Method for differentiating epithelial or basal cells

Abstract

The present disclosure relates to methods of differentiating pluripotent stem cells or lung progenitor cells into epithelial or basal cells. The present disclosure also relates to epithelial or basal cells prepared by such methods, organoids comprising such epithelial or basal cells, and methods of use thereof. The present disclosure also relates to differential media for use therein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 390,454, filed on July 19, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present disclosure relates to methods for differentiating pluripotent stem cells or lung progenitor cells into epithelial or basal cells. The present disclosure also relates to epithelial or basal cells prepared by such methods, organoids comprising such epithelial or basal cells, and methods of using the same. The present disclosure also relates to differentiation medium for use in the above. Background Art

[0003] Pluripotent stem cells (PSCs) are undifferentiated or partially differentiated cells that can differentiate into a variety of other cell types. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells derived from adult somatic cells that are genetically reprogrammed to an embryonic stem cell (ESC)-like state by expressing genes and factors that are important for maintaining the most typical characteristics of embryonic stem cells (ESCs). iPSCs have recently attracted attention in the medical community because they solve many of the obstacles associated with the use of embryonic stem cells and can generate patient-specific PSCs that can be genetically corrected, differentiated into adult cell lineages, and returned to the same patient as an autologous transplant. 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 being used for genetic diseases, iPSCs can also be used for tissue regeneration and disease modeling. Kogut et al., Methods Mol. Biol. 1195:1-12 (2014). Brief description of the invention

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

[0005] In some aspects, the method comprises: (i) culturing LPCs in a basal medium comprising fibroblast growth factor 2 (FGF2), fibroblast growth factor 10 (FGF10), dexamethasone, cyclic adenosine monophosphate (cAMP), a cyclic nucleotide phosphodiesterase inhibitor, and a Rho-associated coiled-coil protein kinase (ROCK) inhibitor; (ii) isolating cells in (i) that have no or low expression of nerve growth factor receptor (NGFR) to form basal progenitor cells; (iii) culturing cells in (ii) in a basal medium comprising FGF10, keratinocyte growth factor (KGF), a transforming growth factor beta type I receptor kinase (ALK5) inhibitor, an inhibitor of phosphorylation of maternal anti-limb paralysis homolog (SMAD), and a ROCK inhibitor; (iv) isolating cells in (iii) that have NGFR expression to form basal cells.

[0006] In some aspects, the method further comprises: (v) culturing the cells with NGFR expression described in (iv) in a basal medium comprising FGF10, KGF, an ALK15 inhibitor, an inhibitor of SMAD phosphorylation, and a ROCK inhibitor. In some aspects, the culturing in (v) is on a surface coated with laminin. In some aspects, the laminin is laminin-521.

[0007] In some aspects, the culture in (i) is in a three-dimensional (3D) matrix. In some aspects, the culture in (ii) is in a 3D matrix. In some aspects, the 3D matrix is

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

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

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

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

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

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

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

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

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

[0017] The present disclosure also provides epithelial cells prepared by the differentiation methods disclosed herein, basal cells prepared by the differentiation methods disclosed herein, organoids comprising the epithelial cells disclosed herein, organoids comprising the basal cells disclosed herein, and certain methods of use thereof.

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

[0019] Some aspects of the present invention are described herein by way of example only and with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is emphasized that the details shown are by way of example only and for the purpose of illustrative discussion of various aspects of the present invention.

[0020] Figure 1 Schematic diagram of the differentiation process described in Example 1.

[0021] Figure 2A is an exemplary whole-cell image of cells at 4X magnification on day 26 of the differentiation protocol described in Example 1.

[0022] Figure 2B-2C The flow cytometric analysis of cell surface markers EpCAM, NKX2-1 and p63 at day 26 of the differentiation process described in Example 1 is shown.

[0023] Figure 3A is an exemplary whole-cell image of cells at 4X magnification on day 33 of the differentiation protocol described in Example 1.

[0024] Figure 3B-3C The flow cytometric analysis of cell surface markers EpCAM, NKX2-1 and p63 at day 33 of the differentiation process described in Example 1 is shown.

[0025] Figure 4A is an exemplary whole-cell image of cells at 4X magnification at day 40 of the differentiation protocol described in Example 1.

[0026] Figure 4B-4E Demonstrates the use of the differentiation protocol described in Example 1 at day 40 to identify EpCAM + 、NKX2-1 + 、NKX2-1 - 、p63 + , CK5 + and NGFR + Flow cytometry gating strategy for cells.

[0027] Figure 4F is an exemplary whole-cell image of cells at 10X magnification at day 40 of the differentiation protocol described in Example 1.

[0028] Figure 4G The differentiation protocol described in Example 1 was demonstrated to be used to identify CD49f + , CK5 + ,CK8 + NGFR + and SOX2 + Flow cytometry gating strategy for cells.

[0029] Figure 5A is an exemplary whole-cell image of cells at 4X magnification at day 45 of the differentiation protocol described in Example 1.

[0030] Figure 5B-5E Demonstrates the use of the differentiation protocol described in Example 1 at day 45 to identify EpCAM + 、NKX2-1 + 、NKX2-1 - 、p63 + , CK5 + and NGFR + Flow cytometry gating strategy for cells.

[0031] Fig. 5F is an exemplary whole-cell image of cells at 10X magnification at day 45 of the differentiation protocol described in Example 1.

[0032] Figure 5G The differentiation protocol described in Example 1 was demonstrated to be used to identify CD49f + , CK5 + ,CK8 + NGFR + and SOX2 + Flow cytometry gating strategy for cells.

[0033] Fig. 6A Demonstrated the identification of EpCAM on day 44 before cryopreservation as described in Example 1 + 、NKX2-1 + 、p63 + , CK5 + and NGFR + Flow cytometry gating strategy for IPS-BC cells.

[0034] Figure 6B Demonstrated identification of EpCAM at day 47 as described in Example 1 + 、NKX2-1 + 、p63 + , CK5 + and NGFR + Flow cytometry gating strategy for control iPS-BC cells.

[0035] Figure 6C Demonstrated identification of EpCAM after thawing from cryopreservation as described in Example 1 + 、NKX2-1 + 、p63 + , CK5 + and NGFR + Flow cytometry gating strategy for IPS-BC cells. DETAILED DESCRIPTION OF THE INVENTION I. General Definitions

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present application (including definitions therein) shall prevail. Unless otherwise required by the context, terms in the singular shall include the plural form, and terms in the plural form shall include the singular form. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes, just as each individual publication or patent application is expressly and individually indicated to be incorporated by reference.

[0037] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. These materials, methods and examples are for illustrative purposes only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0038] In order to further clarify the present disclosure, the following terms and definitions are provided.

[0039] The singular forms "a" and "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein. In some aspects, the terms "a" or "an" mean "single". In other aspects, the terms "a" or "an" include "two or more" or "plurality".

[0040] The term "about" as used herein means approximately, roughly, approximately, or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values. Generally, the term "about" is used herein to modify a numerical value by floating it within a range of 10% (higher or lower) above or below a numerical value.

[0041] Throughout the disclosure of the present invention, various aspects of the present invention are presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity and should not be interpreted as an unchangeable limitation on the scope of the present invention. Therefore, the description of a range should be deemed to have specifically disclosed all possible sub-ranges and single values ​​within the range. For example, a description such as a range of 1 to 6 should be deemed to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and single numbers within the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the range, this rule applies. The listed numerical ranges include the numbers that define the range and cover each integer within the defined range.

