Raft cultures and methods for preparing them

Abstract

Disclosed herein are esophageal raft culture compositions that more closely resemble native organ structures. These esophageal raft cultures can also be innervated by combining enteric neural crest cells. These raft culture compositions are advantageous for purposes such as studying organelle function, development, and organization. Methods for producing such esophageal raft culture compositions are also disclosed herein.

Description

[Technical Field]

[0001] Description of federally funded research and development. This invention was developed with government support under P01 HD093363, awarded by the National Institutes of Health. The government has certain rights to this invention.

[0002] The aspects of this disclosure generally relate to esophageal raft culture compositions and methods for preparing such esophageal raft culture compositions. The raft cultures disclosed herein more closely approximate the structure of natural organs. [Background technology]

[0003] Three-dimensional (3D) cell cultures, such as organoids, are highly promising as models of biological function and development compared to conventional two-dimensional culture systems. These 3D cultures have the potential to more accurately reflect the characteristics of organs found in vivo for applications such as pharmacological behavior, cell signaling, cancer formation and migration, or transplantation / grafting. However, the in vitro formation of organ tissues that mimic the complex structures found in living organisms remains a relatively new field. The following are prior art documents related to the invention of this application (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries): (Prior art document) (Patent Document) (Patent Document 1) International Publication No. 2019 / 074793 (Patent Document 2) International Publication No. 2021 / 041443 (Non-patent literature) (Non-patent document 1) NAKAHARA et al. "Human papillomavirus type 16 E1circumflexE4 contributes to multiple facets of the papillomavirus life cycle," Journal of Virology, 31 October 2005 (31.10.2005), Vol.79,No.20,Pgs.13150-13165.entire document (Non-patent document 2) OHASHI et al, "Epidermal Growth Factor Receptor and Mutant p53 Expand an Esophageal Cellular Subpopulation Capable of Epithelial-to-Mesenchymal Transition through ZEB Transcription Factors," Cancer Research,27 April 2010 (27.04.2010),Vol.70,No.10,Pgs.4147-4184.entire document (Non-patent document 3) SHACHAM-SILVERBERG et al."Generation of esophageal organoids and organotypic raft cultures from human pluripotent stem cells," Methods of Cell Biology,13 May 2020 (13.05.2020),Vol.159,Pgs.1-23.entire document [Overview of the Initiative]

[0004] Currently, there is a need for, for example, more efficient, inexpensive, time-saving, and accurate 3D organ models and methods for producing them. There is also a need for culture preparations that selectively avoid heterogeneous components that may have significant safety and regulatory effects. Esophageal raft cultures and intermediate cell compositions thereof from differentiated anterior foregut cells are disclosed herein. In some embodiments, the intermediate cell composition comprises dorsal anterior foregut cells and / or esophageal progenitor cells. Methods for producing such esophageal raft cultures and intermediate cell compositions are also disclosed herein. In some embodiments, the method comprises differentiating anterior foregut cells into dorsal anterior foregut cells by contacting anterior foregut cells with one or more (e.g., at least one, two, three, or four) or any combination thereof from among EGF pathway activators, BMP pathway inhibitors, FGF pathway activators, or growth stimulants. In some embodiments, the method involves differentiating anterior foregut cells into dorsal anterior foregut cells by contacting them with one or more (e.g., at least one, two, three, or four) of an EGF pathway activator, a BMP pathway inhibitor, or an FGF pathway activator, optionally a neuronal precursor inhibitor, or any combination thereof. In some embodiments, the dorsal anterior foregut cells are then dissociated into single cells and cultured in a first tissue culture vessel to enlarge the dorsal anterior foregut cells and differentiate them into esophageal progenitor cells. In some embodiments, the enlarged esophageal progenitor cells are then dissociated into single cells and cultured in and / or on the surface of an insert member (e.g., a transwell or cell insert), in which case the insert member is placed in a second tissue culture vessel and the insert member includes a surface that is permeable to growth medium but impermeable to cells. In some embodiments, the insert member and the second tissue culture vessel each contain an amount of growth medium such that the esophageal progenitor cells are completely immersed in the growth medium. In some embodiments, esophageal progenitor cells are then cultured in an insert member, in which case the second tissue culture vessel and / or insert member contains an amount of growth medium such that the esophageal progenitor cells are only partially immersed in the growth medium to produce an esophageal raft culture. In some embodiments, the second tissue culture vessel is the same as the first tissue culture vessel.In some embodiments, the partially immersed esophageal progenitor cells or esophageal raft cultures are cultured at the air-liquid interface. In some embodiments, the anterior foregut cells differentiate from embryonic endoderm cells. In some embodiments, the anterior foregut cells or embryonic endoderm cells differentiate from induced pluripotent stem cells. In some embodiments, the induced pluripotent stem cells are human induced pluripotent stem cells.

[0005] In the methods and compositions disclosed herein, esophageal progenitor cells can also be mixed with enteric neural crest cells to prepare innervated esophageal raft cultures.

[0006] The embodiments of the disclosure provided herein are illustrated by the following numbered alternatives. Alternative 1. An in vitro esophageal raft culture comprising: a stratified squamous epithelial layer comprising a basal upper layer and a basal layer, a mesenchymal layer comprising muscle fibers, wherein the stratified squamous epithelium is E-cadherin + , + , , + , + , + , , , , + , + , + , + , + and the basal upper layer is KRT13 + and KRT8 + and the basal layer is SOX2 + , P63 + , and KRT5 + and the mesenchymal layer is FOXF1 + , NKX6-1 + , and vimentin + and the muscle fibers are desmin + An in vitro esophageal raft composition. Alternative 2. The esophageal raft culture according to Alternative 1, wherein the esophageal raft culture lacks a lamina propria or has a reduced lamina propria compared to esophageal tissue from an adult animal of the same species as the raft culture. Alternative 3. The esophageal raft culture according to Alternative 1 or 2, further comprising a growth medium such as DMEM / F12. Alternative 4. Further comprising a tissue culture container and an insert member, wherein the esophageal raft culture is disposed within the insert member and the insert member is disposed within the tissue culture container, An esophageal raft culture according to any one of Alternative Forms 1 to 3, wherein the insert component includes a surface that is permeable to the growth medium but not to cells. Alternative form 5. The esophageal raft culture according to Alternative Form 4, wherein the insert member is coated with an extracellular matrix or its components. Alternative form 6. Esophageal raft culture as described in Alternative form 5, wherein the extracellular matrix or its components are derived from humans. Alternative form 7. Esophageal raft culture according to alternative form 5 or 6, wherein the extracellular matrix or its components include human collagen type IV. Alternative form 8. Esophageal raft culture according to any one of Alternative Forms 5 to 7, wherein the extracellular matrix or its components do not contain rat collagen type I matrix or Matrigel. Alternative form 9. Esophageal raft culture according to any one of Alternative Forms 4 to 8, wherein the insert member and the tissue culture vessel each contain an amount of growth medium such that the esophageal raft culture is completely immersed in the growth medium. Alternative form 10. The esophageal raft culture according to Alternative Form 9, wherein the insert member further comprises an EGF pathway activator, a ROCK inhibitor, a SMAD inhibitor, or any combination thereof, and the tissue culture vessel comprises an EGF pathway activator. Alternative form 11. The insert member does not contain the growth medium, and the tissue culture vessel contains an amount of growth medium such that the esophageal raft culture is partially immersed in the growth medium. Esophageal raft cultures according to any one of Alternative Forms 4-8, wherein stratified squamous epithelium is partially immersed in or not immersed in the growth medium and located at the gas-liquid interface. Alternative form 12. Esophageal raft culture according to Alternative form 11, wherein the tissue culture vessel contains an EGF pathway activator. Alternative form 13. The insert member is 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or Esophageal raft cultures according to any one of Alternative Forms 4 to 12, having pore sizes of 10 μm, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or less, or any pore size within the range defined by any two of the aforementioned sizes. Alternative form 14. An esophageal raft culture according to any one of Alternative Forms 4 to 13, wherein the insert member has a pore size of 3 μm. Alternative form 15. In vitro cell culture, An in vitro cell culture comprising a population of esophageal progenitor cells derived from dorsal anterior foregut cells treated with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or a growth adjuvant (e.g., CultureOne adjuvant), or any combination thereof. Alternative form 16. The cell culture according to alternative form 15, further comprising a growth medium such as keratinocyte SFM or other serum-free medium. Alternative form 17. The cell culture according to Alternative form 16, wherein the growth medium contains an EGF pathway activator, bovine pituitary extract (BPE), or both. Alternative form 18. The EGF pathway activator is at a concentration of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations, or The cell culture according to Alternate Form 17, wherein the BPE is at a concentration of approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or at any concentration within the range defined by any two of the aforementioned concentrations, or both. Alternative form 19. A cell culture according to any one of Alternative Forms 15 to 18, further comprising a tissue culture vessel. Alternative form 20. The cell culture according to alternative form 19, wherein the tissue culture vessel is coated with an extracellular matrix or its components. Alternative form 21. A cell culture according to alternative form 20, wherein the extracellular matrix or its components are derived from humans. Alternative form 22. A cell culture according to alternative form 20 or 21, wherein the extracellular matrix or its components include human collagen type IV. Alternative form 23. A cell culture according to any one of Alternative Forms 20 to 22, wherein the extracellular matrix or its components do not include rat collage type I matrix or Matrigel. Alternative form 24. A cell culture according to any one of Alternative Forms 15 to 23, further comprising a ROCK inhibitor. Alternative form 25. In vitro cell culture, An in vitro cell composition comprising a population of anterior foregut cells treated with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or a growth stimulant, or any combination thereof. Alternative form 26. Cell culture of alternative form 25, further comprising a growth medium such as RPMI, and optionally containing FBS such as 0%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% FBS, or any percentage of FBS within the range defined by any two of the aforementioned percentages. Alternative form 27. The cell culture according to alternative form 25 or 26, further comprising a tissue culture vessel. Alternative form 28. Esophageal raft culture or cell culture according to any one of Alternative Forms 1 to 14 or any one of Alternative Forms 15 to 27, wherein the esophageal raft culture or cell culture has been grown for at least 1, 2, 3, 4, 5, 6, 7, or 8 days. Alternative form 29. The esophageal raft culture or cell culture according to Alternative form 28, wherein the esophageal raft culture or cell culture is derived from human induced pluripotent stem cells. Alternative form 30. The esophageal raft culture or cell culture according to alternative form 28 or 29, wherein the esophageal raft culture or cell culture is not derived from spheroids or organoids. Alternative form 31. A method for producing esophageal raft cultures, (a) Differentiating anterior foregut cells into dorsal anterior foregut cells by contacting them with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or a growth stimulant, or any combination thereof. (b) Dissociating the dorsal anterior foregut cells from step (a) into single cells, (c) Culturing dorsal anterior foregut cells in a first tissue culture vessel to differentiate them into esophageal progenitor cells, (d) Dissociating the esophageal progenitor cells from step (c) into single cells, (e) Culturing esophageal progenitor cells within the insert member, The insert member is placed inside the second tissue culture vessel. The insert component includes a surface that is permeable to the growth medium but not to cells. The insert member and the second tissue culture vessel each contain an amount of growth medium such that the esophageal progenitor cells are completely immersed in the growth medium, and the cells are cultured. (f) A method comprising culturing esophageal progenitor cells in an insert member, wherein the insert member does not contain growth medium, and a second tissue culture vessel contains an amount of growth medium such that the esophageal progenitor cells are partially immersed in the growth medium. Alternative form 32. The method according to alternative form 31, wherein esophageal progenitor cells are dissociated using a dissociative enzyme such as trypsin, chymotrypsin, collagenase, elastase, or Accutase. Alternative form 33. The method according to alternative form 31 or 32, wherein the first tissue culture vessel and / or the second tissue culture vessel are coated with an extracellular matrix or its components. Alternative form 34. The method according to alternative form 33, wherein the extracellular matrix or its components are derived from humans. Alternative form 35. The method according to alternative form 33 or 34, wherein the extracellular matrix or its components include human collagen type IV. Alternative form 36. The method according to any one of Alternative Forms 33 to 35, wherein the extracellular matrix or its components does not include rat collagen type I matrix or Matrigel. The method according to any one of the alternatives 31 to 36, wherein the contact step of alternative 37(a) is performed for at least 1, 2, 3, 4, or 5 days. The method according to any one of the alternative forms 31 to 37, wherein the culture step of alternative form 38(c) is carried out for at least 1, 2, 3, 4, or 5 days. The method according to any one of the alternative forms 31 to 38, wherein the culture step of alternative form 39(e) is carried out for at least 2, 3, 4, 5, 6, 7, or 8 days. The method according to any one of the alternative forms 31 to 39, wherein the culture step of alternative form 40(f) is carried out for at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. Alternative form 41. The method according to any one of Alternative Forms 31 to 40, wherein the dorsal anterior foregut cells of step (c) are cultured with an EGF pathway activator, BPE, a ROCK inhibitor, or any combination thereof. Alternative form 42. The method according to any one of Alternative Forms 31 to 41, wherein the esophageal progenitor cells of step (e) are cultured with an EGF pathway activator, a ROCK inhibitor, a SMAD inhibitor, or any combination thereof in the growth medium of the insert material, and EGF in the growth medium of the second tissue culture vessel. Alternative form 43. The method according to any one of Alternative Forms 31 to 42, wherein the esophageal progenitor cells of step (f) are cultured together with an EGF pathway activator in the growth medium of a second tissue culture vessel. Alternative form 44. The method according to any one of Alternative Forms 31 to 43, wherein the anterior foregut cells are derived from human induced pluripotent stem cells. Alternative form 45. The method according to any one of Alternative Forms 31 to 44, wherein the anterior foregut cells are derived from endoderm cells of the embryo, and the endoderm cells are derived from human induced pluripotent stem cells. Alternative form 46. The method according to alternative form 45, wherein endoderm cells are treated with Wnt3a, FGF4, noggin, or RA, or any combination thereof. Alternative form 47. The method according to alternative form 45 or 46, wherein endoderm cells of the embryo are treated for 1, 2, 3, 4, or 5 days. Alternative form 48. The method according to any one of Alternative Forms 44 to 47, wherein human induced pluripotent stem cells are treated with BMP4 and / or activin A. Alternative form 49. The method according to any one of Alternative Forms 44 to 48, wherein the human induced pluripotent stem cells are treated for 1, 2, 3, 4, or 5 days. Alternative form50. The process involves contacting human induced pluripotent stem cells with BMP4 and / or activin A to differentiate the human induced pluripotent stem cells into endoderm cells of an embryo, The method according to any one of the alternative forms 31 to 49, further comprising contacting the endoderm cells of the embryo with Wnt, FGF4, noggin, or RA, or any combination thereof, to differentiate the endoderm cells of the embryo into the anterior foregut cells of step (a). Alternative form 51. The method according to alternative form 50, wherein the human induced pluripotent stem cells are contacted for 1, 2, 3, 4, or 5 days. Alternative form 52. The method according to alternative form 50 or 51, wherein the endoderm cells of the embryo are brought into contact for 1, 2, 3, 4, or 5 days. Alternative form 53. In vitro esophageal raft composition, A stratified squamous epithelium including the basal layer and basal layer, The mesenchymal layer contains muscle fibers, and The stratified squamous epithelium is E-cadherin + The base upper layer is KRT13 + and KRT8 + The basal layer is SOX2 + ,P63 + , and KRT5 + And, Mesenchymal layer, FOXF1 + NKX6-1 + , and vimentin + Therefore, muscle fibers are desmin + This is an in vitro esophageal raft composition. Alternative form 54. In vitro cell composition, An in vitro cell composition comprising a population of dorsal anterior foregut cells derived from anterior foregut cells treated with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or a growth stimulant, or any combination thereof. Alternative form 55. In vitro cell composition, An in vitro cell composition comprising a population of anterior foregut cells treated with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or a growth stimulant, or any combination thereof. Alternative form 56. Esophageal raft cell composition according to any one of Alternative Forms 1 to 55, wherein the esophageal raft cell composition has a thickness of about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 57. Esophageal raft cell composition according to any one of Alternative Forms 1 to 56, wherein the esophageal raft cell composition has a thickness of about 150, 200, 250, 300, 350, 400, 450, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 58. Esophageal raft composition is approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 cm 2 An esophageal raft cell composition according to any one of Alternative Forms 1 to 57, having a surface area of ​​any two of the aforementioned surface areas. Alternative form 59. Esophageal raft composition is approximately 0.1, 0.5, 1, 1.5, or 2 cm 2An esophageal raft cell composition according to any one of Alternative Forms 1 to 58, having a surface area of ​​any two of the aforementioned surface areas. Alternative form 60. Esophageal raft composition, approximately 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 , 1, 5, or 10 cm 3 An esophageal raft cell composition according to any one of Alternative Forms 1 to 59, having a volume of or any volume within the range defined by any two of the aforementioned volumes. Alternative form 61. Esophageal raft cell composition according to any one of Alternative Forms 1 to 60, wherein the stratified squamous epithelial cell layer has a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 62. Esophageal raft cell composition according to any one of Alternative Forms 1 to 61, wherein the stratified squamous epithelial cell layer has a thickness of approximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 63. The esophageal raft cell composition according to any one of Alternative Forms 1 to 62, wherein the basal upper layer has a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 64. The esophageal raft cell composition according to any one of Alternative Forms 1 to 63, wherein the basal upper layer has a thickness of approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 65. Esophageal raft cell composition according to any one of Alternative Forms 1 to 64, wherein the basal upper layer has a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 66. The esophageal raft cell composition according to any one of Alternative Forms 1 to 65, wherein the basal layer has a thickness of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 67. The esophageal raft cell composition according to any one of Alternative Forms 1 to 66, wherein the mesenchymal layer has a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 68. Esophageal raft cell composition according to any one of Alternative Forms 1 to 67, wherein the mesenchymal layer has a thickness of approximately 100, 150, 200, 250, 300, 350, or 400 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 69. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 68, wherein the EGF pathway activator comprises EGF, TGF-α, AR, BTC, HB-EGF, EPR, tomoreglin, NRG-1, NRG-2, NRG-3, or NRG-4, or any combination thereof. Alternative form 70. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 69, wherein the EGF pathway activator is EGF. Alternative Form 71. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 70, wherein the EGF pathway activator is provided at a concentration of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative form 72. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 71, wherein the EGF pathway activator is provided at a concentration of 100 ng / mL or about 100 ng / mL. Alternative form 73. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 72, wherein the BMP pathway inhibitor comprises noggin, RepSox, LY364947, LDN193189, SB431542, or any combination thereof. Alternative form 74. Esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 73, wherein the BMP pathway inhibitor is noggin. Alternative form 75. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 74, wherein the BMP pathway inhibitor is provided at a concentration of approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative form 76. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 75, wherein the BMP pathway inhibitor is provided at a concentration of 200 ng / mL or approximately 200 ng / mL. Alternative form 77. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 76, wherein the FGF pathway activator comprises FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23, or any combination thereof. Alternative form 78. Esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 77, wherein the FGF pathway activator is FGF10. Alternative form 79. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 78, wherein the FGF pathway activator is provided at a concentration of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative Form 80. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 79, wherein the FGF pathway activator is provided at a concentration of 50 ng / mL or about 50 ng / mL. Alternative form 81. The esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 80, wherein the growth stimulant is a serum-free growth stimulant. Alternative form 82. The esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 81, wherein the adjuvant is a CultureOne adjuvant. Alternative form 83. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 82, wherein the auxiliary agent is provided at a concentration of 1x or approximately 1x. Alternative form 84. Esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 83, wherein the ROCK inhibitor comprises Y-27632, Y-30141, Y-39983, Ki-23095, SLx-2119, thiazovibin, azaindole 1, fasudil, ripasudil, netalusidil, RKI-1447, or GSK429286A, or any combination thereof. Alternative form 85. Esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 84, wherein the ROCK inhibitor is Y-27632. Alternative Form 86. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 85, wherein the ROCK inhibitor is provided at a concentration of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative Form 87. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 86, wherein the ROCK inhibitor is provided at a concentration of 10 μM or about 10 μM. Alternative form 88. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 87, wherein the SMAD inhibitor comprises A-83-01, DMH1, RepSox, LY365947, LY2109761, LY364947, SB431542, SB525334, SB505125, garnicertib, GW788388, LDN-193189, LDN-212854, hesperetin, or any combination thereof. Alternative form 89. The esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 88, wherein the SMAD inhibitors are DMH1 and A-83-01. Alternative Form 90. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 89, wherein the SMAD inhibitor is provided at a concentration of approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative form 91. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 90, wherein the SMAD inhibitor is provided at a concentration of 1 μM or about 1 μM.

