] In some embodiments, the differentiation methods provided herein include certain cell culture conditions, such as cell culture in a particular culture medium.

[0045] As used herein, the terms "cell culture", "culturing cells", "culture", "culturing", and "cultured" refer to the maintenance, growth and / or differentiation of cells in an in vitro environment. The terms "cell culture medium" and "culture medium" refer to compositions for cell culture that contain nutrients that maintain cell viability, support growth, and optionally support differentiation. A cell culture medium can contain one or more of the following: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum replacements, and other components such as growth factors, vitamins.

[0046] In some embodiments, the differentiation methods provided herein refer to a cell culture medium as a "basal culture medium" supplemented with other components (sometimes referred to herein as a "differentiation medium"). As used herein, "basal culture medium" refers to a composition that contains the minimal elements necessary for the maintenance, growth and / or differentiation of cells in an in vitro environment. Examples of basal culture media include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), MEM, Iscove's Modified Dulbecco Medium (IMDM), Glasgow Modified MEM (GMEM), DMEM / F12, Leibovitz L-15, RPMI-1640, CMRL, Ham's F10, and Ham's F12. In some embodiments, the basal culture medium is supplemented with one or more other components such as, for example, amino acids, antibiotics, serum, growth factors. Such components are well known in the art and are further described herein.

[0047] In some embodiments, the differentiation methods provided herein include subculturing cells under certain cell culture conditions, such as in a particular culture medium. As used herein, the terms “subculture,” “subcultured,” and “subculturing” refer to the act of splitting and seeding cells at a lower density onto one or more cell culture surfaces or vessels when the cells have grown to a desired extent. Subculturing typically involves detaching the cells by mechanical or enzymatic means (e.g., incubation with Accutane®) as appropriate prior to seeding the cells at a particular density. Methods for subculturing cells are well known and are further described herein.

[0048] In some embodiments, the culturing and subculturing in the differentiation methods provided herein are performed using one or more substrates coated on the cell culture surface or vessel. Such substrates include vitronectin, gelatin, laminin (e.g., laminin-111, laminin-211, laminin-121, laminin-221, laminin-332, laminin-311, laminin-321, laminin-411, laminin-421, laminin-511, laminin-213, laminin-521, laminin-423, laminin-522, laminin-523, or a combination thereof), fibronectin, collagen (e.g., collagen I, collagen IV, or a combination thereof), elastin, osteopontin, thrombospondin, a mixture of naturally occurring cell line-produced matrices such as Matrigel™, as well as synthetic or artificial surfaces such as polyamine monolayers and carboxy-terminal monolayers, or a combination thereof, but are not limited thereto. In some embodiments, the substrate is laminin-521. Methods for coating substrates onto the cell culture surface or vessel are well known and are further described herein.

[0049] In some embodiments, the culturing and / or subculturing of the differentiation methods provided herein are two-dimensional (2D) cultures. In such cultures, the cells are grown as a monolayer on the cell culture surface or vessel, as appropriate, with the substrate coating described above.

[0050] In some embodiments, the culturing and / or subculturing of the differentiation methods provided herein are performed in a three-dimensional (3D) matrix. Examples of 3D matrices include, but are not limited to, polymers (natural or synthetic), ceramics, composite materials, and combinations thereof. The 3D matrix can be in the form of a hydrogel, a porous 3D scaffold, a rapid prototyping scaffold, a foam, a sponge, a mesh, microparticles, a fibrous network, a mixture of naturally occurring cell line-produced matrices such as Matrigel™, and combinations thereof, for example, a hydrogel filled with microparticles.

[0051] In some embodiments, the differentiation methods provided herein include one or more steps of separating cultured cells having certain specific biochemical properties. As used herein, the terms "isolated" and "isolating" refer to the process of isolating one or more specific cell populations from a heterogeneous mixture of cells.

[0052] In some embodiments, the differentiation methods provided herein include separating cells that do not express NGFR at all or have low expression thereof to form basal progenitor cells. As used herein, the term "do not express NGFR at all" does not include, but is not limited to, detectable expression of NGFR. As used herein, the term "low expression of NGFR" includes, but is not limited to, no detectable expression of NGFR and NGFR expression that is lower than the NGFR expression in control cells that do not express NGFR and / or are not basal progenitor cells.

[0053] In some embodiments, the differentiation methods provided herein include separating cells having NGFR expression to form epithelial cells or basal cells. As used herein, the terms "expression" or "expressing" with respect to NGFR include NGFR expression comparable to that of mature epithelial cells or basal cells, or their precursors or progenitors, and NGFR expression higher than that in control cells that do not express NGFR and / or are not endothelial cells or basal cells, but are not limited thereto.

[0054] Cell separation methods based on marker expression are well known in the art and include, but are not limited to, affinity separation, fluorescence-activated cell sorting (FACS), density gradient centrifugation, immunodensity cell isolation, microfluidic cell sorting, buoyancy-activated cell sorting, aptamer-based cell isolation, complement depletion, etc. Techniques for affinity separation include separation using antibody-coated magnetic beads (e.g., immunomagnetic cell separation), affinity chromatography, cytotoxic agents conjugated to monoclonal antibodies or used in combination with monoclonal antibodies, such as complement and cytotoxins, and "panning" using antibodies bound to solid matrices such as plates, or other convenient techniques, but are not limited thereto. In some embodiments, the cells of the present differentiation method are separated by immunomagnetic cell separation.

[0055] Some embodiments of the differentiation methods provided herein include culturing cells in a basal culture medium containing fibroblast growth factor 2 (FGF2). FGF2, also known as basic fibroblast growth factor or FGF-β, is a growth factor and signaling protein encoded by the FGF2 gene. FGF2 has a wide range of mitogenic and cell survival activities and is involved in diverse biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion.

[0056] In some embodiments, FGF2 is present in the basal culture medium at a concentration of about 100 ng / mL to about 400 ng / mL, or any value or range of values therebetween, such as, for example, about 200 ng / mL to about 400 ng / mL, about 250 ng / mL to about 400 ng / mL, about 300 ng / mL to about 400 ng / mL, about 100 ng / mL to about 300 ng / mL, about 200 ng / mL to about 300 ng / mL, about 250 ng / mL to about 300 ng / mL, about 100 ng / mL to about 250 ng / mL, about 100 ng / mL to about 250 ng / mL, or about 100 ng / mL to about 200 ng / mL. In some embodiments, FGF2 is present in the basal culture medium at a concentration of about 100 ng / mL, about 200 ng / mL, about 250 ng / mL, about 300 ng / mL, or about 400 ng / mL. In some embodiments, FGF2 is present in the basal culture medium at a concentration of about 250 ng / mL.