[0042] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numbers that define the range. When a numerical range is listed, it should be understood that each intermediate integer value between the upper and lower limits listed in the range and each fractional value thereof, as well as each subrange between these values, is also specifically disclosed. The upper and lower limits of any range may be independently included in the range or excluded from the range, and any range that includes the upper limit, the lower limit, both or neither is also included in the disclosure of the present invention. Therefore, the ranges listed herein should be understood to be a shorthand representation of all numerical values ​​in the range, including the endpoint values ​​listed. For example, a range of 1 to 10 should be understood to include any number, combination of numbers or subrange in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0043] When a numerical value is explicitly listed, it should be understood that the numerical value of the quantity or amount approximately the same as the listed numerical value is also within the scope of the present disclosure. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. On the contrary, when different elements or groups of elements are disclosed separately, their combinations are also disclosed. When any element of the invention disclosure is disclosed as having multiple alternatives, examples of the invention disclosure in which each alternative is excluded individually or in any combination with other alternatives are also disclosed; more than one element of the invention disclosure can have such exclusions, and all combinations of elements with such exclusions are also disclosed herein.

[0044] As used herein, the term "and / or" should be considered as a specific disclosure of the presence or absence of the other of the two specified features or components. Thus, the term "and / or" as used in phrases such as "A and / or B" 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 cover the following: 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).

[0045] It should be understood that wherever herein aspects are described with the language "comprising," similar aspects described with "consisting of" and / or "consisting essentially of" are also provided. II. Differentiation Methods

[0046] The present disclosure relates to methods for differentiating pluripotent stem cells (PSCs) or lung progenitor cells (PLCs) into epithelial or basal cells. Such methods result in, for example, improved epithelial or basal cell viability, yield and / or differentiation properties.

[0047] As used herein, the term "differentiation" and "differentiating" refers 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 or basal cells). In general, differentiation of pluripotent stem cells can be achieved, for example, by changing the culture conditions of the cells (such as changing the stimulatory agent in the culture medium or the physical state of the cells).

[0048] As used herein, the terms "pluripotent stem cell," "pluripotent stem cells," and "PSC" refer to young or immature cells that can develop into more mature or specialized cells, such as epithelial or basal cells.

[0049] In some aspects, 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 aspects, PSCs are from humans. In some aspects, PSCs are from animals. In some aspects, the animals are sheep, pigs, or primates.

[0050] As used herein, the terms "induced pluripotent stem cell," "induced pluripotent stem cells" and "iPSC" refer to cells generated from differentiated adult, neonatal or fetal cells that have been induced or reprogrammed to become pluripotent stem cells.

[0051] As used herein, the term "lung progenitor cell" or "LPC" refers to a multipotent cell that can differentiate into a variety of cell types of the respiratory system, including but not limited to type I and II pneumocytes, alveolar interstitial cells, smooth muscle cells, alveolar epithelial cells, endothelial cells, and erythrocytes. Lung progenitor cells are committed to the lung lineage, retain the ability to self-renew, and can be identified by, for example, 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 aspects, LPCs according to the present disclosure do not express certain markers, or are negative or have low expression of certain markers, such as markers of endothelial cells (such as CD144, CD31), markers of hematopoietic cells (such as CD43, CD45, CD235a or CD41a) and / or markers of pluripotent stem cells (such as TRA1-60).

[0052] As used herein, the term "epithelial cell", "epithelial cells" or "epithelium" refers to a single cell or cell colony that lines the inner wall of a hollow organ, and constitutes a gland and the outer surface of the body. Epithelial cells may include squamous epithelial cells, columnar epithelial cells, adenomatous epithelial cells or transitional epithelial cells. Epithelial cells may be arranged in a single layer or may be arranged 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.

[0053] In some aspects, the cells in the differentiation methods provided herein express one or more markers (e.g., cell surface markers, mRNA, proteins, epigenetic features) or have the expression of one or more markers (e.g., cell surface markers, mRNA, proteins, epigenetic features), which are usually associated with epithelial cells (e.g., T1α, EpCAM, CD49f, and CD104). In some aspects, the cells express one or more markers (e.g., cell surface markers, mRNA, proteins, epigenetic features) or have the expression of one or more markers (e.g., cell surface markers, mRNA, proteins, epigenetic features), which are usually associated with basal epithelial cells. The basal epithelial cells are, for example, integrin α6 subunits (ITGA6), integrin β4 subunits (ITGB4), keratin 14 (KRT14), KRT15, ​​KRT5, and p63. In some aspects, the cell expresses or has expression of one or more markers (e.g., cell surface markers, mRNA, proteins, epigenetic features) that are typically associated with differentiated epithelial cells (e.g., KRT4, KRT6, and KRT8). In some aspects, the cell expresses or has expression of one or more markers (e.g., cell surface markers, mRNA, proteins, epigenetic features) that are typically associated with airway epithelial cells, such as Hes-related family basic helix-loop-helix transcription factor 2 (HEY2) containing a YRPW motif, nerve growth factor receptor (NGFR), and bone morphogenetic protein 7 (BMP7). In some aspects, the cells express or have expression of one or more of the following markers, such as cell surface markers, mRNAs, proteins, epigenetic features, such as cyclin-dependent kinase inhibitor 2B (CDKN2B), Cbp / P300-interacting transactivator 2 with a glutamate / aspartate-rich carboxyl-terminal domain (CITED2), cellular repressor of E1A-stimulated gene 1 (CREG1), inhibitor of DNA binding 1 (ID1), mitogen-activated protein kinase 6 (MAP2K6), insulin-like growth factor binding protein 3 (IGFBP3), and insulin-like growth factor binding protein 5 (IGFBP5).

[0054] In some aspects, the cells in the differentiation methods provided herein have no expression or low expression of one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic features) commonly 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 aspects, the cells have no expression or low expression of one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic features) commonly associated with pluripotent stem cells, such as Lin-28 homolog A (LIN28A), NANOG, POU5F1 / OCT4, and SRY-related HMG box 2 (SOX2). In some aspects, the cells have no expression or low expression of one or more markers (e.g., cell surface markers, mRNAs, proteins, epigenetic features) typically associated with terminally differentiated epithelial cells, such as cystic fibrosis transmembrane conductance regulator (CFTR), forkhead box J1 (FOXJ1), loricrin (IVL), keratin 1 (KRT1), KRT10, KRT20, LOR, mucin 1 (MUC1), MUC5AC, secretoglobulin family 1A member 1 (SCGB1A1), surfactant protein B (SFTPB), and surfactant protein D (SFTPD).

[0055] In some aspects, the cells in the differentiation methods provided herein have no or low expression of NGFR and are basal precursor cells. In some aspects, the cells in the differentiation methods provided herein have no or low expression of NGFR and are epithelial cells or basal cells.

[0056] As used herein, "basal cells" or "BCs" are epithelial progenitor cells that are present in the airway epithelium starting from the trachea and gradually decreasing in number until the respiratory bronchioles. They are responsible for normal cell replacement and epithelial remodeling when the lungs are injured. Basal cells can regenerate the airway epithelium by serving as precursors of important specialized epithelial cell types including secretory cells (SC) and multiciliated cells (MCC). Basal cells are usually the deepest cells of stratified epithelia and multilayered epithelia.