[0007] Additional embodiments of the Disclosure provided herein are described by the following alternatively numbered alternative forms. Alternative form 1. In vitro esophageal raft culture, A stratified squamous epithelium including the basal layer and basal layer, The mesenchymal layer contains muscle fibers, and Stratified squamous epithelium contains E-cadherins + The base upper layer is KRT13 + and KRT8 + The basal layer is SOX2 + ,P63 + , and KRT5 + And, Mesenchymal layer, FOXF1 + NKX6-1 + , and vimentin + Therefore, muscle fibers are desmin + This is an in vitro esophageal raft composition. Alternative form 2. The esophageal raft culture described in Alternative Form 1, wherein the esophageal raft culture lacks lamina propria or has reduced lamina propria compared to esophageal tissue derived from adult animals of the same species as the raft culture. Alternative form 3. Esophageal raft culture according to alternative form 1 or 2, further comprising growth medium and optionally DMEM / F12. Alternative form 4. The esophageal raft culture is located within and / or on the surface of an insert member that has a surface permeable to the growth medium but not to cells, and the insert member is placed in a tissue culture vessel. An esophageal raft culture according to any one of Alternative Forms 1 to 3, wherein, optionally, the esophageal raft culture is placed on a surface that is permeable to the growth medium but impermeable to cells. Alternative form 5. The esophageal raft culture according to Alternative Form 4, wherein at least a portion of the insert member, optionally, has a surface that is permeable to growth medium but impermeable to cells, coated with an extracellular matrix or its components. Alternative form 6. Esophageal raft culture as described in Alternative form 5, wherein the extracellular matrix or its components are derived from humans. Alternative form 7. Esophageal raft culture according to alternative form 5 or 6, wherein the extracellular matrix or its components include human collagen type IV. Alternative form 8. Esophageal raft culture according to any one of Alternative Forms 5 to 7, wherein the extracellular matrix or its components do not contain rat collagen type I matrix or Matrigel. Alternative form 9. Esophageal raft culture according to any one of Alternative Forms 4 to 8, wherein the insert member and / or tissue culture vessel contains an amount of growth medium such that the esophageal raft culture is completely immersed in the growth medium. Alternative form 10. The esophageal raft culture according to Alternative Form 9, wherein the growth medium contained within the insert member further comprises an EGF pathway activator, a ROCK inhibitor, a SMAD inhibitor, or any combination thereof, and the growth medium contained within the tissue culture vessel comprises an EGF pathway activator. Alternative form 11. The tissue culture vessel and / or insert member contains an amount of growth medium such that the esophageal raft culture is only partially immersed in the growth medium. Esophageal raft culture according to any one of Alternative Forms 4 to 8, wherein stratified squamous epithelium is partially immersed in or not immersed in the growth medium, forming and / or located at a gas-liquid interface. Alternative form 12. Esophageal raft culture according to Alternative form 11, wherein the growth medium contained in the tissue culture vessel contains an EGF pathway activator. Alternative form 13. The permeable surface of the insert member is 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 Esophageal raft cultures according to any one of Alternative Forms 4 to 12, having pore sizes of 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or less, or approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or less, or any pore size within the range defined by any two of the aforementioned sizes. Alternative form 14. An esophageal raft culture according to any one of Alternative Forms 4 to 13, wherein the permeable surface of the insert member has a pore size of 3 μm. Alternative form 15. Esophageal raft culture according to any one of Alternative Forms 1 to 14, wherein the esophageal raft culture substantially does not contain neural progenitor cells and / or βIII-tubulin+ neurons. Alternative form 16. Esophageal raft culture according to any one of Alternative Forms 1 to 14, wherein the esophageal raft culture further comprises enteric neural crest cells (ENCCs), neural progenitor cells, and / or βIII-tubulin+ neurons, and optionally the neural progenitor cells are SOX10+, so that the esophageal raft culture is a neurally innervated esophageal raft culture. Alternative form 17. An esophageal raft culture according to any one of Alternative Forms 1 to 16, wherein the esophageal raft culture does not have angiogenesis, blood vessels, and / or endothelial cells. Alternative form 18. In vitro cell culture, An in vitro cell culture comprising a population of esophageal progenitor cells derived from dorsal anterior foregut cells treated with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or any combination thereof. Alternative form 19. The cell culture described in Alternative form 18, wherein dorsal anterior foregut cells are treated with a neuronal precursor inhibitor, optionally with CultureOne adjuvant, or cytarabine. Alternative form 20. A cell culture according to alternative form 18 or 19, further comprising growth medium, optionally serum-free medium, and optionally keratinocyte SFM. Alternative form 21. The cell culture according to Alternative form 20, wherein the growth medium contains an EGF pathway activator or bovine pituitary extract (BPE), or both. Alternative form 22. The EGF pathway activator is at a concentration of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations, or The cell culture according to Alternate Form 21, wherein the BPE is at a concentration of approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or at any concentration within the range defined by any two of the aforementioned concentrations, or both. Alternative form 23. A cell culture according to any one of Alternative Forms 18 to 22, wherein the cell culture is located inside and / or on the surface thereof. Alternative form 24. The cell culture according to alternative form 23, wherein at least a portion of the tissue culture vessel is coated with extracellular matrix or its viable elements, and the population of esophageal progenitor cells is on or in contact with the portion. Alternative form 25. A cell culture according to alternative form 24, wherein the extracellular matrix or its components are derived from humans. Alternative form 26. A cell culture according to alternative form 24 or 25, wherein the extracellular matrix or its components include human collagen type IV. Alternative form 27. A cell culture according to any one of Alternative Forms 24 to 27, wherein the extracellular matrix or its components do not contain rat collage type I matrix or Matrigel. Alternative form 28. A cell culture according to any one of Alternative Forms 18 to 27, further comprising a ROCK inhibitor. Alternative form 29. A cell culture according to any one of Alternative Forms 18-28, further comprising enteric neural crest cells. Alternative form 30. In vitro cell culture, An in vitro cell composition comprising a population of anterior foregut cells treated with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or any combination thereof. Alternative form 31. The cell culture according to Alternative form 30, wherein anterior foregut cells are further treated with a neuronal precursor inhibitor, optionally a CultureOne adjuvant, or cytarabine. Alternative form 32. A cell culture according to alternative form 30 or 31, further comprising growth medium, optionally RPMI, optionally FBS, optionally 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5% FBS, or any percentage of FBS within the range defined by any two of the aforementioned percentages. Alternative form 33. A cell culture according to any one of alternative forms 30 to 32, wherein the cell culture is located inside and / or on the surface of a tissue culture vessel. Alternative form 34. Esophageal raft culture or cell culture according to any one of Alternative Forms 1 to 17 or any one of Alternative Forms 18 to 33, wherein the esophageal raft culture or cell culture has been grown for at least 1, 2, 3, 4, 5, 6, 7, or 8 days. Alternative form 35. The esophageal raft culture or cell culture according to alternative form 34, wherein the esophageal raft culture or cell culture is derived from human induced pluripotent stem cells. Alternative form 36. The esophageal raft culture or cell culture according to alternative form 34 or 35, wherein the esophageal raft culture or cell culture is not derived from spheroids or organoids. Alternative form 37. A method for producing esophageal raft cultures, (a) Differentiating anterior foregut cells into dorsal anterior foregut cells by contacting them with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or any combination thereof. (b) Dissociating the dorsal anterior foregut cells from step (a) into single cells, (c) Culturing dorsal anterior foregut cells in a first tissue culture vessel to differentiate them into esophageal progenitor cells, (d) Dissociating the esophageal progenitor cells from step (c) into single cells, (e) Culturing esophageal progenitor cells within and / or on the surface of the insert member, The insert member is placed inside the second tissue culture vessel. The insert component includes a surface that is permeable to the growth medium but not to cells. The insert member and the second tissue culture vessel each contain an amount of growth medium such that the esophageal progenitor cells are completely immersed in the growth medium, and the cells are cultured. (f) A method comprising culturing esophageal progenitor cells in an insert member, wherein the second tissue culture vessel and / or insert member contains an amount of growth medium such that the esophageal progenitor cells are only partially immersed in the growth medium. Alternative form 38. The method according to alternative form 37, wherein anterior foregut cells are further contacted with a neuronal precursor inhibitor, optionally a CultureOne adjuvant, or cytarabine. Alternative form 39. The method according to alternative form 37 or 38, wherein esophageal progenitor cells are dissociated using a dissociating enzyme, optionally trypsin, chymotrypsin, collagenase, elastase, or Accutase. Alternative form 40. The method according to any one of Alternative Forms 37 to 39, wherein at least a portion of the first tissue culture vessel and / or the second tissue culture vessel is coated with an extracellular matrix or its components. Alternative form 41. The method according to alternative form 40, wherein the extracellular matrix or its components are derived from humans. Alternative form 42. The method according to alternative form 40 or 41, wherein the extracellular matrix or its components include human collagen type IV. Alternative form 43. The method according to any one of Alternative Forms 40 to 42, wherein the extracellular matrix or its components does not include rat collagen type I matrix or Matrigel. The method according to any one of the alternative forms 37 to 43, wherein the contact step of alternative form 44(a) is performed for at least 1, 2, 3, 4, or 5 days. The method according to any one of the alternative forms 37 to 44, wherein the culture step of alternative form 45(c) is carried out for at least 1, 2, 3, 4, or 5 days. The method according to any one of the alternative forms 37 to 45, wherein the culture step of alternative form 46(e) is carried out for at least 2, 3, 4, 5, 6, 7, or 8 days. The method according to any one of the alternative forms 37 to 46, wherein the culture step of alternative form 47(f) is carried out for at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. Alternative form 48. The method according to any one of Alternative Forms 37 to 47, wherein the dorsal anterior foregut cells of step (c) are cultured with an EGF pathway activator, BPE, a ROCK inhibitor, or any combination thereof. Alternative form 49. The method according to any one of Alternative Forms 37 to 48, wherein the esophageal progenitor cells of step (e) are cultured with an EGF pathway activator, a ROCK inhibitor, a SMAD inhibitor, or any combination thereof in the growth medium of the insert material, and EGF in the growth medium of the second tissue culture vessel. Alternative form 50. The method according to any one of Alternative Forms 37 to 49, wherein the esophageal progenitor cells of step (f) are cultured together with an EGF pathway activator in the growth medium of a second tissue culture vessel. Alternative form 51. The method according to any one of Alternative Forms 37 to 50, wherein the anterior foregut cells are derived from human induced pluripotent stem cells. Alternative form 52. The method according to any one of Alternative Forms 37 to 51, wherein the anterior foregut cells are derived from endoderm cells of the embryo, and the endoderm cells are derived from human induced pluripotent stem cells. Alternative form 53. The method according to alternative form 52, wherein endoderm cells are treated with Wnt3a, FGF4, Noggin, or RA, or any combination thereof, to differentiate the endoderm cells into anterior foregut cells. Alternative form 54. The method according to alternative form 53, wherein endoderm cells are further treated with a neuronal precursor inhibitor, optionally with CultureOne adjuvant, or with cytarabine. Alternative form 55. The method according to any one of Alternative Forms 52-54, wherein endoderm cells of the embryo are treated for 1, 2, 3, 4, or 5 days. Alternative form 56. The method according to any one of Alternative Forms 51 to 55, wherein human induced pluripotent stem cells are treated with BMP4 and / or activin A to differentiate the human induced pluripotent stem cells into endoderm cells. Alternative form 57. The method according to alternative form 56, wherein human induced pluripotent stem cells are further treated with a neuronal precursor inhibitor, optionally a CultureOne adjuvant, or cytarabine. Alternative form 58. The method according to any one of Alternative Forms 51 to 57, wherein human induced pluripotent stem cells are treated for 1, 2, 3, 4, or 5 days. Alternative form59. The process involves contacting human induced pluripotent stem cells with BMP4 and / or activin A to differentiate them into endoderm cells of the embryo, The method according to any one of the alternative forms 37 to 58, further comprising contacting endoderm cells with Wnt, FGF4, noggin, or RA, or any combination thereof, to differentiate the endoderm cells into anterior foregut cells of step (a). Alternative form 60. The method according to alternative form 59, wherein human induced pluripotent stem cells and / or endoderm cells are further contacted with a neural precursor inhibitor, optionally a CultureOne adjuvant, or cytarabine. Alternative form 61. The method according to alternative form 59 or 60, wherein human induced pluripotent stem cells are exposed for 1, 2, 3, 4, or 5 days. Alternative form 62. The method according to any one of Alternative Forms 59 to 61, wherein endoderm cells of the embryo are brought into contact for 1, 2, 3, 4, or 5 days. Alternative form 63. The method according to any one of Alternative Forms 37 to 62, wherein the esophageal raft culture substantially does not contain neural progenitor cells and / or βIII-tubulin+ neurons. Alternative form 64. The method according to any one of Alternative Forms 37 to 63, further comprising combining the dissociated esophageal progenitor cells of step (d) with enteric neural crest cells (ENCC), and culturing the combined esophageal progenitor cells and ENCC according to steps (e) and (f) to produce a neurally innervated esophageal raft culture. Alternative form 65. The method according to alternative form 64, wherein the neurally innervated esophageal raft culture comprises enteric neural crest cells (ENCCs), neural progenitor cells and / or βIII-tubulin+ neurons, and optionally the neural progenitor cells are SOX10+. Alternative form 66. The method according to any one of Alternative Forms 37 to 65, wherein the esophageal raft culture does not contain angiogenesis, blood vessels, and / or endothelial cells. Alternative form 67. In vitro cell composition, An in vitro cell composition comprising a population of dorsal anterior foregut cells derived from anterior foregut cells treated with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or any combination thereof. Alternative form 68. In vitro cell composition, An in vitro cell composition comprising a population of anterior foregut cells treated with an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or any combination thereof. Alternative form 69. The cell composition according to alternative form 68, wherein a population of anterior foregut cells is further treated with a neuronal precursor inhibitor, optionally a CultureOne adjuvant, or cytarabine. Alternative form 70. In vitro esophageal raft composition, A stratified squamous epithelium including the basal layer and basal layer, The mesenchymal layer contains muscle fibers, and Stratified squamous epithelium contains E-cadherins + The base upper layer is KRT13 + and KRT8 + The basal layer is SOX2 + ,P63+ , and KRT5 + And, Mesenchymal layer, FOXF1 + NKX6-1 + , and vimentin + Therefore, muscle fibers are desmin + This is an in vitro esophageal raft composition. Alternative form 71. The esophageal raft cell composition according to Alternative Form 70, wherein the esophageal raft cell composition substantially does not contain neural progenitor cells and / or βIII-tubulin+ neurons. Alternative form 72. The esophageal raft cell composition according to Alternative Form 70, wherein the esophageal raft culture is a neurally innervated esophageal raft culture, the esophageal raft cell composition further comprises enteric neural crest cells (ENCCs), neural progenitor cells, and / or βIII-tubulin+ neurons, and optionally the neural progenitor cells are SOX10+. Alternative form 73. Esophageal raft cell culture composition according to any one of Alternative Forms 70 to 72, wherein the esophageal raft cell composition does not contain angiogenesis, blood vessels, and / or endothelial cells. Alternative form 74. Esophageal raft cell composition according to any one of Alternative Forms 1 to 73, wherein the esophageal raft cell composition has a thickness of about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 75. Esophageal raft cell composition according to any one of Alternative Forms 1 to 74, wherein the esophageal raft cell composition has a thickness of about 150, 200, 250, 300, 350, 400, 450, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 76. Esophageal raft composition is approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 cm 2 An esophageal raft cell composition according to any one of Alternative Forms 1 to 75, having a surface area of ​​any two of the aforementioned surface areas. Alternative form 77. Esophageal raft composition is approximately 0.1, 0.5, 1, 1.5, or 2 cm 2 An esophageal raft cell composition according to any one of Alternative Forms 1 to 76, having a surface area of ​​any two of the aforementioned surface areas. Alternative form 78. Esophageal raft composition, approximately 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 , 1, 5, or 10 cm 3 An esophageal raft cell composition according to any one of Alternative Forms 1 to 77, having a volume of or any volume within the range defined by any two of the aforementioned volumes. Alternative form 79. Esophageal raft cell composition according to any one of Alternative Forms 1 to 78, wherein the stratified squamous epithelial cell layer has a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 80. Esophageal raft cell composition according to any one of Alternative Forms 1 to 79, wherein the stratified squamous epithelial cell layer has a thickness of approximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 81. The esophageal raft cell composition according to any one of Alternative Forms 1 to 80, wherein the basal upper layer has a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 82. The esophageal raft cell composition according to any one of Alternative Forms 1 to 81, wherein the basal upper layer has a thickness of approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 83. The esophageal raft cell composition according to any one of Alternative Forms 1 to 82, wherein the basal upper layer has a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 84. The esophageal raft cell composition according to any one of Alternative Forms 1 to 83, wherein the basal layer has a thickness of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 85. Esophageal raft cell composition according to any one of Alternative Forms 1 to 84, wherein the mesenchymal layer has a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 86. The esophageal raft cell composition according to any one of Alternative Forms 1 to 85, wherein the mesenchymal layer has a thickness of approximately 100, 150, 200, 250, 300, 350, or 400 μm, or any thickness within the range defined by any two of the aforementioned thicknesses. Alternative form 87. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 86, wherein the EGF pathway activator comprises EGF, TGF-α, AR, BTC, HB-EGF, EPR, tomoreglin, NRG-1, NRG-2, NRG-3, or NRG-4, or any combination thereof. Alternative form 88. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 87, wherein the EGF pathway activator is EGF. Alternative form 89. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 88, wherein the EGF pathway activator is provided at a concentration of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative form 90. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 89, wherein the EGF pathway activator is provided at a concentration of 100 ng / mL or about 100 ng / mL. Alternative Form 91. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 90, wherein the BMP pathway inhibitor comprises noggin, RepSox, LY364947, LDN193189, SB431542, or any combination thereof. Alternative form 92. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 91, wherein the BMP pathway inhibitor is noggin. Alternative form 93. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 92, wherein the BMP pathway inhibitor is provided at a concentration of approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative form 94. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 93, wherein the BMP pathway inhibitor is provided at a concentration of 200 ng / mL or approximately 200 ng / mL. Alternative form 95. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 94, wherein the FGF pathway activator comprises FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23, or any combination thereof. Alternative form 96. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 95, wherein the FGF pathway activator is FGF10. Alternative form 97. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 96, wherein the FGF pathway activator is provided at a concentration of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative Form 98. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 97, wherein the FGF pathway activator is provided at a concentration of 50 ng / mL or approximately 50 ng / mL. Alternative form 99. Esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 98, wherein the ROCK inhibitor comprises Y-27632, Y-30141, Y-39983, Ki-23095, SLx-2119, thiazovibin, azaindole 1, fasudil, ripasudil, netalusidil, RKI-1447, or GSK429286A, or any combination thereof. Alternative form 100. Esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative forms 1 to 99, wherein the ROCK inhibitor is Y-27632. Alternative Form 101. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 100, wherein the ROCK inhibitor is provided at a concentration of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative Form 102. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 101, wherein the ROCK inhibitor is provided at a concentration of 10 μM or about 10 μM. Alternative form 103. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 102, wherein the SMAD inhibitor comprises A-83-01, DMH1, RepSox, LY365947, LY2109761, LY364947, SB431542, SB525334, SB505125, garnicertib, GW788388, LDN-193189, LDN-212854, hesperetin, or any combination thereof. Alternative form 104. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 103, wherein the SMAD inhibitors are DMH1 and A-83-01. Alternative Form 105. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 104, wherein the SMAD inhibitor is provided at a concentration of approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or at any concentration within the range defined by any two of the aforementioned concentrations. Alternative Form 106. An esophageal raft culture, cell culture, method, or esophageal raft cell composition according to any one of Alternative Forms 1 to 105, wherein the SMAD inhibitor is provided at a concentration of 1 μM or about 1 μM. [Brief explanation of the drawing]

