Method for differentiating AT2 cells

Abstract

The present disclosure relates to a method for differentiating pluripotent stem cells or lung progenitor cells into alveolar type 2 (AT2) cells. The present disclosure also relates to AT2 cells produced by such a method, organoids containing such AT2 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 / 390,463, filed Jul. 19, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates to a method of differentiating pluripotent stem cells or lung progenitor cells into alveolar type 2 (AT2) cells. The present disclosure also relates to AT2 cells produced by such methods, organoids containing such AT2 cells, and methods of using the same. The present disclosure also relates to a differentiation medium 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 PSCs derived from adult somatic cells that have been genetically reprogrammed into an ESC - like state by the expression of genes and factors important for maintaining the properties that define embryonic stem cells (ESCs). iPSCs have attracted 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 a method of differentiating pluripotent stem cells (PSCs) or lung progenitor cells (LPCs) into alveolar type 2 (AT2) cells.

[0005] In some embodiments, the method comprises: (i) culturing LPCs in a base culture medium comprising a glycogen synthase kinase 3 (GSK3) inhibitor, keratinocyte growth factor (KGF), fibroblast growth factor 10 (FGF10), and a γ-secretase inhibitor; (ii) subculturing the cells of (i) in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cyclic adenosine monophosphate (cAMP), an inhibitor of cyclic nucleotide phosphodiesterase, and a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor for about 24 to about 48 hours; (iii) culturing the cells of (ii) in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cyclic adenosine monophosphate (cAMP), and an inhibitor of cyclic nucleotide phosphodiesterase for about 7 days; (iv) isolating the cells of (iii) having expression of epithelial cell adhesion molecule (EpCAM) and / or carboxypeptidase M (CPM); (v) subculturing the cells of (iv) having expression of EpCAM and / or CPM in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor; and (vi) isolating the cells of (v) having expression of surfactant protein C (SFTPC) to form AT2 cells.

[0006] In some embodiments, the subculture of (ii) is performed in a two-dimensional (2D) matrix. In some embodiments, the 2D matrix is Matrigel®.

[0007] In some embodiments, the subculture of (v) is performed in a three-dimensional (3D) matrix. In some embodiments, the 3D matrix is Matrigel®.

[0008] In some embodiments, the method comprises: (i) culturing LPCs in a basal culture medium comprising a GSK3 inhibitor, KGF, FGF10, and a γ-secretase inhibitor; (ii) isolating the cells of (i) that have expression of EpCAM and / or CPM; (iii) subculturing the cells of (ii) that have expression of EpCAM and / or CPM in a basal culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor for about 24 to about 48 hours; (iv) culturing the cells of (iii) in a basal culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterase, which does not contain or essentially does not contain a ROCK inhibitor, for about 7 days; and (v) isolating the cells of (iv) that have expression of SFTPC to form AT2 cells.

[0009] In some embodiments, the subculture of (iii) is performed in a 3D matrix. In some embodiments, the 3D matrix is Matrigel®.

[0010] In some embodiments, the method comprises: (i) culturing LPCs in a basal culture medium comprising a GSK3 inhibitor, KGF, FGF10, and a γ-secretase inhibitor; (ii) subculturing the cells of (i) in a basal culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor; (iii) separating the cells of (ii) having expression of EpCAM and / or CPM; (iv) subculturing the cells of (iii) having expression of EpCAM and / or CPM in a basal culture medium comprising KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor; (v) culturing the cells of (iv) in a basal culture medium comprising KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a GSK3 inhibitor, the basal culture medium not containing or essentially not containing a ROCK inhibitor; and (vi) separating the cells of (v) having expression of SFTPC to form AT2 cells.

[0011] In some embodiments, the subculture of (iv) is for about 2 days to about 4 days, and / or the culture of (v) is for about 2 days to about 4 days.

[0012] In some embodiments, the subculture of (iv) is for about 2 days, and / or the culture of (v) is for about 2 days.

[0013] In some embodiments, the subculture of (iv) and / or the culture of (v) are repeated before the separation of (vi).

[0014] In some embodiments, the repeated subculture of (iv) is for about 4 days, and the repeated culture of (v) is for about 4 days.

[0015] In some embodiments, the subculture of (ii) is performed in a 2D matrix. In some embodiments, the 2D matrix is Matrigel®.

[0016] In some embodiments, the subculture of (iv) is performed in a 3D matrix. In some embodiments, the 3D matrix is Matrigel®.

[0017] In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, CHIR99021 is present in the culture medium at a concentration of about 3 μM.

[0018] In some embodiments, FGF10 is present in the culture medium at a concentration of about 10 ng / mL.

[0019] In some embodiments, KGF is present in the culture medium at a concentration of about 10 ng / mL.

[0020] In some embodiments, the γ-secretase inhibitor is N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). In some embodiments, DAPT is present in the culture medium at a concentration of about 20 μM.

[0021] In some embodiments, dexamethasone is present in the culture medium at a concentration of about 50 nM.

[0022] In some embodiments, cAMP is present in the culture medium at a concentration of about 100 μM.

[0023] 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.

[0024] In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, Y-27632 is present in the culture medium at a concentration of about 10 μM.

[0025] The present disclosure also provides AT2 cells produced by the differentiation methods disclosed herein, organoids comprising the AT2 cells disclosed herein, and certain methods of using the same.

[0026] The present disclosure also provides a specific differentiation medium. In some embodiments, the differentiation medium comprises a basal culture medium, a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor. In some embodiments, the differentiation medium comprises a basal culture medium, about 3 μM CHIR99021, about 10 ng / mL KGF, about 10 ng / mL FGF10, about 50 nM dexamethasone, about 100 μM cAMP, about 100 μM IBMX, and about 10 μM Y-27632.

[0027] The present disclosure also provides a differentiation medium comprising a basal culture medium, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor. In some embodiments, the differentiation medium comprises a basal culture medium, about 10 ng / mL KGF, about 10 ng / mL FGF10, about 50 nM dexamethasone, about 100 μM cAMP, about 100 μM IBMX, and about 10 μM Y-27632.

[0028] Some aspects of the invention are described herein with reference to the accompanying drawings, which are for illustrative purposes only. Although the drawings are referred to in detail herein, it should be emphasized that the details shown are for purposes of illustration and for the purpose of explicitly discussing aspects of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029]

Figure 1

Figure 2A

Figure 2B

Figure 3

Modes for Carrying Out the Invention

[0030] I. General Definitions Unless otherwise defined, all technical and scientific terms used in this specification 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, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents, and other references mentioned in this specification 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.

[0031] 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 from the claims.

[0032] To further define the present disclosure, the following terms and definitions are provided.

[0033] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates 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".

[0034] 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 numerical value above and below the indicated value by a variation of up to (higher or lower) 10 percent.