[0057] In some aspects, the basal cells according to the present disclosure have one or more markers (e.g., cell surface markers, mRNA, proteins, epigenetic features), including but not limited to CD49f, EpCAM, NGFR, NKX2-1, p63 and CK5. In some aspects, the basal cells according to the present disclosure do not express certain markers, 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).

[0058] Methods for generating lung progenitor cells (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.

[0059] In some aspects, the differentiation methods provided herein include certain cell culture conditions, such as culturing cells in certain culture media.

[0060] As used herein, the terms "cell culture", "cell culturing", "culture", "culturing" and "cultured" refer to the maintenance, growth and / or differentiation of cells in an in vitro environment. The terms "cell culture medium", "cell culture media", "culture medium" and "culture media" refer to a composition for culturing cells that contains nutrients that maintain cell viability, support proliferation and, optionally, differentiation. A cell culture medium may contain one or more of the following: one or more salts, one or more buffers, one or more amino acids, glucose or other sugars, one or more antibiotics, serum or serum substitutes, and other components such as growth factors, vitamins, and the like.

[0061] In some aspects, the differentiation methods provided herein refer to the cell culture medium as a "basal medium" (sometimes referred to as a "differentiation medium" or "differentiation medium") supplemented with other components. As used herein, "basal culture medium" or "basal culture medium" refers to a composition containing the most basic elements required 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's medium (DMEM), MEM, Iskoff's modified Dulbecco's medium (IMDM), Glasgow modified MEM (GMEM), DMEM / F12, Leibovitz L-15, RPMI-1640, CMRL, Ham F10, and Ham F12. In some aspects, the basal culture medium is supplemented with one or more other components, such as one or more amino acids, one or more antibiotics, serum, one or more growth factors. Such components are well known in the art and are further described herein.

[0062] In some aspects, the differentiation methods provided herein include certain cell culture conditions, such as passage of cells in certain culture media. As used herein, the terms "passage," "passaged," and "passaging" refer to the act of subdividing cells at lower concentrations and inoculating them onto one or more cell culture surfaces or containers when the cells have proliferated to a desired degree. Passaging generally involves mechanical or enzymatic methods (e.g., in vitro) prior to inoculation (optionally inoculating cells at a certain density). Methods for passaging cells are well known and further described herein.

[0063] In some aspects, the culture and passage in the differentiation method provided herein are carried out under the condition of coating one or more substrates on the cell culture surface or container. Such substrates include, but are not limited to, 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 matrix mixture produced by a naturally occurring cell line (such as Matrigel TM ) and synthetic or artificial surfaces (e.g., polyamine monolayers and carboxyl-terminated monolayers) or combinations thereof. In some aspects, the substrate is laminin-521. Methods for coating substrates onto cell culture surfaces or containers are well known and further described herein.

[0064] In some aspects, the culture and / or passage in the differentiation method provided herein is in two-dimensional (2D) culture. In such culture, cells are grown as a monolayer on a cell culture surface or container, and the cell culture surface or container optionally has a substrate coating as described above.

[0065] In some aspects, the culture and / or passage in the differentiation method provided herein is 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 a hydrogel, a porous three-dimensional scaffold, a rapid prototyping scaffold, a foam, a sponge, a net, a microparticle, a fibrous network, a matrix mixture produced by a naturally occurring cell line (such as Matrigel, TM ) and combinations thereof, such as microparticle-loaded hydrogels.

[0066] In some aspects, the differentiation methods provided herein include one or more steps of isolating cultured cells having certain biochemical characteristics. As used herein, the terms "separated" and "separating" refer to the process of isolating one or more specific cell populations from a heterogeneous mixture of cells.

[0067] In some aspects, the differentiation methods provided herein include isolating cells that do not express or underexpress NGFR to form basal precursor cells. As used herein, the term "no expression" of NGFR includes, but is not limited to, undetectable expression of NGFR. As used herein, the term "underexpression" of NGFR includes, but is not limited to, undetectable expression of NGFR, and NGFR expression is lower than NGFR expression in control cells that do not express NGFR and / or are not basal precursor cells.

[0068] In some aspects, the differentiation methods provided herein include isolating cells having NGFR expression to form epithelial or basal cells. As used herein, the term "expression" of NGFR includes, but is not limited to, NGFR expression comparable to mature epithelial or basal cells or their precursors or progenitor cells, and NGFR expression higher than NGFR expression in control cells that do not express NGFR and / or are not endothelial or basal cells.

[0069] Cell separation methods based on the expression of markers are well known in the art, including but not limited to affinity separation, fluorescence activated cell sorting (FACS), density gradient centrifugation, immunodensity cell separation, microfluidic cell sorting, buoyancy activated cell sorting, cell separation based on aptamers, complement depletion, etc. Affinity separation techniques include but are not limited to separation using antibody-coated magnetic beads (e.g., immunomagnetic cell separation), affinity chromatography, cytotoxic agents (e.g., complement and cytotoxins) connected to or used in conjunction with monoclonal antibodies, and "panning" or other convenient techniques using antibodies attached to solid matrices (e.g., culture plates). In some aspects, the cells of the differentiation method of the present invention are separated by immunomagnetic cell separation.

[0070] Some aspects of the differentiation method provided herein are included in culturing cells in a basal medium containing fibroblast growth factor 2 (FGF2). FGF2, also known as basic fibroblast growth factor or FGF-β, is a growth factor and signal transduction protein encoded by the FGF2 gene. It has a wide range of mitogenic and cell survival activities, and participates in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion.

[0071] In some respects, FGF2 exists with the concentration of about 100ng / mL to about 400ng / mL (or any numerical value or numerical range) in basal medium, and the concentration includes such as about 200ng / mL to about 400ng / mL, about 250ng / mL to about 400ng / mL, about 300ng / mL to about 400ng / mL, about 100ng / mL to about 300ng / mL, about 200ng / mL to about 300ng / mL, about 250ng / mL to about 300ng / mL, about 100ng / mL to about 250ng / mL, about 100ng / mL to about 250ng / mL or about 100ng / mL to about 200ng / mL. In some respects, FGF2 exists with the concentration of about 100ng / mL, about 200ng / mL, about 250ng / mL, about 300ng / mL or about 400ng / mL in basal medium. In some aspects, FGF2 is present in the basal medium at a concentration of about 250 ng / mL.

[0072] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing fibroblast growth factor 10 (FGF10). FGF10 is a paracrine signaling molecule that was first discovered during the development of limb buds and organogenesis. FGF10 turns on the development of limbs and is involved in the branching of morphogenesis in multiple organs (such as lungs, skin, ears, and salivary glands).

[0073] In some aspects, FGF10 is present in the basal medium in (i) at a concentration of about 25ng / mL to about 200ng / mL (or any numerical value or numerical range thereof), for example, including about 50ng / mL to about 200ng / mL, about 100ng / mL to about 200ng / mL, about 150ng / mL to about 200ng / mL, about 25ng / mL to about 150ng / mL, about 50ng / mL to about 150ng / mL, about 100ng / mL to about 150ng / mL, about 25ng / mL to about 100ng / mL, about 50ng / mL to about 100ng / mL or about 25ng / mL to about 50ng / mL. In some aspects, FGF10 is present in the basal medium in (i) at a concentration of about 25ng / mL, about 50ng / mL, about 100ng / mL, about 150ng / mL or about 200ng / mL. In some aspects, FGF10 is present in the basal medium in (i) at a concentration of about 100 ng / mL.