[0008] In addition to the features described herein, additional features and variations will readily become apparent from the following drawings and descriptions of exemplary embodiments. It should be understood that these drawings illustrate embodiments and are not intended to limit the scope. [Figure 1] This specification shows a schematic embodiment for producing the esophageal raft culture described herein. [Figure 2] This document describes an embodiment of an esophageal raft culture containing epithelium and mesenchyme. The apical layer of the raft culture expresses the esophageal epithelial markers SOX2 and P63, as well as the common epithelial marker E-cadherin (Ecad) (Panel A). The epithelium is stratified squamous epithelium expressing basal (KRT5) and suprabasal (KRT13 and KRT8) layer markers in separate layers (Panel B). The two distinct layers can be identified by hematoxylin / eosin staining (Panel C). The basal layer of the raft culture expresses the mesenchymal markers FOXF1, NKX6-1 (Panel D), and vimentin (Panel E). The mesenchyme expresses the differentiated muscle fiber marker (desmin) (Panel F). Cell nuclei are labeled with DAPI as a common marker. Scale bar is 100 μm. A schematic diagram of an esophageal raft culture with distinct epithelial and mesenchymal layers possessing characteristic markers is also shown (Panel G). [Figure 3A] This specification shows a schematic embodiment for producing esophageal raft cultures, in which an optional neuronal precursor inhibitor (e.g., CultureOne adjuvant [Cult1]) is added from days 0 to 9. The addition of neuronal precursor inhibitors prevents the expansion of neuronal cell types during differentiation of esophageal raft cultures, which may be undesirable for some purposes. [Figure 3B]Immunofluorescence images of embodiments showing esophageal raft cultures differentiated using a neuronal precursor inhibitor (e.g., CultureOne adjuvant) from either day 0-9 or day 6-9 are shown. Esophageal raft cultures were stained with 1) hematoxylin and eosin, 2) SOX2, 3) E-cadherin (Ecad), 4) a mixture of SOX, Ecad, and DAPI, or 5) βIII-tubulin and DAPI. The dashed line indicates the mesenchymal-epithelial boundary. The arrows indicate the presence of neuronal cell types in the mesenchyme of raft cultures treated with CultureOne alone from day 6 onward. These neuronal cell types are not observed in cultures grown with CultureOne from day 0-9. Neuronal cell types are indicated by the expression of SOX2 and βIII-tubulin (arrows) in the mesenchymal layer. [Figure 4A] A schematic diagram of an embodiment for producing nerve-innervated esophageal raft cultures is shown. Esophageal progenitor cells and enteric neural crest cells (ENCCs) are differentiated separately from hPSCs and co-cultured on a cell insert to generate esophageal raft cultures with mesenchyme innervated by enteric nerves. [Figure 4B] Immunofluorescence images of embodiments showing nerve-innervated esophageal raft cultures are shown. Nerve-innervated esophageal raft cultures were stained with 1) SOX2, P63, Ecad, and DAPI, 2) KRT5, KRT13, and DAPI, 3) GFP (expressed by ENCC), vimentin, KRT8, and DAPI, or 4) GFP (expressed by ENCC), βIII-tubulin, KRT8, and DAPI. ENCC-innervated esophageal raft cultures express esophageal epithelial markers SOX2, P63, KRT5, KRT13, and KRT8, as well as the common epithelial marker E-cadherin (Ecad). GFP-expressing ENCC (arrow) innervates the esophageal raft culture mesenchyme indicated by vimentin expression. GFP-expressing ENCC co-localizes with the nerve marker βIII-tubulin, indicating their differentiation into enteric nerves. [Modes for carrying out the invention]

[0009] This specification discloses iPSC-derived esophageal raft cultures and esophageal raft cell compositions. In some embodiments, the esophageal raft cultures and esophageal raft cell compositions described herein are produced from human iPSCs. In some embodiments, the esophageal raft cultures and esophageal raft cell compositions include esophageal epithelium and esophageal mesenchyme. In some embodiments, the esophageal raft cultures and esophageal raft cell compositions can be grown to eliminate neuronal cell types by culturing them with neuronal precursor inhibitors (i.e., compounds that inhibit the growth and / or differentiation of neural progenitor cells and neuronal cell types). In some embodiments, the esophageal raft cultures and esophageal raft compositions can be grown to innervate by culturing the esophageal raft culture or its precursor, for example, esophageal progenitor cells, with enteric neural crest cells (ENCCs) that differentiate into neuronal cell types. Methods for preparing esophageal raft cultures and esophageal raft cell compositions are also disclosed herein. In some embodiments, the esophageal raft cultures and esophageal raft cell compositions are different from esophageal organoids. By manipulating factors that control embryonic organogenesis, in vitro methods have been developed to induce the stepwise differentiation of PSCs into embryonic germline cells, and subsequently into specific cell types such as epithelial cells, mesenchymal cells, muscle cells, nerve cells, and vascular cells. Methods for producing esophageal organoids are explored in International Publication No. 2019 / 074793 (the entire publication expressly incorporated herein by reference). Further methods for producing other organoid and intermediate cell types (e.g., endoderm and anterior foregut spheroid) can be found in U.S. Patents 9,719,068 and 10,174,289, and International Publications 2011 / 140411, 2015 / 183920, 2016 / 061464, 2017 / 192997, 2018 / 106628, 2018 / 200481, 2018 / 085615, 2018 / 085622, 2018 / 085623, 2018 / 226267, and 2020 / 023245, each of which is expressly incorporated herein in whole by reference.Methods for creating other organoid types innervated using enteric neural crest cells (ENCCs) are explored in International Publication No. 2016 / 061464 (the entire publication expressly incorporated herein by reference).

[0010] The esophagus actively facilitates the passage of food from the oral cavity and pharynx to the stomach. It consists of stratified squamous epithelium, mesenchymal layers, muscular layers, and the enteric nervous system, which senses stretch and controls peristalsis. Congenital disorders such as esophageal atresia are caused by genetic mutations that result in narrowing or discontinuity of the esophagus. Other diseases, such as esophageal cancer, eosinophilic esophagitis, achalasia, and other motility disorders, affect the esophagus later in life. Tracheal and esophageal disorders are common in humans and difficult to accurately model in mice. The need for improved esophageal and other gastrointestinal models is clear.

[0011] term In the embodiments for carrying out the invention described below, reference is made to the accompanying drawings which form part of this specification. In the drawings, unless the context otherwise indicates, similar symbols typically identify similar components. The exemplary embodiments described in the embodiments for carrying out the invention, drawings, and claims are not intended to limit. Other embodiments may be used and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily apparent that the embodiments of this disclosure generally described herein and illustrated in the drawings can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly intended herein.

[0012] Unless otherwise defined, technical and scientific terms used herein have the same meanings as they are generally understood when read in light of this disclosure by a person skilled in the art to which this disclosure belongs. For the purposes of this disclosure, the following terms are defined below.

[0013] This disclosure uses positive language to describe numerous embodiments. This disclosure also includes embodiments in which subject matter such as substances or materials, methods, processes and conditions, protocols, or procedures is completely or partially excluded.

[0014] The articles "a" and "an" are used herein to refer to one or more (e.g., at least one) grammatical objects of the article. For example, "an element" means one or more elements.

[0015] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by about 10% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0016] Throughout this specification, unless the context requires otherwise, the terms “comprise,” “comprises,” and “comprising” mean to encompass the described process or element or group of processes or elements, but not to exclude any other process or element or group of processes or elements. “Consisting of” means to include and be limited to what follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the enumerated elements are necessary or essential, and other elements may or may not be present. “Essentially consisting of” means to include any elements enumerated after this phrase, and is limited to other elements that do not interfere with or contribute to the activity or action of the enumerated elements as identified in this disclosure. Thus, the phrase “essentially consisting of” indicates that the enumerated elements are necessary or essential, but other elements are optional and may or may not be present, depending on whether they essentially affect the activity or action of the enumerated elements.

[0017] As used herein, the terms “individual,” “subject,” or “patient” have their obvious and ordinary meanings as understood in light of this specification and mean human or non-human mammals, e.g., dogs, cats, mice, rats, cattle, sheep, pigs, goats, non-human primates, or birds, e.g., chickens, and any other vertebrates or invertebrates. The term “mammal” is used in its ordinary biological sense. Specifically, this includes, but is not limited to, monkeys (chimpanzees, apes, monkeys) and primates, including humans, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, and others.

[0018] As used herein, the terms “effective dose” or “effective amount” have their obvious and ordinary meanings as understood in light of this specification and refer to the amount of the enumerated composition or compound that produces an observable effect. The actual dose level of the active ingredient in the active composition of the subject matter of this disclosure can be modified to administer an amount of the active composition or compound that is effective in achieving a desired response for a particular subject and / or application. The selected dose level depends on a variety of factors, including but not limited to the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimum dose is administered, and the dose is gradually increased to a minimum effective dose in the absence of dose-limiting toxicity. Determination and adjustment of effective doses, and evaluation of when and how such adjustments should be made, are contemplated herein.

[0019] As used herein, the terms “function” and “functional” have their obvious and ordinary meanings as understood in light of this specification, and refer to biological, enzymatic, or therapeutic functions.

[0020] As used herein, the term “inhibit” has its obvious and ordinary meaning as understood herein and may mean reduction or prevention of biological activity. Reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or at least about 10%, 20%, 30%, 40%, 50% The percentage may be %, 60%, 70%, 80%, 90%, or 100%, or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or less, or a percentage that is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or less, or a quantity that is within the range defined by any two of the aforementioned values. As used herein, the term “delay” has its obvious and ordinary meaning as understood herein and refers to the slowing, postponement, or delay of a biological event to a later time than otherwise expected. The delay is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least approximately 0%, 10%, 20%, 30%, 40%, 50%. The percentages may be 60%, 70%, 80%, 90%, 100%, or 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or less, or percentages that are approximately 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or less, or quantities within the range defined by any two of the aforementioned values. The terms inhibition and delay do not necessarily indicate 100% inhibition or delay. Partial inhibition or delay may be achieved.

[0021] As used herein, the term “isolated” has its obvious and ordinary meaning as understood in light herein, and means (1) a substance and / or entity that has been separated from at least some of the components to which it was assembled when it was first produced (whether in a natural and / or experimental setting), and / or (2) a substance and / or entity produced, prepared and / or manufactured by human hands. The isolated substances and / or entities are 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or equal to 100%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100%, at least 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100%, It can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100%, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or less than 100%, or about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or less than 100% (or the range including and / or extending to the aforementioned values).In some embodiments, the isolated active substance is 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or including and / or extending to the aforementioned values), or about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure, or at least 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially It is 100% or 100% pure, or at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure, or 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or less than 100%, or about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or less than 100% pure. As used herein, “isolated” substance may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multicellular organism or tissue.

[0022] As used herein, “in vivo” is given its obvious and ordinary meaning as understood in light herein and refers to the implementation of a method within a living organism, typically an animal, a mammal including humans, and a plant, as opposed to a tissue extract or a dead organism.

[0023] As used herein, “exvivo” is given its obvious and ordinary meaning as understood in light of this specification and refers to the implementation of a method outside of a living organism with little to no alteration to its natural state.

[0024] As used herein, “in vitro” is given its obvious and ordinary meaning as understood in light of this specification and refers to the execution of a method outside of a biological state (e.g., in a petri dish or test tube).

[0025] As used herein, the terms “nucleic acid” or “nucleic acid molecule” have their obvious and ordinary meanings as understood herein, and refer to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those occurring naturally in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease activity, and exonuclease activity. Nucleic acid molecules may consist of monomers that are naturally occurring nucleotides (such as DNA and RNA), analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have alterations to the sugar moiety and / or pyrimidine or purine base moiety. Sugar modifications may include, for example, the substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar may be functionalized as an ether or ester. Furthermore, the entire sugar moiety can be replaced with sterically and electronically similar structures such as aza-sugars and carbocyclic sugar analogs. Examples of modifications to the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutions. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoranilideates, or phosphoramidates. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. “Oligocyte” can be used interchangeably with nucleic acids and can refer to either double-stranded or single-stranded DNA or RNA.Nucleic acids (or nucleic acids) are contained in nucleic acid vectors or constructs (e.g., plasmids, viruses, retroviruses, lentiviruses, bacteriophages, cosmids, fosmids, phagemids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or human artificial chromosomes (HACs)) that can be used for amplification and / or expression of nucleic acids (or nucleic acids) in various biological systems. Typically, vectors or constructs also contain elements including, but not limited to, promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multicloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.

[0026] A nucleic acid or nucleic acid molecule may contain one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences may be adjacent to or between extra nucleic acids, e.g., linkers, repeats, or restriction enzyme sites, on the same nucleic acid or nucleic acid molecule, or be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 nucleotides long, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs in length, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs in length. It is at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs long, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, It can be concatenated to any other sequence of any length within the range defined by any two of the aforementioned lengths, which is 90, 95, 100, 150, 200, or 300 base pairs or less, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs or less.As used herein, the term “downstream” on a nucleic acid has its obvious and ordinary meaning as understood herein, and, if the nucleic acid is double-stranded, refers to the sequence on the strand containing the coding sequence (sense strand) that follows the 3' end of the preceding sequence. As used herein, the term “upstream” on a nucleic acid has its obvious and ordinary meaning as understood herein, and, if the nucleic acid is double-stranded, refers to the sequence on the strand containing the coding sequence (sense strand) that precedes the 5' end of the following sequence.As used herein, the term “grouped” on nucleic acids has its obvious and ordinary meaning as understood herein, and refers to two or more sequences that occur in close proximity, either directly or with an extra nucleic acid in between, e.g., a linker, repeat, or restriction enzyme site, or sequences of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs in length. or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs long, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs long. Is it a base length, or at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base lengths, or is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, Or any other sequence that is 300 nucleotides or less in length, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 nucleotides or less in length, or any length within the range defined by any two of the aforementioned lengths, but generally refers to any other sequence that does not contain a sequence encoding a functional or catalytic polypeptide, protein, or protein domain.

[0027] The nucleic acids described herein include nucleic acid bases. Primary, standard, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleic acid bases include, but are not limited to, purines, pyrimidines, modified nucleic acid bases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

[0028] As used herein, the terms “peptide,” “polypeptide,” and “protein” have their obvious and common meanings as understood herein and refer to macromolecules composed of amino acids linked by peptide bonds. Numerous functions of peptides, polypeptides, and proteins are known in the art, including, but not limited to, enzymes, structural, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, though not always, produced biologically by ribosome complexes using nucleic acid templates, although chemosynthesis is also available. By manipulating nucleic acid templates, peptides, polypeptides, and proteins, mutations such as substitution, deletion, truncation, addition, replication, or fusion of two or more peptides, polypeptides, or proteins can be performed.These fusions of two or more peptides, polypeptides, or proteins are adjacent to or between extra amino acids, e.g., linkers, repeats, epitopes, or tags, into the same molecule, or are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs long, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs in length, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or The base pairs are 300 base pairs long, or at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 base pairs long, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80 It can be concatenated to any other sequence of any length within the range defined by any two of the aforementioned lengths, which is 85, 90, 95, 100, 150, 200, or 300 nucleotides or less in length, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 nucleotides or less in length, or any other sequence of any length within the range defined by any two of the aforementioned lengths. As used herein, the term “downstream” on a polypeptide has its obvious and ordinary meaning as understood herein and refers to the sequence following the C-terminus of the preceding sequence.As used herein, the term “upstream” on a polypeptide has its obvious and ordinary meaning as understood herein, and refers to the sequence preceding the N-terminus of a subsequent sequence.

[0029] Where used herein, the term “purity” of any given substance, compound, or material has its obvious and ordinary meaning as understood in light of this specification and refers to the actual amount of the substance, compound, or material relative to the expected amount. For example, a substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure (including all decimals in between). Purity may be affected by undesirable impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell fragments, small molecules, degradation products, solvents, carriers, vehicles, or contaminants, or any combination thereof. In some embodiments, a substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasmas, pyrogens, bacterial endotoxins, and exogenous active substances. Purity can be measured using techniques including, but not limited to, electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin-layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-Vis spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetric analysis, or titration, or any combination thereof.

[0030] Where used herein, the term “yield” for any given substance, compound, or material has its obvious and ordinary meaning as understood herein and refers to the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material is 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, or about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% The yield is either 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96, 97, 98, 99%, or 100% or less, or 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96, 97, 98, 99%, or 100% or less (including all decimals in between). The yield may be affected by the efficiency of the reaction or process, undesirable side reactions, decomposition, the quality of inputs, compounds, or materials, or the loss of desired substances, compounds, or materials during any step of production.

[0031] Some embodiments described herein relate to pharmaceutical compositions comprising, essentially consisting of, or comprising an effective amount of the cell composition described herein and a pharmaceutically acceptable carrier, excipient, or combination thereof. The pharmaceutical compositions described herein are suitable for human and / or veterinary use.

[0032] As used herein, “pharmaceutically acceptable” means, in its obvious and ordinary meaning as understood herein, a carrier, excipient, and / or stabilizer that is nontoxic or has an acceptable level of toxicity to cells or mammals to which it is exposed at the dosage and concentration used. As used herein, “pharmaceutically acceptable,” “diluent,” “excipient,” and / or “carrier” means, in its obvious and ordinary meaning as understood herein, and is intended to include any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders, etc., suitable for administration to human, cat, dog, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or carrier approved by a federal, state, or other regulatory body for use in animals, including humans and non-human mammals such as cats and dogs, or listed in the United States Pharmacopeia or other commonly recognized pharmacopoeias. The terms diluent, excipient, and / or “carrier” may refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutically acceptable diluents, excipients, and / or carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin. Water, physiological saline, and aqueous dextrose and glycerol solutions can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutically acceptable diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffer solution.Physiologically acceptable carriers may also include one or more of the following: antioxidants, e.g., ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids; carbohydrates, e.g., glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugar alcohols, e.g., mannitol or sorbitol; salt-forming counterions, e.g., sodium; and nonionic surfactants, e.g., TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The compositions may also contain small amounts of wetting agents, fillers, emulsifiers, or pH buffers, if desired. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. The formulations should be suitable for the mode of administration.

[0033] Cryoprotective agents are cell composition additives used to improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Examples of cryoprotective agents include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Cryoprotective agents can be used as part of a cryopreservation medium containing other components such as nutrients (e.g., albumin, serum, bovine serum, fetal bovine serum [FCS]) to enhance the viability of cells after thawing.In these cryopreservation media, at least one cryoprotectant is present in amounts of 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or approximately 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 1%. 2%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or at least 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or at least about 0.01%, 0. 0.5%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, It can be found at concentrations of 40%, 50%, 60%, 70%, 80%, or 90% or less, or at concentrations of approximately 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or less, or at concentrations of any percentage within the range defined by any two of the aforementioned numbers.

[0034] Further excipients having desirable properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be present in residues or impurities from the manufacturing process, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivators, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell fragments, nucleic acids, peptides, amino acids, or growth medium components, or any combination thereof.The amount of excipients in the composition is 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, or approximately 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%. , 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, or at least 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, or at least approximately 0%, 0.1%, 0.2%, 0.3%, Is it 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, or 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, Percentages may be found to be 80%, 90%, 95%, 100% w / w or less, or approximately 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or less, or any percentage within the range defined by any two of the aforementioned numbers.

[0035] The term “pharmaceutically acceptable salt” has its obvious and ordinary meaning as understood herein and includes, but is not limited to, relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including analgesics, therapeutic agents, and other materials. Examples of pharmaceutically acceptable salts include salts derived from mineral acids, such as hydrochloric acid and sulfuric acid, and salts derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of inorganic bases suitable for salt formation include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts may also be formed using suitable organic bases, including those that are non-toxic and sufficiently strong to form such salts. For example, such a class of organic bases may include, but are not limited to, mono-, di-, and trialkylamines (including methylamine, dimethylamine, and triethylamine), mono-, di-, or trihydroxyalkylamines (including mono-, di-, and triethanolamine), amino acids (including glycine, arginine, and lysine), guanidine, N-methylglucosamine, N-methylglucamine, L-glutamine, N-methylpiperazine, morpholine, ethylenediamine, N-benzylphenethylamine, and trihydroxymethylaminoethane.

[0036] The appropriate formulation depends on the chosen route of administration. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques exist in the art for administering compounds, including but not limited to intestinal, oral, rectal, topical, sublingual, buccal, intraocular, epidural, cutaneous, aerosol, and parenteral delivery (including intramuscular, subcutaneous, intra-arterial, intra-venous, intra-portal, intra-articular, intradermal, peritoneal, intramedullary, subarachnoid, direct intraventricular, intraperitoneal, intranasal, or intraocular injection). Pharmaceutical compositions are generally tailored to a specific intended route of administration.

[0037] As used herein, “carrier” means a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or incorporation of a compound into cells, tissues, and / or organs of the body, in its obvious and ordinary sense as understood herein.

[0038] As used herein, “diluent” has its obvious and ordinary meaning as understood in light of this specification and refers to a component in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the volume of a potent drug that is too small in mass to manufacture and / or administer. It may also be a liquid for dissolving a drug administered by injection, ingestion, or inhalation. Common forms of diluents in the art, but not limited to, are buffered aqueous solutions such as phosphate-buffered saline that mimic the composition of human blood.

[0039] As used herein, the term “raft culture” has its obvious and ordinary meaning as understood herein and refers to a three-dimensional cell culture comprising two or more cell types having a cellular organization and function that more closely approximates that of a natural organ tissue. In some embodiments, as the name suggests, the culture is grown and maintained at a gas-liquid interface where a portion of the culture is exposed to a gaseous or atmospheric environment, while another portion is located on or within a layer of liquid growth medium. Diffusion allows nutrients from the growth medium to access the exposed cells. The gas-liquid interface promotes the differentiation of the raft culture into a stratified epithelial layer, which is found in all organs (including the lungs, esophagus, and skin), including those exposed to ambient air in vivo. In some embodiments described herein, the raft culture also includes a mesenchymal layer or mesenchyme.

