Improved methods for preparing different mesodermal cell types

Abstract

Disclosed herein are improved in vitro methods for generating visceral mesoderm cell types and their subtypes from pluripotent cells.These methods can be used to produce improved foregut and hindgut derived organoids that contain enriched mesenchyme, which enhances the survival, growth and maturation of organoids both in vitro culture and in vivo transplantation.

Description

[Technical Field]

[0001] (Statement regarding federally sponsored research and development) This invention was made with government support under HD093363 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 264,927, filed December 3, 2021.

[0003] FIELD OF THE INVENTION Aspects of the present disclosure relate generally to new and improved methods for differentiating visceral mesoderm and its subtypes from pluripotent stem cells. [Background technology]

[0004] During early fetal development, from embryonic day (E) 8.5 to E9.5 in mice, corresponding to days 17–23 of human gestation, a series of inductive tissue interactions between the definitive endoderm (DE) and its surrounding splanchnic mesoderm (SM, SpM) progressively patterns the naive foregut tube into distinct progenitor domains. These domains further develop into distinct visceral organs, including the trachea, lung, esophagus, stomach, liver, pancreas, and proximal small intestine. The DE gives rise to the epithelial lining and parenchyma of the respiratory and digestive tracts, while the SM gives rise to mesenchymal tissues such as smooth muscle, fibroblasts, and mesentery surrounding the visceral organs. This foregut patterning defines the landscape of the thoracic and abdominal cavities and sets the relative positions of various organs. Disruption of this process can lead to life-threatening congenital birth defects. Summary of the Invention

[0005] For example, there is currently a need for a better understanding of mesoderm differentiation during embryogenesis and improved methods for differentiating mesoderm in vitro using pluripotent stem cells (PSCs), e.g., patient-derived PSCs, with applications in genetics, drug screening, personalized medicine, and the production of improved organoids for transplantation.

[0006] Disclosed herein are methods for producing retinoic acid-responsive lateral plate mesoderm cells (RA-LPM).

[0007] Disclosed herein are methods for producing retinoic acid-responsive splanchnic mesoderm cells (RA-SpM).

[0008] Disclosed herein are methods for producing septum transversum (STM) and mesothelial cells.

[0009] Disclosed herein are methods for producing liver fibroblasts (LF).

[0010] Disclosed herein are methods for producing gastric mesenchyme cells (GM).

[0011] Disclosed herein are methods for producing hedgehog-responsive lateral plate mesoderm cells (HH-LPM).

[0012] Disclosed herein are methods for producing hedgehog-responsive splanchnic mesoderm cells (HH-SpM).

[0013] Disclosed herein are methods for producing esophageal mesenchyme cells (EM).

[0014] Disclosed herein are methods for producing respiratory mesenchyme cells (RM).

[0015] Disclosed herein are retinoic acid-responsive lateral plate mesoderm cells (RA-LPM), hedgehog-responsive lateral plate mesoderm cells (HH-LPM), visceral mesoderm cells, retinoic acid-responsive visceral mesoderm cells (RA-SpM), hedgehog-responsive visceral mesoderm cells (HH-SpM), septum transversum (STM) and mesothelial cells, liver fibroblasts, gastric mesenchymal cells (GM), respiratory mesenchymal cells (RM), and esophageal mesenchymal cells (EM) produced by any of the methods disclosed herein.