[0074] In some aspects, FGF10 is present in the basal medium in (iii) and / or (iv) at a concentration of about 5ng / mL to about 35ng / mL (or any numerical value or numerical range thereof), for example, including about 15ng / mL to about 35ng / mL, about 20ng / mL to about 35ng / mL, about 25ng / mL to about 35ng / mL, about 5ng / mL to about 25ng / mL, about 15ng / mL to about 25ng / mL, about 20ng / mL to about 25ng / mL, about 5ng / mL to about 20ng / mL, about 15ng / mL to about 20ng / mL or about 5ng / mL to about 15ng / mL. In some aspects, FGF10 is present in the basal medium in (iii) and / or (iv) at a concentration of about 5ng / mL, about 15ng / mL, about 20ng / mL, about 25ng / mL or about 35ng / mL. In some aspects, FGF10 is present in the basal medium in (iii) and / or (iv) at a concentration of about 20 ng / mL.

[0075] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing dexamethasone, an anti-inflammatory glucocorticoid that has a range of effects on cell proliferation and differentiation.

[0076] In some aspects, dexamethasone is present in a concentration of about 20nM to about 80nM (or any numerical value or numerical range thereof) in a basal medium, and the concentration, for example, includes about 40nM to about 80nM, about 50nM to about 80nM, about 60nM to about 80nM, about 20nM to about 60nM, about 40nM to about 60nM, about 50nM to about 60nM, about 20nM to about 50nM, about 40nM to about 50nM or about 20nM to about 40nM. In some aspects, dexamethasone is present in a concentration of about 20nM, about 40nM, about 50nM, about 60nM or about 80nM in a basal medium. In some aspects, dexamethasone is present in a concentration of about 50nM in a basal medium.

[0077] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing cyclic adenosine monophosphate (cAMP). cAMP helps mobilize glucose and fatty acid stores and is involved in the function and differentiation of many different cell types.

[0078] In some aspects, cAMP is present in a concentration of about 25 μM to about 200 μM (or any numerical value or numerical range thereof) in a basal medium, and the concentration, for example, includes 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, cAMP is present in a concentration of about 25 μM, 50 μM, 100 μM, 150 μM or 200 μM in a basal medium. In some aspects, cAMP is present in a concentration of about 100 μM in a basal medium.

[0079] Some aspects of the differentiation method provided herein are included in culturing cells in a basal medium containing a cyclic nucleotide phosphodiesterase inhibitor. Cyclic nucleotide phosphodiesterase is an enzyme family that can hydrolyze the phosphodiester bond in cyclic adenosine monophosphate and cyclic guanosine monophosphate to inhibit their pulmonary vasodilation properties. In some aspects, the cyclic nucleotide phosphodiesterase inhibitor is theophylline, 3-isobutyl-1-methylxanthine (IBMX) or Ro20-1724. In some aspects, the cyclic nucleotide phosphodiesterase inhibitor is IBMX.

[0080] In some aspects, cyclic nucleotide phosphodiesterase inhibitors (e.g., IBMX) are present in a concentration of about 25 μM to about 200 μM (or any value or range thereof) in a basal medium, for example, including 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, cyclic nucleotide phosphodiesterase inhibitors (e.g., IBMX) are present in a concentration of about 25 μM, 50 μM, 100 μM, 150 μM or 200 μM in a basal medium. In some aspects, cyclic nucleotide phosphodiesterase inhibitors (e.g., IBMX) are present in a concentration of about 100 μM in a basal medium.

[0081] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing a Rho-associated kinase (ROCK) inhibitor. ROCK is a serine / threonine kinase that functions as a downstream effector of Rho kinase, wherein Rho kinase has three subtypes (RhoA, RhoB, and RhoC). A "ROCK inhibitor" can, for example, reduce ROCK expression and / or ROCK activity. 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, dominant negative ROCK variants, siRNA, shRNA, miRNA, and antisense nucleic acids targeting ROCK. Other examples of ROCK inhibitors include, but are not limited to, thiazovivin, Y-27632, Fasudil, AR122-86, Y-30141, WF-536, HA-1077, hydroxy-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)-cyclohexanecarboxylic acid amide, and ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated herein by reference in its entirety. In some aspects, the ROCK inhibitor is Y-27632.

[0082] In some aspects, ROCK inhibitor (e.g., Y-27632) is present in a concentration of about 1 μM to about 20 μM (or any numerical value or numerical range thereof) in a basal medium, and the concentration includes, for example, 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 aspects, the ROCK inhibitor (e.g., Y-27632) is present in a concentration of about 1 μM, about 5 μM, about 10 μM, about 15 μM or about 20 μM in a basal medium. In some aspects, the ROCK inhibitor (e.g., Y-27632) is present in a concentration of about 10 μM in a basal medium.

[0083] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing keratinocyte growth factor (KGF). KGF is a member of the heparin-binding fibroblast growth factor family with a unique target cell specificity pattern. KGF has mitogenic activity on epithelial cells and is produced by cells of mesenchymal origin.

[0084] In some aspects, KGF is present in a concentration of about 5ng / mL to about 35ng / mL (or any numerical value or numerical range thereof) in a basal medium, for example, including about 15ng / mL to about 35ng / mL, about 20ng / mL to about 35ng / mL, about 25ng / mL to about 35ng / mL, about 5ng / mL to about 25ng / mL, about 15ng / mL to about 25ng / mL, about 20ng / mL to about 25ng / mL, about 5ng / mL to about 20ng / mL, about 15ng / mL to about 20ng / mL, or about 5ng / mL to about 15ng / mL. In some aspects, KGF is present in a concentration of about 5ng / mL, about 15ng / mL, about 20ng / mL, about 25ng / mL, or about 35ng / mL in a basal medium. In some aspects, KGF is present in a concentration of about 20ng / mL in a basal medium.

[0085] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing a transforming growth factor-β receptor I kinase (ALK5) inhibitor. ALK5 is a serine / threonine protein kinase that forms a heterologous complex with the type II TGF-β receptor when bound to TGF-β, transducing the TGF-β signal from the cell surface to the cytoplasm. This protein is involved in the regulation of numerous physiological and pathological processes, including cell cycle arrest of 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, such as inhibiting or reducing the transduction of TGF-β signals from the cell surface to the cytoplasm.

[0086] Examples of ALK5 inhibitors include, but are not limited to, A83-01 (Xcess Biosciences (San Diego, Calif.)), ALK5 inhibitor II (Enzo, Farmingdale, NY), 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-βRⅠ kinase inhibitor V (EMD Chemicals, Gibstown, NJ), TGF-β receptor inhibitor I (EMD Chemicals, Gibstown, NJ), transforming growth factor-β receptor inhibitor IV (EMD Chemicals, Gibstown, NJ), TGF-β receptor inhibitor VII (EMD Chemicals, Gibstown, NJ), TGF-β receptor inhibitor VIII (EMD Chemicals, Gibstown, NJ), TGF-β receptor inhibitor II (EMD Chemicals, Gibstown, NJ), TGF-β RI kinase inhibitor IV (EMD Chemicals, Gibstown, NJ), and TGF-β receptor inhibitor III (EMD Chemicals, Gibstown, NJ). In some aspects, the ALK5 inhibitor is A83-01.

[0087] In some aspects, the ALK5 inhibitor (e.g., A83-01) is present in a basal medium at a concentration of about 0.2 μM to about 2 μM (or any value or range thereof), for example, 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 aspects, the ALK5 inhibitor (e.g., A83-01) is present in a basal 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 aspects, the ALK5 inhibitor (e.g., A83-01) is present in a basal medium at a concentration of about 1 μM.