[0035] Throughout this disclosure, various aspects of the invention are presented in a 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. An enumerated numerical range includes the numbers defining the range and each integer within the defined range.

[0036] 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, along with each subrange between such values. The upper and lower limits of any range can be included in the range independently or excluded from the range, and each range, whether one limit is included, neither limit is included, or both limits are included, is also encompassed within the scope of the present disclosure. Accordingly, ranges described herein are understood to be 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 subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0037] When values are explicitly recited, values of an amount or quantity 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 subcombination 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.

[0038] 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).

[0039] It is understood that when an aspect is described in the expression "comprising" herein, other similar aspects described in the words "consisting of" and / or "consisting essentially of" are also provided.

[0040] II. Differentiation methods The present disclosure relates to an improved method for differentiating pluripotent stem cells (PSCs) or lung progenitor cells (LPCs) into alveolar type 2 (AT2) cells. Such a method provides, for example, an improvement in cell viability, yield and / or characteristics of the differentiated cells.

[0041] As used herein, the terms "differentiate" and "differentiating" refer to the process of inducing or reprogramming a young or immature cell (e.g., a pluripotent stem cell) into a more mature or specialized cell (e.g., an AT2 cell). 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.

[0042] 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., AT2 cells).

[0043] 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, pig, or primate.

[0044] 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.

[0045] 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 lung lineage-specified and retain self-renewal capacity 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, 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.

[0046] In some embodiments, the LPC is derived from a PSC (e.g., iPSC). Methods of generating LPC 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.

[0047] As used herein, the term “alveolar type II cell” or “AT2 cell” refers to a cell that lines the alveoli and produces surfactant (e.g., surfactant protein C (SFTPC)) that enables proper gas exchange upon lung inflation. AT2 cells contain electron-dense organelles with a lamellar structure (lamellar bodies). As used herein, AT2 cells include mature AT2 cells, AT2 progenitor cells, and AT2 precursor cells.

[0048] In some embodiments, the AT2 cells produced by the differentiation methods provided herein express one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic signatures) typical of AT2 cells. Examples of such markers include, but are not limited to, Nkx2.1, SFTPC, SFTPB, lysophosphatidylcholine acyltransferase 1 (LPCAT1), SFTPA2, SFTPD, ATP-binding cassette subfamily A member 2 (ABCA2), napsin A aspartic peptidase 1 (NAPSA1), progastricsin (PGC), Solute Carrier family 34 member 2 (SLC34A2), epithelial cell adhesion molecule (EpCAM), and carboxypeptidase M (CPM). In some embodiments, the marker is EpCAM. In some embodiments, the marker is CPM. In some embodiments, the marker is SFTPC.

[0049] As used herein, the terms “expression,” “expresses,” or “express” with respect to a marker (e.g., EpCAM, CPM, and / or SFTPC) include, but are not limited to, the expression of a detectable marker, the expression of a marker equal to or greater than that of mature AT2 cells, or their precursor or progenitor, and the expression of a marker that exceeds that of control cells that do not express the marker and are not AT2 cells.

[0050] In some embodiments, the AT2 cells produced by the differentiation methods provided herein have certain morphological features typical of AT2 cells. Examples of such features include, but are not limited to, a cuboidal shape, lamellar bodies, and / or microvilli when analyzed by microscopy, such as an electron microscope.

[0051] In some embodiments, the differentiation methods provided herein include certain cell culture conditions, such as cell culture in a particular culture medium.

[0052] 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 a composition for cell culture that contains nutrients that maintain cell viability, support growth, and optionally support differentiation. The 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, etc.

[0053] In some embodiments, the differentiation methods provided herein refer to cell culture media as "basal culture media" supplemented with other components. As used herein, "basal culture media" refers to a composition containing 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 media are supplemented with one or more other components such as, for example, amino acids, antibiotics, serum, growth factors, etc. Such components are well known in the art and are further described herein.

[0054] In some embodiments, the cell culture media or basal culture media of the provided differentiation methods "essentially do not contain" or "do not contain" certain components (e.g., ROCK inhibitors such as Y-27632). As used herein, the term "essentially do not contain" is known in the art and, as measured by the methods further described herein, refers to a culture medium that contains at least 95%, 96%, 97%, 98%, or 99% less of a particular component, or has undetectable amounts of that component (e.g., ROCK inhibitors such as Y-27632). The terms "does not comprise" and "do not comprise" are known in the art and, as measured by the methods further described herein, refer to a culture medium that does not contain a particular component (e.g., ROCK inhibitors such as Y-27632), or has undetectable amounts of that component.

[0055] In some embodiments, the differentiation methods provided herein include certain cell culture conditions such as subculturing cells 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®) at a particular cell density, as appropriate, prior to seeding. Methods for subculturing cells are well known and are further described herein.

[0056] 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®, and 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 cell culture surfaces or vessels are well known and are further described herein.

[0057] In some embodiments, the culturing and / or subculturing of the differentiation methods provided herein are performed in a two-dimensional (2D) matrix. In such embodiments, the cells are grown as a monolayer, as appropriate, on a cell culture surface or vessel having the substrate coating described above.

[0058] 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.

[0059] In some embodiments, the differentiation methods provided herein include one or more steps of separating cultured cells having certain specific biochemical characteristics. 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.

[0060] In some embodiments, the differentiation methods provided herein include separating cells having expression of EpCAM and / or CPM. In some embodiments, the differentiation methods provided herein include separating cells having expression of SFTPC.

[0061] 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, and the like. Techniques for affinity separation include separation using antibody-coated magnetic beads (e.g., immunomagnetic cell separation), affinity chromatography, cytotoxic agents that bind to monoclonal antibodies or are used in combination with monoclonal antibodies, such as complement and cytotoxins, and "panning" using antibodies bound to a solid matrix such as a plate, 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.

[0062] Some embodiments of the differentiation methods provided herein include culturing and / or passaging cells in a basal culture medium containing a glycogen synthase kinase 3 (GSK3) inhibitor. GSK3 is a serine / threonine protein kinase that mediates the addition of phosphate molecules to certain serine and threonine amino acids of cellular substrates (e.g., glycogen synthase). This phosphorylation typically results in inhibition of the substrate. GSK3 is also involved in the control of the cellular response to damaged DNA and the phosphorylation of Ci in the Wnt signaling and Hedgehog (Hh) pathways, targeting proteolysis to the inactive form.