[0016] The embodiments of the present disclosure provided herein are illustrated by the following numbered alternatives: 1. A method for producing retinoic acid-responsive lateral plate mesoderm cells (RA-LPM), comprising: A method comprising contacting a mesoprimitive streak cell with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, and a retinoic acid (RA) signaling pathway activator, thereby differentiating the mesoprimitive streak cell into an RA-LPM. 2. The method of Alternative 1, wherein the mesoprimitive streak cells are contacted with A83-01, Wnt-C59 (C59), BMP4, RA, or any combination thereof. 3. The method of alternative 1 or 2, wherein the medial primitive streak cells are contacted for a time sufficient to differentiate the medial primitive streak cells into RA-LPM, and / or for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or a time that is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the foregoing times, inclusive of 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours. 4. The method of any one of alternatives 1 to 3, wherein the mesoprimitive streak cells are contacted for a period of time that is at or about 24 hours. 5. A method for producing retinoic acid-responsive visceral mesoderm cells (RA-SpM), comprising: A method comprising contacting RA-LPM with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, and an RA signaling pathway activator, thereby differentiating the RA-LPM into RA-SpM. 6. The method of Alternative 5, wherein the RA-LPM is RA-LPM produced by the method of any one of Alternatives 1-4. 7. The method of alternative 5 or 6, wherein RA-LPM is contacted with A83-01, C59, BMP4, bFGF (FGF2), RA, or any combination thereof. 8. The RA-LPMs are cultured for a time sufficient to differentiate the RA-LPMs into RA-SpMs, and / or for about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours. , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or any time within a range defined by any two of the above times, inclusive: 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours. 9. The method of any one of alternatives 5 to 8, wherein the RA-LPM is contacted for a period of time that is at or about 48 hours. 10. A method for producing septum transversum (STM) and mesothelial cells, comprising contacting RA-SpM with a retinoic acid signaling pathway activator and a BMP signaling pathway activator, thereby differentiating the RA-SpM into STM and mesothelial cells. 11. The method of alternative 10, wherein the RA-SpM is an RA-SpM according to any one of alternatives 5 to 9. 12. The method of alternative 10 or 11, wherein the visceral mesoderm cells are contacted with RA, BMP4, or both. 13. Incubate the RA-SpM for a time sufficient to differentiate the RA-SpM into STMs and mesothelial cells, and / or for about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 hours. 13. The method of any one of alternatives 10-12, wherein the contacting is for a period of time that is 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 hours, or any period of time within a range defined by any two of the foregoing periods, inclusive of 60-84 hours, 60-72 hours, 72-84 hours, or 70-74 hours. 14. The method of any one of alternatives 10 to 13, wherein the RA-SpM is contacted for a period of time that is at or about 72 hours. 15. A method for producing liver fibroblasts (LF), comprising contacting RA-SpM with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and a Wnt signaling pathway activator, thereby differentiating the RA-SpM into liver fibroblasts. 16. The method of alternative 15, wherein the RA-SpM is an RA-SpM according to any one of alternatives 5 to 9. 17. The method of alternative 15 or 16, wherein the RA-SpM is contacted with RA, BMP4, CHIR99021, or any combination thereof. 18. The method of any one of alternatives 15-17, wherein the RA-SpM are contacted for a time sufficient to differentiate the RA-SpM into liver fibroblasts, and / or for 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 hours, or for a time that is about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 hours, or any time within a range defined by any two of the foregoing times, inclusive of 60-84 hours, 60-72 hours, 72-84 hours, or 70-74 hours. 19. The method of any one of alternatives 15 to 18, wherein the RA-SpM is contacted for a period of time that is at or about 72 hours. 20. A method for producing gastric mesenchymal cells (GM), comprising: a) contacting RA-SpM with a retinoic acid signaling pathway activator and an HH signaling pathway activator; b) contacting the cells obtained in step a) with a retinoic acid signaling pathway activator, a BMP signaling pathway inhibitor, and an HH signaling pathway activator; This method allows RA-SpM to differentiate into GM. 21. The method according to alternative example 20, wherein the RA-SpM is RA-SpM produced by the method according to any one of alternative examples 5 to 9. 22. The method of alternative 20 or 21, wherein the RA-SpM is contacted for step a) for 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or for any time within a range defined by any two of the above-mentioned times, inclusive: 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours. 23. The method according to any one of alternatives 20 to 22, wherein the RA-SpM is contacted for step a) for a period of time that is at or about 48 hours. 24. The method of any one of alternatives 20-23, wherein the cells obtained in step a) are contacted with the cells obtained in step b) for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or for a time that is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the above-mentioned times, including 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours. 25. The method according to any one of alternatives 20 to 24, wherein the cells obtained in step a) are contacted for a period of time that is at or about 24 hours for step b). 26. The method of any one of alternatives 20-25, wherein the RA-SpM is contacted with RA, Noggin, PMA, or any combination thereof. 