[0088] Some aspects of the differentiation methods provided herein include culturing cells in a basal medium containing an inhibitor that inhibits the phosphorylation of maternal anti-limb paralysis protein (SMAD). SMAD proteins (receptor-regulated or R-SMAD (SMAD1, 2, 3, 5 and 8) and SMAD4) form a heterologous complex that acts as a transcription factor after entering the nucleus, including inducing the expression of a series of proteins. The phosphorylation of SMAD has a significant impact on the structural and functional characteristics 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, Lerdelimumab, Metelimumab, GC-I008, AP-12009, AP-110I4, L Y550410, 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, dolsomorphin dihydrochloride, and derivatives and / or variants thereof. In some aspects, the inhibitor of SMAD phosphorylation is DMH-1.

[0089] In some aspects, the inhibitor of SMAD phosphorylation (e.g., DMH-1) is present in a concentration of about 0.1 μM to about 1.5 μM (or any numerical value or numerical range thereof) in a basal medium, for example, including 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 aspects, the inhibitor of SMAD phosphorylation (e.g., DMH-1) is present in a concentration of about 0.1 μM, about 0.2 μM, about 0.5 μM, about 1 μM or about 1.5 μM in a basal medium. In some aspects, an inhibitor of SMAD phosphorylation (eg, DMH-1) is present in the basal medium at a concentration of about 0.5 μM.

[0090] In some aspects, the differentiation methods provided herein comprise:

[0091] (i) culturing LPCs in a basal medium containing FGF2, FGF10, dexamethasone, cAMP, a cyclic nucleotide phosphodiesterase inhibitor, and a ROCK inhibitor;

[0092] (ii) optionally isolating cells with no or low NGFR expression in (i) to form basal precursor cells;

[0093] (iii) culturing the cells in (ii) in a basal medium comprising FGF10, KGF, an ALK5 inhibitor, an inhibitor of SMAD phosphorylation, and a ROCK inhibitor;

[0094] (iv) optionally isolating the cells having NGFR expression in (iii) to form epithelial or basal cells.

[0095] In some aspects, the differentiation methods provided herein comprise:

[0096] (i) culturing LPCs in a basal medium containing FGF2, FGF10, dexamethasone, cAMP, a cyclic nucleotide phosphodiesterase inhibitor, and a ROCK inhibitor;

[0097] (ii) isolating cells with no or low NGFR expression in (i) to form basal progenitor cells;

[0098] (iii) culturing the cells in (ii) in a basal medium comprising FGF10, KGF, an ALK5 inhibitor, an inhibitor of SMAD phosphorylation, and a ROCK inhibitor;

[0099] (iv) isolating the cells expressing NGFR in (iii) to form epithelial or basal cells.

[0100] In some aspects, the method further comprises:

[0101] (v) culturing the cells expressing NGFR in (iii) in a basal medium comprising FGF10, KGF, an ALK5 inhibitor, an inhibitor of SMAD phosphorylation, and a ROCK inhibitor.

[0102] In some respects, the culture in (v) is on the surface of coating laminin. In some respects, 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 its combination. In some respects, the laminin is laminin-521.

[0103] In some aspects, the culture in (i) and / or (iii) is in a 3D matrix. In some aspects, the 3D matrix is III. Other aspects

[0104] The present disclosure also relates to epithelial or basal cells prepared by any of the differentiation methods disclosed herein.

[0105] The present disclosure also relates to an organoid comprising epithelial or basal cells prepared by any differentiation method disclosed herein. As used herein, the term "organoid" refers to a differentiated or partially differentiated 3D cell structure derived from PSC (e.g., iPSC) or LPC, which is self-organized by dense aggregation of cells in a controlled space. Such structures can be constructed to replicate most of the complexity of an organ, or to express specific aspects thereof, for example, to produce only certain types of cells.

[0106] Methods for maintaining differentiated epithelial or basal cells and organoids are well known and include culturing the cells or organoids in a cell culture medium as described herein and / or cryopreservation. Methods for preparing organoids generally include culturing cells in a 3D matrix. Examples of 3D matrices include, but are not limited to, polymers (natural or synthetic), ceramics, composite materials, 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, a microparticle, a fibrous network, a matrix mixture produced by a naturally occurring cell line (e.g., Matrigel). TM ) and combinations thereof, such as microparticle-loaded hydrogels.

[0107] The disclosure also relates to certain methods of using such epithelial and basal cells.In some aspects, the disclosure provides a method of treating a lung disease comprising administering epithelial cells or basal cells or organoids prepared by any of the differentiation methods disclosed herein.

[0108] The present disclosure also relates to a differentiation medium for differentiating epithelial cells or basal cells from PSCs or LPCs.

[0109] In some aspects, the differentiation medium comprises basal medium, FGF2, FGF10, dexamethasone, cAMP, cyclic nucleotide phosphodiesterase inhibitor (e.g., IBMX) and ROCK inhibitor (e.g., Y-27632). In some aspects, the differentiation medium comprises about 100ng / mL to about 400ng / mL FGF2, about 25ng / mL to about 200ng / mL FGF10, about 20nM to about 80nM dexamethasone, about 25μM to about 200μM cAMP, about 25μM to about 200μM cyclic nucleotide phosphodiesterase inhibitor (e.g., IBMX) and about 1μM to about 20μM ROCK inhibitor (e.g., Y-27632). In some aspects, the differentiation medium comprises about 250 ng / mL FGF2, about 100 ng / mL FGF10, about 50 nM dexamethasone, about 100 μM cAMP, about 100 μM cyclic nucleotide phosphodiesterase inhibitor (eg, IBMX), and about 10 μM ROCK inhibitor (eg, Y-27632).

[0110] In some aspects, the differentiation medium comprises a basal 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 aspects, the differentiation medium comprises a basal medium, about 5ng / mL to about 35ng / mL FGF10, about 5ng / mL to about 35ng / mL KGF, about 0.2μM to about 2μM ALK5 inhibitor (e.g., A83-01), about 0.1μM to about 1.5μM SMAD phosphorylation inhibitor (e.g., DMH-1) and about 1μM to about 20μM ROCK inhibitor (e.g., Y-27632). In some aspects, the differentiation medium comprises basal medium, about 20 ng / mL FGF10, about 20 ng / mL KGF, about 1 μM ALK5 inhibitor (e.g., A83-01), about 0.5 μM inhibitor of SMAD phosphorylation (e.g., DMH-1), and about 10 μM ROCK inhibitor (e.g., Y-27632). Example

[0111] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion. Example 1 Differentiation of iPSCs into LPCs Materials and methods