[0040] As used herein, the term “insert member” means any structure or container having at least a surface on which cells can grow, in the obvious and ordinary sense as understood in light herein, the surface or at least a portion thereof that is permeable to an aqueous medium but not to cells, and which can be positioned within the separate container or other structure so that cells are exposed to the environment of both the insert member and the separate container or other structure (however, cells may be exposed to the environment of the separate container or other structure over a surface or at least a portion thereof that is permeable to an aqueous medium but not to cells, and which is not necessarily in direct contact). As used herein, a common example of an insert member is a transwell, which is a tissue culture vessel having a permeable surface, in which an aqueous medium may be contained within one or more of the internal volumes of the transwell or the internal volumes of a separate tissue culture vessel, exchange between the two aqueous mediums may occur over the permeable surface, and which can be positioned within a separate, generally larger volume tissue culture vessel so that cells may be in contact on the permeable surface or at least a portion thereof so that cells are exposed to the aqueous medium in the transwell and the aqueous medium in the separate container. However, alternative structures for the insert member are conceivable, such as in which the insert member is fixed to a separate container and a channel or other opening is available to access the internal volume of the separate container, or in which either or both of the transwell or the separate container have no conventional internal volume and contact between the aqueous medium in the insert member and the separate container occurs via alternative means such as microfluidic channels. As disclosed herein, cells can be grown on the surface or a portion thereof of the insert member in a separate container such that the cells are only partially immersed in the gas-liquid interface (i.e., there is little or no aqueous medium in the insert member).

[0041] As used herein, the terms "%w / w" or "%wt / wt" have their obvious and ordinary meanings as understood herein and refer to a percentage expressed by multiplying the weight of an ingredient or drug by 100 relative to the total weight of a composition. The terms "v / v%" or "vol / vol%" have their obvious and ordinary meanings as understood herein and refer to a percentage expressed by multiplying the liquid volume of a compound, substance, ingredient, or drug by 100 relative to the total liquid volume of a composition.

[0042] stem cells As used herein, the term “totipotent stem cell” (also known as omnipotent stem cell) has its obvious and ordinary meaning as understood herein and refers to a stem cell capable of differentiating into embryonic and extraembryonic cell types. Such cells can construct a complete living organism. These cells are produced from the fusion of an egg and a sperm cell. Cells produced by the first few divisions of a fertilized egg are also totipotent.

[0043] The term “embryonic stem cell (ESC),” also commonly abbreviated as ES cell, when used herein, has its obvious and ordinary meaning as understood herein, referring to pluripotent cells derived from the inner cell mass of a blastocyst, which is an early-stage embryo. For the purposes of this disclosure, the term “ESC” is used more broadly to also include, in some cases, embryonic germ cells.

[0044] As used herein, the term “pluripotent stem cell (PSC)” has its obvious and ordinary meaning as understood herein and encompasses any cell that can differentiate into any of the body’s nearly all cell types, namely any cell that can differentiate into any of the three germ layers (embryonic epithelium), including the endoderm (endoderm, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, genitourinary tract), and ectoderm (epidermal tissue and nervous system). PSCs may be descendants of inner cell mass cells of a preimplantation blastocyst, or they may be obtained through the induction of non-pluripotent cells, such as adult somatic cells, by forcing the expression of certain genes. Pluripotent stem cells may be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including humans, rodents, pigs, and cattle.

[0045] The term "induced pluripotent stem cell (iPSC)," also commonly abbreviated as iPS cells, has, as used herein, its obvious and ordinary meaning as understood herein, and refers to a type of pluripotent stem cell artificially derived from normally non-pluripotent cells, such as adult somatic cells, by inducing the "forced" expression of certain genes. hiPSC refers to human iPSCs. In several methods known in the art, iPSCs can be induced by transfection of non-pluripotent cells, such as adult fibroblasts, with certain stem cell-related genes. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes may include the master transcription factors Oct-3 / 4 (POU5F1) and Sox2, but other genes may enhance the efficiency of induction. After 3-4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically, and are typically isolated through morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include first-generation iPSCs, second-generation iPSCs in mice, and human induced pluripotent stem cells. In some methods, retroviral systems are used to transform human fibroblasts into pluripotent stem cells using four key genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In other methods, lentiviral systems are used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28.Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4(POU5F1), certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15), certain members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof. Other methods for producing induced pluripotent stem cells that are conventionally known in the field are also conceivable.

[0046] As used herein, the term “progenitor cell” has its obvious and ordinary meaning as understood herein, and one or more progenitor cells encompass any cells that can be used in the methods herein, which either regenerate themselves or acquire the ability to differentiate into one or more specialized cell types. In some embodiments, progenitor cells are pluripotent or capable of becoming pluripotent. In some embodiments, progenitor cells are subjected to treatment with an extrinsic factor (e.g., a growth factor) to acquire pluripotency. In some embodiments, progenitor cells may be totipotent (or omnipotent) stem cells, pluripotent stem cells (induced or uninduced), polypotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells may be derived from an embryo, infant, child, or adult. In some embodiments, progenitor cells may be somatic cells subjected to treatment such that pluripotency is conferred via genetic engineering or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), epiblast stem cells (EpiSCs), and induced pluripotent stem cells.

[0047] In some embodiments, one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, the pluripotent stem cells are derived from embryonic stem cells, which are derived from totipotent cells of an early mammalian embryo and are capable of undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of a blastocyst, which is an early stage embryo. Methods for inducing embryonic stem cells from blastocytes are well known in the art. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described herein, but it will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells.

[0048] Further stem cells that may be used in embodiments of this disclosure include, but are not limited to, those provided by or listed in databases hosted by the National Stem Cell Bank (NSCB), Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF), WISC cell Bank at the Wi Cell Research Institute, University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC), Novocell, Inc. (San Diego, California), Cellartis AB (Goteborg, Sweden), ES Cell International Pte Ltd (Singapore), Technion at the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Examples of embryonic stem cells that can be used in embodiments of this disclosure include, but are not limited to, SA01 (SA001), SA02 (SA002), ES01 (HES-1), ES02 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6), BG01 (BGN-01), BG02 (BGN-02), BG03 (BGN-03), TE03 (13), TE04 (14), TE06 (16), UCOl (HSF1), UC06 (HSF6), WA01 (HI), WA07 (H7), WA09 (H9), WA13 (H13), and WA14 (H14).Examples of human pluripotent cell lines include, but are not limited to, TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cells.

[0049] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the terms “differentiation” or “targeted differentiation” describe the process by which less specialized cells become a specific specialized target cell type. The specialization of the specialized target cell type can be determined by any appropriate method that can be used to define or alter the fate of the initial cell. Exemplary methods include, but are not limited to, genetic engineering, chemical treatment, protein treatment, and nucleic acid treatment.

[0050] In some embodiments, adenoviruses can be used to transport the four required genes, resulting in iPSCs substantially identical to embryonic stem cells. Since none of the adenovirus's own genes bind to the target host, the risk of tumor formation is eliminated. In some embodiments, non-viral-based techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without any viral transfection system, albeit with very low efficiency. In other embodiments, direct protein delivery is used to generate iPSCs, thus eliminating the need for viruses or gene modification. In some embodiments, mouse iPSC generation is possible using a similar methodology: repeated treatment of cells with a specific protein directed to the cells via a polyarginine anchor was sufficient to induce pluripotency. In some embodiments, the expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0051] As used herein, the term “feeder cell” has its obvious and ordinary meaning as understood herein and refers to a cell that supports the growth of pluripotent stem cells by secreting growth factors into the culture medium or presenting them on its cell surface, etc. Feeder cells are generally adherent cells and can be denatured. For example, feeder cells can be denatured by irradiation (e.g., gamma rays), mitomycin-C treatment, electrical pulses, or mild chemical fixation (e.g., with formaldehyde or glutaraldehyde). However, feeder cells do not necessarily need to be denatured. Feeder cells may serve purposes such as secreting growth factors, presenting growth factors on their cell surface, detoxifying the culture medium, or synthesizing extracellular matrix proteins. In some embodiments, feeder cells are homogeneous or heterogeneous to the supported target stem cells, which may have implications in downstream applications. In some embodiments, feeder cells are mouse cells. In some embodiments, feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human precipitous fibroblasts, human dermal fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal myocytes, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, a conditioned medium prepared from the feeder cells is used instead of, or in combination with, the feeder cell co-culture. In some embodiments, the feeder cells are not used during the proliferation of the target stem cells.

[0052] Differentiation of PSC and endoderm In some embodiments, PSCs such as ESCs and iPSCs undergo stepwise directional differentiation, first to the definitive endoderm (DE), then to the anterior / foregut lineage, and then to esophageal tissue. In some embodiments, PSCs such as ESCs and iPSCs undergo non-stepwise directional differentiation, with the simultaneous addition of molecules to promote DE formation (e.g., growth factors, ligands) and molecules for subsequent tissue formation.

[0053] The endoderm of the embryo gives rise to the intestinal tract. The anterior DE forms the foregut and its associated organs, including the esophagus, lungs, stomach, liver, and pancreas, while the posterior DE forms the midgut and hindgut, which in turn form the small and large intestines and part of the genitourinary system. Studies using mouse, chicken, and frog embryos suggest that the establishment of the anterior-posterior pattern in the DE at the gastrula stage is a prerequisite for subsequent foregut and hindgut development. Wnt and FGF signaling pathways are important for facilitating the fate of either the posterior endoderm / hindgut or the anterior endoderm / foregut.

[0054] Methods for directing the differentiation of DE cells into esophageal tissue in vitro are explored in International Publication No. 2019 / 074793 (the entire publication expressly incorporated herein by reference). In some embodiments, directed differentiation is achieved by selectively activating certain signaling pathways in iPSCs and / or DE cells. In some embodiments, the signaling pathways include, but are not limited to, the EGF signaling pathway, the Wnt signaling pathway, the Wnt / APC signaling pathway, the FGF signaling pathway, the TGF-β signaling pathway, the BMP signaling pathway, the Notch signaling pathway, the Hedgehog signaling pathway, the LKB signaling pathway, and the Par polarity signaling pathway, all of which are active in esophageal or gastrointestinal development.

[0055] Methods for producing endoderm from pluripotent cells (e.g., iPSCs or ESCs) are applicable to the methods described herein. In some embodiments, the pluripotent cells are derived from a morula. In some embodiments, the pluripotent stem cells are stem cells. Examples of stem cells used in these methods include, but are not limited to, embryonic stem cells. Embryonic stem cells may be derived from the embryonic intracellular mass or embryonic germline. Embryonic stem cells or germ cells may be derived from a variety of animal species, including, but not limited to, various mammalian species, including humans. In some embodiments, human embryonic stem cells are used to produce endoderm. In some embodiments, human embryonic germ cells are used to produce endoderm. In some embodiments, iPSCs are used to produce endoderm. In some embodiments, human iPSCs (hiPSCs) are used to produce endoderm.

[0056] In some embodiments, embryonic stem cells or germ cells or iPSCs are 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours, or approximately 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 1 50 hours, 180 hours, 240 hours, 300 hours, or at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours, or at least approximately 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 1 Treatment with one or more small molecule compounds, activators, inhibitors, or growth factors for a period of time of 20 hours, 150 hours, 180 hours, 240 hours, 300 hours, or within 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, or within approximately 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, or 300 hours, or any time within the range defined by any two of the aforementioned times, for example, 6 hours to 300 hours, 24 hours to 120 hours, 48 ​​hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours. In some embodiments, two or more small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the two or more small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0057] In some embodiments, embryonic stem cells or germ cells or iPSCs are 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / mL, 10000 ng / mL, or 15000 ng / mL, or approximately 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 1 50 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / mL, 10000 ng / mL, or 15000 ng / mL, or at least 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 70 It is either 00 ng / mL, 10000 ng / mL, or 15000 ng / mL, or at least about 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / mL, 10000 ng / mL, or 15000 ng / mL, or 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL L, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / mL, 10000 ng / mL, or 15000 ng / mL or less, or approximately 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL,The sample is treated with one or more small molecule compounds, activators, inhibitors, or growth factors at concentrations of 5000 ng / mL, 7000 ng / mL, 10000 ng / mL, or 15000 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 10 ng / mL to 15000 ng / mL, 100 ng / mL to 5000 ng / mL, 500 ng / mL to 2000 ng / mL, 10 ng / mL to 2000 ng / mL, or 1000 ng / mL to 15000 ng / mL. In some embodiments, the concentration of one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, the concentration of one or more small molecule compounds, activators, inhibitors, or growth factors changes during the course of treatment. In some embodiments, two or more small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, two or more small molecule compounds, activators, inhibitors, or growth factors may be present at different concentrations.

[0058] In some embodiments, ESCs, germ cells, or iPSCs, or any downstream cell type, are cultured in a growth medium that supports stem cell growth. In some embodiments, the stem cell growth medium is serum-free medium, RPMI 1640, DMEM, DMEM / F12, Advanced DMEM / F12, or keratinocyte SFM medium. In some embodiments, the stem cell growth medium contains fetal bovine serum (FBS).In some embodiments, the stem cell growth medium is 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or approximately 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 1% 0%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or at least 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or at least approximately 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7% , 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or less, or about 0%, FBS is included in concentrations of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or less, or in any percentage within the range defined by any two of the aforementioned values, for example, 0% to 20%, 0.2% to 10%, 2% to 5%, 0% to 5%, or 2% to 20%. In some embodiments, the stem cell growth medium does not contain heterogeneous components. In some embodiments, the growth medium includes one or more small molecule compounds, activators, inhibitors, or growth factors.

[0059] In some embodiments, a cell population enriched with endoderm cells is used. In some embodiments, the endoderm cells are isolated or substantially purified. In some embodiments, the isolated or substantially purified endoderm cells express one or more of the SOX17, FOXA2, or CXRC4 markers (e.g., at least one or three) to a higher degree than one or more of the OCT4, AFP, TM, SPARC, or SOX7 markers (e.g., at least one, three, or five).

[0060] In some embodiments, endoderm cells and hESCs are treated with one or more growth factors. Such growth factors may include growth factors from the TGF-β superfamily. In some embodiments, one or more growth factors include the nodal / activin and / or BMP subgroup of the TGF-β superfamily of growth factors. In some embodiments, one or more growth factors are selected from the group consisting of nodal, activin A, activin B, BMP4, Wnt3a, or any combination of these growth factors.

[0061] In some embodiments, activin-induced endoderm (DE) can undergo further anterior endoderm patterning, foregut identification, and morphogenesis depending on FGF, Wnt, BMP, or retinoic acid, or any combination thereof, and an esophageal culture system that promotes esophageal growth, morphogenesis, and cell differentiation. In some embodiments, human PSCs are directed to efficiently differentiate into esophageal epithelium and mesenchyme in vitro. It is understood that molecules such as growth factors may be added at any developmental stage to promote specific types of gastrointestinal tissue formation.

[0062] In some embodiments, targeted differentiation is achieved by selectively activating and / or inhibiting a particular signaling pathway in PSCs, DEs, or any downstream cell types. In some embodiments, the signaling pathways include, but are not limited to, the Wnt pathway, FGF pathway, BMP pathway, retinoic acid pathway, EGF pathway, Rho kinase (ROCK) pathway, or SMAD pathway, or any combination thereof. It will be understood by those skilled in the art that differentiation according to this disclosure can be driven by altering the concentration, expression, or function of any one of the signaling pathways disclosed herein. In some embodiments, inhibition or activation of the signaling pathway can be brought about by using cellular components associated with the signaling pathway, such as innate inhibitors, antagonists, activators, or agonists of the pathway. In some embodiments, siRNAs and / or shRNAs targeting cellular components associated with the signaling pathways are used to inhibit or activate these pathways.

[0063] In some embodiments, pluripotent stem cells, endoderm cells, anterior foregut cells, dorsal anterior foregut cells, esophageal progenitor cells, and / or esophageal raft cells are brought into contact with a Wnt pathway activator. In some embodiments, the Wnt pathway activator comprises a Wnt protein. In some embodiments, the Wnt protein comprises a recombinant Wnt protein. In some embodiments, the Wnt pathway activator comprises Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML284, IQ-1, WAY262611, or any combination thereof. In some embodiments, the Wnt pathway activator comprises a GSK3 signaling pathway inhibitor. In some embodiments, the Wnt pathway activator includes CHIR99021, CHIR98014, AZD2858, BIO, AR-A014418, SB216763, SB415286, aloysin, indirubin, alsterpaulone, kaempaulone, lithium chloride, TDZD8, or TWS119, or any combination thereof. In some embodiments, the cells are not treated with the Wnt pathway activator. The Wnt pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors described herein.

[0064] Fibroblast growth factor (FGF) is a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGF is a heparin-binding protein, and its interaction with cell surface-associated heparan sulfate proteoglycans has been shown to be essential for FGF signaling. FGF plays a crucial role in the proliferation and differentiation processes of a wide variety of cells and tissues. In humans, 22 members of the FGF family have been identified, all of which are structurally related signaling molecules. Members FGF1-FGF10 all bind to the fibroblast growth factor receptor (FGFR). FGF1 is also known as acidic fibroblast growth factor (bFGF), and FGF2 is also known as basic fibroblast growth factor (bFGF). Members FGF11, FGF12, FGF13, and FGF14, also known as FGF homologs 1-4 (FHF1-FHF4), have been shown to have distinct functional differences compared to FGF. These factors exhibit strikingly similar sequence homology, yet they do not bind to FGFR and are involved in intracellular processes unrelated to FGF. This group is also known as "iFGF." Members FGF15–FGF23 are newer and less characterized. FGF15 is a mouse ortholog of human FGF19 (therefore, human FGF15 does not exist). Human FGF20 was identified based on its homology to Xenopus FGF-20 (XFGF-20). In contrast to the local activity of other FGFs, FGF15 / FGF19, FGF21, and FGF23 have more systemic effects.

[0065] In some embodiments, pluripotent stem cells, endoderm cells, anterior foregut cells, dorsal anterior foregut cells, esophageal progenitor cells, and / or esophageal raft cells are brought into contact with an FGF pathway activator. In some embodiments, the FGF pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF pathway activator comprises one or more of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15 / FGF19), FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, or FGF23. In some embodiments, the cells are not treated with the FGF pathway activator. The FGF pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0066] In some embodiments, pluripotent stem cells, endoderm cells, anterior foregut cells, dorsal anterior foregut cells, esophageal progenitor cells, and / or esophageal raft cells are contacted with a bone morphogenetic protein (BMP) pathway activator or BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises a BMP protein. In some embodiments, the BMP protein is a recombinant BMP protein. In some embodiments, the BMP pathway activator comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof. In some embodiments, the BMP pathway inhibitor comprises Noggin, RepSox, LY364947, LDN193189, SB431542, or any combination thereof. In some embodiments, the cells are not treated with the BMP pathway activator or BMP pathway inhibitor. The BMP pathway activators or BMP pathway inhibitors provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0067] In some embodiments, pluripotent stem cells, endoderm cells, anterior foregut cells, dorsal anterior foregut cells, esophageal progenitor cells, and / or esophageal raft cells are brought into contact with a retinoic acid pathway activator. In some embodiments, the retinoic acid pathway activator includes retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, or AM580, or any combination thereof. In some embodiments, the cells are not treated with the retinoic acid pathway activator. The retinoic acid pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0068] In some embodiments, pluripotent stem cells, endoderm cells, anterior foregut cells, dorsal anterior foregut cells, esophageal progenitor cells, and / or esophageal raft cells are brought into contact with an epidermal growth factor (EGF) pathway activator. In some embodiments, the EGF pathway activator includes EGF, TGF-α, AR, BTC, HB-EGF, EPR, tomoregulin, NRG-1, NRG-2, NRG-3, or NRG-4, or any combination thereof. In some embodiments, the cells are not treated with the EGF pathway activator. The EGF pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0069] In some embodiments, pluripotent stem cells, endoderm cells, anterior foregut cells, dorsal anterior foregut cells, esophageal progenitor cells, and / or esophageal raft cells are brought into contact with a ROCK inhibitor (ROCKi). In some embodiments, ROCKi includes Y-27632, Y-30141, Y-39983, Ki-23095, SLx-2119, thiazovibin, azaindole-1, fasudil, ripasudil, netalusidil, RKI-1447, or GSK429286A, or any combination thereof. In some embodiments, the cells are not treated with ROCKi. The ROCKi provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0070] In some embodiments, pluripotent stem cells, endoderm cells of the embryo, anterior foregut cells, dorsal anterior foregut cells, esophageal progenitor cells, and / or esophageal raft cells are contacted with a transforming growth factor-beta (TGF-β) pathway activator or a TGF-β pathway inhibitor. In some embodiments, the TGF-β family includes bone morphogenesis proteins (BMPs), differentiation factors (GDFs), anti-Müllerian hormones, activin, and the nodal pathway. In some embodiments, the TGF-β pathway activator includes TGF-β1, TGF-β2, TGF-β3, activin A, activin B, nodal, BMPs, IDE1, IDE2, or any combination thereof. In some embodiments, the TGF-β pathway inhibitors include A-83-01, DMH1, RepSox, LY365947, LY2109761, LY364947, SB431542, SB525334, SB505125, garnicertib, GW788388, LDN-193189, LDN-212854, hesperetin, or any combination thereof. In some embodiments, cells are not treated with the TGF-β pathway activator or TGF-β pathway inhibitor. The TGF-β pathway activators or TGF-β pathway inhibitors provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0071] In some embodiments, pluripotent stem cells, endoderm cells, anterior foregut cells, dorsal anterior foregut cells, esophageal progenitor cells, and / or esophageal raft cells are brought into contact with a SMAD pathway inhibitor. In some embodiments, the SMAD pathway inhibitor is a TGF-β pathway inhibitor. In some embodiments, the SMAD pathway inhibitor includes A-83-01, DMH1, RepSox, LY365947, LY2109761, LY364947, SB431542, SB525334, SB505125, garnicertib, GW788388, LDN-193189, LDN-212854, hesperetin, or any combination thereof. In some embodiments, the cells are not treated with the SMAD pathway inhibitor. The SMAD pathway inhibitors provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0072] In some embodiments, cells are differentiated by a “one-step” process. For example, pluripotent stem cells are directly treated with one or more molecules (e.g., activin A) that can differentiate pluripotent stem cells into DE cultures, in combination with additional molecules (e.g., FGF4, Wnt, Noggin, RA) that can promote targeted differentiation of DE cultures.