[0063] As used herein, "GSK3 inhibitor" refers to a compound that inhibits one or more GSK3 enzymes. The family of GSK3 enzymes is well known and numerous variants have been described (e.g., Schaffer et al., Gene, 302:73-81, 2003). Specific examples of GSK3 inhibitors include, but are not limited to, kenpaullone, 1-azakenpaullone, CHIR99021, CHIR98014, AR-A014418, CT99021, CT20026, SB415286, SB216763, AR-A014418, lithium, SB 415286, and TDZD-8.Further exemplary GSK3 inhibitors include BIO (2’Z,3’E)-6-bromindirubin-3’-oxime (GSK3 inhibitor IX); BIO-acetoxime (2’Z,3’E)-6-bromindirubin-3’-acetoxime (GSK3 inhibitor X); (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3 inhibitor XIII); pyridocarbazole-cyclopentadienylruthenium complex (GSK3 inhibitor XV); TDZD-8, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3β inhibitor I); 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3β inhibitor II); OTDZT 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (GSK3β inhibitor III); α-4-dibromoacetophenone (GSK3β inhibitor VII); AR-AO 14418 N-(4-methoxybenzyl)-N’-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3β inhibitor VIII); 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK3β inhibitor XI); TWS1 19-pyrrolopyrimidine compound (GSK3β inhibitor XII); L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form (GSK3β inhibitor XIII); 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β inhibitor VI); AR-AO 144-18; SB216763; and SB415286, but are not limited thereto. In some embodiments, the GSK3 inhibitor is CHIR99021.

[0064] In some embodiments, the GSK3 inhibitor (e.g., CHIR99021) is present in the basal culture medium at a concentration of about 0.5 μM to about 6 μM, or any value or range of values thereof, such as a concentration comprising about 1 μM to about 6 μM, about 3 μM to about 6 μM, about 0.5 μM to about 3 μM, about 1 μM to about 3 μM, or about 0.5 μM to about 1 μM. In some embodiments, the GSK3 inhibitor (e.g., CHIR99021) is about 0.5 μM, about 1 μM, about 3 μM or about 6 μM. In some embodiments, the GSK3 inhibitor (e.g., CHIR99021) is about 3 μM.

[0065] Some embodiments of the differentiation methods provided herein include culturing and / or passaging 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 mesoderm-derived cells.

[0066] In some embodiments, KGF is present in the basal culture medium at a concentration of about 2 ng / mL to about 20 ng / mL, or any value or range of values thereof, such as a concentration comprising about 5 ng / mL to about 20 ng / mL, about 10 ng / mL to about 20 ng / mL, about 2 ng / mL to about 10 ng / mL, about 5 ng / mL to about 10 ng / mL, or about 2 ng / mL to about 5 ng / mL. In some embodiments, KGF is present in the basal culture medium at a concentration of about 2 ng / mL, about 5 ng / mL, about 10 ng / mL, or about 20 ng / mL. In some embodiments, KGF is present in the basal culture medium at a concentration of about 10 ng / mL.

[0067] Some embodiments of the differentiation methods provided herein include culturing and / or passaging 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 lung, skin, ear and salivary gland.

[0068] In some embodiments, FGF10 is present in the basal culture medium at a concentration of about 2 ng / mL to about 20 ng / mL, or any value or range of values therebetween, such as, for example, about 5 ng / mL to about 20 ng / mL, about 10 ng / mL to about 20 ng / mL, about 2 ng / mL to about 10 ng / mL, about 5 ng / mL to about 10 ng / mL, or about 2 ng / mL to about 5 ng / mL. In some embodiments, FGF10 is present in the basal culture medium at a concentration of about 2 ng / mL, about 5 ng / mL, about 10 ng / mL, or about 20 ng / mL. In some embodiments, FGF10 is present in the basal culture medium at a concentration of about 10 ng / mL.

[0069] Some embodiments of the differentiation methods provided herein include culturing and / or passaging cells in a basal culture medium containing a γ-secretase inhibitor. As used herein, the term “γ-secretase” refers to any protein or protein complex that exhibits γ-secretase activity, which catalyzes the cleavage of a γ-secretase cleavage sequence at a γ-secretase cleavage site in a substrate having a γ-secretase cleavage sequence to produce a substrate cleavage product, but is not limited thereto. In some embodiments, γ-secretase is a protein complex consisting of one or more of the following subunits: presenilin, nicastrin, γ-secretase subunit APH-1, and γ-secretase subunit PEN-2.

[0070] As used herein, the term "γ-secretase inhibitor", or "GSI", refers to any substance or compound that binds to, for example, γ-secretase or the γ-secretase pathway and partially or completely inhibits, reduces, prevents, delays activation, inactivates, desensitizes, or downregulates the activity or expression thereof. Examples of γ-secretase inhibitors include recombinant versions of γ-secretase proteins, such as versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, small molecule chemicals, etc., but are not limited thereto. In some embodiments, the γ-secretase inhibitor reduces the expression and / or function of a subunit of γ-secretase (e.g., presenilin, nicastrin, APH-1, or PEN-2).

[0071] More specific examples of γ-secretase inhibitors include, but are not limited to, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester), talsaclidine (Hock et al., 2003), xanomeline, L-689660, L-685458, McN-A-343, CDD-0097, phentylamine, MG132, WPE-111-31C, MW-11-36C / 26A, MW-167, CM-265, lactacystin, DNPS1, DAM, LY-450139, PF-5212362, BMS-708163, MK-0752, ELN-318463, BMS-299897, LY-411575, BMS-906024, PF-3084014, RO4929097, and LY3039478. In some embodiments, the γ-secretase inhibitor is DAPT.

[0072] Some embodiments of the differentiation methods provided herein include culturing and / or passaging cells in a basal culture medium containing dexamethasone. Dexamethasone is an anti-inflammatory glucocorticoid that has various effects on cell growth and differentiation.

[0073] 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.

[0074] Some embodiments of the differentiation methods provided herein include culturing and / or passaging 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.

[0075] 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.

[0076] Some aspects of the differentiation methods provided herein include culturing and / or passaging cells in a basal culture medium containing an inhibitor of cyclic nucleotide phosphodiesterase. Cyclic nucleotide phosphodiesterase is a family of enzymes that hydrolyze the phosphodiester bonds of cyclic adenosine monophosphate and cyclic guanosine monophosphate, thereby inhibiting their pulmonary vasodilatory properties. 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.

[0077] 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.

[0078] Some aspects of the differentiation methods provided herein include culturing and / or passaging 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, reduce the expression and / or the 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 to these. In some aspects, the ROCK inhibitor is Y-27632.

[0079] 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.

[0080] In some embodiments, the γ-secretase inhibitor (e.g., DAPT) is present in the basal culture medium at a concentration of about 5 μM to about 40 μM, or any value or range of values thereof, such as about 10 μM to about 40 μM, about 20 μM to about 40 μM, about 5 μM to about 20 μM, about 10 μM to about 20 μM, or about 5 μM to about 10 μM. In some embodiments, the γ-secretase inhibitor (e.g., DAPT) is present in the culture medium at a concentration of about 5 μM, about 10 μM, about 20 μM or about 40 μM. In some embodiments, the γ-secretase inhibitor (e.g., DAPT) is present in the basal culture medium at a concentration of about 20 μM.