[0057] Some embodiments of the differentiation methods provided herein include culturing cells in a basal culture medium containing fibroblast growth factor 10 (FGF10). FGF10 is a paracrine signaling molecule first identified in limb bud and organogenesis development. FGF10 initiates limb development and is involved in the morphogenetic branching of multiple organs such as the lungs, skin, ears, and salivary glands.

[0058] In some embodiments, FGF10 is present in the basal culture medium of (i) at a concentration of about 25 ng / mL to about 200 ng / mL, or any value or range of values thereof, for example, about 50 ng / mL to about 200 ng / mL, about 100 ng / mL to about 200 ng / mL, about 150 ng / mL to about 200 ng / mL, about 25 ng / mL to about 150 ng / mL, about 50 ng / mL to about 150 ng / mL, about 100 ng / mL to about 150 ng / mL, about 25 ng / mL to about 100 ng / mL, about 50 ng / mL to about 100 ng / mL, or about 25 ng / mL to about 50 ng / mL. In some embodiments, FGF10 is present in the basal culture medium of (i) at a concentration of about 25 ng / mL, about 50 ng / mL, about 100 ng / mL, about 150 ng / mL, or about 200 ng / mL. In some embodiments, FGF10 is present in the basal culture medium of (i) at a concentration of about 100 ng / mL.

[0059] In some embodiments, FGF10 is present in the basal culture medium of (iii) and / or (iv) at a concentration of about 5 ng / mL to about 35 ng / mL, or any value or range of values thereof, for example, about 15 ng / mL to about 35 ng / mL, about 20 ng / mL to about 35 ng / mL, about 25 ng / mL to about 35 ng / mL, about 5 ng / mL to about 25 ng / mL, about 15 ng / mL to about 25 ng / mL, about 20 ng / mL to about 25 ng / mL, about 5 ng / mL to about 20 ng / mL, about 15 ng / mL to about 20 ng / mL, or about 5 ng / mL to about 15 ng / mL. In some embodiments, FGF10 is present in the basal culture medium of (iii) and / or (iv) at a concentration of about 5 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, or about 35 ng / mL. In some embodiments, FGF10 is present in the basal culture medium of (iii) and / or (iv) at a concentration of about 20 ng / mL.

[0060] Some embodiments of the differentiation methods provided herein include culturing cells in a basal culture medium containing dexamethasone. Dexamethasone is an anti-inflammatory glucocorticoid that has various effects on cell growth and differentiation.

[0061] In some embodiments, dexamethasone is present in the basal culture medium at a concentration of about 20 nM to about 80 nM, or any value or range of values therebetween, such as, for example, about 40 nM to about 80 nM, about 50 nM to about 80 nM, about 60 nM to about 80 nM, about 20 nM to about 60 nM, about 40 nM to about 60 nM, about 50 nM to about 60 nM, about 20 nM to about 50 nM, about 40 nM to about 50 nM, or about 20 nM to about 40 nM. In some embodiments, dexamethasone is present in the basal culture medium at a concentration of about 20 nM, about 40 nM, about 50 nM, about 60 nM, or about 80 nM. In some embodiments, dexamethasone is present in the basal culture medium at a concentration of about 50 nM.

[0062] Some embodiments of the differentiation methods provided herein include culturing cells in a basal culture medium containing cyclic adenosine monophosphate (cAMP). cAMP promotes the mobilization of glucose and fatty acid accumulation and is involved in the function and differentiation of many different cell types.

[0063] In some embodiments, cAMP is present in the basal culture medium at a concentration of about 25 μM to about 200 μM, or any value or range of values therebetween, such as, for example, about 50 μM to about 200 μM, about 100 μM to about 200 μM, about 150 μM to about 200 μM, about 25 μM to about 150 μM, about 50 μM to about 150 μM, about 100 μM to about 150 μM, about 25 μM to about 100 μM, about 50 μM to about 100 μM, or about 25 μM to about 50 μM. In some embodiments, cAMP is present in the basal culture medium at a concentration of about 25 μM, 50 μM, 100 μM, 150 μM, or 200 μM. In some embodiments, cAMP is present in the basal culture medium at a concentration of about 100 μM.

[0064] Some aspects of the differentiation methods provided herein involve culturing cells in a basal culture medium containing an inhibitor of cyclic nucleotide phosphodiesterase. Cyclic nucleotide phosphodiesterases are a family of enzymes that hydrolyze the phosphodiester bonds of cyclic adenosine monophosphate and cyclic guanosine monophosphate, thereby inhibiting their vasodilatory properties in the lungs. In some aspects, the inhibitor of cyclic nucleotide phosphodiesterase is theophylline, 3-isobutyl-1-methylxanthine (IBMX), or Ro 20-1724. In some aspects, the inhibitor of cyclic nucleotide phosphodiesterase is IBMX.

[0065] In some aspects, the inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX) is present in the basal culture medium at a concentration of about 25 μM to about 200 μM, or any value or range of values therebetween, such as about 50 μM to about 200 μM, about 100 μM to about 200 μM, about 150 μM to about 200 μM, about 25 μM to about 150 μM, about 50 μM to about 150 μM, about 100 μM to about 150 μM, about 25 μM to about 100 μM, about 50 μM to about 100 μM, or about 25 μM to about 50 μM. In some aspects, the inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX) is present in the basal culture medium at a concentration of about 25 μM, 50 μM, 100 μM, 150 μM, or 200 μM. In some aspects, the inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX) is present in the basal culture medium at a concentration of about 100 μM.

[0066] Some aspects of the differentiation methods provided herein include culturing cells in a basal culture medium containing a Rho-associated kinase (ROCK) inhibitor. ROCK is a serine / threonine kinase that acts as a downstream effector of Rho kinase and has three isoforms (RhoA, RhoB, RhoC). A “ROCK inhibitor” can, for example, decrease the expression and / or activity of ROCK. Examples of ROCK inhibitors include, but are not limited to, polynucleotides, polypeptides, and small molecules. More specific examples of ROCK inhibitors include, but are not limited to, anti-ROCK antibodies that target ROCK, and dominant negative ROCK variants, siRNA, shRNA, miRNA, and antisense nucleic acids. Examples of other ROCK inhibitors include thiazovivin, Y-27632, fasudil, AR122-86, Y-30141, WF-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N′-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, and the ROCK inhibitors disclosed in U.S. Patent No. 8,044,201, which is hereby incorporated by reference in its entirety, but are not limited thereto. In some aspects, the ROCK inhibitor is Y-27632.