[0112] The following reagents and materials were used: Iscove's Modified Dulbecco's Medium (IMDM) (Thermo Fisher Scientific; cat#12440053) Ham's F-12 medium supplemented with L-glutamine ( cat#10-080-CV) GlutaMAX TM Supplements (Thermo Fisher Scientific; cat# 35050061) B-27 Supplement (50X) (Thermo Fisher Scientific; cat#17504044) Bovine serum albumin (BSA); 7.5% in Dulbecco's phosphate buffered saline (DPBS) (Sigma Aldrich; cat# A8412-100ML) N-2 supplement (Thermo Fisher Scientific; cat#17502048) L-Ascorbic acid (Sigma-Aldrich; cat#A4544-25G) 1-Methylthioglycerol (MTG) (Sigma-Aldrich; cat#M1753-100ML) (InvivoGen; cat#ant-pm-1) IBMX (3-isobutyl-1-methylxanthine, Sigma-Aldrich; cat# I5879) 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt (cAMP, Sigma-Aldrich; cat# B7880-100MG) SAGM TM Small Airway Epithelial Cell Growth Medium Kit (Lonza, cat#CC-3118) Dexamethasone powder (Sigma-Aldrich; cat# D4902-25 MG) Recombinant human fibroblast growth factor-2 (FGF-2) (Peprotech; cat# AF-100-18B) Recombinant human keratinocyte growth factor (KGF; FGF-7) (Peprotech; cat# AF-100-19-50UG) Recombinant human fibroblast growth factor-10 (FGF-10) (Peprotech; cat# AF-100-26-25UG) DAPT (γ-secretase inhibitor IX) (Millipore Sigma #565770–10MG) Dulbecco's Modified Eagle's Medium (DMEM) / F12 medium (Thermo Fisher Scientific; cat#11320033) Y-27632 (Rho-associated coiled-coil kinase (ROCK) inhibitor) (STEMCELL TM Technologies; cat#72304) Dispase II (Thermo Fisher Scientific; cat#17105041) TrypLE TM Express (Thermo Fisher Scientific; cat#12604013) DMEM, high glucose, GlutaMAX TM Supplement, pyruvate (Thermo Fisher Scientific; cat#10569010) PneumaCult TM Ex Plus(STEMCELL TM Technologies; cat#05040) Hydrocortisone stock solution (STEMCELL TM Technologies; cat#07926) GFR basement membrane matrix, without LDEV ( cat#354230) TGF-βRI Kinase Inhibitor IV (A-83-01) (Millipore Sigma; cat# 616454) BMP inhibitor II, DMH1 (Millipore Sigma; cat# 203646) EZSolution CHIR99021 (GSK-3 inhibitor) (BioVision; cat#1748-5) CellAdherE Laminin-521 (STEMCELL TM Technologies; cat# 200-0117) STEMdiff TM Definitive Endoderm Kit (STEMCELL TM Technologies; cat#05110)

[0113] The reagents were prepared as follows:

[0114] ascorbic acid : A 50 mg / mL ascorbic acid solution was prepared by dissolving 500 mg ascorbic acid in tissue culture grade water. The solution was sterile filtered and stored in 500 μL aliquots at -20°C for up to 6 months.

[0115] MTG : Prepare a 13 μL / mL MTG solution by adding 26 μL MTG to 2 mL IMDM immediately before preparing the complete serum-free differentiation medium described below.

[0116] Recombinant human FGF-2 (rhFGF-2) : rhFGF-2 was reconstituted with sterile water to a concentration of 1 mg / mL and diluted with 0.1% BSA / phosphate buffered saline (PBS) to a concentration of 250 μg / mL. The solution was stored at -80°C for up to 3 months.

[0117] Recombinant human KGF (rhKGF) : rhKGF was reconstituted with PBS to a concentration of 0.1 mg / mL and diluted with 0.1% BSA / PBS to a concentration of 20 μg / mL. This solution was stored at -80°C for up to 3 months.

[0118] Recombinant human FGF-10 (rhFGF-10) : rhFGF-10 was reconstituted with 5 mM sodium phosphate to a concentration of 0.1 mg / mL and diluted with 0.1% BSA / PBS to a concentration of 20 μg / mL. This solution was stored at -80°C for up to 3 months.

[0119] DAPT : DAPT was reconstituted in sterile dimethyl sulfoxide (DMSO) to a concentration of 20 mM. The solution was aliquoted and stored at -20°C for up to 3 months.

[0120] Y-27632 : Y-27632 was reconstituted in sterile PBS to a concentration of 10 mM.

[0121] IBMX : Reconstitute IBMX in 200% pure ethanol to a concentration of 0.1 M. Aliquot the solution and store at -20°C for up to 1 year.

[0122] 10X cAMP / IBMX : A 10X cAMP / IBMX solution was prepared by mixing 50 mL cSFDM, 21.5 mg cAMP, and 500 μL 0.1 M IBMX. The solution was sterile filtered with a 0.22 μM filter and stored at 4°C in the dark for up to 1 month.

[0123] Dexamethasone : Reconstitute dexamethasone to a concentration of 1 mM in 63.7 mL of molecular biology grade ethanol. Store this solution at -20°C for up to 2 years. Then make a 100 μM dexamethasone solution by mixing 500 μL of 1 mM dexamethasone with 4.5 mL of molecular biology grade ethanol. Aliquot this solution into 100 μL volumes and store at -20°C for up to 1 year.

[0124] 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 two weeks, or aliquoted and stored at -20°C for up to 6 months.

[0125] A83-01 : TGF-βRI kinase inhibitor IV (A-83-01) was reconstituted in an appropriate volume of DMSO to a concentration of 2 mM.

[0126] DMH1 : DMH1 was reconstituted in DMSO to a concentration of 2 mM.

[0127] Complete serum-free differentiation medium (cSFDM) : 375mL IMDM, 125mL Ham's F-12 medium, 5mL GlutaMAX TM supplement, 5 mL B-27 supplement, 3.3 mL 7.5% BSA, 2.5 mL N-2 supplement, 500 μL of 50 mg / mL ascorbic acid, 1.5 mL of 13 μL / mL MTG, and 500 μL Mix and sterile filter. Store the medium at 4°C protected from light for up to 1 month.

[0128] Airway Differentiation Medium (ADM) : 45 mL of cSFDM basal solution, 5 mL of 10XcAMP / 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.

[0129] Complete PneumaCult TM Ex Plus Medium :10mL PneumaCult TM Ex Plus50X supplement, 0.5mL hydrocortisone stock solution and 1mL Mix to 490 mL PneumaCult TM Ex Plus basal medium is sterile filtered.

[0130] 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, and 10 μM Y-27632 were mixed and sterile filtered.

[0131] Basal Cell Maturation Medium : Will completely PneumaCult TM Ex Plus medium, 1 μM A-83-01, 1 μM DMH1 and 10 μM Y-27632 were mixed and sterile filtered.

[0132] DS / SB medium (front-end medium) cSFDM medium was supplemented with 2 μM doxormorphine and 10 μM SB431542 and then sterile filtered.

[0133] CBRa medium (pulmonary progenitor cell-directed medium) : cSFDM medium, 3 μM CHIR99021 and 10 ng / mL BMP-4 were mixed and sterile filtered. On the day of medium change, retinoic acid (RA) was freshly added to prepare CBRa medium with a final concentration of 100 nM. The medium was stored at 4°C for up to 1 week.

[0134] LPC Isolation Buffer : CMF-HBSS, 2% FBS, 25 mM HEPES, 2 mM EDTA, and 10 μM Y-27632 were mixed and sterile filtered.