[0081] In some embodiments, the differentiation method provided herein comprises culturing and / or subculturing cells in a basal culture medium containing a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, and a γ-secretase inhibitor (e.g., DAPT). In some embodiments, the basal culture medium contains from about 0.5 μM to about 6 μM of a GSK3 inhibitor (e.g., CHIR99021), from about 2 ng / mL to about 20 ng / mL of KGF, from about 2 ng / mL to about 20 ng / mL of FGF10, and from about 5 μM to about 40 μM of a γ-secretase inhibitor (e.g., DAPT). In some embodiments, the basal culture medium contains about 3 μM of a GSK3 inhibitor (e.g., CHIR99021), about 10 ng / mL of KGF, about 10 ng / mL of FGF10, and about 20 μM of a γ-secretase inhibitor (e.g., DAPT).

[0082] In some embodiments, the differentiation method provided herein comprises culturing and / or subculturing cells in a basal culture medium containing a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a ROCK inhibitor (e.g., Y-27632). In some embodiments, the basal culture medium contains from about 0.5 μM to about 6 μM of a GSK3 inhibitor (e.g., CHIR99021), from about 2 ng / mL to about 20 ng / mL of KGF, from about 2 ng / mL to about 20 ng / mL of FGF10, from about 20 nM to about 80 nM of dexamethasone, from about 25 μM to about 200 μM of cAMP, from about 25 μM to about 200 μM of an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and from about 1 μM to about 20 μM of a ROCK inhibitor (e.g., Y-27632). In some embodiments, the basal culture medium contains about 3 μM of a GSK3 inhibitor (e.g., CHIR99021), about 10 ng / mL of KGF, about 10 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). In some embodiments, culturing and / or subculturing in such a culture medium is for about 24 hours to about 48 hours, or any value or range of values within that range, such as about 24 hours to about 36 hours, about 24 hours to about 30 hours, about 30 hours to about 48 hours, about 30 hours to about 36 hours, or about 36 hours to about 48 hours, etc. In some embodiments, culturing and / or subculturing is about 24 hours, about 30 hours, about 36 hours, or about 48 hours.

[0083] In some embodiments, the differentiation method provided herein includes culturing and / or subculturing cells in a basal culture medium containing a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX). In some embodiments, the basal culture medium contains from about 0.5 μM to about 6 μM of a GSK3 inhibitor (e.g., CHIR99021), from about 2 ng / mL to about 20 ng / mL of KGF, from about 2 ng / mL to about 20 ng / mL of FGF10, from about 20 nM to about 80 nM of dexamethasone, from about 25 μM to about 200 μM of cAMP, and from about 25 μM to about 200 μM of an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX). In some embodiments, the basal culture medium contains about 3 μM of a GSK3 inhibitor (e.g., CHIR99021), about 10 ng / mL of KGF, about 10 ng / mL of FGF10, about 50 nM of dexamethasone, about 100 μM of cAMP, and about 100 μM of an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX). In some embodiments, culturing and / or subculturing in such culture medium is for about 3 days to about 10 days, or any value or range of values within that range, e.g., about 5 days to about 10 days, about 7 days to about 10 days, about 5 days to about 10 days, about 7 days to about 10 days, or about 7 days to about 10 days, etc. In some embodiments, the culturing and / or subculturing is for about 3 days, about 5 days, about 7 days, or about 10 days.

[0084] In some embodiments, the differentiation method provided herein includes subculturing cells in a basal culture medium containing a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a ROCK inhibitor (e.g., Y-27632). In some embodiments, the subculturing is for about 24 hours to about 48 hours.

[0085] In some embodiments, the differentiation method provided herein is as follows: (i) Culturing LPC in a basal culture medium containing a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, and a γ-secretase inhibitor (e.g., DAPT); (ii) Subculturing the cells of (i) in a basal culture medium containing a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a ROCK inhibitor (e.g., Y-27632); (iii) Culturing the cells of (ii) in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX); (iv) Separating the cells of (iii) having the expression of EpCAM and / or CPM; (v) Subculturing the cells of (iv) having the expression of EpCAM and / or CPM in a basal culture medium containing a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a ROCK inhibitor (e.g., Y-27632); and (vi) Separating the cells of (v) having the expression of SFTPC to form AT2 cells.

[0086] In some embodiments, the subculture of (ii) is from about 24 hours to about 48 hours. In some embodiments, the culture of (iii) is about 7 days. In some embodiments, the subculture of (ii) is performed in a 2D matrix. In some embodiments, the 2D matrix is Matrigel®. In some embodiments, the subculture of (v) is performed in a 3D matrix. In some embodiments, the 3D matrix is Matrigel®.

[0087] In some embodiments, the differentiation method provided herein comprises culturing cells in a basal culture medium that contains a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and that does not contain, or is essentially free of, a ROCK inhibitor. In some embodiments, the culturing is for about 7 days.

[0088] In some embodiments, the differentiation method provided herein is as follows: (i) culturing LPCs in a basal culture medium that contains a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, and a γ-secretase inhibitor (e.g., DAPT); (ii) isolating the cells of (i) that have high expression of EpCAM and / or CPM; (iii) passaging the cells of (ii) that have high expression of EpCAM and / or CPM in a basal culture medium that contains a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a ROCK inhibitor (e.g., Y-27632); (iv) culturing the cells of (iii) in a basal culture medium that contains a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and that does not contain, or is essentially free of, a ROCK inhibitor; and (v) isolating the cells of (iv) that have high expression of SFTPC to form AT2 cells.

[0089] In some embodiments, the subculture of (iii) is about 24 hours to about 48 hours. In some embodiments, the culture of (iv) is about 7 days. In some embodiments, the subculture of (iii) is performed in a 3D matrix. In some embodiments, the 3D matrix is Matrigel®.

[0090] In some embodiments, the differentiation method provided herein includes subculturing cells in a basal culture medium containing KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a ROCK inhibitor (e.g., Y-27632). In some embodiments, following such subculture, the cells are cultured in a basal culture medium containing KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a GSK3 inhibitor (e.g., CHIR99021), which does not contain or is essentially free of a ROCK inhibitor.