[0067] In some embodiments, the ROCK inhibitor (e.g., Y-27632) is present in the basal culture medium at a concentration of about 1 μM to about 20 μM, or any value or range of values thereof, such as about 1 μM to about 15 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, about 5 μM to about 20 μM, about 5 μM to about 15 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 10 μM to about 15 μM, or about 15 μM to about 20 μM. In some embodiments, the ROCK inhibitor (e.g., Y-27632) is present in the basal culture medium at a concentration of about 1 μM, about 5 μM, about 10 μM, about 15 μM, or about 20 μM. In some embodiments, the ROCK inhibitor (e.g., Y-27632) is present in the basal culture medium at a concentration of about 10 μM.

[0068] Some embodiments of the differentiation methods provided herein include culturing cells in a basal culture medium containing keratinocyte growth factor (KGF). KGF is a member of the heparin-binding fibroblast growth factor family that has a unique pattern of target cell specificity. KGF has mitogenic activity for epithelial cells and is produced by mesenchyme-derived cells.

[0069] In some embodiments, KGF is present in the basal culture medium at a concentration of about 5 ng / mL to about 35 ng / mL, or any value or range of values thereof, such as about 15 ng / mL to about 35 ng / mL, about 20 ng / mL to about 35 ng / mL, about 25 ng / mL to about 35 ng / mL, about 5 ng / mL to about 25 ng / mL, about 15 ng / mL to about 25 ng / mL, about 20 ng / mL to about 25 ng / mL, about 5 ng / mL to about 20 ng / mL, about 15 ng / mL to about 20 ng / mL, or about 5 ng / mL to about 15 ng / mL. In some embodiments, KGF is present in the basal culture medium at a concentration of about 5 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, or about 35 ng / mL. In some embodiments, KGF is present in the basal culture medium at a concentration of about 20 ng / mL.

[0070] Some aspects of the differentiation methods provided herein involve culturing cells in a basal culture medium containing a TGF-β receptor I kinase (ALK5) inhibitor. ALK5 is a serine / threonine protein kinase that, upon binding to TGF-β, forms a heteromeric complex with the type II TGF-β receptor and transmits the TGF-β signal from the cell surface to the cytoplasm. This protein is involved in the control of many physiological and pathological processes, including cell cycle arrest in epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression, and carcinogenesis. As used herein, the term "ALK5 inhibitor" refers to a molecule that inhibits or reduces the activity of ALK5, e.g., a molecule that inhibits or reduces the transmission of the TGF-β signal from the cell surface to the cytoplasm.

[0071] Examples of ALK5 inhibitors include, but are not limited to, A83-01 (Xcess Biosciences (San Diego, Calif.)), ALK5 inhibitor II (Enzo, Farmingdale, N.Y.), ALK5i (Axxora, San Diego, Calif.), SD208 (R&D systems (MN)), TGF-β inhibitor SB431542 (Xcess Biosciences (San Diego, Calif.)), ITD-1 (Xcess Biosciences (San Diego, Calif.)), LY2109761 (Xcess Biosciences (San Diego, Calif.)), LY2157299 (Xcess Biosciences (San Diego, Calif.)), TGF-β RI kinase inhibitor V (EMD Chemicals, Gibstown, N.J.), TGF-β receptor inhibitor I (EMD Chemicals, Gibstown, N.J.), TGF-β receptor inhibitor IV (EMD Chemicals, Gibstown, N.J.), TGF-β receptor inhibitor VII (EMD Chemicals, Gibstown, N.J.), TGF-β receptor inhibitor VIII (EMD Chemicals, Gibstown, N.J.), TGF-β receptor inhibitor II (EMD Chemicals, Gibstown, N.J.), TGF-β RI kinase inhibitor IV (EMD Chemicals, Gibstown, N.J.), and TGF-β receptor inhibitor III (EMD Chemicals, Gibstown, N.J.). In some embodiments, the ALK5 inhibitor is A83-01.

[0072] In some embodiments, the ALK5 inhibitor (e.g., A83-01) is present in the basal culture medium at a concentration of about 0.2 μM to about 2 μM, or any value or range therebetween, such as about 0.2 μM to about 1.5 μM, about 0.2 μM to about 1 μM, about 0.2 μM to about 0.5 μM, about 0.5 μM to about 2 μM, about 0.5 μM to about 1.5 μM, about 0.5 μM to about 1 μM, about 1 μM to about 2 μM, about 1 μM to about 1.5 μM, or about 1.5 μM to about 2 μM. In some embodiments, the ALK5 inhibitor (e.g., A83-01) is present in the basal culture medium at a concentration of about 0.2 μM, about 0.5 μM, about 1 μM, about 1.5 μM, or about 2 μM. In some embodiments, the ALK5 inhibitor (e.g., A83-01) is present in the basal culture medium at a concentration of about 1 μM.

[0073] Some aspects of the differentiation methods provided herein involve culturing cells in a basal culture medium containing an inhibitor of phosphorylation of SMAD (suppressor of mothers against decapentaplegic). SMAD proteins (receptor-regulated SMADs or R-SMADs (SMAD1, 2, 3, 5, and 8) and SMAD4) enter the nucleus, where they act as transcription factors and form heterocomplexes that induce the expression of a wide range of proteins. Phosphorylation of SMAD strongly affects the structural and functional properties of SMAD. Inhibitors of SMAD phosphorylation include, but are not limited to, SB431542, LDN-193189, Noggin PD169316, SB203580, LY364497, A77-01, A83-01, BMP4, GW788388, GW6604, SB-505124, Reldemizumab, Metelimumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364497, LY2109761, SB-505124, E-616452 (ALK inhibitor RepSox), SD-208, SMI6, NPC-30345, Ki26894, SB-23580, SD-093, Activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride, and derivatives and / or variants thereof. In some aspects, the inhibitor of SMAD phosphorylation is DMH-1.

[0074] In some embodiments, a SMAD phosphorylation inhibitor (e.g., DMH-1) is present in the basal culture medium at a concentration of about 0.1 μM to about 1.5 μM, or any value or range of values therebetween, such as about 0.2 μM to about 1.5 μM, about 0.5 μM to about 1.5 μM, about 1 μM to about 1.5 μM, about 0.1 μM to about 1 μM, about 0.2 μM to about 1 μM, about 0.5 μM to about 1 μM, about 0.1 μM to about 0.5 μM, about 0.2 μM to about 0.5 μM, or about 0.1 μM to about 0.2 μM. In some embodiments, a SMAD phosphorylation inhibitor (e.g., DMH-1) is present in the basal culture medium at a concentration of about 0.1 μM, about 0.2 μM, about 0.5 μM, about 1 μM, or about 1.5 μM. In some embodiments, a SMAD phosphorylation inhibitor (e.g., DMH-1) is present in the basal culture medium at a concentration of about 0.5 μM.