[0073] In some embodiments, pluripotent stem cells are prepared from somatic cells. In some embodiments, pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, pluripotent stem cells are cryopreserved. In some embodiments, somatic cells are cryopreserved. In some embodiments, pluripotent stem cells are prepared from PBMCs. In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, PBMCs are grown on a feeder cell substrate. In some embodiments, PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, PBMCs are grown on an irradiated MEF feeder cell substrate.

[0074] In some embodiments, pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) are expanded in cell culture. In some embodiments, pluripotent stem cells (e.g., iPSCs) are expanded in Matrigel. In some embodiments, pluripotent stem cells are expanded in a cell culture containing a ROCK inhibitor (e.g., Y-27632). In some embodiments, pluripotent stem cells (e.g., iPSCs) are differentiated into endoderm cells. Pluripotent stem cells (e.g., iPSCs) are differentiated into endoderm cells by contacting them with activin A, BMP4, or both. In some embodiments, pluripotent stem cells (e.g., iPSCs) are administered at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 1 60, 170, 180, 190, or 200 ng / mL, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 1 30, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 The substance is brought into contact with activin A at a concentration of 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or with any concentration within the range defined by any two of the aforementioned concentrations, for example, activin A at concentrations of 10-200 ng / mL, 10-100 ng / mL, 100-200 ng / mL, or 50-150 ng / mL.In some embodiments, pluripotent stem cells (e.g., iPSCs) are administered at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 1 40, 150, 160, 170, 180, 190, or 200 ng / mL, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 4 The cells are brought into contact with BMP4 at concentrations of 0, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, BMP4 at concentrations of 1-200 ng / mL, 1-100 ng / mL, 25-200 ng / mL, 1-80 ng / mL, or 25-100 ng / mL. In some embodiments, pluripotent stem cells are differentiated into endoderm cells in a culture medium containing growth serum.In some embodiments, the culture medium contains 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5% of growth serum, or any percentage within the range defined by any two of the aforementioned percentages, for example, 0-2%, 1-2.5%, 1.5-2.5%, 1.5-2%, or 0.5-2% of growth serum. In some embodiments, endoderm cells are differentiated into anterior foregut cells in a culture medium containing FBS. In some embodiments, the culture medium includes FBS in the following percentages: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5%, or any percentage within the range defined by any two of the aforementioned percentages, for example, FBS in the following percentages: 0-2%, 1-2.5%, 1.5-2.5%, 1.5-2%, or 0.5-2%. In some embodiments, pluripotent stem cells are differentiated in growth medium in which the FBS concentration is gradually increased by changing the medium containing two or more of the following FBS concentrations: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5%. In some embodiments, pluripotent stem cells are differentiated stepwise in medium containing 0%, 0.2%, and 2% FBS. In addition to these embodiments, pluripotent stem cells (e.g., iPSCs) can be differentiated into endoderm cells by any other method known in the art.

[0075] In some embodiments, endoderm cells are differentiated into anterior foregut cells. In some embodiments, anterior foregut cells are grown as a monolayer. In some embodiments, endoderm cells are differentiated into anterior foregut cells by contacting them with one or more (e.g., at least one, two, three, or four) of the following: Wnt protein or pathway activator, FGF protein or pathway activator, BMP pathway inhibitor, or retinoic acid pathway activator, or any combination thereof. In some embodiments, the Wnt protein or pathway activator is Wnt3a. In some embodiments, the FGF protein or pathway activator is FGF4. In some embodiments, the BMP pathway inhibitor is noggin. In some embodiments, the retinoic acid pathway activator is retinoic acid.In some embodiments, Wnt protein or pathway activator, FGF protein or pathway activator, BMP pathway inhibitor, or retinoic acid pathway activator, or any combination thereof, is 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL, or approximately 0, 100, 120, 140, 160, 1 80, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL, or at least 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL, or at least about 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL, or 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL It is provided in concentrations that are less than or equal to 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations, for example, concentrations of 0-600 ng / mL, 0-200 ng / mL, 200-500 ng / mL, or 200-600 ng / mL.In some embodiments, the Wnt protein or pathway activator, FGF protein or pathway activator, BMP pathway inhibitor, or retinoic acid pathway activator, or any combination thereof, is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or approximately 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or at least 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or at least At most, approximately 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2. It is provided in concentrations of 8, 2.9, or 3.0 μM or less, or approximately 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM or less, or any concentration within the range defined by any two of the aforementioned concentrations, for example, concentrations of 0 to 3.0 μM, 1.0 to 3.0 μM, 0 to 2.0 μM, or 1.5 to 3.0 μM.In some embodiments, endoderm cells are exposed to one or more (e.g., at least one, two, three, or four) of Wnt protein or pathway activators, FGF protein or pathway activators, BMP pathway inhibitors, or retinoic acid pathway activators, or any combination thereof, for 1, 2, 3, 4, 5, 6, 7, or 8 days, or about 1, 2, 3, 4, 5, 6, 7, or 8 days, or at least about 1, 2, 3, 4, 5, 6, 7, or 8 days, or within 1, 2, 3, 4, 5, 6, 7, or 8 days, or within about 1, 2, 3, 4, 5, 6, 7, or 8 days. In some embodiments, endoderm cells are differentiated into anterior foregut cells without contact with one or more (e.g., at least one, two, three, or four) of the following: Wnt protein or pathway activator, FGF protein or pathway activator, BMP pathway inhibitor, or retinoic acid pathway activator, or any combination thereof. In some embodiments, endoderm cells are differentiated into anterior foregut cells without contact with one or more (e.g., at least one, two, three, or four) of the following: Wnt3a, FGF4, noggin, or retinoic acid, or any combination thereof. In some embodiments, endoderm cells are differentiated into anterior foregut cells in a culture medium containing growth serum. In some embodiments, the culture medium contains 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5% of growth serum, or any percentage within the range defined by any two of the aforementioned percentages, for example, 0-2%, 1-2.5%, 1.5-2.5%, 1.5-2%, or 0.5-2% of growth serum. In some embodiments, endoderm cells are differentiated into anterior foregut cells in a culture medium containing FBS.In some embodiments, the culture medium includes FBS in the following percentages: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5%, or any percentage within the range defined by any two of the aforementioned percentages, for example, FBS in the following percentages: 0-2%, 1-2.5%, 1.5-2.5%, 1.5-2%, or 0.5-2%.

[0076] In addition to these embodiments, endoderm cells can be differentiated into anterior foregut cells by any other method known in the art.

[0077] Formation of dorsal anterior foregut cells Methods for producing anterior foregut cells from pluripotent stem cells, either disclosed herein or known in the art, are applicable to the methods described herein. In some embodiments, methods known in the art for producing anterior foregut spheroids can be applied to the production of anterior foregut cells. In some embodiments, anterior foregut cells are obtained as a cell monolayer during differentiation by eliminating spontaneously generated anterior foregut spheroids. In some embodiments, anterior foregut cells are obtained by dissociating spontaneously generated anterior foregut spheroids into single cells and plating the dissociated single cells into a monolayer. In some embodiments, anterior foregut cells are obtained both as a cell monolayer and by dissociating spontaneously generated anterior foregut spheroids into single cells.

[0078] In some embodiments, anterior foregut cells are differentiated into dorsal anterior foregut cells expressing SOX2, HNF1β, or both. In some embodiments, anterior foregut cells are differentiated into dorsal anterior foregut cells by contacting them with one or more (e.g., at least one, two, or three) epidermal growth factor (EGF) pathway activators, BMP pathway inhibitors, or FGF pathway activators, or any combination thereof. In some embodiments, anterior foregut cells are further contacted with growth promoters. In some embodiments, anterior foregut cells are differentiated into dorsal anterior foregut cells by contacting them with one or more (e.g., at least one, two, three, or four) EGF pathway activators, BMP pathway inhibitors, FGF pathway activators, or growth promoters, or any combination thereof. In some embodiments, anterior foregut cells are further contacted with neuronal precursor inhibitors (i.e., compounds that inhibit the growth and / or differentiation of neuronal precursors and neuronal cell types). In some embodiments, anterior foregut cells are differentiated into dorsal anterior foregut cells by contacting them with one or more (e.g., at least one, two, three, or four) of the following: an EGF pathway activator, a BMP pathway inhibitor, an FGF pathway activator, or a neuronal precursor inhibitor, or any combination thereof. In some embodiments, the EGF pathway activator includes EGF, TGF-α, AR, BTC, HB-EGF, EPR, tomoregulin, NRG-1, NRG-2, NRG-3, or NRG-4, or any combination thereof. In some embodiments, the BMP pathway inhibitor includes noggin, RepSox, LY364947, LDN193189, SB431542, or any combination thereof. In some embodiments, the FGF pathway activating agent includes FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15 / FGF19), FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, or FGF23, or any combination thereof.In some embodiments, the growth stimulant is a serum-free growth stimulant, for example, any one of those known in the art, optionally a CultureOne stimulant. In some embodiments, the neuronal precursor inhibitor comprises a CultureOne stimulant or cytarabine. In some embodiments, anterior foregut cells are contacted with EGF, noggin, or FGF4, or any combination thereof (including all three), to differentiate them into dorsal anterior foregut cells. In some embodiments, anterior foregut cells are contacted with EGF, noggin, FGF4, or a neuronal precursor inhibitor, or any combination thereof (including all four), to differentiate them into dorsal anterior foregut cells.

[0079] In some embodiments, anterior foregut cells are brought into contact with an EGF pathway activator. In some embodiments, the EGF pathway activator is or contains EGF. In some embodiments, anterior foregut cells are brought into contact with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 , 180, 190, or 200 ng / mL, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or at least approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 , 140, 150, 160, 170, 180, 190, or 200 ng / mL, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 The cells are brought into contact with an EGF pathway activator (e.g., EGF) at a concentration of 10, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 10-200 ng / mL, 10-150 ng / mL, or 50-200 ng / mL. In some embodiments, the anterior foregut cells are brought into contact with an EGF pathway activator (e.g., EGF) at a concentration of 100 ng / mL, about 100 ng / mL, at least 100 ng / mL, at least about 100 ng / mL, 100 ng / mL or less, or about 100 ng / mL or less.

[0080] In some embodiments, anterior foregut cells are brought into contact with a BMP pathway inhibitor. In some embodiments, the BMP pathway inhibitor is noggin or contains noggin. In some embodiments, anterior foregut cells are brought into contact with 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 2 60, 270, 280, 290, or 300 ng / mL, or at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or at least approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 , 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL or less, or approximately 100, 110, 120, 130, 140, 150, 160 The cells are brought into contact with a BMP pathway inhibitor (e.g., noggin) at a concentration of 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL or less, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 100-300 ng / mL, 100-250 ng / mL, or 150-300 ng / mL. In some embodiments, the anterior foregut cells are brought into contact with a BMP pathway inhibitor (e.g., noggin) at a concentration of 200 ng / mL, about 200 ng / mL, at least 200 ng / mL, at least about 200 ng / mL, 200 ng / mL or less, or about 200 ng / mL or less.

[0081] In some embodiments, anterior foregut cells are brought into contact with an FGF pathway activator. In some embodiments, the FGF pathway activator is or contains FGF10. In some embodiments, anterior foregut cells are brought into contact with 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95 or 100 ng / mL, or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng / mL, or at least approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, The FGF pathway activator (e.g., FGF10) is brought into contact with the FGF pathway activator at a concentration of 75, 80, 85, 90, 95, or 100 ng / mL, or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL or less, or at a concentration of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 5 to 100 ng / mL, 5 to 75 ng / mL, or 25 to 100 ng / mL. In some embodiments, anterior foregut cells are exposed to an FGF pathway activator (e.g., FGF10) at a concentration of 50 ng / mL, about 50 ng / mL, at least 50 ng / mL, at least about 50 ng / mL, 50 ng / mL or less, or about 50 ng / mL or less.

[0082] In some embodiments, anterior foregut cells are brought into contact with a growth stimulant. In some embodiments, the growth stimulant is a serum-free growth stimulant, such as one commonly known in the art. In some embodiments, the growth stimulant is or contains CultureOne stimulant (GIBCO, Carlsbad, CA, USA). In some embodiments, anterior foregut cells are brought into contact with a concentration of 0.25x, 0.5x, 0.75x, 1x, 1.25x, 1.5x, 1.75x, or 2x, according to the manufacturer's recommended concentration, or about 0.25x, 0.5x, 0.75x, 1x, 1.25x, 1.5x, 1.75x, or 2x, or at least 0.25x, 0.5x, 0.75x, 1x, 1.25x, 1.5x, 1.75x, or 2x, or at least The cells are brought into contact with a growth stimulant (e.g., CultureOne) at a concentration of approximately 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2 times, or 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2 times or less, or approximately 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2 times or less. In some embodiments, the anterior foregut cells are brought into contact with a growth stimulant (e.g., CultureOne) at a concentration of 1, approximately 1, at least 1, at least approximately 1, 1 times or less, or approximately 1 times or less. In some embodiments, CultureOne stimulant or other growth stimulants are used to inhibit the growth of neural progenitor cells during the culture of the anterior foregut cells.

[0083] In some embodiments, anterior foregut cells are brought into contact with a neuronal precursor inhibitor. In some embodiments, the neuronal precursor inhibitor is or comprises CultureOne adjuvant or cytarabine. In some embodiments, anterior foregut cells are brought into contact with a neuronal precursor inhibitor at concentrations of 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2, or about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2, or at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2, or at least about 0.25 The cells are brought into contact with the neuronal precursor inhibitor (e.g., CultureOne or cytarabine) at a concentration that is 0.5 times, 0.75 times, 1 time, 1.25 times, 1.5 times, 1.75 times, or 2 times, or 0.25 times, 0.5 times, 0.75 times, 1 time, 1.25 times, 1.5 times, 1.75 times, or 2 times or less, or about 0.25 times, 0.5 times, 0.75 times, 1 time, 1.25 times, 1.5 times, 1.75 times, or 2 times or less. In some embodiments, the anterior foregut cells are brought into contact with the neuronal precursor inhibitor (e.g., CultureOne or cytarabine) at a concentration that is 1 times, about 1 times, at least 1 times, at least about 1 times, 1 times or less, or about 1 times or less. In some embodiments, CultureOne adjuvants, cytarabine, or other neural precursor inhibitors are used to inhibit the growth of neural precursor cells during the culture of pluripotent stem cells, endoderm, and / or anterior foregut cells.

[0084] In some embodiments, the anterior foregut cells are subjected to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or at least approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or at least approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, Contact with one or more (e.g., at least one, two, three, or four) of the following: EGF pathway activators, BMP pathway inhibitors, FGF pathway activators, or growth promoters, for 4, 5, 6, 7, or 8 days, or for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or for 1, 2, 3, 4, 5, 6, 7, or 8 days, or within a range defined by any two of the aforementioned values, for example, 12 hours to 8 days, 1 to 8 days, or 2 to 6 days.In some embodiments, the anterior foregut cells are subjected to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or at least approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, 4 , 6, 7, or 8 days, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or within 1, 2, 3, 4, 5, 6, 7, or 8 days, or within approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or within 1, 2, 3, 4, 5, 6, 7, or 8 days, or within a range defined by any two of the aforementioned values, for example, 12 hours to 8 days, 1 to 8 days, or 2 to 6 days, exposure to one or more (e.g., at least one, two, three, or four) of the EGF pathway activators, BMP pathway inhibitors, FGF pathway activators, or neurotransmitter inhibitors.

[0085] Formation of esophageal progenitor cells Methods for producing dorsal anterior foregut cells disclosed herein or otherwise known in the art are applicable to the methods disclosed herein.

[0086] In some embodiments, dorsal anterior foregut cells are enlarged and differentiated into esophageal progenitor cells. In some embodiments, dorsal anterior foregut cells are dissociated into a single-cell suspension. In some embodiments, dorsal anterior foregut cells are dissociated using a dissociating enzyme. In some embodiments, the dissociating enzyme is one or more (e.g., at least one, two, three, four, five) of trypsin, chymotrypsin, collagenase, elastase, or Accutase, or any combination thereof or comprising them. In some embodiments, the single-cell suspension of dorsal anterior foregut cells is plated onto a tissue culture vessel (e.g., a tissue culture plate) or a portion thereof (in particular, the surface in contact with the cells) coated with an extracellular matrix or its components or mimetic. In some embodiments, the extracellular matrix or its components or mimetic is homogeneous with respect to the dorsal anterior foregut cells. In some embodiments, the dorsal anterior foregut cells are of human origin, and the extracellular matrix or its components or mimetic is of human origin. In some embodiments, the extracellular matrix or its components or mimetic is collagen type IV. In some embodiments, Collagen IV is human collagen type IV. In some embodiments, collagen type IV is derived from human placenta. In some embodiments, the extracellular matrix or its components or mimics does not include rat collagen type I matrix or Matrigel, or both.

[0087] In some embodiments, dorsal anterior foregut cells are cultured in growth medium within a tissue culture vessel. In some embodiments, the growth medium comprises an EGF pathway activator, bovine pituitary extract (BPE), or a ROCK inhibitor, or any combination thereof (including all three). In some embodiments, dorsal anterior foregut cells are brought into contact with an EGF pathway activator, BPE, or a ROCK inhibitor, or any combination thereof (including all three). In some embodiments, the growth medium is serum-free medium. In some embodiments, the growth medium is keratinocyte SFM (GIBCO, Carlsbad, CA, USA). In some embodiments, the growth medium comprises EGF, BPE, or a ROCK inhibitor, or any combination thereof. In some embodiments, the EGF pathway activator comprises EGF, TGF-α, AR, BTC, HB-EGF, EPR, tomoregulin, NRG-1, NRG-2, NRG-3, or NRG-4, or any combination thereof. In some embodiments, the ROCK inhibitor includes Y-27632, Y-30141, Y-39983, Ki-23095, SLx-2119, thiazovibin, azaindole 1, fasudil, ripasudil, netalusdil, RKI-1447, or GSK429286A, or any combination thereof. In some embodiments, the growth medium includes EGF, BPE, or Y-27632, or any combination thereof (including all three). In some embodiments, dorsal anterior foregut cells are brought into contact with EGF, BPE, or Y-27632, or any combination thereof (including all three).

[0088] In some embodiments, dorsal anterior foregut cells are brought into contact with an EGF pathway activator. In some embodiments, the EGF pathway activator is or contains EGF. In some embodiments, the dorsal anterior foregut cells are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 The EGF pathway activator (e.g., EGF) is brought into contact with the EGF pathway activator at a concentration of 8, 19, or 20 ng / mL, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL or less, or at a concentration of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 1-10 ng / mL, 10-20 ng / mL, 5-15 ng / mL, or 8-12 ng / mL. In some embodiments, dorsal anterior foregut cells are brought into contact with an EGF pathway activator (e.g., EGF) at a concentration of 10 ng / mL, about 10 ng / mL, at least 10 ng / mL, at least about 10 ng / mL, 10 ng / mL or less, or about 10 ng / mL or less.

[0089] In some embodiments, dorsal anterior foregut cells are brought into contact with BPE. In some embodiments, dorsal anterior foregut cells are brought into contact with BPE at a concentration of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL, or at least approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μ BPE is brought into contact with the BPE at a concentration of g / mL, or 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL or less, or at a concentration of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 5-50 μg / mL, 20-100 μg / mL, 20-60 μg / mL, or 10-50 μg / mL. In some embodiments, dorsal anterior foregut cells are brought into contact with BPE at a concentration of 30 μg / mL, about 30 μg / mL, at least 30 μg / mL, at least about 30 μg / mL, 30 μg / mL or less, or about 30 μg / mL or less.