[0091] In some embodiments, the differentiation method provided herein is as follows: (i) culturing LPC in a basal culture medium containing a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, and a γ-secretase inhibitor (e.g., DAPT); (ii) subculturing the cells of (i) in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a ROCK inhibitor (e.g., Y-27632); (iii) separating the cells of (ii) having high expression of EpCAM and / or CPM; (iv) subculturing the cells of (iii) having high expression of EpCAM and / or CPM in a basal culture medium containing KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a ROCK inhibitor (e.g., Y-27632); culturing the cells of (v)(iv) in a basal culture medium containing KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX), and a GSK3 inhibitor (e.g., CHIR99021), which is a basal culture medium not containing a ROCK inhibitor; and (vi) separating the cells of (v) having high expression of SFTPC to form AT2 cells.

[0092] In some embodiments, the subculture of (iv) is from about 2 days to about 4 days. In some embodiments, the culture of (v) is from about 2 days to about 4 days. In some embodiments, the subculture of (iv) is about 2 days, and the culture of (v) is about 2 days.

[0093] In some embodiments, the subculture of (iv) and / or the culture of (v) is repeated before the separation of (vi). In some embodiments, the repeated subculture of (iv) is about 4 days, and / or the repeated culture of (v) is about 4 days. In some embodiments, the subculture of (ii) is performed in a 2D matrix. In some embodiments, the 2D matrix is Matrigel®. In some embodiments, the subculture of (iv) is performed in a 3D matrix. In some embodiments, the 3D matrix is Matrigel®.

[0094] III. Other Embodiments The present disclosure also relates to AT2 cells produced by any of the differentiation methods disclosed herein.

[0095] The present disclosure also relates to organoids comprising the AT2 cells 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 densely 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, e.g., to produce only a particular type of cell.

[0096] Methods for maintaining differentiated AT2 cells and organoids are well known and include culturing the cells or organoids and / or cryopreserving them in the cell culture media described herein. 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-derived matrices such as Matrigel™, and combinations thereof, e.g., a hydrogel filled with microparticles.

[0097] The present disclosure also relates to certain methods of using such AT2 cells and organoids. In some aspects, the present disclosure provides a method of treating a lung disease comprising administering an AT2 cell or organoid made by any of the differentiation methods disclosed herein.

[0098] The present disclosure also relates to certain differentiation media for differentiating AT2 cells from PSCs or LPCs.

[0099] In some embodiments, the differentiation medium comprises a basal culture medium, a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, and a γ-secretase inhibitor (e.g., DAPT). In some embodiments, the differentiation medium comprises a basal culture medium, a GSK3 inhibitor (e.g., CHIR99021) at about 0.5 μM to about 6 μM, KGF at about 2 ng / mL to about 20 ng / mL, FGF10 at about 2 ng / mL to about 20 ng / mL, and a γ-secretase inhibitor (e.g., DAPT) at about 5 μM to about 40 μM. In some embodiments, the differentiation medium comprises a basal culture medium, a GSK3 inhibitor (e.g., CHIR99021) at about 3 μM, KGF at about 10 ng / mL, FGF10 at about 10 ng / mL, and a γ-secretase inhibitor (e.g., DAPT) at about 20 μM.

[0100] In some embodiments, the differentiation medium comprises a basal culture medium, a GSK3 inhibitor (e.g., CHIR99021), KGF, 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 medium comprises a basal culture medium, a GSK3 inhibitor (e.g., CHIR99021) at about 0.5 μM to about 6 μM, KGF at about 2 ng / mL to about 20 ng / mL, FGF10 at about 2 ng / mL to about 20 ng / mL, dexamethasone at about 20 nM to about 80 nM, cAMP at about 25 μM to about 200 μM, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX) at about 25 μM to about 200 μM, and a ROCK inhibitor (e.g., Y-27632) at about 1 μM to about 20 μM. In some embodiments, the differentiation medium comprises a basal culture medium, a GSK3 inhibitor (e.g., CHIR99021) at about 3 μM, KGF at about 10 ng / mL, FGF10 at about 10 ng / mL, dexamethasone at about 50 nM, cAMP at about 100 μM, an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX) at about 100 μM, and a ROCK inhibitor (e.g., Y-27632) at about 10 μM.

[0101] In some embodiments, the differentiation medium comprises a basal culture medium, a GSK3 inhibitor (e.g., CHIR99021), KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX). The differentiation medium may or may not essentially contain a ROCK inhibitor (e.g., Y-27632). In some embodiments, the differentiation medium comprises a basal culture medium, a GSK3 inhibitor (e.g., CHIR99021) at about 0.5 μM to about 6 μM, KGF at about 2 ng / mL to about 20 ng / mL, FGF10 at about 2 ng / mL to about 20 ng / mL, dexamethasone at about 20 nM to about 80 nM, cAMP at about 25 μM to about 200 μM, and an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX) at about 25 μM to about 200 μM. In some embodiments, the differentiation medium comprises a basal culture medium, a GSK3 inhibitor (e.g., CHIR99021) at about 3 μM, KGF at about 10 ng / mL, FGF10 at about 10 ng / mL, dexamethasone at about 50 nM, cAMP at about 100 μM, and an inhibitor of cyclic nucleotide phosphodiesterase (e.g., IBMX) at about 100 μM.

Example

[0102] Here, with the above description, reference is made to the following examples which illustrate some embodiments of the invention in a non-limiting manner. [Example 1]

[0103] Differentiation from LPC to AT2 cells An experiment was conducted to differentiate alveolar type 2 (AT2) cells from lung progenitor cells (LPC) using the following protocol and reagents.

[0104] 1. Concentration of LPC The reagents used were as follows:

[0105] Iscove's Modified Dulbecco's Medium (IMDM) (Thermo Fisher Scientific; catalog number 12440053)

[0106] Ham's F-12 medium supplemented with L-glutamine (Corning®; Catalog No. 10-080-CV)

[0107] GlutaMAX™ Supplement (Thermo Fisher Scientific; Catalog No. 35050061)

[0108] B-27 Supplement (50X) (Thermo Fisher Scientific; Catalog No. 17504044)

[0109] Bovine Serum Albumin (BSA); 7.5% in Dulbecco's Phosphate Buffered Saline (DPBS) (Sigma Aldrich; Catalog No. A8412-100ML)

[0110] N-2 Supplement (Thermo Fisher Scientific; Catalog No. 17502048)

[0111] L-Ascorbic acid (Sigma Aldrich; Catalog No. A4544-25G)

[0112] 1-Thioglycerol (MTG) (Sigma-Aldrich; Catalog No. M1753-100ML)

[0113] Primocin® (InvivoGen; Catalog No. ant-pm-1)

[0114] IBMX (3-Isobutyl-1-methylxanthine, Sigma-Aldrich; Catalog No. I5879)

[0115] 8-Bromo-cyclic Adenosine Monophosphate Sodium Salt (cAMP, Sigma-Aldrich; Catalog No. B7880-100MG)

[0116] Dexamethasone powder (Sigma-Aldrich; Catalog No. D4902-25 MG)

[0117] CHIR99021 (Catalog No. 1991-1 from Biovision)

[0118] Recombinant human keratinocyte growth factor (KGF; FGF-7) (Peprotech; Catalog No. AF-100-19-50UG)

[0119] Recombinant human fibroblast growth factor-10 (FGF-10) (Peprotech; Catalog No. AF-100-26-25UG)

[0120] DAPT (γ-secretase inhibitor IX) (Millipore Sigma #565770 - 10MG)

[0121] Dulbecco's Modified Eagle Medium (DMEM) / F12 Medium (Thermo Fisher Scientific; Catalog No. 11320033)

[0122] Y-27632 (Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor) (STEMCELL™ Technologies; Catalog No. 72304)

[0123] The reagents were prepared as follows:

[0124] 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.