[0075] In some embodiments, the differentiation method provided herein is as follows:

[0076] (i) culturing LPC in a basal culture medium containing FGF2, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor;

[0077] (ii) optionally, separating the cells of (i) that do not express or have low expression of NGFR to form basal progenitor cells;

[0078] (iii) culturing the cells of (ii) in a basal culture medium containing FGF10, KGF, an ALK5 inhibitor, a SMAD phosphorylation inhibitor, and a ROCK inhibitor; and

[0079] (iv) optionally, separating the cells of (iii) that have expression of NGFR to form epithelial cells or basal cells.

[0080] In some embodiments, the differentiation method provided herein is as follows:

[0081] (i) Culturing LPCs in a basal culture medium containing FGF2, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor;

[0082] (ii) Separating the cells of (i) that do not express NGFR at all or have low expression thereof to form basal progenitor cells;

[0083] (iii) Culturing the cells of (ii) in a basal culture medium containing FGF10, KGF, an ALK5 inhibitor, an inhibitor of SMAD phosphorylation, and a ROCK inhibitor; and

[0084] (iv) Separating the cells of (iii) that have NGFR expression to form epithelial cells or basal cells.

[0085] In some embodiments, the method comprises:

[0086] (v) Further comprising culturing the cells of (iii) that have NGFR expression in a basal culture medium containing FGF10, KGF, an ALK5 inhibitor, an inhibitor of SMAD phosphorylation, and a ROCK inhibitor.

[0087] In some embodiments, the culture of (v) is performed on a laminin-coated surface. In some embodiments, the laminin is laminin-111, laminin-211, laminin-121, laminin-221, laminin-332, laminin-311, laminin-321, laminin-411, laminin-421, laminin-511, laminin-213, laminin-521, laminin-423, laminin-522, laminin-523, or a combination thereof. In some embodiments, the laminin is laminin-521.

[0088] In some embodiments, the culture of (i) and / or (iii) is in a 3D matrix. In some embodiments, the 3D matrix is Matrigel®.

[0089] III. Other Aspects The present disclosure also relates to epithelial cells or basal cells produced by any of the differentiation methods disclosed herein.

[0090] The present disclosure also relates to organoids comprising epithelial cells or basal cells produced by any of the differentiation methods disclosed herein. As used herein, the term "organoid" refers to a differentiated or partially differentiated 3D cell organism derived from PSCs (e.g., iPSCs) or LPCs that self-organizes by tightly accumulating cells in a controlled space. Such organisms can be made to reproduce much of the complexity of an organ, or to represent a selected aspect thereof, for example, by producing only a particular type of cell.

[0091] Methods for maintaining differentiated epithelial cells or basal cells and organoids are well known and include culturing the cells or organoids in the cell culture media described herein and / or cryopreservation. Methods for making organoids typically include culturing the cells in a 3D matrix. Examples of 3D matrices include, but are not limited to, polymers (natural or synthetic), ceramics, composites, or combinations thereof. The 3D matrix can be in the form of a hydrogel, a porous 3D scaffold, a rapid prototyping scaffold, a foam, a sponge, a mesh, microparticles, a fibrous network, a mixture of naturally occurring cell line-produced matrices such as Matrigel™, and combinations thereof, for example, a hydrogel filled with microparticles.

[0092] The present disclosure also relates to certain methods of using such epithelial cells and basal cells. In some aspects, the present disclosure provides a method of treating a lung disease comprising administering an epithelial cell or basal cell or organoid produced by any of the differentiation methods disclosed herein.

[0093] The present disclosure also relates to a differentiation medium for differentiating epithelial cells or basal cells from PSCs or LPCs.

[0094] In some embodiments, the differentiation medium comprises a basal culture medium, FGF2, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a ROCK inhibitor (e.g., Y-27632). In some embodiments, the differentiation culture medium comprises about 100 ng / mL to about 400 ng / mL of FGF2, about 25 ng / mL to about 200 ng / mL of FGF10, about 20 nM to about 80 nM of dexamethasone, about 25 μM to about 200 μM of cAMP, about 25 μM to about 200 μM of an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and about 1 μM to about 20 μM of a ROCK inhibitor (e.g., Y-27632). In some embodiments, the differentiation medium comprises about 250 ng / mL of FGF2, about 100 ng / mL of FGF10, about 50 nM of dexamethasone, about 100 μM of cAMP, about 100 μM of an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and about 10 μM of a ROCK inhibitor (e.g., Y-27632).

[0095] In some embodiments, the differentiation medium comprises a basal culture medium, FGF10, KGF, an ALK5 inhibitor (e.g., A83-01), an inhibitor of SMAD phosphorylation (e.g., DMH-1), and a ROCK inhibitor (e.g., Y-27632). In some embodiments, the differentiation culture medium comprises a basal culture medium, about 5 ng / mL to about 35 ng / mL of FGF10, about 5 ng / mL to about 35 ng / mL of KGF, about 0.2 μM to about 2 μM of an ALK5 inhibitor (e.g., A83-01), about 0.1 μM to about 1.5 μM of an inhibitor of SMAD phosphorylation (e.g., DMH-1), and about 1 μM to about 20 μM of a ROCK inhibitor (e.g., Y-27632). In some embodiments, the differentiation medium comprises a basal culture medium, about 20 ng / mL of FGF10, about 20 ng / mL of KGF, about 1 μM of an ALK5 inhibitor (e.g., A83-01), about 0.5 μM of an inhibitor of SMAD phosphorylation (e.g., DMH-1), and about 10 μM of a ROCK inhibitor (e.g., Y-27632).