[0135] An experiment was performed to differentiate lung progenitor cells (LPCs) from human induced pluripotent stem cells (iPSCs) (BJRiPS IPSCs provided by JintangDu) using the following protocol and reagents. Coating Plate preparation

[0136] Thaw on ice overnight and aliquot into 1.5 mL microcentrifuge tubes according to the dilution factor provided by the manufacturer. Store the aliquots at -20°C. On the day of cell seeding, dilute with cold DMEM / F-12 medium according to the dilution factor provided by the manufacturer. Make enough volume to coat the wells / plates for cell seeding. Next, use 1.5 mL per well of Coat the wells of a 6-well plate with 1 mL of DMEM / F-12 solution and incubate at 37°C for at least 1 hour. Rinse the wells with 1 mL of DMEM / F-12 medium before adding cells. iPSC single cell seeding

[0137] BJRiP (iPSC) cultured in a 6-well plate was washed with PBS. 1 mL of The cells were detached and incubated at 37°C for 5-7 minutes. The cells were then gently resuspended and pelleted at 200xg for 5 minutes. The iPSC pellet was resuspended in 15 mL of mTeSR1 medium with 10 μM Y-27632 to form a single cell suspension using a P1000 pipette. The iPSC pellet was then centrifuged using a Moxi TM Cell Counter ( Technology Company) and seeded the coated cells at 2E6 cells per well. The volume per well was then increased to 3 mL with mTeSR1 medium containing 10 μM Y-27632. Definitive endoderm (DE) differentiation (Day 0)

[0138] DMEM / F12 and STEMDiff TM Definitive Endoderm Basal Medium was warmed to 37°C. Supplement MR and Supplement CJ were thawed on ice and placed in STEMdiff TM Dilute 1:100 in endoderm basal medium. Wash the iPSC wells twice with 1 mL warm DMEM / F12 and then wash with 2 mL STEMDiff containing supplements MR and CJ. TM The definitive endoderm basal medium was replaced and the cells were then incubated at 37°C for approximately 24 hours. Definitive endoderm (DE) differentiation (Day 1-2)

[0139] CMEM / F12 and STEMDiff TM Definitive Endoderm Basal Medium was pre-warmed to 37°C. Supplement CJ was thawed on ice and used in STEMdiff TM Dilute 1:100 in endoderm basal medium. Add about 2 mL of medium to each well and incubate the cells at 37°C for about 24 hours. Change the medium again on day 2 and continue culturing at 37°C. Harvesting cells for passaging and flow cytometry QC

[0140] Remove the culture medium from the 6-well plate and wash the wells twice with 1 mL of PBS. Remove the PBS and add 1.5 mL of Accutase to each well. TM Incubate the plate at 37°C for approximately 4-5 minutes or until the cells detach easily. Dilute the harvested cells with an equal volume of DMEM / F12 medium. Pipette the cells up and down with a P1000 pipette to separate cell clumps into single cells.

[0141] Then, filter the cells through a 30 μM filter to remove debris and spin at 200 g for 5 minutes. Aspirate the medium and add approximately 5 mL of FACS wash buffer or DMEM / F12. Pipette the cells up and down with a P1000 pipette to separate cell clumps into single cells and use Moxi TM Cell Counter ( Technology companies) are counted.

[0142] Approximately 250,000 to 300,000 cells were set aside for flow cytometry quality control (QC) assays. The remaining cells were spun down and resuspended in DMEM / F12 medium to a concentration of approximately 1 million cells per mL. The cells were plated at approximately 208,000 cells / cm 2 Inoculation to coating of 6-well plates. Anterior foregut endoderm (AFE) differentiation (Day 3)

[0143] 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 plates were incubated at 37°C for 24 hours. For the first 24-48 hours of cell seeding, Y-27632 (10 μM final concentration) was added to the medium. The medium was changed in the same manner every 24 hours until day 6.

[0144] Aspirate the spent medium from the cells and gently wash the cells twice with approximately 1 mL of DMEM / F12 medium. Add 2 mL of CBRa medium to the wells and incubate the cells at 37°C for 48 hours. Replace the medium again on days 8, 10, and 12. Example 2 Differentiation of LPCs into basal cells

[0145] The following reagents and materials were used: Fluorescein isothiocyanate (FITC) anti-human CD326 (Ep-CAM) (BioLegend; Cat# 324204) PE anti-human CD26 (BioLegend; cat#302706) BV421 anti-human CD47 (BioLegend; cat#323116) FITC Mouse IgG2b, κ Isotype Control (BioLegend; cat#400310) PE Mouse IgG2a, κ isotype control (BioLegend; cat#400212) BV421 Mouse IgG1, κ isotype control (BioLegend; cat#400158) GFR basement membrane matrix, without LDEV ( cat#354230) UltraComp eBeads TM Compensation beads (Thermo Fisher; cat#01-2222-41) Ghost Dye TM Red780 (Tonbo Biosciences; cat#13-0865-T100)

[0146] Prepare the following reagents:

[0147] Sorting Wash Buffer : CMF-PBS, 1% FBS, 1 mM EDTA and 25 mM HEPES were mixed and sterile filtered.

[0148] LPC Isolation Buffer : CMF-HBSS, 2% FBS, 2 mM EDTA, 25 mM HEPES, and 10 μM Y-27632 were mixed and sterile filtered. EPCAM / CD47 / CD26 surface staining :

[0149] Cells were harvested according to the protocol disclosed in Example 1. 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, and one drop of UltraComp eBeads TM Compensation beads for phycoerythrin (PE) single staining control, BV421 anti-human CD47 single staining control, Ghost Dye TM Red780 single staining control, and with Ghost Dye TM Isotype controls (FITC, PE and / or BV421) for Red780. Approximately 14E6 cells were stained with GhostDye TMRed780 was used to stain EpCAM, CD47, and CD26. After counting the cells, the cells were washed with PBS and centrifuged at 200 x g for 5 minutes at 8°C. Meanwhile, Ghost Dye was added to the cells with PBS. TM Red780 was diluted to 1:750-1:1000. Aspirate the supernatant and add 100 μL Ghost Dye TM Red780 and incubate at room temperature for 10 minutes. After incubation, wash the cells once with 1 mL of sorting wash buffer and then centrifuge at 200 x g for 5 minutes at 8°C.

[0150] To the appropriate samples, add the following and stain the cells for 30 min on ice or at 4 °C: 0.8 μL FITC anti-human CD-326 single stain control per 100 μL LPC sorting buffer (for up to 1E6 cells) 2.5 μL BV421 anti-human CD47 single stain control per 100 μL LPC sorting buffer (for up to 1E6 cells) 0.2 μL FITC mouse IgG2b, κ isotype control + 0.6 μL PE mouse IgG2a, κ isotype control + 1.2 μL BV421 mouse IgG1, κ isotype control per 100 μL LPC sorting buffer (for up to 1.5E6 cells) 2.5 μL BV421 anti-human CD47 + 0.8 μL FITC per 100 μL LPC sorting buffer Anti-human CD-326 + 2.5 μL PE anti-human CD26 (for up to 1.5E6 cells)

[0151] The supernatant was then aspirated and LPC sorting buffer was added to the sample to a final concentration of approximately 1E6-3E6 cells / mL. TM Before running cells at confluency, filter the cells using a flow cytometer with a 40 μM filter to remove clumps from the cell suspension. BD FACSAria TM Fusion for CD47hi / CD27-sorting :

[0152] The parameters used were as follows: FSC: 260; SSC: 270; AF488: 350; PE: 360; APC-Cy7: 440; BV421: 275; and FSC scaled area: 0.3.