[0125] MTG: Immediately before 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.

[0126] Recombinant human KGF: KGF was reconstituted in sterile phosphate buffered saline (PBS) at 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.

[0127] Recombinant human FGF-10: FGF-10 was reconstituted to a concentration of 0.1 mg / mL with 5 mM sodium phosphate and diluted to a concentration of 20 μg / mL with 0.1% BSA / PBS. The solution was stored at -80 °C for up to 3 months.

[0128] DAPT: DAPT was reconstituted to a concentration of 20 mM with sterile dimethyl sulfoxide (DMSO). The solution was aliquoted and stored at -20 °C for up to 3 months.

[0129] Y-27632: Y-27632 was reconstituted to a concentration of 10 mM with sterile PBS.

[0130] 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.

[0131] Concentrated medium (CFKD medium): To cSFDM, CHIR99021 (C) at a final concentration of 3 μM, FGF-10 (F) at 10 ng / mL, KGF (K) at 10 ng / mL, and DAPT (D) at 20 μM were added.

[0132] The BU-NGST, an induced pluripotent stem cell (iPSC)-derived reporter cell line developed in the Kotton laboratory at Boston University, was used. These cells have a green fluorescent protein (GFP) reporter and a TdTomato (TDT) reporter targeted to the Nkx2.1 locus and the surfactant protein C (SFTPC) locus, respectively. Carboxypeptidase M (CPM) can be used as an alternative for the selection of Nkx2.1+ cells.

[0133] Concentration was performed by aspirating the medium from the cells seeded in the 6-well plate and adding 2 mL of CFKD medium to each well. The plate was incubated at 37 °C for 24 hours. The medium was similarly changed every 24 hours for 7 days.

[0134] 2. Cell Recovery for 2D Subculture and Quality Control Next, the cells were recovered for two-dimensional (2D) subculture and quality control evaluation.

[0135] The following reagents were used:

[0136] Accutase™ (Biolegend; catalog number 423201)

[0137] Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free (Corning®; catalog number 354230)

[0138] Gentle Cell Dissociation Reagent (GCDR) (STEMCELL™ Technologies, catalog number 07174)

[0139] IBMX (3-isobutyl-1-methylxanthine, Sigma-Aldrich; catalog number I5879)

[0140] The reagents were prepared as follows:

[0141] IBMX: IBMX was reconstituted to a concentration of 0.1 M with 200 proof ethanol. The solution was aliquoted and stored at -20 °C for up to 1 year.

[0142] 10x cAMP / IBMX: The 10x cAMP / IBMX solution was made 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.

[0143] Dexamethasone: Dexamethasone was reconstituted at a concentration of 1 mM in 63.7 mL of molecular biology grade ethanol. The solution was stored at -20°C for up to 2 years. A 100 μM dexamethasone solution was prepared by mixing 500 μL of 1 mM dexamethasone with 4.5 mL of molecular biology grade ethanol. The solution was dispensed in 100 μL aliquots and stored at -20°C for up to 1 year.

[0144] Y-27632: Y-27632 was reconstituted in PBS at a concentration of 10 mM. The solution was dispensed and stored at -20°C for up to 6 months.

[0145] CK+DCI+F Medium: cSFDM medium was supplemented with 3 μM CHIR99021 (C), 10 ng / mL KGF (K), 10 ng / mL FGF-10 (F), 50 nM dexamethasone (D), and 1x cAMP / IBMX (CI). On the day of cell passage or seeding, the medium was supplemented with 10 μM Y-27632.

[0146] Preparation of Matrigel® Plate Coating Matrigel® was thawed overnight on ice and dispensed into 1.5 mL microtubes according to the dilution factor specified by the manufacturer. The aliquots were stored at -20°C. On the day of cell seeding, Matrigel® was diluted in cold DMEM / F-12 medium according to the dilution factor 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.

[0147] Cell Recovery for Passage and Flow Cytometry QC The medium was removed from the 6-well plate, and the wells were washed with 2 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 15 - 20 minutes or until the cells detached easily. The harvested cells were diluted with an equal volume of DMEM / F12 medium supplemented with 0.25% BSA. The cells were pipetted with a p1000 pipettor to disperse the cell clumps into single cells.

[0148] Next, the cells were filtered through a 30 μm filter to remove debris and centrifuged at 200 g for 5 minutes. The medium was aspirated, and approximately 2 - 3 mL of CK+DCI+F medium containing 10 μM Y-27632 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). Additional CK+DCI+F medium containing 10 μM Y-27632 was added to achieve a cell concentration of approximately 1 - 2 million cells / mL.

[0149] 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 supplemented with 10% fetal bovine serum (FBS) to a concentration of approximately 1 million cells / mL. The cells were seeded at approximately 208,000 cells / cm 2 onto a 6-well plate coated with Matrigel®.

[0150] After incubation, the medium was aspirated, and 2 mL of CK+DCI+F medium 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 for 7 - 10 days.

[0151] 3. Sort by EpCAM+ / GFP by FACS and passage in 3D Next, the cells were sorted by fluorescence-activated cell sorting (FACS) based on the expression of EpCAM / GFP or EpCAM / CPM. The sorting medium was prepared by supplementing cSFDM with 10 ng / mL KGF (K), 10 ng / mL FGF-10 (F), 50 nM dexamethasone (D), 1x cAMP / IBMX (CI), and 10 μM Y-27632 (Y) (FK+DCI+Y medium).

[0152] The medium was removed from the seeded plates above, and 2 mL of PBS was added to each well. The PBS was removed, and 1.5 mL of Accutase™ was added to each well. The plate was incubated at 37 °C for approximately 15 - 20 minutes or until the cells detached easily. The harvested cells were diluted with an equal volume of DMEM / F12 medium supplemented with 0.25% BSA. The cells were pipetted with a p1000 pipettor to disperse the cell clumps into single cells. The cells were filtered through a 30 μM filter to remove debris and centrifuged at 200 g for 5 minutes. The medium was removed, and DMEM / F12 medium containing BSA or LPC sorting buffer was added. The cells were counted with a Moxi™ cell counter (ORFLOW® technology). Approximately 250,000 - 300,000 cells were sorted for the flow cytometry QC assay. The remaining cells were centrifuged and resuspended in DMEM / F12 medium supplemented with 10% FBS to a concentration of approximately 1 million cells / mL.