Example

[0096] Below, with the above description, refer to the examples showing some embodiments of the present invention in a non-limiting manner. [Example 1] Differentiation from iPS to LPC Materials and Methods

[0097] The reagents and materials used are as follows: Iscove's Modified Dulbecco's Medium (IMDM) (Thermo Fisher Scientific; Catalog number 12440053) Ham's F-12 medium supplemented with L-glutamine (Corning®; Catalog number 10-080-CV) GlutaMAX™ Supplement (Thermo Fisher Scientific; Catalog number 35050061) B-27 Supplement (50X) (Thermo Fisher Scientific; Catalog number 17504044) Bovine Serum Albumin (BSA); 7.5% in Dulbecco's Phosphate Buffered Saline (DPBS) (Sigma Aldrich; Catalog number A8412-100ML) N-2 Supplement (Thermo Fisher Scientific; Catalog number 17502048) L-Ascorbic acid (Sigma Aldrich; Catalog number A4544-25G) 1-Thioglycerol (MTG) (Sigma-Aldrich; Catalog number M1753-100ML) Primocin® (InvivoGen; Catalog number ant-pm-1) IBMX (3-Isobutyl-1-methylxanthine, Sigma-Aldrich; Catalog number I5879) 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt (cAMP, Sigma-Aldrich; Catalog number B7880-100MG) SAGM™ Small Airway Epithelial Cell Growth Medium BulletKit (Lonza, Catalog number CC-3118) Dexamethasone powder (Sigma - Aldrich; Catalog number D4902 - 25MG) Recombinant human fibroblast growth factor - 2 (FGF - 2) (Peprotech; Catalog number AF - 100 - 18B) Recombinant human keratinocyte growth factor (KGF; FGF - 7) (Peprotech; Catalog number AF - 100 - 19 - 50UG) Recombinant human fibroblast growth factor - 10 (FGF - 10) (Peprotech; Catalog number AF - 100 - 26 - 25UG) DAPT (γ - secretase inhibitor IX) (Millipore Sigma #565770 - 10MG) Dulbecco's Modified Eagle Medium (DMEM) / F12 medium (Thermo Fisher Scientific; Catalog number 11320033) Y - 27632 (Rho - associated coiled - coil containing protein kinase (ROCK) inhibitor) (STEMCELL™ Technologies; Catalog number 72304) Dispase II (Thermo Fisher Scientific; Catalog number 17105041) TrypLE™ Express (Thermo Fisher Scientific; Catalog number 12604013) DMEM, high glucose, GlutaMAX™ supplement, pyruvate (Thermo Fisher Scientific; Catalog number 10569010) PneumaCult™ Ex Plus (STEMCELL™ Technologies; Catalog number 05040) Hydrocortisone stock solution (STEMCELL™ Technologies; Catalog number 07926) Matrigel® GFR basement membrane matrix, LDEV - free (Corning®; Catalog number 354230) TGF - β RI kinase inhibitor IV (A - 83 - 01) (Millipore Sigma; Catalog number 616454) BMP inhibitor II, DMH1 (Millipore Sigma; Catalog No. 203646) EZSolution CHIR99021 (GSK-3 inhibitor) (BioVision; Catalog No. 1748-5) CellAdhere Laminin-521 (STEMCELL™ Technologies; Catalog No. 200-0117) STEMdiff™ Definitive Endoderm Kit (STEMCELL™ Technologies; Catalog No. 05110)

[0098] The reagents were prepared as follows:

[0099] Ascorbic acid: A 50 mg / mL ascorbic acid solution was prepared by dissolving 500 mg of ascorbic acid in tissue culture grade water. The solution was sterile filtered, aliquoted into 500 μL portions and stored at -20 °C for up to 6 months.

[0100] MTG: Immediately prior to preparing the complete serum-free differentiation medium as discussed below, 26 μL of MTG was added to 2 mL of IMDM to prepare a 13 μL / mL MTG solution.

[0101] Recombinant human FGF-2 (rhFGF-2): rhFGF-2 was reconstituted in sterile water to a concentration of 1 mg / mL and diluted to a concentration of 250 μg / mL in 0.1% BSA / phosphate buffered saline (PBS). This solution was stored at -80 °C for up to 3 months.

[0102] Recombinant human KGF (rhKGF): rhKGF was reconstituted in PBS to a concentration of 0.1 mg / mL and diluted to a concentration of 20 μg / mL in 0.1% BSA / PBS. The solution was stored at -80 °C for up to 3 months.

[0103] Recombinant human FGF-10 (rhFGF-10): rhFGF-10 was reconstituted in 5 mM sodium phosphate to a concentration of 0.1 mg / mL and diluted to a concentration of 20 μg / mL in 0.1% BSA / PBS. The solution was stored at -80 °C for up to 3 months.

[0104] DAPT: DAPT was reconstituted in sterile dimethyl sulfoxide (DMSO) at a concentration of 20 mM. The solution was aliquoted and stored at -20 °C for up to 3 months.

[0105] Y-27632: Y-27632 was reconstituted in sterile PBS at a concentration of 10 mM.

[0106] IBMX: IBMX was reconstituted in 200 proof ethanol at a concentration of 0.1 M. The solution was aliquoted and stored at -20 °C for up to 1 year.

[0107] 10X cAMP / IBMX: The 10X cAMP / IBMX solution was prepared by mixing 50 mL of cSFDM, 21.5 mg of cAMP, and 500 μL of 0.1 M IBMX. The solution was sterile filtered through a 0.22 μM filter and stored in the dark at 4 °C for up to 1 month.

[0108] Dexamethasone: Dexamethasone was reconstituted in 63.7 mL of molecular biology grade ethanol to a concentration of 1 mM. The solution was stored at -20 °C for up to 2 years. Next, a 100 μM dexamethasone solution was prepared by mixing 500 μL of 1 mM dexamethasone and 4.5 mL of molecular biology grade ethanol. The solution was aliquoted in volumes of 100 μL and stored at -20 °C for up to 1 year.

[0109] Dispase II: Dispase II was dissolved in DMEM medium at 2 mg / mL. The solution was sterile filtered and stored at 4 °C for up to 2 weeks or aliquoted and stored at -20 °C for up to 6 months.

[0110] A83-01: The TGF-β RI kinase inhibitor IV (A-83-01) was reconstituted in an appropriate amount of DMSO at a concentration of 2 mM.

[0111] DMH1: DMH1 was reconstituted in DMSO at a concentration of 2 mM.

[0112] Complete Serum-Free Differentiation Medium (cSFDM): 375 mL of IMDM, 125 mL of Ham's F-12 medium, 5 mL of GlutaMAX™ supplement, 5 mL of B-27 supplement, 3.3 mL of 7.5% BSA, 2.5 mL of N-2 supplement, 500 μL of 50 mg / mL ascorbic acid, 1.5 mL of 13 μL / mL MTG, and 500 μL of Primocin® were mixed and sterile filtered. The medium was stored at 4°C in the dark for up to 1 month.

[0113] Airway Differentiation Medium (ADM): 45 mL of cSFDM base, 5 mL of 10X cAMP / IBMX, 50 μL of 250 μg / mL rhFGF2, 500 μL of 10 μg / mL rhFGF10, 25 μL of 100 μM dexamethasone, and 50 μL of 10 mM Y-27632 were mixed and sterile filtered. The medium was stored at 4°C in the dark for up to 1 month.