[0090] In some embodiments, dorsal anterior foregut cells are brought into contact with a ROCK inhibitor. In some embodiments, the ROCK inhibitor is or includes Y-27632. In some embodiments, the dorsal anterior foregut cells are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 ROCK inhibitors (e.g., Y-27632) are brought into contact with a concentration of 5, 16, 17, 18, 19, or 20 μM, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM or less, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 1-20 μM, 1-15 μM, or 5-20 μM. In some embodiments, dorsal anterior foregut cells are exposed to a ROCK inhibitor (e.g., Y-27632) at a concentration of 10 μM, about 10 μM, at least 10 μM, at least about 10 μM, 10 μM or less, or about 10 μM or less.

[0091] In some embodiments, dorsal anterior foregut cells are cultured on a coated tissue culture vessel for 1, 2, 3, 4, 5, 6, 7, or 8 days, or about 1, 2, 3, 4, 5, 6, 7, or 8 days, or at least about 1, 2, 3, 4, 5, 6, 7, or 8 days, or within 1, 2, 3, 4, 5, 6, 7, or 8 days, or within a range defined by any two of the aforementioned values, for example, 1–8, 2–6, 4–8, or 1–4 days, to differentiate into esophageal progenitor cells.

[0092] In some embodiments, dorsal anterior foregut cells are enlarged and differentiated into esophageal progenitor cells. In some embodiments, the dorsal anterior foregut cells are dissociated into a single-cell suspension. In some embodiments, the single-cell suspension of dorsal anterior foregut cells is plated onto a tissue culture vessel (e.g., a tissue culture plate) or a portion thereof (in particular, the surface in contact with the cells) coated with an extracellular matrix or its components or mimics. In some embodiments, the dorsal anterior foregut cells are cultured in a growth medium within the tissue culture vessel. In some embodiments, the growth medium comprises an EGF pathway activator, bovine pituitary extract (BPE), or a ROCK inhibitor, or any combination thereof (including one, two, or all three). In some embodiments, the dorsal anterior foregut cells are brought into contact with an EGF pathway activator, BPE, or a ROCK inhibitor, or any combination thereof (including one, two, or all three). In some embodiments, the growth medium is serum-free medium. In some embodiments, dorsal anterior foregut cells are contacted with EGF, BPE, or Y-27632, or any combination thereof (including one, two, or all three). In some embodiments, dorsal anterior foregut cells are contacted with an EGF pathway activator. In some embodiments, the EGF pathway activator is EGF or contains EGF. In some embodiments, dorsal anterior foregut cells are contacted with an EGF pathway activator (e.g., EGF) at a concentration of 10 ng / mL, about 10 ng / mL, at least 10 ng / mL, at least about 10 ng / mL, 10 ng / mL or less, or about 10 ng / mL or less. In some embodiments, dorsal anterior foregut cells are contacted with BPE. In some embodiments, dorsal anterior foregut cells are contacted with BPE at a concentration of 30 μg / mL, about 30 μg / mL, at least 30 μg / mL, at least about 30 μg / mL, 30 μg / mL or less, or about 30 μg / mL or less. In some embodiments, dorsal anterior foregut cells are contacted with a ROCK inhibitor. In some embodiments, the ROCK inhibitor is or comprises Y-27632.In some embodiments, dorsal anterior foregut cells are exposed to a ROCK inhibitor (e.g., Y-27632) at a concentration of 10 μM, about 10 μM, at least 10 μM, at least about 10 μM, 10 μM or less, or about 10 μM or less. In some embodiments, dorsal anterior foregut cells are cultured on a coated tissue culture vessel for 1, 2, 3, 4, 5, 6, 7, or 8 days, or about 1, 2, 3, 4, 5, 6, 7, or 8 days, or at least about 1, 2, 3, 4, 5, 6, 7, or 8 days, or within 1, 2, 3, 4, 5, 6, 7, or 8 days, or within a range defined by any two of the aforementioned values, for example, 1–8, 2–6, 4–8, or 1–4 days, to differentiate into esophageal progenitor cells.

[0093] In some embodiments, the resulting esophageal progenitor cells express SOX2, P63, or HNF1β, or any combination thereof. In some embodiments, the resulting esophageal progenitor cells express SOX2 at higher levels compared to dorsal anterior foregut cells.

[0094] Formation of esophageal raft cells In some embodiments, esophageal progenitor cells differentiated from dorsal anterior foregut cells and expanded on a coated tissue culture vessel are dissociated into a single-cell suspension. In some embodiments, the expanded esophageal progenitor cells are dissociated using a dissociation enzyme. In some embodiments, the dissociation enzyme is one or more (e.g., at least one, two, three, four, or five) of trypsin, chymotrypsin, collagenase, elastase, or Accutase, or any combination thereof or comprising them. In some embodiments, the single-cell suspension of expanded esophageal progenitor cells is plated onto an insert member (e.g., transwell or cell insert) or a portion thereof (in particular, the portion in contact with the cells or the permeable surface of the insert member) coated with an extracellular matrix or its components or mimics. In some embodiments, the insert member includes a surface that is permeable to the growth medium but not to the cells. In some embodiments, the permeable surface of the insert member is 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10 μm, or 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or less, or a pore size of approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or less, or any pore size within the range defined by any two of the aforementioned sizes, for example, having pore sizes of 0.1 to 10 μm, 0.1 to 5 μm, 1 to 5 μm, or 2 to 8 μm. In some embodiments, the permeable surface of the insert member has a pore size of 3 μm or approximately 3 μm. In some embodiments, the extracellular matrix or its components or mimics are homogeneous with respect to esophageal progenitor cells. In some embodiments, the esophageal progenitor cells are of human origin, and the extracellular matrix or its components or mimics are of human origin.In some embodiments, the extracellular matrix or its components or mimics are type IV collagen. In some embodiments, type IV collagen is human type IV collagen. In some embodiments, type IV collagen is derived from human placenta. In some embodiments, the extracellular matrix or its components or mimics do not include rat type I collagen matrix or Matrigel, or both. In some embodiments, enlarged esophageal progenitor cells are cultured in growth medium. In some embodiments, the growth medium is Advanced DMEM / F12. In some embodiments, the insert member is placed in a tissue culture vessel.

[0095] In some embodiments, the growth medium contained within the insert member comprises one or more (e.g., one, two, or three) of the following: an EGF pathway activator, a ROCK inhibitor, and a SMAD pathway inhibitor. In some embodiments, the EGF pathway activator comprises EGF, TGF-α, AR, BTC, HB-EGF, EPR, tomoregulin, NRG-1, NRG-2, NRG-3, or NRG-4, or any combination thereof. In some embodiments, the ROCK inhibitor comprises Y-27632, Y-30141, Y-39983, Ki-23095, SLx-2119, thiazovibin, azaindole-1, fasudil, ripasudil, netalusdil, RKI-1447, or GSK429286A, or any combination thereof. In some embodiments, the SMAD pathway inhibitor includes A-83-01, DMH1, RepSox, LY365947, LY2109761, LY364947, SB431542, SB525334, SB505125, garnicertib, GW788388, LDN-193189, LDN-212854, hesperetin, or any combination thereof. In some embodiments, the growth medium contained within the insert includes EGF, Y-27632, A-83-01, or DMH1, or any combination thereof (including all four).

[0096] In some embodiments, esophageal progenitor cells are brought into contact with an EGF pathway activator. In some embodiments, the EGF pathway activator is or contains EGF. In some embodiments, esophageal progenitor cells are brought into contact with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 1 90 or 200 ng / mL, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or at least approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, or 200 ng / mL, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 The cells are brought into contact with an EGF pathway activator (e.g., EGF) at a concentration of 160, 170, 180, 190, or 200 ng / mL or less, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 10-200 ng / mL, 10-100 ng / mL, 100-200 ng / mL, 50-150 ng / mL, or 80-120 ng / mL. In some embodiments, esophageal progenitor cells are brought into contact with an EGF pathway activator (e.g., EGF) at a concentration of 100 ng / mL, about 100 ng / mL, at least 100 ng / mL, at least about 100 ng / mL, 100 ng / mL or less, or about 100 ng / mL or less.

[0097] In some embodiments, esophageal progenitor cells are brought into contact with a ROCK inhibitor. In some embodiments, the ROCK inhibitor is or includes Y-27632. In some embodiments, the esophageal progenitor cells are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 ROCK inhibitors (e.g., Y-27632) are brought into contact with a concentration of 8, 19, or 20 μM, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM or less, or at a concentration of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 1-20 μM, 1-10 μM, 10-20 μM, 5-15 μM, or 8-12 μM. In some embodiments, esophageal progenitor cells are exposed to a ROCK inhibitor (e.g., Y-27632) at a concentration of 10 μM, about 10 μM, at least 10 μM, at least about 10 μM, 10 μM or less, or about 10 μM or less.

[0098] In some embodiments, esophageal progenitor cells are brought into contact with a SMAD pathway inhibitor. In some embodiments, the SMAD pathway inhibitor is DMH1 and A-83-01 or comprises them. In some embodiments, esophageal progenitor cells are brought into contact with 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, 0.1, 0.5, The cells are brought into contact with the SMAD pathway inhibitor (e.g., DMH1 and A-83-01) at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM or less, or at a concentration of approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1 to 10 μM, 0.5 to 2 μM, 0.1 to 2 μM, or 0.5 to 5 μM. In some embodiments, the esophageal progenitor cells are brought into contact with the SMAD pathway inhibitor (e.g., DMH1 and A-83-01) at a concentration of 1 μM (e.g., 1 μM for each of DMH1 and A-83-01), approximately 1 μM, at least 1 μM, at least approximately 1 μM, 1 μM or less, or approximately 1 μM or less.

[0099] In some embodiments, enlarged esophageal progenitor cells are brought into contact with one or more (e.g., one, two, or three) of the EGF pathway activator, ROCK inhibitor, and SMAD pathway inhibitor in the insert. In some embodiments, the tissue culture vessel contains EGF. In some embodiments, the enlarged esophageal progenitor cells are cultured in the insert for a number of days that is 1, 2, 3, 4, 5, 6, 7, or 8 days, or about 1, 2, 3, 4, 5, 6, 7, or 8 days, or at least about 1, 2, 3, 4, 5, 6, 7, or 8 days, or within 1, 2, 3, 4, 5, 6, 7, or 8 days, or within about 1, 2, 3, 4, 5, 6, 7, or 8 days, or within a range defined by any two of the aforementioned values, for example, 1 to 8, 2 to 6, 4 to 8, or 1 to 4 days.

[0100] In some embodiments, enlarged esophageal progenitor cells are differentiated into esophageal raft cultures. In some embodiments, after the enlarged esophageal progenitor cells are brought into contact with one or more of EGF, ROCK inhibitors, and SMAD inhibitors in an insert member, the growth medium in the insert member is removed, and the tissue culture vessel contains an amount of growth medium such that the esophageal raft cultures are only partially immersed in the growth medium, so that the esophageal raft cultures are at the gas-liquid interface. In some embodiments, the esophageal raft cultures are cultured at the gas-liquid interface. In some embodiments, the tissue culture vessel contains EGF. In some embodiments, the esophageal raft cultures are kept at the gas-liquid interface for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or at least approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or within 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or approximately 5, 6, 7, 8, 9, 10, The culture is incubated for a number of days that is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or within a range defined by any two of the aforementioned values, for example, 5 to 30 days, 5 to 20 days, 10 to 25 days, 10 to 30 days, or 20 to 30 days. In some embodiments, culturing the esophageal raft culture at the gas-liquid interface matures the esophageal raft culture.

[0101] In some embodiments, esophageal progenitor cells differentiated from dorsal anterior foregut cells and expanded on a coated tissue culture vessel are dissociated into a single-cell suspension. In some embodiments, the expanded single-cell suspension of esophageal progenitor cells is plated onto an insert member (e.g., a transwell or cell insert) or a portion thereof (particularly the portion in contact with the cells or the permeable surface of the insert member) coated with an extracellular matrix or its components or mimics. In some embodiments, the insert member includes a surface that is permeable to the growth medium but impermeable to the cells. In some embodiments, the permeable surface of the insert member is 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10 μm, or 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or less, or a pore size of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or less, or any pore size within the range defined by any two of the aforementioned sizes, for example, having pore sizes of 0.1 to 10 μm, 0.1 to 5 μm, 1 to 5 μm, or 2 to 8 μm. In some embodiments, the permeable surface of the insert member has a pore size of 3 μm or about 3 μm. In some embodiments, the insert member is placed in a tissue culture vessel. In some embodiments, the growth medium contained in the insert member contains one or more (e.g., one, two, or three) of the following: an EGF pathway activator, a ROCK inhibitor, and a SMAD pathway inhibitor. In some embodiments, esophageal progenitor cells are brought into contact with the EGF pathway activator. In some embodiments, the EGF pathway activator is or contains EGF.In some embodiments, esophageal progenitor cells are contacted with an EGF pathway activator (e.g., EGF) at a concentration of 100 ng / mL, about 100 ng / mL, at least 100 ng / mL, at least about 100 ng / mL, 100 ng / mL or less, or about 100 ng / mL or less. In some embodiments, esophageal progenitor cells are contacted with a ROCK inhibitor. In some embodiments, the ROCK inhibitor is Y-27632 or contains it. In some embodiments, esophageal progenitor cells are contacted with a ROCK inhibitor (e.g., Y-27632) at a concentration of 10 μM, about 10 μM, at least 10 μM, at least about 10 μM, 10 μM or less, or about 10 μM or less. In some embodiments, esophageal progenitor cells are contacted with a SMAD pathway inhibitor. In some embodiments, the SMAD pathway inhibitor is DMH1 and A-83-01 or contains them. In some embodiments, esophageal progenitor cells are contacted with a SMAD pathway inhibitor (e.g., DMH1 and A-83-01) at a concentration of 1 μM (e.g., 1 μM each of DMH1 and A-83-01), about 1 μM, at least 1 μM, at least about 1 μM, 1 μM or less, or about 1 μM or less. In some embodiments, enlarged esophageal progenitor cells are contacted with one or more (e.g., one, two, or three) of an EGF pathway activator, a ROCK inhibitor, and a SMAD pathway inhibitor in an insert member. In some embodiments, the tissue culture vessel contains EGF. In some embodiments, enlarged esophageal progenitor cells are cultured in an insert for 1, 2, 3, 4, 5, 6, 7, or 8 days, or about 1, 2, 3, 4, 5, 6, 7, or 8 days, or at least about 1, 2, 3, 4, 5, 6, 7, or 8 days, or within 1, 2, 3, 4, 5, 6, 7, or 8 days, or within about 1, 2, 3, 4, 5, 6, 7, or 8 days, or within a range defined by any two of the aforementioned values, for example, 1 to 8, 2 to 6, 4 to 8, or 1 to 4 days.In some embodiments, enlarged esophageal progenitor cells are differentiated into esophageal raft cultures. In some embodiments, after the enlarged esophageal progenitor cells are brought into contact with one or more of EGF, ROCK inhibitors, and SMAD inhibitors in an insert member, the growth medium in the insert member is removed, and the tissue culture vessel contains an amount of growth medium such that the esophageal raft cultures are only partially immersed in the growth medium, so that the esophageal raft cultures are at the gas-liquid interface. In some embodiments, the esophageal raft cultures are cultured at the gas-liquid interface. In some embodiments, the tissue culture vessel contains EGF. In some embodiments, the esophageal raft cultures are kept at the gas-liquid interface for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or at least approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or within 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or approximately 5, 6, 7, 8, 9, 10, The culture is incubated for a number of days that is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or within a range defined by any two of the aforementioned values, for example, 5 to 30 days, 5 to 20 days, 10 to 25 days, 10 to 30 days, or 20 to 30 days. In some embodiments, culturing the esophageal raft culture at the gas-liquid interface matures the esophageal raft culture.

[0102] In some embodiments, dissociated esophageal progenitor cells are combined with enteric neural crest cells (ENCCs), and the combined esophageal progenitor cells and ENCCs are cultured to form a neurite-supplied esophageal raft culture. In some embodiments, the neurite-supplied esophageal raft culture comprises enteric neural crest cells (ENCCs), neural progenitor cells, and / or βIII-tubulin+ neurons. In some embodiments, the neural progenitor cells are SOX10+. In some embodiments, the esophageal progenitor cells and ENCCs are combined by slow centrifugation or other methods that aggregate the cells without excessive disruption. In some embodiments, the ENCCs are isolated as single cells derived from neurospheres that may be derived from pluripotent stem cells. Methods for producing ENCCs are generally known in the art, and methods for combining them with organoids are explored in International Publication No. 2016 / 061464 (which is expressly incorporated herein in its entirety by reference).

[0103] Exemplary method for esophageal raft culture In some embodiments, esophageal raft cultures are prepared from anterior foregut cells. In some embodiments, esophageal raft cultures and anterior foregut cells are originally prepared from iPSCs. In some embodiments, the iPSCs are hiPSCs. In some embodiments, anterior foregut cells are differentiated from iPSCs by one or more of the methods disclosed herein. In some embodiments, the method includes culturing iPSCs under conditions that differentiate them into endoderm cells and culturing endoderm cells under conditions that differentiate endoderm cells into anterior foregut cells. In some embodiments, the method includes culturing iPSCs with TGF-β superfamily growth factors to differentiate them into endoderm cells and culturing endoderm cells with one or more (e.g., at least one, two, three, or four) of Wnt protein or pathway activators, FGF protein or activators, BMP pathway inhibitors, or retinoic acid pathway activators to differentiate the endoderm cells into anterior foregut cells. In some embodiments, the method includes culturing iPSCs with activin A or BMP4, or both, to differentiate the iPSCs into endoderm cells, and culturing the endoderm cells with one or more of Wnt3a, FGF4, noggin, or retinoic acid (e.g., at least one, two, three, or four) to differentiate the endoderm cells into anterior foregut cells.In some embodiments, iPSCs are used at concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 19, 0, or 200 ng / mL, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, at least approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 1 The iPSCs are brought into contact with activin A at a concentration of 20, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or with any concentration within the range defined by any two of the aforementioned concentrations, for example, 10-200 ng / mL, 10-100 ng / mL, 100-200 ng / mL, or 50-150 ng / mL. In some embodiments, the iPSCs are cultured with 100 ng / mL of activin A.In some embodiments, iPSC is expressed in a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 1 60, 170, 180, 190, or 200 ng / mL, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 10 Is it 0, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or is it 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or is it approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, The cells are cultured with BMP4 at concentrations of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or with any concentration within the range defined by any two of the aforementioned concentrations, for example, 1-200 ng / mL, 1-100 ng / mL, 25-200 ng / mL, 1-80 ng / mL, or 25-100 ng / mL. In some embodiments, the iPSCs are cultured with 50 ng / mL of BMP4. In some embodiments, the iPSCs are cultured with activin A or BMP4, or both, for 1, 2, 3, 4, or 5 days. In some embodiments, endoderm cells are cultured with one or more of Wnt3a, FGF4, noggin, or retinoic acid, or any combination thereof (e.g., at least one, two, three, or four), each at a concentration sufficient to differentiate the endoderm cells into anterior foregut cells.In some embodiments, endoderm cells are expressed in concentrations of 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL, or approximately 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 5 40, 560, 580, or 600 ng / mL, or at least 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL, or at least approximately 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL, or 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL or less, or approximately 0, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 4 Culture with Wnt3a, FGF4, noggin, or retinoic acid, or any combination thereof (e.g., at least one, two, three, or four) at concentrations of 0, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 0-600 ng / mL, 0-200 ng / mL, 200-500 ng / mL, or 200-600 ng / mL.In some embodiments, endoderm cells are expressed in sizes of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or approximately 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or at least 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or at least approximately 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1 0.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM or less, or approximately 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, Culture with one or more of Wnt3a, FGF4, noggin, or retinoic acid (e.g., at least one, two, three, or four), or any combination thereof, at concentrations of 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 μM, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0-3.0 μM, 1.0-3.0 μM, 0-2.0 μM, or 1.5-3.0 μM.In some embodiments, endoderm cells are cultured with 500 ng / mL or about 500 ng / mL of Wnt3a, 500 ng / mL or about 500 ng / mL of FGF4, 200 ng / mL or about 200 ng / mL of noggin, and 2 μM or about 2 μM of retinoic acid. In some embodiments, endoderm cells are cultured with one or more of Wnt3a, FGF4, noggin, or retinoic acid (e.g., at least one, two, three, or four) for 1, 2, 3, 4, or 5 days.