[0153] Before running samples, run all compensation single color controls and calculate the compensation matrix. All isotype controls and LPC samples were run on a BD FACSAria with the appropriate compensation matrix. TMRun on fusion. Then prepare a collection tube with 5-6mL LPC sorting buffer and sort the cells based on single cell gating (FSC-H vs FSC-w and SSC-H vs SSC-W), live cells in the APC-Cy7 channel (Ghost Dye Red780 low), EpCAM+ in the AF488 channel, and CD47hi / CD26- in the PE / BV421 channel. For each sample, 480,000 cells per 12-well plate (3D) were also sorted using a purity mask. In 3D Re-plating cells

[0154] After the cells were collected, they were centrifuged at 200 x g for 5 min at 8°C. The culture medium was aspirated and undiluted GFR to a concentration of about 600 cells / μL. On ice, about 45-50 μL of droplets were placed and spread on the bottom of each well of a 12-well plate. The droplets were then 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 replaced on day 16 of differentiation. Differentiation Day 17-Day 24 (LPC to basal cell (BS) differentiation Day 1 to Day 9)

[0155] Aspirate the medium from each well and add 1 mL of ADM. Then incubate the cells at 37°C for 48-72 hours. Replace the medium every 24-72 hours until day 26 of differentiation. Differentiation day 26 (LPC to BS differentiation day 10)

[0156] Aspirate the medium, add 2 mg / mL dispase to cover the droplets (usually 1 mL / well), and then incubate at 37°C for 60 minutes until the cells are fully grown. Completely lyse. Gently pipette up and down the cells 3-5 times. Use a P1000 pipette to transfer the dissociated organoids to a new 15-mL conical tube and add an equal volume of DMEM. Centrifuge the spheres at 300xg for 2 minutes at 4°C. Aspirate the supernatant and add 1 mL of TrypLE TM . TrypLE TM Transfer the cells to the wells of a 12-well plate and incubate at 37°C for 10-12 minutes. Incubate the cells until they are completely dissociated. Add an equal volume of DMEM / F12 + 10% FBS to each well. Then collect the dissociated cells, filter using a 30 μM filter to remove debris, and place in a Moxi TM Cell Counter ( Approximately 100,000 cells were counted on a 3D An exemplary image of intact cells differentiated at day 26 is shown in Figure 2A The QC graphs of cell surface markers EpCAM, NKX2-1 and p63 on day 26 are shown in Figure 2B-2C On differentiation days 28 and 30, approximately 1 mL of medium was replaced for all wells, and cells were maintained at 37°C for 24-48 hours. From 3D Harvest AE2 from culture for NGFR sorting and QC (Day 31-40)

[0157] Cells were harvested according to the protocol described in Example 1 and sorted for NGFR using magnetic labeling and magnetic separation according to the manufacturer's protocol. Prior to seeding, laminin-521-coated plates were prepared by incubating 5 μg / mL of laminin-521 solution in each well of a 6-well plate at 37°C for 2 hours or at 4°C overnight. After sorting, NGFR-positive cells were plated at approximately 5,000 cells / cm 2 The cells were plated on laminin-521 plates at a density of 1.54 × 10 cells / mL and incubated at 37 °C for 24 h. An exemplary image of cells on day 33 of differentiation is shown in Figure 3A (4X magnification) and Figure 3F (10X magnification). QC diagrams of cell surface markers EpCAM, NKX2-1, and p63 on day 33 are shown in Figure 3B-3C .

[0158] The medium was replaced every 24 hours until approximately 95-100% confluence. An exemplary image of cells at day 40 of differentiation is shown in Figure 4A (4X magnification) and Figure 4F (10X magnification). QC images of cell surface markers EpCAM, NKX2-1, p63, CK5, and NGFR on day 40 are shown in Figure 4B-4E The QC diagrams of cell surface markers CD49f, p63, CK8, NGFR, and SOX2 on day 40 are shown in Figure 4G . Basal cell expansion (Day 41+)

[0159] Laminin-521 coated plates were prepared as described above. Spent media was removed from the wells and cells were washed once with PBS. Cells were harvested as described above and plated at approximately 5,000 cells / cm on laminin-521 coated plates using basal cell maturation media. 2 The cells were seeded at a cell density of 100 μg / mL. The cells were then incubated at 37°C for 24 hours. The medium was replaced every 24 hours until approximately 95-100% confluence. An exemplary image of cells on day 45 of differentiation is shown in Figure 5A (4X magnification) and Fig. 5F(10X magnification). QC images of cell surface markers EpCAM, NKX2-1, p63, CK5, and NGFR on day 45 are shown in Figure 5B-5E The QC diagrams of cell surface markers CD49f, p63, CK8, NGFR, and SOX2 on day 45 are shown in Figure 5G .

[0160] Fig. 6A Shown is the flow cytometry gating strategy to identify EpCAM+, NKX2-1+, p63+, CK5+, and NGFR+ iPS-BC cells on day 44 before cryopreservation. Figure 6B Flow cytometry gating strategy to identify EpCAM+, NKX2-1+, p63+, CK5+, and NGFR+ control iPS-BC cells at day 47 is shown. Figure 6C The flow cytometry gating strategy to identify EpCAM+, NKX2-1+, p63+, CK5+, and NGFR+ iPS-BC cells after cryopreservation thawing is shown.

[0161] All publications, patents and patent applications mentioned in this application are hereby incorporated by reference in their entirety into this specification to the same extent as if each publication, patent or patent application were individually and specifically indicated to be incorporated by reference into this application. In addition, the citation or identification of any reference in this application should not be construed as an admission that the reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

1. A method for differentiating lung progenitor cells (LPC) into epithelial or basal cells, comprising: (i) culturing LPCs in a basal medium containing fibroblast growth factor 2 (FGF2), fibroblast growth factor 10 (FGF10), dexamethasone, cyclic adenosine monophosphate (cAMP), a cyclic nucleotide phosphodiesterase inhibitor, and a Rho-associated coiled-coil kinase (ROCK) inhibitor; (ii) isolating cells that do not express or low express nerve growth factor receptor (NGFR) in (i) to form basal progenitor cells; (iii) culturing the cells in (ii) in a basal medium comprising FGF10, keratinocyte growth factor (KGF), a transforming growth factor beta type I receptor kinase (ALK5) inhibitor, an inhibitor of phosphorylation of maternal anti-limbic homolog (SMAD), and a ROCK inhibitor; and (iv) isolating the cells expressing NGFR in (iii) to form epithelial or basal cells.

2. The method of claim 1, further comprising: (v) culturing the cells expressing NGFR described in (iv) in a basal medium comprising FGF10, KGF, an ALK15 inhibitor, an inhibitor of SMAD phosphorylation, and a ROCK inhibitor.

3. The method of claim 2, wherein the culturing in (v) is on a laminin-coated surface. The method of claim 3 , wherein the laminin is laminin-521.

5. The method of any one of claims 1-4, wherein the culturing in (i) is in a three-dimensional (3D) matrix.

6. The method of any one of claims 1-5, wherein the culturing in (ii) is in a 3D matrix.

7. The method of claim 5 or 6, wherein the 3D matrix is 8. The method of any one of claims 1-7, wherein FGF2 is present in the culture medium at a concentration of about 250 ng / mL.

9. The method of any one of claims 1-8, wherein FGF10 is present in the culture medium in (i) at a concentration of about 100 ng / mL.

10. The method of any one of claims 1-9, wherein FGF10 is present in the culture medium in (ii) and (iv) at a concentration of about 20 ng / mL.

11. The method of any one of claims 1-10, wherein dexamethasone is present in the culture medium at a concentration of about 50 nM.

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

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

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

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

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

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

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

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

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

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

22. Endothelial cells prepared by the method of any one of claims 1-21.

23. Basal cells prepared by the method of any one of claims 1-21.

24. An organoid comprising the endothelial cells of claim 22.

25. An organoid comprising the basal cell of claim 23.

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

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

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