[0114] Complete PneumaCult™ Ex Plus Medium: 10 mL of PneumaCult™ Ex Plus 50X supplement, 0.5 mL of hydrocortisone stock solution, and 1 mL of Primocin® were mixed into 490 mL of PneumaCult™ Ex Plus basal medium and sterile filtered.

[0115] Complete Proximal Airway Stem Cell Maturation Medium (PASC Medium): SAGM medium (Lonza), 1 μM A8301, 0.5 μM DHM-1, 20 ng / ml FGF10, 20 ng / ml FGF7, 10 μM Y-27632 were mixed and sterile filtered.

[0116] Basal Cell Maturation Medium: Complete PneumaCult™ Ex Plus Medium, 1 μM A-83-01, 1 μM DMH1 and 10 μM Y-27632 were mixed and sterile filtered.

[0117] DS / SB Medium (Anteriorization Medium): cSFDM medium was supplemented with 2 μM dorsomorphin and 10 μM SB431542. This was then sterile filtered.

[0118] CBRa medium (lung progenitor cell specification medium): cSFDM medium, 3 μM CHIR99021, and 10 ng / mL BMP-4 were mixed and sterile filtered. Retinoic acid (RA) was added fresh, and CBRa medium was prepared at a final concentration of 100 nM on the medium change day. The medium was stored at 4°C for up to 1 week.

[0119] LPC sorting buffer: CMF-HBSS, 2% FBS, 25 mM HEPES, 2 mM EDTA, and 10 μM Y-27632 were mixed and sterile filtered.

[0120] An experiment to differentiate lung progenitor cells (LPCs) from human induced pluripotent stem cells (iPSCs) (BJRiPS iPSCs were provided by Jintang Du) was conducted using the following protocol and reagents.

[0121] Preparation of Matrigel® plate coating Matrigel® was thawed overnight on ice and aliquoted into 1.5 mL microtubes according to the dilution ratio specified by the manufacturer. The aliquots were stored at -20°C. On the day of cell seeding, Matrigel® was diluted with cold DMEM / F-12 medium according to the dilution ratio specified by the manufacturer to an amount sufficient to coat the wells / plates used for cell seeding. Next, the wells of a 6-well plate were coated with 1.5 mL of Matrigel® / DMEM F-12 solution per well and incubated at 37°C for at least 1 hour. Before adding the cells, the wells were rinsed with 1 mL of DMEM / F-12 medium.

[0122] Seeding of iPSC single cells BJRiP (iPS cells) cultured in a 6-well plate were washed with PBS. Using 1 mL of Accutase (registered trademark) in each well, the cells were incubated at 37 °C for 5 to 7 minutes to detach them. Then, the cells were gently resuspended and pelleted at 200×g for 5 minutes. The iPSC pellet was resuspended in 15 mL of mTeSR1 medium supplemented with 10 μM Y-27632, and a single-cell suspension was created using a P1000 tip. Then, the cells were counted using a Moxi (trademark) cell counter (ORFLOW (registered trademark) Technology) and seeded onto a Matrigel (registered trademark)-coated 6-well plate at 2E6 cells per well. Then, using mTeSR1 medium supplemented with 10 μM Y-27632, the volume per well was increased to 3 mL.

[0123] Differentiation into definitive endoderm (DE) (day 0) DMEM / F12 and STEMDiff (trademark) definitive endoderm basal medium were warmed to 37 °C. Supplements MR and CJ were thawed on ice and diluted 1:100 in STEMdiff (trademark) definitive endoderm basal medium. The iPSC wells were washed twice with 1 mL of warmed DMEM / F12 and then exchanged with 2 mL of STEMDiff (trademark) definitive endoderm basal medium containing supplements MR and CJ. The cells were incubated at 37 °C for approximately 24 hours.

[0124] Differentiation of definitive endoderm (DE) (days 1 - 2) DMEM / F12 and STEMDiff (trademark) definitive endoderm basal medium were warmed to 37 °C. Supplement CJ was thawed on ice and diluted 1:100 in STEMdiff (trademark) definitive endoderm basal medium. Approximately 2 mL of medium was added to each well, and the cells were incubated at 37 °C for approximately 24 hours. On day 2, the medium was replaced again, and the culture was continued at 37 °C.

[0125] Cell recovery for subculture and flow cytometry QC The medium was removed from the 6-well plate, and the wells were washed twice with 1 mL of PBS. The PBS was removed, and 1.5 mL of Accutase™ was added to each well. The plate was incubated at 37 °C for approximately 4 - 5 minutes or until the cells detached easily. The collected cells were diluted with an equal volume of DMEM / F12 medium. The cells were pipetted with a p1000 pipettor to disperse the cell clumps into single cells.

[0126] The cells were then filtered through a 30 μM filter to remove debris and centrifuged at 200 g for 5 minutes. The medium was aspirated, and approximately 5 mL of FACS wash buffer or DMEM / F12 was added. The cells were pipetted with a p1000 pipettor to disperse the cell clumps into single cells and counted using a Moxi™ cell counter (ORFLOW® technology).

[0127] For the flow cytometry quality control (QC) assay, approximately 250,000 - 300,000 cells were sorted. The remaining cells were centrifuged and resuspended in DMEM / F12 medium to a concentration of approximately 1 million cells / mL. The cells were seeded onto a Matrigel® - coated 6-well plate at a density of approximately 208,000 cells / cm 2 ².

[0128] Differentiation of anterior foregut endoderm (AFE) (day 3) After incubation, the medium was aspirated, and 2 mL of AFE DS / SB medium supplemented with 2 mL of Y-27632 was added to each well. The plate was incubated at 37 °C for 24 hours. Y-27632 was added to the medium (final concentration 10 μM) during the first 24 - 48 hours after cell seeding. The medium was replaced similarly every 24 hours until day 6.

[0129] The medium used was aspirated from the cells, and the cells were gently washed twice with approximately 1 mL of DMEM / F12 medium. 2 mL of CBRa medium was added to the wells, and the cells were incubated at 37 °C for 48 hours. The medium was replaced again on days 8, 10, 12, and 14. [Example 2]

[0130] Differentiation of LPCs into basal cells The reagents and materials used were as follows: Fluorescein isothiocyanate (FITC) anti-human CD326 (Ep-CAM) (BioLegend; catalog number 324204) PE anti-human CD26 (BioLegend; catalog number 302706) BV421 anti-human CD47 (BioLegend; catalog number 323116) FITC mouse IgG2b, k isotype control (BioLegend; catalog number 400310) PE mouse IgG2a, k isotype control (BioLegend; catalog number 400212) BV421 mouse IgG1, k isotype control (BioLegend; catalog number 400158) Matrigel® GFR basement membrane matrix, LDEV-free (Corning®; catalog number 354230) UltraComp eBeads™ compensation beads (Thermo Fisher; catalog number 01-2222-41) Ghost Dye® Red780 (Tonbo Biosciences; catalog number 13-0865-T100)

[0131] The following reagents were prepared:

[0132] Sorting wash buffer: CMF-PBS, 1% FBS, 1 mM EDTA, and 25 mM HEPES were mixed and sterile filtered.