[0104] In some embodiments, esophageal raft cultures are prepared from anterior foregut cells produced by one or more of the methods disclosed herein. In some embodiments, esophageal raft cultures are prepared by culturing anterior foregut cells under conditions that differentiate them into dorsal anterior foregut cells, culturing dorsal anterior foregut cells under conditions that differentiate them into esophageal progenitor cells, and culturing esophageal progenitor cells under conditions that differentiate them into esophageal raft cultures. In some embodiments, anterior foregut cells are cultured as a monolayer. In some embodiments, anterior foregut cells are not cultured as spheroids. In some embodiments, esophageal progenitor cells are cultured to expand before culturing them under conditions that differentiate them into esophageal raft cultures. In some embodiments, the conditions for differentiating esophageal progenitor cells into esophageal raft cultures include culturing esophageal progenitor cells at a gas-liquid interface.

[0105] In some embodiments, esophageal raft cultures are prepared from anterior foregut cells produced by one or more of the methods disclosed herein. In some embodiments, esophageal raft cultures are prepared by culturing anterior foregut cells with one or more (e.g., at least one, two, three, or four) of EGF pathway activators, BMP pathway inhibitors, FGF pathway activators, or growth promoters, or by culturing anterior foregut cells with one or more (e.g., at least one, two, three, or four) of EGF pathway activators, BMP pathway inhibitors, or FGF pathway activators, optionally, a neuronal precursor inhibitor, or any combination thereof, thereby differentiating the anterior foregut cells into dorsal anterior foregut cells, and dissociating the dorsal anterior foregut cells into single cells. The esophageal progenitor cells are prepared by differentiating intestinal cells into esophageal progenitor cells by culturing dorsal anterior foregut cells in a first tissue culture vessel containing a ROCK inhibitor, dissociating the esophageal progenitor cells into single cells, culturing the esophageal progenitor cells in and / or on the surface of an insert member (e.g., transwell) placed in a second tissue culture vessel (the insert member includes a surface that is permeable to growth medium but impermeable to cells, and the insert member and the second tissue culture vessel contain an amount of growth medium such that the esophageal progenitor cells are completely immersed in the growth medium), culturing the esophageal progenitor cells at the gas-liquid interface, and differentiating the esophageal progenitor cells into an esophageal raft culture. In some embodiments, the second tissue culture vessel is the same as the first tissue culture vessel. In some embodiments, the anterior foregut cells are contacted with EGF, noggin, and FGF10. In some embodiments, the anterior foregut cells are contacted with EGF, noggin, FGF10, and CultureOne adjuvants or some other neurotransmitter inhibitors (e.g., cytarabine).In some embodiments, the anterior foregut cells are 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 , 180, 190, or 200 ng / mL, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or at least approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 , 140, 150, 160, 170, 180, 190, or 200 ng / mL, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 The EGF pathway activator (e.g., EGF) is brought into contact with the EGF pathway activator (e.g., EGF) at a concentration of 10, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 10-200 ng / mL, 10-150 ng / mL, or 50-200 ng / mL.In some embodiments, the anterior foregut cells are 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 2 60, 270, 280, 290, or 300 ng / mL, or at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or at least approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 , 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL or less, or approximately 100, 110, 120, 130, 140, 150, 160 The BMP pathway inhibitor (e.g., noggin) is brought into contact with the BMP pathway inhibitor at a concentration of 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, at concentrations of 100-300 ng / mL, 100-250 ng / mL, or 150-300 ng / mL.In some embodiments, the anterior foregut cells are expressed in a concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95 or 100 ng / mL, or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng / mL, or at least approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, The FGF pathway activator (e.g., FGF10) is brought into contact with the FGF pathway activator at a concentration of 75, 80, 85, 90, 95, or 100 ng / mL, or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL or less, or at a concentration of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 5 to 100 ng / mL, 5 to 75 ng / mL, or 25 to 100 ng / mL. In some embodiments, the anterior foregut cells are concentrated at concentrations of 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2 times the manufacturer's recommended concentration, or approximately 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2 times, or at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2 times, or at least approximately 0.2 A concentrated additive that is 5 times, 0.5 times, 0.75 times, 1 time, 1.25 times, 1.5 times, 1.75 times, or 2 times, or 0.25 times, 0.5 times, 0.75 times, 1 time, 1.25 times, 1.5 times, 1.75 times, or 2 times or less, or approximately 0.25 times, 0.5 times, 0.75 times, 1 time, 1.25 times, 1.5 times, 1.75 times, or 2 times or less, is brought into contact with a growth stimulant (e.g., CultureOne) or another neuronal precursor inhibitor.In some embodiments, the growth stimulant or neuronal precursor inhibitor is provided in a 1:1 ratio. In some embodiments, anterior foregut cells are cultured as a monolayer. In some embodiments, anterior foregut cells are not cultured as spheroids. In some embodiments, dorsal anterior foregut cells are cultured in a first tissue culture vessel on an extracellular matrix or its components or mimics. In some embodiments, esophageal progenitor cells are cultured in and / or on the surface of an insert member on an extracellular matrix or its components or mimics. In some embodiments, esophageal progenitor cells are cultured in an insert member with one or more (e.g., at least one, two, or three) of EGF pathway activators, ROCK inhibitors, or SMAD inhibitors, or any combination thereof, and with EGF in a second tissue culture vessel. In some embodiments, the gas-liquid interface includes removing growth medium from the insert member so that the second tissue culture vessel and / or insert member contains an amount of growth medium such that the esophageal progenitor cells are only partially immersed in the growth medium. In some embodiments, esophageal progenitor cells are brought into contact with EGF, Y-27632, DMH1, and A-83-01.In some embodiments, esophageal progenitor cells are present in 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 1 90 or 200 ng / mL, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or at least approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, or 200 ng / mL, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL or less, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 The EGF pathway activator (e.g., EGF) is brought into contact with the EGF pathway activator (e.g., EGF) at a concentration of 160, 170, 180, 190, or 200 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 10-200 ng / mL, 10-100 ng / mL, 100-200 ng / mL, 50-150 ng / mL, or 80-120 ng / mL.In some embodiments, the esophageal progenitor cells are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 ROCK inhibitors (e.g., Y-27632) are brought into contact with a concentration of 8, 19, or 20 μM, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM or less, or at a concentration of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 1-20 μM, 1-10 μM, 10-20 μM, 5-15 μM, or 8-12 μM. In some embodiments, the esophageal progenitor cells are 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. The SMAD inhibitor (e.g., DMH1 and A-83-01) is brought into contact with the SMAD inhibitor (e.g., DMH1 and A-83-01) at a concentration of 0.1-10 μM, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1-10 μM, 0.5-2 μM, 0.1-2 μM, or 0.5-5 μM (for example, for DMH1 and A-83-01 respectively).

[0106] In some embodiments, esophageal raft cultures are prepared from anterior foregut cells produced by one or more of the methods disclosed herein. In some embodiments, esophageal raft cultures are prepared by culturing anterior foregut cells with one or more of EGF, Noggin, FGF10, or CultureOne adjuvants (e.g., at least one, two, three, or four) to differentiate the anterior foregut cells into dorsal anterior foregut cells, dissociating the dorsal anterior foregut cells into single cells, culturing the dorsal anterior foregut cells in a first tissue culture vessel containing Y-27632 to differentiate the dorsal anterior foregut cells into esophageal progenitor cells, dissociating the esophageal progenitor cells into single cells, culturing the esophageal progenitor cells in and / or on the surface of an insert member (e.g., transwell) placed in a second tissue culture vessel (the insert member includes a surface that is permeable to growth medium but impermeable to cells, and the insert member and the second tissue culture vessel contain an amount of growth medium such that the esophageal progenitor cells are completely immersed in the growth medium), and culturing the esophageal progenitor cells at the gas-liquid interface to differentiate the esophageal progenitor cells into esophageal raft cultures. In some embodiments, the second tissue culture vessel is the same as the first tissue culture vessel. In some embodiments, anterior foregut cells are cultured as a monolayer. In some embodiments, anterior foregut cells are not cultured as spheroids. In some embodiments, dorsal anterior foregut cells are cultured on collagen type IV in the first tissue culture vessel. In some embodiments, esophageal progenitor cells are cultured on collagen type IV in and / or on the surface of the insert member. In some embodiments, esophageal progenitor cells are cultured in and / or on the surface of the insert member together with one or more of EGF, Y-27632, DMH1, or A-83-01 (e.g., at least one, two, three, or four), and together with EGF in the second tissue culture vessel. In some embodiments, the gas-liquid interface includes removing growth medium from the insert member so that the second tissue culture vessel and / or the insert member contains an amount of growth medium such that the esophageal progenitor cells are only partially immersed in the growth medium.In some embodiments, the anterior foregut cells are 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or at least approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 18 0, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL or less, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 13 Culture the cells with one or more of EGF, noggin, or FGF10 (e.g., at least one, two, or three) at concentrations of 0, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL or less, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 10-300 ng / mL, 10-200 ng / mL, 100-200 ng / mL, or 50-200 ng / mL. In some embodiments, the anterior foregut cells are cultured with 100 ng / mL or about 100 ng / mL of EGF.In some embodiments, anterior foregut cells are cultured with 200 ng / mL or approximately 200 ng / mL of noggin. In some embodiments, anterior foregut cells are cultured with 50 ng / mL or approximately 50 ng / mL of FGF10. In some embodiments, anterior foregut cells are cultured with 1x CultureOne adjuvant. In some embodiments, anterior foregut cells are cultured for 1, 2, 3, 4, or 5 days. In some embodiments, dorsal anterior foregut cells are dissociated using Accutase. In some embodiments, the dorsal anterior foregut cells are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 The cells are cultured in a first tissue culture vessel containing Y-27632 at concentrations of 5, 16, 17, 18, 19, or 20 μM, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM or less, or at concentrations of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 1–20 μM, 5–15 μM, or 8–12 μM. In some embodiments, dorsal anterior foregut cells are cultured in the first tissue culture vessel with 10 μM or approximately 10 μM of Y-27632. In some embodiments, dorsal anterior foregut cells are cultured in a first tissue culture vessel at 1.5 μg type IV collagen / cm³. 2The cells are cultured on a culture surface area. In some embodiments, esophageal progenitor cells are cultured in and / or on the surface of an insert member that includes a surface that is permeable to the growth medium but impermeable to the cells. In some embodiments, the permeable surface of the insert member is 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm, or at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or The pore size is 10 μm, or 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or less, or approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or less, or any pore size within the range defined by any two of the aforementioned sizes, for example, pore sizes of 0.1 to 10 μm, 0.1 to 5 μm, 5 to 10 μm, or 1 to 5 μm. In some embodiments, the permeable surface of the insert member has a pore size of 3 μm, approximately 3 μm, at least 3 μm, at least approximately 3 μm, 3 μm or less, or approximately 3 μm or less.In some embodiments, the esophageal progenitor cells are 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 2 70, 280, 290, or 300 ng / mL, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or at least approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL or less, or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, EGF, Y-27632, DMH1, or A-83-01 are cultured together with one or more of them (e.g., at least one, two, three, or four) in an insert member and / or on its surface at concentrations of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng / mL or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 10-300 ng / mL, 10-200 ng / mL, 100-200 ng / mL, or 50-200 ng / mL.In some embodiments, esophageal progenitor cells are 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM It is either 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at least approximately 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or 0 It is cultured in and / or on the surface of the insert material together with one or more of EGF, Y-27632, DMH1, or A-83-01 (e.g., at least one, two, three, or four) at concentrations of 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM or less, or at any concentration within the range defined by any two of the aforementioned concentrations, for example, 0.1-20 μM, 5-15 μM, 0.1-4 μM, or 8-12 μM. In some embodiments, esophageal progenitor cells are cultured in and / or on the surface of the insert with 100 ng / mL or about 100 ng / mL of EGF. In some embodiments, esophageal progenitor cells are cultured in and / or on the surface of the insert with 10 μM or about 10 μM of Y-27632. In some embodiments, esophageal progenitor cells are cultured in and / or on the surface of the insert with 1 μM or about 1 μM of DMH1. In some embodiments, esophageal progenitor cells are cultured in and / or on the surface of the insert material with 1 μM or approximately 1 μM of A-83-01. In some embodiments, esophageal progenitor cells are cultured in and / or on the surface of the insert material with one or more of EGF, Y-27632, DMH1, or A-83-01 (e.g., at least one, two, three, or four). In some embodiments, esophageal progenitor cells are cultured with 1.5 μg type IV collagen / cm³ 2 The cells are cultured within and / or on the surface of the insert material with a culture surface area. In some embodiments, the esophageal progenitor cells are cultured at the gas-liquid interface in a second tissue culture vessel with 100 ng / mL or about 100 ng / mL of EGF. In some embodiments, the esophageal progenitor cells are cultured at the gas-liquid interface for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0107] In some embodiments, the esophageal raft culture is produced by one or more of the methods described herein. In some embodiments, the esophageal raft culture is a human esophageal raft culture. In some embodiments, the esophageal raft culture is derived from human cells. In some embodiments, the esophageal raft culture is derived from human iPSCs. In some embodiments, the esophageal raft culture is not derived from spheroids or organoids. In some embodiments, the esophageal raft culture is not cultured on the extracellular matrix or its components or mimics. In some embodiments, the esophageal raft culture is not cultured on rat collagen type I matrix or Matrigel, or both. In some embodiments, the esophageal raft culture is not cultured with heterologous components. In some embodiments, the esophageal raft culture is cultured on human collagen type IV. In some embodiments, the esophageal raft culture is not cultured on feeder cell substrate. In some embodiments, the esophageal raft culture is not cultured on mouse fibroblasts. In some embodiments, the esophageal raft culture is not cultured on irradiated mouse fibroblasts.

[0108] Characteristics of esophageal raft cultures In some embodiments, the esophageal raft culture is produced by one or more of the methods described herein. In some embodiments, the esophageal raft culture includes a stratified squamous epithelial cell layer. In some embodiments, the stratified squamous epithelial cell layer includes a suprabaddicular and basal lamina. In some embodiments, the stratified squamous epithelial cell layer is positive for E-cadherin (Ecad). In some embodiments, the suprabaddicular layer is positive for keratin 13 (keratin13, KRT13) or keratin 8 (keratin8, KRT8), or both. In some embodiments, the basal lamina is positive for one or more (e.g., at least one, two, or three) of the sex-determining region Y-box 2 (SOX2), tumor protein P63 (p63), or keratin 5 (keratin5, KRT5), or any combination thereof. In some embodiments, the esophageal raft culture includes a mesenchymal layer. In some embodiments, the mesenchymal layer includes muscle fibers. In some embodiments, the mesenchymal layer is positive for one or more of the following (e.g., at least one, two, or three): forkhead box protein F1 (FOXF1), homeobox protein Nkx-6.1 (NKX6-1), or vimentin, or any combination thereof. In some embodiments, muscle fibers are positive for desmin. In some embodiments, the esophageal raft culture lacks lamina propria or has reduced or substantially reduced lamina propria compared to esophageal tissue from adult animals of the same species as the esophageal raft culture (e.g., reduced by 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, the esophageal raft culture is substantially devoid of neural progenitor cells and / or βIII-tubulin+ neurons. In some embodiments, the esophageal raft culture further comprises enteric neural crest cells (ENCCs). In some embodiments, the esophageal raft culture further comprises ENCCs, neural progenitor cells, and / or βIII-tubulin+ neurons, such that the esophageal raft culture is a neurite-supplied raft culture. In some embodiments, the neural progenitor cells are SOX10+.

[0109] In some embodiments, the esophageal raft culture produced by any of the methods disclosed herein is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least about 1, 5, 10, 20, 30, 40, 50, Having a thickness of 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or any thickness within the range defined by any two of the aforementioned lengths, for example, a thickness of 1 to 500 μm, 10 to 300 μm, 50 to 100 μm, 1 to 100 μm, or 100 to 500 μm. In some embodiments, the esophageal raft culture produced by any of the methods disclosed herein is 150, 200, 250, 300, 350, 400, 450, or 500 μm, or about 150, 200, 250, 300, 350, 400, 450, or 500 μm, or at least 150, 200, 250, 300, 350, 400, 450, or 500 μm, or at least about 150, 200, 250, Having a thickness of 300, 350, 400, 450, or 500 μm, or 150, 200, 250, 300, 350, 400, 450, or 500 μm or less, or a thickness of approximately 150, 200, 250, 300, 350, 400, 450, or 500 μm or less, or any thickness within the range defined by any two of the aforementioned thicknesses, for example, a thickness of 150 to 500 μm, 150 to 350 μm, or 250 to 500 μm.

[0110] In some embodiments, the esophageal raft culture produced by any of the methods disclosed herein is 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 cm 2 or approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 cm 2 or at least 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 cm 2 or at least approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 cm 2 or 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 cm 2 The following, or approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 cm 2 The following surface areas, or any surface area within the range defined by any two of the aforementioned surface areas, for example, 0.1 to 100 cm². 2 , 10-80cm 2 , 20-40cm 2 , 0.1~30cm 2 , or 50-100cm 2 It has a surface area of ​​0.1, 0.5, 1, 1.5, or 2 cm. In some embodiments, the esophageal raft culture produced by any of the methods disclosed herein has a surface area of ​​0.1, 0.5, 1, 1.5, or 2 cm. 2 or approximately 0.1, 0.5, 1, 1.5, or 2 cm 2 or at least 0.1, 0.5, 1, 1.5, or 2 cm 2 or at least approximately 0.1, 0.5, 1, 1.5, or 2 cm 2 Either 0.1, 0.5, 1, 1.5, or 2 cm 2 The following, or approximately 0.1, 0.5, 1, 1.5, or 2 cm 2The following surface area, or any surface area within the range defined by any two of the aforementioned surface areas, for example, 0.1 to 2 cm 2 , 0.1 to 1 cm 2 , or 0.5 to 2 cm 2 has a surface area of

[0111] In some embodiments, an esophageal raft culture produced by any of the methods disclosed herein is 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 , 1, 5, or 10 cm 3 or is about 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 , 1, 5, or 10 cm 3 or is at least 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 , 1, 5, or 10 cm 3 or is at least about 10 -5 , 10 -4 , 10 -3 , 10 -2 , 10[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​, or 1-10cm 3 It has the volume of .

[0112] In some embodiments, the stratified squamous epithelium layer of esophageal raft cultures produced by any of the methods disclosed herein is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least about 1, 5, 10, 20, 30, 40 , 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or any thickness within the range defined by any two of the aforementioned lengths, for example, a thickness of 1 to 500 μm, 20 to 200 μm, 50 to 100 μm, 1 to 100 μm, or 100 to 500 μm.In some embodiments, the stratified squamous epithelial cell layer of esophageal raft cultures produced by any of the methods disclosed herein is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm, or approximately 50, 60, 70, 80, 90, 100, 110 , 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm, or at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm, or at least approximately 50, 60, Is it 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm, or is it 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm or less? , or thicknesses of approximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 μm or less, or any thickness within the range defined by any two of the aforementioned thicknesses, for example, thicknesses of 50-250 μm, 50-150 μm, or 100-250 μm.

[0113] In some embodiments, the basal upper layer of esophageal raft cultures produced by any of the methods disclosed herein is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least about 1, 5, 10, 20, 30, 40, 5 Having a thickness of 0, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or any thickness within the range defined by any two of the lengths mentioned above, for example, a thickness of 1 to 500 μm, 20 to 200 μm, 50 to 100 μm, 1 to 100 μm, or 100 to 500 μm.In some embodiments, the basal upper layer of the esophageal raft culture produced by any of the methods disclosed herein is 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 190, 190, or 200 μm, or about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 190, 190, or 200 μm, or at least 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 190, 190, or 200 μm, or at least about 80, 90, 100 , 120, 130, 140, 150, 160, 170, 190, 190, or 200 μm, or 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 190, 190, or 200 μm or less, or a thickness of approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 190, 190, or 200 μm or less, or any thickness within the range defined by any two of the aforementioned thicknesses, for example, a thickness of 80-200 μm, 80-150 μm, or 100-200 μm.

[0114] In some embodiments, the basal layer of esophageal raft cultures produced by any of the methods disclosed herein is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least about 1, 5, 10, 20, 30, 40, 5 Having a thickness of 0, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or any thickness within the range defined by any two of the lengths mentioned above, for example, a thickness of 1 to 500 μm, 20 to 200 μm, 50 to 100 μm, 1 to 100 μm, or 100 to 500 μm. In some embodiments, the basal layer of the esophageal raft culture produced by any of the methods disclosed herein is 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μm, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μm, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μm, or at least about 10, 20, 30 , 50, 60, 70, 80, 90, or 100 μm, or 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μm or less, or a thickness of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μm or less, or any thickness within the range defined by any two of the aforementioned thicknesses, for example, having a thickness of 10 to 100 μm, 10 to 50 μm, or 50 to 100 μm.