[0153] Subsequently, cells were sorted based on GFP expression using a Sony Biotechnology (MA900) cell sorter. BU-NGST iPSCs (CL00366) and BU-NGST LPCs were used as control cells. The cells were centrifuged and resuspended in 1:750 Ghost Dye™ Red780 / PBS and incubated in the dark for 10 minutes. Subsequently, an equal volume of DMEM / F12 supplemented with 10% FBS was added to neutralize the Ghost Dye™. The cells were centrifuged at 200 g for 5 minutes at 4°C and resuspended in LPC FACS Sort Buffer. Subsequently, LPCs were sorted based on single cell gates (FSC-H vs FSC-w and SSC-H vs SSC-W) for live cells (low Ghost Dye™ Red780) in the APC-Cy7 channel; GFP+ and purity or semi-purity masks for the desired number of cells.

[0154] 4. Passage of Enriched LPCs into 3D Matrigel® Culture After sorting and collecting the LPCs, the cells were centrifuged at 200 g for 5 minutes at 4°C. The medium was aspirated from the cell pellet. Undiluted Matrigel® Growth Factor Reduced (GFR) basement membrane matrix or Matrigel® for Organoid Culture (Corning®) was added to obtain a cell density of approximately 350 - 400 cells / μL, and the cells were resuspended. Approximately 45 - 50 μL of the suspension was added to each well of a 12-well plate. The suspension was allowed to solidify at 37°C for at least 20 minutes. After the suspension had completely polymerized, 1 mL of pre-warmed FK+DCI+Y medium was added to each well.

[0155] The cells began to form epithelial spheroids within several days to one week after culturing. The spheroids were grown for 10 - 12 days until they reached a size of approximately 100 - 150 μm.

[0156] 5. 3D Culture, Medium Exchange Cycle Protocol for CHIR99021 (CHIR) The cells were maintained in FK-DCI+Y medium for 48 hours. Then, CHIR99021 was added back to the medium and Y-27632 was removed (FK-DCI+C medium). After 48 hours in this medium, an incubation cycle of 4 days in medium without CHIR99021 was performed, followed by incubation for 4 days in medium containing CHIR99021.

[0157] Specifically, the medium was carefully aspirated from each well of a 12-well plate, and approximately 1 - 1.2 mL of medium (regardless of the presence or absence of CHIR99021) was added to each well. The plate was incubated at 37 °C for 48 hours. This medium exchange was initiated using the "CHIR cycling" method described above, with 2 days off (without CHIR99021) and 2 days on (with CHIR99021), followed by repeating 3 - 4 days off and 3 - 4 days on until passage, and repeated every other day.

[0158] 6. Recovery of AT2 from 3D Matrigel® culture for passage and QC (approx. day 40) The following reagents were used:

[0159] Dispase II (Thermo Fisher Scientific; catalog number 17105041). Dispase II was made into 2 mg / mL DMEM medium. The medium was sterile filtered and stored at 4 °C for a maximum of 2 weeks, or aliquoted and frozen at -20 °C for a maximum of 2 - 3 months.

[0160] TrypLE™ Express (Thermo Fisher Scientific; catalog number 12604013)

[0161] Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free (Corning®; catalog number 354230)

[0162] Matrigel (registered trademark) for Organoid Culture (Corning (registered trademark) #356255)

[0163] 7. 3D Matrigel (registered trademark) Culture Recovery Protocol The medium was aspirated from the plate and 1 mL of 2 mg / mL Dispase II was added. The plate was incubated at 37 °C for about 1 hour until the Matrigel (registered trademark) was completely dissolved. The Matrigel (registered trademark) pellet was gently transferred to the incubation and gently pipetted 3 - 5 times every 10 minutes to promote dispersion. The cells were transferred to a new 15 mL conical tube and centrifuged at 200 g at 8 °C for 2 - 3 minutes. The supernatant was aspirated and an appropriate amount of TrypLE (trademark) Express was added to resuspend all the cells / spheroids. The cells were incubated at 37 °C for about 15 minutes and pipetted occasionally every 5 minutes. Then, a 1:1 volume of DMEM / F12 + 0.25% BSA was added to dilute and inactivate Tryp (trademark) LE. The cells were filtered through a 30 μM filter.

[0164] Subsequently, the cells were counted using a Moxi (trademark) cell counter (ORFLOW (registered trademark) Technology). Approximately 250,000 - 300,000 cells were recovered for FACS QC. The cells were centrifuged and resuspended in 1M / m DMEM / F12 + 10% FBS. The cells were passaged for downstream AT2 analysis by flow cytometry. For the remaining cells, the following sorting protocol was used. At the first passage where SFPTC+ cells occupy more than 15% of GFP+ cells, sort into 3D culture with TDT.

[0165] 8. Sorting Protocol for AT2 (SFPTC+) Organoids TDT+-based organoids were sorted using a Sony Biotechnology (MA900) cell sorter. The sorter was calibrated and initialized according to the manufacturer's standard operating protocol. The following control cells were thawed and prepared: BU-NGST iPSC (CL00366) or BU-NGST LPC for GFP control.

[0166] Cells were centrifuged, resuspended in 1:750 Ghost Dye™ Red780 / PBS, and incubated in the dark for 10 minutes. Subsequently, an equal volume of DMEM / F12 + 10% FBS was added to neutralize the Ghost Dye™. Cells were centrifuged and resuspended in LPC FACS Sort Buffer. LPCs were sorted into LPC FACS Sort Buffer based on the following criteria: 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), GFP+ / TDT+, and purity or semi-purity masks for the desired number of cells.

[0167] Cells were seeded at a cell density of 350 - 400 cells / μL in 50 μL of Matrigel® GFR or Matrigel® for Organoid Culture for further 3D culture.

[0168] 9. Culture and Subculture of AT2 Organoids After sorting with TDT+ and performing the second subculture, the TDT+ organoid population exceeded 50%. Cells were subcultured in 3D culture while continuing the cycle of CHIR99021 (CHIR cycling) as described above.

[0169] The expression of the SFTPC reporter was evaluated during differentiation. Figure 1 shows the results of flow cytometry on days 17, 34, 45, 59, 72, and 84. The differentiated cells had strong expression of the SFTPC reporter, indicating activation of the promoter. The cells needed to continue cycling of CHIR99021 to enhance the AT2 program and acquire stability of Nkx2.1 and SFTPC expression.