[0133] LPC sorting buffer: CMF-HBSS, 2% FBS, 2 mM EDTA, 25 mM HEPES, and 10 μM Y-27632 were mixed and sterile filtered.

[0134] EPCAM / CD47 / CD26 surface staining: Cells were harvested according to the protocol disclosed in Example 1. The cells were counted and prepared for staining. Approximately 150,000 - 200,000 cells were stained with FITC anti-human CD-326 as a single-staining control, one drop of UltraComp eBeads™ calibration beads for phycoerythrin (PE) single-staining control, BV421 anti-human CD47 single-staining control, Ghost Dye™ Red780 single-staining control, and isotype control (FITC, PE, and / or BV421) together with Ghost Dye™ Red780. Approximately 14E6 cells were stained with EpCAM, CD47, and CD26 together with Ghost Dye™ Red780. After counting the cells, they were washed with PBS and centrifuged at 200×g for 5 minutes at 8°C. At the same time, Ghost Dye™ Red780 was diluted with PBS at 1:750 - 1:1000. The supernatant was aspirated, 100 μL of Ghost Dye™ Red780 was added, and the cells were incubated at room temperature for 10 minutes. After incubation, the cells were washed once with 1 mL of sort wash buffer and then centrifuged at 200×g for 5 minutes at 8°C.

[0135] To appropriate samples, the following were added and the cells were stained on ice or at 4°C for 30 minutes: · 0.8 μL of FITC anti-human CD-326 single-staining control per 100 μL of LPC sort buffer (for up to 1E6 cells) · 2.5 μL of BV421 anti-human CD47 single-staining control per 100 μL of LPC sort buffer (for up to 1E6 cells) · 0.2 μL of FITC mouse IgG2b, k isotype control + 0.6 μL of PE mouse IgG2a, k isotype control + 1.2 μL of BV421 mouse IgG1, k isotype control per 100 μL of LPC sort buffer (for up to 1.5E6 cells) · 2.5 μL of BV421 anti-human CD47 + 0.8 μL of FITC anti-human CD-326 + 2.5 μL of PE anti-human CD26 per 100 μL of LPC sort buffer (for up to 1.5E6 cells)

[0136] Next, the supernatant was aspirated, and LPC sorting buffer was added to the sample to a final concentration of approximately 1E6 - 3E6 cells / mL. Prior to running the cells on the BD FACSAria™ Fusion, the cell suspension was filtered using a 40 μM flow tube with a filter to remove aggregates from the cell suspension.

[0137] Sorting of CD47hi / CD27- by BD FACSAria™ Fusion: The parameters used were as follows: FSC: 260; SSC: 270; AF488: 350; PE: 360; APC-Cy7: 440; BV421: 275; and FSC scaling region: 0.3.

[0138] All compensation single-color controls were run, and a correction matrix was calculated prior to running the samples. All isotype controls and LPC samples were run on the BD FACSAria™ Fusion using the appropriate correction matrix. Next, collection tubes were prepared with 5 - 6 mL of LPC sorting buffer added, and the cells were sorted based on: single cell gate (FSC-H vs FSC-w and SSC-H vs SSC-W); live cells in the APC-Cy7 channel (low Ghost Dye Red780); EpCAM+ in the AF488 channel; and CD47hi / CD26- in the PE / BV421 channels. Also, for each sample, 480,000 cells per 12-well plate (3D) were sorted with a purity mask.

[0139] Re-seeding of cells into 3D Matrigel® After the cells were harvested, they were centrifuged at 200×g for 5 minutes at 8°C. The medium was aspirated, and on ice, undiluted Matrigel® GFR was added to reach a concentration of approximately 600 cells / μL. Approximately 45 - 50 μL droplets were dropped onto the bottom of each well of a 12-well plate on ice and spread. Then, the droplets were incubated at 37°C for 15 - 20 minutes, 1 mL of ADM was added to each well, and then incubated at 37°C for 48 - 72 hours. The medium was not changed on day 16 of differentiation.

[0140] Day 17 to 24 of differentiation (Day 1 to 9 of differentiation from LPC to basal cell (BS)) The medium was aspirated from each well and 1 mL of ADM was added. Then the cells were incubated at 37 °C for 48 - 72 hours. The medium was changed every 24 - 72 hours until day 26 of differentiation.

[0141] Day 26 of differentiation (Day 10 of differentiation from LPC to BS) The medium was aspirated and 2 mg / mL dispase was added to cover the droplets (typically 1 mL / well), then incubated at 37 °C for 60 minutes until Matrigel® was completely dissolved. The cells were gently pipetted 3 - 5 times. The dispersed organoids were transferred to a new 15 mL conical tube using a p1000 pipette and an equal volume of DMEM was added. The spheres were centrifuged at 300 × g for 2 minutes at 4 °C. The supernatant was aspirated and 1 mL of TrypLE™ was added per dispersed droplet. TrypLE™ and the cells were transferred to one well of a 12 - well plate and incubated at 37 °C for 10 - 12 minutes. Incubation was continued until the cells were completely dispersed. An equal volume of DMEM / F12 + 10% FBS was added to each well. Then, the dispersed cells were collected, filtered using a 30 μM filter to remove debris, and counted using a Moxi™ cell counter (ORFLOW® Technokogy). Approximately 100,000 cells were used for QC and the remaining cells were re - seeded onto 3D Matrigel®. A typical image of all cells on day 26 of differentiation is shown in Figure 2A. QC plots of cell surface markers EpCAM, NKX2 - 1, and p63 on day 26 are shown in Figures 2B - 2C. On days 28 and 30 of differentiation, approximately 1 mL of medium was exchanged in all wells and the cells were maintained at 37 °C for 24 - 48 hours.

[0142] Recovery of AE2 from 3D Matrigel® cultures for sorting and QC of NGFR (days 31 - 40) Cells were harvested according to the protocol described in Example 1 and sorted using magnetic labeling and magnetic separation of NGFR according to the manufacturer's protocol. Prior to seeding, a laminin-521-coated plate was prepared by placing a 5 μg / mL laminin-521 solution in each well of a 6-well plate and incubating either at 37 °C for 2 hours or overnight at 4 °C. After sorting, NGFR-positive cells were seeded onto the laminin-521 plate at a cell density of approximately 5,000 cells / cm 2 and incubated at 37 °C for 24 hours. Exemplary images of the cells on day 33 of differentiation are shown in FIGS. 3A (4× magnification) and 3F (10× magnification). QC plots of the cell surface markers EpCAM, NKX2-1, and p63 on day 33 are shown in FIGS. 3B-3C.