[0115] In some embodiments, the mesenchymal layer of esophageal raft cultures produced by any of the methods disclosed herein is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or at least about 1, 5, 10, 20, 30, 40, 5 Having a thickness of 0, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm, or 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or a thickness of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μm or less, or any thickness within the range defined by any two of the lengths mentioned above, for example, a thickness of 1 to 500 μm, 20 to 200 μm, 50 to 100 μm, 1 to 100 μm, or 100 to 500 μm. In some embodiments, the mesenchymal layer of esophageal raft cultures produced by any of the methods disclosed herein is 100, 150, 200, 250, 300, 350, or 400 μm, or about 100, 150, 200, 250, 300, 350, or 400 μm, or at least 100, 150, 200, 250, 300, 350, or 400 μm, or at least about 100, 150, 20 Having a thickness of 0, 250, 300, 350, or 400 μm, or 100, 150, 200, 250, 300, 350, or 400 μm or less, or approximately 100, 150, 200, 250, 300, 350, or 400 μm or less, or any thickness within the range defined by any two of the aforementioned thicknesses, for example, 100-400 μm, 100-200 μm, or 200-400 μm.

[0116] In some embodiments, the esophageal raft culture is produced by one or more of the methods described herein. In some embodiments, the esophageal raft culture contains neural structures. In some embodiments, the esophageal raft culture contains cells expressing neural markers such as SOX2 or βIII-tubulin.

[0117] In some embodiments, the esophageal raft culture does not contain angiogenesis, blood vessels, and / or endothelial cells.

[0118] Transplantation and treatment methods In some embodiments, the esophageal raft cultures or esophageal raft cell compositions described herein are transplanted or grafted into a host organism, for example, as a therapeutic or experimental model. In some embodiments, transplantation is performed on the raft culture for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 4 7, 48, 49, or 50 days, or at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or within 50 days, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,The culture is performed after culturing for a number of days that is 47, 48, 49, or 50 days, or any number of culture days within the range defined by any two of the aforementioned number of days, for example, 1 to 50 days, 10 to 40 days, 20 to 30 days, 1 to 30 days, or 20 to 50 days. In some embodiments, the raft culture is sufficiently mature for transplantation and / or study a number of days before esophageal organoids prepared by other methods known in the art reach the same or similar state of maturity, and the number of days is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 3 5, 36, 37, 38, 39, or 40 days, or approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or at least within approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, Or within approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or any number of days within the range defined by any two of the aforementioned number of days, e.g., 5-40 days, 10-40 days, 20-30 days, or 5-30 days. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is an immunodeficient mammal. In some embodiments, the host organism is an immunodeficient mouse. In some embodiments, the host organism is a monkey, cat, dog, hamster,or rat. In some embodiments, the host organism is an immunodeficient monkey, cat, dog, hamster, or rat. In some embodiments, the host organism is a human. In some embodiments, the host organism is an immunodeficient human. In some embodiments, the host organism is an immunocompetent human. In some embodiments, the host organism is an immunocompetent human treated with an immunosuppressant. In some embodiments, the raft culture is autologous to the host organism. In some embodiments, the raft culture is homogeneous to the host organism. In some embodiments, the host organism is a mammal requiring an esophageal transplant or graft. In some embodiments, the host organism is a human requiring an esophageal transplant or graft.

[0119] In some embodiments, esophageal raft cultures or esophageal raft cell compositions function as clinically beneficial tissues that can be used to study or treat a variety of different pathological conditions, including but not limited to achalasia, Barrett's esophagus, esophageal cancer, gastroesophageal reflux disease (GERD), dysphagia, heartburn, eosinophilic esophagitis, paraesophageal hernia, or esophageal perforation. In some embodiments, esophageal raft cultures or esophageal raft cell compositions are used to evaluate pharmacological behavior, cell signaling, peristalsis, cancer formation and migration, or transplantation / grafting, or any combination thereof. [Examples]

[0120] Some aspects of the embodiments discussed herein are further disclosed in the following examples, and these examples are not intended to limit the scope of this disclosure. Those skilled in the art will understand that many other embodiments, as described herein and in the claims, are also included within the scope of this disclosure.

[0121] Example 1. Generation of esophageal raft cultures containing epithelium and mesenchyme. Esophageal raft cultures were produced according to the exemplary schematic diagram shown in Figure 1.

[0122] Human PSCs (hPSCs) were cultured on hESC-compatible Matrigel-coated 10cm plates and differentiated into esophageal monolayers (day 9). From day 0 to 6, the growth medium containing the provided adjuvants was changed daily. From day 0 to 1, hPSCs were cultured in RPMI1640 supplemented with 50 ng / mL BMP4 (R&D Systems) and 100 ng / mL activin A (R&D Systems). From day 1 to 2, hPSCs were cultured in RPMI1640 supplemented with 100 ng / mL activin A and 0.2% fetal bovine serum (FBS). From day 2 to 3, hPSCs were cultured in RPMI1640 supplemented with 100 ng / mL activin A and 2% FBS. At the end of day 3, hPSCs were differentiated into endoderm cells.

[0123] Next, endoderm cells were differentiated into anterior foregut monolayer cells according to the following procedure: From day 3 to 5, endoderm cells were cultured in RPMI1640 supplemented with 500 ng / mL Wnt3a (R&D systems), 500 ng / mL FGF4 (R&D systems), 200 ng / mL noggin (BMP inhibitor, R&D systems), and 2% FBS. From day 5 to 6, endoderm cells were cultured in RPMI1640 supplemented with 500 ng / mL FGF4, 200 ng / mL noggin, 2 μM retinoic acid (RA, Sigma), and 2% FBS. At the end of day 6, endoderm cells were differentiated into anterior foregut monolayer cells.

[0124] Next, anterior foregut monolayer cells were differentiated into dorsal anterior foregut cells and esophageal progenitor cells. From day 6 to day 9, anterior foregut monolayer cells were cultured in Advanced DMEM / F12 supplemented with 100 ng / mL EGF (R&D Systems), 200 ng / mL noggin, and 50 ng / mL FGF10 to pattern them into dorsal anterior foregut cells.

[0125] Optionally, in addition to other growth factors added daily as provided herein, 1x CultureOne adjuvant (Cult1) (GIBCO) may be added on days 6–9 (shown in Figure 1). Alternatively, Figure 3A shows 1x CultureOne added optionally from day 0 to day 9. CultureOne was removed from the culture after day 9. Adding CultureOne can reduce neuronal congestion, and adding it earlier further reduces neuronal congestion in the mesenchymal layer of the raft culture. Alternative neuronal precursor inhibitors such as cytarabine (ara-C) are considered. In other embodiments, the CultureOne adjuvant is not included in the culture conditions.

[0126] On day 9, the differentiated dorsal anterior foregut monolayer was dissociated into a single-cell suspension using Accutase, and a collagen type IV (human placental-derived) coated plate (1.5 μg collagen / cm³) was placed in serum-free medium (SFM) (GIBCO, Carlsbad, CA, USA) supplemented with 10 μM Y-27632 (ROCK inhibitor). 2 ) Approximately 1.8 × 10 4 cells / cm 2 Then, in order to differentiate them into esophageal progenitor cells, the cells were cultured for 5-6 days, changing the growth medium every other day, until they reached confluence.

[0127] Once the esophageal progenitor cells reached confluence, they were dissociated into single-cell suspensions using 0.05% trypsin-EDTA and coated with collagen type IV (1.5 μg collagen / cm³). 2Cells were cultured on a 3 μm pore size polycarbonate membrane cell insert (Corning). For the first 5 days of culture on the cell insert, cells were cultured daily in fresh medium in the upper (insert) and lower (plate) compartments. The upper compartment was supplied with Advanced DMEM / F12 supplemented with 100 ng / mL EGF, 10 μM Y-27632, 1 μM DMH1, and 1 μM A83-01 (SMAD inhibitor). The lower compartment was supplied with Advanced DMEM / F12 supplemented with 100 ng / mL EGF. After 5 days, the cells were moved to the gas-liquid interface and cultured daily in fresh medium only in the bottom compartment. In this way, the epithelium of the raft culture, which is the more apical layer, is exposed to air.

[0128] Cells at all stages were cultured under standard 37°C, 5% CO2 incubation conditions.

[0129] Example 2. Observation of esophageal raft cultures Esophageal raft cultures produced by the method described herein could be differentiated and cultured in 10 cm plates rather than in 24-well plates. There was no spheroid / organoid stage prior to differentiation into raft cultures. Instead, cells were cultured in a monolayer and then in cell inserts. Feeder cell matrix (e.g., rat collagen type I matrix with irradiated mouse fibroblasts) was not required to culture the raft cultures. Instead, the plates and inserts, particularly the surface in contact with the cells or the permeable surface of the inserts, were coated with human-derived collagen type IV. Matrigel and other basement membrane matrices were also not required. This is an important consideration for scaling to larger cultures and cGMP production. The gas-liquid interface initiated approximately two weeks after the start of esophageal differentiation. In comparison, previous protocols for esophageal raft cultures and organoids involved gas-liquid interface initiation approximately 40 days after the start of differentiation.

[0130] Importantly, the esophageal raft cultures described herein contain both epithelium and mesenchyme, whereas previous raft cultures and organoids lacked mesenchyme. A small population of mesenchymal progenitor cells was present in the monolayer at days 6–9, but the mesenchymal population expanded during culture on collagen type IV coated plates in supplemented keratinocyte SFM medium at days 9–14 (as described in Example 1). By using the entire monolayer instead of collecting only spontaneously arising spheroids as in previous protocols, it was possible to expand the small precursor population that would otherwise be lost. Figure 2 shows the morphology of the esophageal raft culture. The stratified squamous epithelium, indicated by E-cadherin, is subdivided into the superbasal layer indicated by keratin 13 (KRT13) and keratin 8 (KRT8), and the basal layer indicated by SOX2, P63, and keratin 5 (KRT5). Beneath the epithelium, the mesenchyme, indicated by the mesenchymal cell markers FOXF1, NKX6-1, and vimentin, contains differentiated muscle cells (desmin).

[0131] Figure 3B shows immunofluorescence images comparing esophageal raft cultures treated with CultureOne adjuvant at either day 0–9 or day 6–9 of culture. The presence of potentially undesirable neuronal cell types is indicated by the expression of SOX2 (arrow) or βIII-tubulin (arrow) in the Ecad-negative mesenchymal layer. SOX2 is normally expressed in Ecad+ esophageal epithelium.

[0132] Example 3. Differentiation and co-culture of enteric neural crest cells (ENCCs) into esophageal raft cultures. Esophageal raft cultures can be innervated by combining them with enteric neural crest cells (ENCCs) during culture and differentiation (Figure 4A).

[0133] hPSCs were cultured on hESC-compatible Matrigel-coated plates and treated with collagenase IV (500 U / mL, Gibco) in mTeSR1 at 37°C for 60–90 minutes to detach colonies. The cells were then washed with DMEM / F-12 (Gibco) and transferred to 15 mL conical tubes. Once the cells had pelleted at the bottom of the tubes, the DMEM / F-12 was removed, the cells were gently pulverized, and resuspended in nerve induction medium. The neuronal induction medium consisted of Neurobasal Medium (Gibco) supplemented with a 1:1 ratio of DMEM / F12-GlutaMAX (Gibco), B27 adjuvant (0.5x, Gibco), N2 adjuvant (0.5x, Gibco), pen-strep (1x, Gibco), insulin (5 μg / mL, Sigma-Aldrich), FGF2 (20 ng / mL, R&D Systems), and EGF (20 ng / mL, R&D Systems). Cells were cultured on non-tissue culture treated 60 mm Petri dishes (Fisherbrand). The neuronal induction medium was changed daily for 5 days, and 2 μM retinoic acid (RA) was added to the medium on days 4 and 5 for posteriorizing. On day 6, suspended neurospheres were collected and incubated for another 4 days in nerve induction medium (RA-free) with human fibronectin (HFN) coated plates (3 μg / cm³ diluted in PBS). 2The cells were cultured on Corning (Esophageal Raft) plates. Confluent cells were then collected by a short Accutase treatment of 2-3 minutes and cultured again on HFN-coated plates for a further 4 days in RA-free nerve induction medium. At this stage, the cells were again collected and counted by a short Accutase treatment, recombined with esophageal progenitor cells (day 13 of the esophageal raft culture differentiation process disclosed herein), and cultured on cell inserts. The ENCC-esophageal progenitor cell seeding ratio was approximately 2:1. The co-culture was maintained under the same conditions as the esophageal culture lacking ENCC. The upper compartment was supplied with Advanced DMEM / F12 supplemented with 100 ng / mL EGF, 10 μM Y-27632, 1 μM DMH1, and 1 μM A83-01 (SMAD inhibitor). The lower compartment was supplied with Advanced DMEM / F12 supplemented with 100 ng / mL EGF. After 5 days, the cells are moved to the gas-liquid interface and cultured daily in fresh medium only in the bottom compartment.

[0134] Figure 4B shows typical markers of innervated esophageal raft cultures. The raft cultures express typical esophageal epithelial markers (SOX2, P63, KRT5, KRT13, and KRT8) and the common epithelial marker E-cadherin (Ecad). The integrated GFP-expressing ENCC, indicated by the arrow, innervates the esophageal raft culture mesenchyme, indicated by the expression of vimentin and the neuronal marker βIII-tubulin. βIII-tubulin expression is co-localized with GFP expression, indicating that the innervating nerves arose from hPSC-GFP that directly differentiated into ENCCs, rather than from neuronal interference during esophageal raft differentiation.

[0135] In at least some of the embodiments described above, one or more elements used in the embodiments may be used interchangeably in other embodiments unless such substitution is technically feasible. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter as defined by the appended claims.

[0136] With regard to the use of substantially any plural and / or singular terms herein, those skilled in the art can convert from plural to singular and / or singular to plural as appropriate to the context and / or use. Various singular / plural substitutions may be explicitly stated herein for clarity.

[0137] Regarding the use of "e.g.", it is understood to mean "for example," and therefore is a non-restrictive example.

[0138] In general, it will be understood by those skilled in the art that the terms used herein, in particular in the appended claims (e.g., in the text of the appended claims), are generally intended to be “non-limiting” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” and the term “including” should be interpreted as “including but not limited to,” etc.). If a particular number of claims to be introduced is intended, such intent will be explicitly stated in the claims, and if such statement is not present, such intent will not be present, as will be understood by those skilled in the art. For example, for the sake of understanding, the following appended claims may include the use of the introductory phrases “at least one” and “one or more” to introduce the claims. However, the use of such phrases should not be interpreted as implying that the introduction of a claim by the indefinite article "a" or "an" limits any particular claim containing such introduced claim to embodiments containing only one such claim (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim descriptions. In addition, even if a specific number of claims being introduced is explicitly stated, a person skilled in the art will recognize that such a statement should be interpreted as meaning at least the number stated (for example, the explicit statement "two claims" without other modifying phrases means at least two claims or two or more claims). Furthermore, when a rule similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in a sense that a person skilled in the art would understand the rule (for example, "a system having at least one of A, B, and C" includes, but is not limited to, a system having only A, only B, only C, both A and B, both A and C, both B and C, and / or a system having both A, B, and C, etc.).In these cases where a rule similar to “at least one of A, B, or C” is used, such a construction is generally intended in a sense that a person skilled in the art would understand the rule to be (for example, “a system having at least one of A, B, or C” includes, but is not limited to, A only, B only, C only, A and B together, A and C together, B and C together, and / or a system having A, B, and C together). A person skilled in the art will further understand that substantially any disjunctive word and / or phrase presenting two or more alternative terms should be understood, whether in a description, claim, or drawing, to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase “A or B” is understood to include the possibilities of “A” or “B” or “A and B”.

[0139] In addition, if any feature or aspect of the present disclosure is described in relation to the Markush group, a person skilled in the art will recognize that the present disclosure also describes any individual member or subgroup of a member of the Markush group.

[0140] For all purposes, including providing written explanations, as will be understood by those skilled in the art, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. Any enumerated scope can be readily recognized as fully explaining and enabling that the same scope may be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can readily be decomposed into a lower third, middle third, upper third, etc. As will be understood by those skilled in the art, words such as “maximum,” “at least,” “greater than,” and “less than” include the number stated and refer to scopes that can be decomposed into subscopes discussed later herein. Finally, as will be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0141] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to limit, and the true scope and spirit are indicated by the following claims.

[0142] All references cited herein, including but not limited to published and unpublished applications, patents, and references, are incorporated in their entirety by reference and are thus part of this Specified Publication and Patent or Patent Application incorporated by Reference to the extent that they conflict with any disclosure contained herein, this Specified Publication and Patent or Patent Application incorporated by Reference is intended to supersede and / or take precedence over any such conflicting material.

Claims

1. An in vitro esophageal raft culture composition, A stratified squamous epithelium including the basal layer and basal layer, The mesenchymal layer contains muscle fibers, and The stratified squamous epithelial layer is E-cadherin ＋ The base upper layer is KRT13 ＋ and KRT8 ＋ The base layer is SOX2 ＋ , P63 ＋ , and KRT5 ＋ And, The mesenchyme is FOXF1 ＋ , NKX6-1 ＋ , and vimentin ＋ , and the muscle fibers are desmin ＋ , an in vitro esophageal raft culture composition.

2. A method for producing esophageal raft cultures, (a) A step of differentiating anterior foregut cells into dorsal anterior foregut cells, the step being: Activating the EGF pathway in the anterior foregut cells, Inhibiting the BMP pathway in the anterior foregut cells, or Activating the FGF pathway in the aforementioned anterior foregut cells, A differentiation process performed by executing one or more of the following, (b) A step of dissociating the dorsal anterior foregut cells from step (a) into single cells, (c) A step of culturing the dorsal anterior foregut cells in a first tissue culture vessel in order to differentiate the dorsal anterior foregut cells into esophageal progenitor cells, (d) A step of dissociating the esophageal progenitor cells from step (c) into single cells, (e) A step of culturing the esophageal progenitor cells inside and / or on the surface thereof, The insert member is placed inside the second tissue culture vessel. The insert member includes a surface that is permeable to the growth medium but not to cells. The insert member and the second tissue culture vessel each contain an amount of growth medium such that the esophageal progenitor cells are completely immersed in the growth medium, and the process is to culture the cells. (f) A method comprising the step of culturing the esophageal progenitor cells in and / or on the surface thereof, wherein the second tissue culture vessel and / or insert member contains an amount of growth medium such that the esophageal progenitor cells are partially immersed in the growth medium, thereby producing an esophageal raft culture.

3. The method according to claim 2, further comprising the step of inhibiting the neural precursor of the anterior foregut cell.

4. The method according to claim 2 or 3, wherein at least a portion of the first tissue culture vessel and / or the insert member is coated with an extracellular matrix or its components.

5. The method according to any one of claims 2 to 4, wherein the differentiation step in (a) is carried out over a period of at least one day.

6. The method according to any one of claims 2 to 5, wherein the culturing step in (c) is carried out for at least one day.

7. The method according to any one of claims 2 to 6, wherein the culturing step in (e) is carried out over a period of at least two days.

8. The method according to any one of claims 2 to 7, wherein the culturing step in (f) is carried out for at least 10 days.

9. In step (c) above, Activating the epidermal growth factor (EGF) pathway in the dorsal anterior foregut cells, Culturing bovine pituitary extract (BPE) from the dorsal anterior foregut cells, or Inhibiting Rho kinase (ROCK) in the dorsal anterior foregut cells, The method according to any one of claims 2 to 8, further comprising the step of performing one or more of the steps.

10. In step (e) above, In the growth medium of the insert member, Activating the EGF pathway, Inhibiting ROCK, or Inhibiting SMAD, A process that performs one or more of the following, and The process of culturing the tissue in the second tissue culture vessel together with EGF in the growth medium, The method according to any one of claims 2 to 9, further comprising:

11. The method according to any one of claims 2 to 10, further comprising step (f) of activating the EGF pathway of the esophageal progenitor cells in the growth medium of the second tissue culture vessel.

12. The method according to any one of claims 2 to 11, wherein the anterior foregut cells are derived from human induced pluripotent stem cells.

13. The process involves contacting human induced pluripotent stem cells with BMP4 and / or activin A to differentiate the human induced pluripotent stem cells into endoderm cells of an embryo, The method according to any one of claims 2 to 12, further comprising contacting the endoderm cells of the embryo with Wnt, FGF4, noggin, or RA, or any combination thereof, to differentiate the endoderm cells of the embryo into anterior foregut cells of step (a).

14. The method according to claim 13, further comprising the step of inhibiting the neural precursor of the human induced pluripotent stem cell and / or the endoderm cell of the embryo.

15. The method according to any one of claims 2 to 14, wherein the esophageal raft culture substantially does not contain neural progenitor cells and / or βIII-tubulin+ neurons.

16. The method according to any one of claims 2 to 15, further comprising the steps of: combining the dissociated esophageal progenitor cells of step (d) with enteric neural crest cells (ENCCs); and culturing the combined esophageal progenitor cells and ENCCs according to steps (e) and (f) to produce a neurally innervated esophageal raft culture.

17. The method according to claim 16, wherein the nerve-supplied esophageal raft culture comprises ENCC, neural progenitor cells and / or βIII-tubulin+ nerve cells.

18. Esophageal raft culture produced by the method according to any one of claims 2 to 17.

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