[0170] The expression of AT2-related genes during the differentiation protocol was also evaluated compared to the control of primary cultured AT2 cells. The results are shown in Figures 2A - 2B for the SFTPC gene (Figure 2A) and the LPCAT1 gene (Figure 2B). The differentiated cells strongly induced AT2-related genes over time.

[0171] The SFTPC protein was also detected by flow cytometry in the differentiated cells. The results of flow cytometry on day 94 of the differentiated cells (iPS-AT2) are shown in Figure 3 together with the results of the control primary cultured AT2 cells.

[0172] All publications, patents, and patent applications mentioned in this application are hereby incorporated 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 herein. 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

1. A method for differentiating lung progenitor cells (LPCs) into alveolar type 2 (AT2) cells, comprising the following steps: (i) culturing LPCs in a basal culture medium containing a glycogen synthase kinase 3 (GSK3) inhibitor, a keratinocyte growth factor (KGF), a fibroblast growth factor 10 (FGF10), and a γ-secretase inhibitor; (ii) subculturing the cells of (i) in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, dexamethasone, cyclic adenosine monophosphate (cAMP), an inhibitor of cyclic nucleotide phosphodiesterase, and a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor for about 24 to about 48 hours; (iii) culturing the cells of (ii) in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterase for about 7 days; (iv) separating the cells of (iii) having the expression of epithelial cell adhesion molecule (EpCAM) and / or carboxypeptidase M (CPM); (v) subculturing the cells of (iv) having the expression of EpCAM and / or CPM in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor; and (vi) separating the cells of (v) having the expression of surfactant protein C (SFTPC) to form AT2 cells.

2. The method according to claim 1, wherein the subculture of (ii) is performed in a two-dimensional (2D) matrix.

3. The method according to claim 2, wherein the 2D matrix is Matrigel®.

4. The method according to any one of claims 1 to 3, wherein the subculture of (v) is performed in a three-dimensional (3D) matrix.

5. The method according to claim 4, wherein the 3D matrix is Matrigel®.

6. A method for differentiating LPCs into AT2 cells, comprising the following steps: (i) culturing LPCs in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, and a γ-secretase inhibitor; (ii) separating the cells of (i) having the expression of EpCAM and / or CPM. (iii) culturing the cells of (ii) having EpCAM and / or CPM expression in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor for about 24 to about 48 hours; (iv) culturing the cells of (iii) in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterase, which does not contain a ROCK inhibitor or essentially does not contain a ROCK inhibitor, for about 7 days; and (v) separating the cells of (iv) having SFTP C expression to form AT2 cells, a method comprising. (Claim 7) (iii) The method according to claim 6, wherein the subculture of is performed in a 3D matrix. (Claim 8) (7) The method according to claim 7, wherein the 3D matrix is Matrigel (registered trademark). (Claim 9) (9) A method for differentiating LPCs into AT2 cells, comprising: (i) culturing LPCs in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, and a γ-secretase inhibitor; (ii) subculturing the cells of (i) in a basal culture medium containing a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor; (iii) separating the cells of (ii) having EpCAM and / or CPM expression; (iv) subculturing the cells of (iii) having EpCAM and / or CPM expression in a basal culture medium containing KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor; (v) culturing the cells of (iv) in a basal culture medium containing KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a GSK3 inhibitor, which does not contain a ROCK inhibitor or essentially does not contain a ROCK inhibitor; and (vi) separating the cells of (v) having SFTP C expression to form AT2 cells, a method comprising. (Claim 10) (16) The method according to claim 9, wherein the subculture of (iv) is about 2 to about 4 days and / or the culture of (v) is about 2 to about 4 days. (Claim 11) The method according to claim 9 or 10, wherein the subculture of (iv) is about 2 days and / or the culture of (v) is about 2 days. **Claim 12** The method according to any one of claims 9 to 11, wherein the subculture of (iv) and / or the culture of (v) is repeated before the isolation of (vi). **Claim 13** The method according to claim 12, wherein the repeated subculture of (iv) is about 4 days and / or the repeated culture of (v) is about 4 days. **Claim 14** The method according to any one of claims 9 to 13, wherein the subculture of (ii) is carried out in a 2D matrix. **Claim 15** The method according to claim 14, wherein the 2D matrix is Matrigel (registered trademark). **Claim 16** The method according to any one of claims 9 to 15, wherein the subculture of (iv) is carried out in a 3D matrix. **Claim 17** The method according to claim 16, wherein the 3D matrix is Matrigel (registered trademark). **Claim 18** The method according to any one of claims 1 to 17, wherein the GSK3 inhibitor is CHIR99021. **Claim 19** The method according to claim 18, wherein CHIR99021 is present in the culture medium at a concentration of about 3 μM. **Claim 20** The method according to any one of claims 1 to 19, wherein FGF10 is present in the culture medium at a concentration of about 10 ng / mL. **Claim 21** The method according to any one of claims 1 to 20, wherein KGF is present in the culture medium at a concentration of about 10 ng / mL. **Claim 22** The method according to any one of claims 1 to 21, wherein the γ-secretase inhibitor is N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). **Claim 23** The method according to claim 22, wherein DAPT is present in the culture medium at a concentration of about 20 μM. **Claim 24** The method according to any one of claims 1 to 23, wherein dexamethasone is present in the culture medium at a concentration of about 50 nM. **Claim 25** The method according to any one of claims 1 to 24, wherein cAMP is present in the culture medium at a concentration of about 100 μM. **Claim 26** The method according to any one of claims 1 to 25, wherein the inhibitor of cyclic nucleotide phosphodiesterase is 3-isobutyl-1-methylxanthine (IBMX). **Claim 27** The method according to claim 26, wherein IBMX is present in the culture medium at a concentration of about 100 μM. **Claim 28** The method according to any one of claims 1 to 27, wherein the ROCK inhibitor is Y-27632.

29. The method according to claim 28, wherein Y-27632 is present in the culture medium at a concentration of about 10 μM.

30. AT2 cells produced by the method according to any one of claims 1 to 29.

31. An organoid comprising the AT2 cells according to claim 30.

32. A differentiation medium comprising a basal culture medium, a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor.

33. A differentiation medium comprising a basal culture medium, about 3 μM of CHIR99021, about 10 ng / mL of KGF, about 10 ng / mL of FGF10, about 50 nM of dexamethasone, about 100 μM of cAMP, about 100 μM of IBMX, and about 10 μM of Y-27632.

34. A differentiation medium comprising a basal culture medium, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterase, and a ROCK inhibitor.

35. A differentiation medium comprising a basal culture medium, about 10 ng / mL of KGF, about 10 ng / mL of FGF10, about 50 nM of dexamethasone, about 100 μM of cAMP, about 100 μM of IBMX, and about 10 μM of Y-27632.

Images