[0143] The medium was changed every 24 hours until the cells reached an approximately 95-100% confluent state. Exemplary images of the cells on day 40 of differentiation are shown in FIGS. 4A (4× magnification) and 4F (10× magnification). QC plots of the cell surface markers EpCAM, NKX2-1, p63, CK5, and NGFR on day 40 are shown in FIGS. 4B-4E. QC plots of the cell surface markers CD49f, p63, CK8, NGFR, and SOX2 on day 40 are shown in FIG. 4G.

[0144] Proliferation of basal cells (after day 41) A laminin-521-coated plate was prepared as described above. The medium used was removed from the wells and the cells were washed once with PBS. The cells were harvested as described above and seeded onto the laminin-521-coated plate at a density of approximately 5,000 cells / cm 2At the cell density of , seeding was performed using the basal maturation medium. The cells were then incubated at 37 °C for 24 hours. The medium was changed every 24 hours until reaching an approximately 95 - 100% confluent state. Exemplary images of the cells on the 45th day of differentiation are shown in FIGS. 5A (4x magnification) and 5F (10x magnification). QC plots of the cell surface markers EpCAM, NKX2-1, p63, CK5, and NGFR on the 45th day are shown in FIGS. 5B - 5E. QC plots of the cell surface markers CD49f, p63, CK8, NGFR, and SOX2 on the 45th day are shown in FIG. 5G.

[0145] FIG. 6A shows a flow cytometry gating strategy for identifying iPS-BC cells of EpCAM + , NKX2-1 + , p63 + , CK5 + , and NGFR + before cryopreservation on the 44th day. FIG. 6B shows a flow cytometry gating strategy for identifying control iPS-BC cells of EpCAM + , NKX2-1 + , p63 + , CK5 + , and NGFR + on the 47th day. FIG. 6C shows a flow cytometry gating strategy for identifying iPS-BC cells of EpCAM + , NKX2-1 + , p63 + , CK5 + , and NGFR + after thawing from cryopreservation.

[0146] All publications, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Further, any citation or identification of a reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. When section headings are used, they should not necessarily be construed as limiting.

Claims

**Claim 1** A method for differentiating lung progenitor cells (LPCs) into epithelial cells or basal cells, comprising: (i) culturing the LPCs in a basal culture medium comprising fibroblast growth factor 2 (FGF2), fibroblast growth factor 10 (FGF10), dexamethasone, cyclic adenosine monophosphate (cAMP), an inhibitor of cyclic nucleotide phosphodiesterase, and a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor; (ii) separating cells of (i) that do not express nerve growth factor receptor (NGFR) or express it at low levels to form basal progenitor cells; (iii) culturing the cells of (ii) in a basal culture medium comprising FGF10, keratinocyte growth factor (KGF), an inhibitor of transforming growth factor β type I receptor kinase (ALK5), an inhibitor of phosphorylation of SMAD (suppressor of mothers against decapentaplegic), and a ROCK inhibitor; and (iv) separating the cells of (iii) having NGFR expression to form epithelial cells or basal cells. **Claim 2** The method according to claim 1, further comprising (v) culturing the cells of (iv) having NGFR expression in a basal culture medium comprising FGF10, KGF, an ALK5 inhibitor, an inhibitor of phosphorylation of SMAD, and a ROCK inhibitor. **Claim 3** The method according to claim 2, wherein the culture of (v) is performed on a laminin-coated surface. **Claim 4** The method according to claim 3, wherein the laminin is laminin-521. **Claim 5** The method according to any one of claims 1 to 4, wherein the culture of (i) is performed in a three-dimensional (3D) matrix. **Claim 6** The method according to any one of claims 1 to 5, wherein the culture of (ii) is performed in a 3D matrix. **Claim 7** The method according to claim 5 or 6, wherein the 3D matrix is Matrigel®. **Claim 8** The method according to any one of claims 1 to 7, wherein FGF2 is present in the culture medium at a concentration of about 250 ng / mL. **Claim 9** The method according to any one of claims 1 to 8, wherein FGF10 is present in the culture medium of (i) at a concentration of about 100 ng / mL. **Claim 10** The method according to any one of claims 1 to 9, wherein FGF10 is present in the culture media of (ii) and (iv) at a concentration of about 20 ng / mL. **Claim 11** The method according to any one of claims 1 to 10, wherein dexamethasone is present in the culture medium at a concentration of about 50 nM.

12. The method according to any one of claims 1 to 11, wherein cAMP is present in the culture medium at a concentration of about 100 μM.

13. The method according to any one of claims 1 to 12, wherein the inhibitor of cyclic nucleotide phosphodiesterase is 3-isobutyl-1-methylxanthine (IBMX).

14. The method according to claim 13, wherein IBMX is present in the culture medium at a concentration of about 100 μM.

15. The method according to any one of claims 1 to 14, wherein the ROCK inhibitor is Y-27632.

16. The method according to claim 15, wherein Y-27632 is present in the culture medium at a concentration of about 10 μM.

17. The method according to any one of claims 1 to 16, wherein KGF is present in the culture medium at a concentration of about 20 ng / mL.

18. The method according to any one of claims 1 to 17, wherein the ALK5 inhibitor is A83-01.

19. The method according to claim 18, wherein A83-01 is present in the culture medium at a concentration of about 1 μM.

20. The method according to any one of claims 1 to 19, wherein the inhibitor of SMAD phosphorylation is DMH-1.

21. The method according to claim 20, wherein DMH-1 is present in the culture medium at a concentration of about 0.5 μM.

22. An epithelial cell produced by the method according to any one of claims 1 to 21.

23. A basal cell produced by the method according to any one of claims 1 to 21.

24. An organoid comprising the epithelial cell according to claim 22.

25. An organoid comprising the basal cell according to claim 23.

26. A differentiation medium comprising a basal culture medium, FGF10, KGF, an ALK5 inhibitor, an inhibitor of SMAD phosphorylation, and a ROCK inhibitor.

27. A differentiation medium comprising a basal culture medium, about 20 ng / mL of FGF10, about 20 ng / mL of KGF, about 1 μM of A83-01, about 0.5 μM of DMH-1, and about 10 μM of Y-27632.

Images