27. A method for producing hedgehog-responsive lateral plate mesoderm cells (HH-LPM), comprising: A method comprising contacting a mesoprimitive streak cell with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an RA signaling pathway activator, and a hedgehog (HH) signaling pathway activator, thereby differentiating the mesoprimitive streak cell into an HH-LPM. 28. The method of alternative 27, wherein the mesoprimitive streak cells are contacted with A83-01, C59, BMP4, RA, PMA, or any combination thereof. 29. The method of alternative 27 or 28, wherein the mesoprimitive streak cells are contacted for a time sufficient to differentiate the mesoprimitive streak cells into HH-LPM, and / or for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or a time that is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the above-mentioned times, inclusive of 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours. 30. The method of any one of alternatives 27-29, wherein the mesoprimitive streak cells are contacted for a period of time that is at or about 24 hours. 31. A method for producing hedgehog-responsive visceral mesoderm cells (HH-SpM), comprising: A method comprising contacting HH-LPM with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, an RA signaling pathway activator, and an HH signaling pathway activator, thereby differentiating the HH-LPM into HH-SpM. 32. The method described in Alternative Example 31, wherein the HH-LPM is HH-LPM produced by the method described in any one of Alternative Examples 27 to 30. 33. The method of alternative 31 or 32, wherein HH-LPM is contacted with A83-01, C59, BMP4, bFGF, RA, PMA, or any combination thereof. 34. The HH-LPM is cultured for a time sufficient to differentiate the HH-LPM into HH-SpM, and / or for about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours. 34. The method of any one of alternatives 31-33, wherein the contacting is for a period of time that is 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or any period of time within a range defined by any two of the foregoing periods, inclusive of 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours. 35. The method of any one of alternatives 31-34, wherein the HH-LPM is contacted for a period of time that is at or about 48 hours. 36. A method for producing esophageal mesenchymal cells (EM), comprising: a) contacting HH-SpM with a retinoic acid signaling pathway activator and an HH signaling pathway activator; b) contacting the cells obtained in step a) with a retinoic acid signaling pathway activator, a BMP signaling pathway inhibitor, and an HH signaling pathway activator; This method allows HH-SpM to differentiate into EM. 37. The method according to alternative example 36, wherein the HH-SpM is HH-SpM produced by the method according to any one of alternative examples 31 to 35. 38. The method of alternative 36 or 37, wherein the HH-SpM is contacted for step a) for 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or for a time that is about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or any time within a range defined by any two of the above-mentioned times, inclusive: 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours. 39. The method according to any one of alternatives 36 to 38, wherein the HH-SpM is contacted for step a) for a period of time that is at or about 48 hours. 40. The method of any one of alternatives 36-39, wherein the cells obtained in step a) are contacted with the cells obtained in step b) for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or for a time that is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the above-mentioned times, inclusive of 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours. 41. The method according to any one of alternatives 36 to 40, wherein the cells obtained in step a) are contacted for a period of time that is 24 hours or about 24 hours for step b). 42. The method of any one of alternatives 36-41, wherein the HH-SpM is contacted with RA, Noggin, PMA, or any combination thereof. 43. A method for producing respiratory mesenchymal cells (RM), comprising: a) contacting HH-SpM with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and a hedgehog (HH) signaling pathway activator; b) contacting the cells obtained in step a) with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, an HH signaling pathway activator, and a Wnt signaling pathway activator; This method allows HH-SpM to differentiate into RM. 44. The method described in alternative example 43, wherein the HH-SpM is HH-SpM produced by the method described in any one of alternative examples 31 to 35. 45. The method of alternative 43 or 44, wherein the HH-SpM is contacted for step a) for 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or for a time that is about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or any time within a range defined by any two of the above-mentioned times, inclusive: 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours. 46. ​​The method according to any one of alternatives 43 to 45, wherein the HH-SpM is contacted for step a) for a period of time that is at or about 48 hours. 47. The method of any one of alternatives 43-46, wherein the cells obtained in step a) are contacted with step b) for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or for a time that is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the above-mentioned times, inclusive of 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours. 48. The method according to any one of alternatives 43 to 47, wherein the cells obtained in step a) are contacted for a period of time that is 24 hours or about 24 hours for step b). 49. The method of any one of alternatives 43-48, wherein the HH-SpM is contacted with RA, BMP4, PMA, CHIR99021, or any combination thereof. 50. The method of any one of alternatives 1-49, wherein the intermediate primitive streak cells are human intermediate primitive streak cells. 51. The method of any one of alternatives 1-50, wherein the mesoprimitive streak cells are differentiated from pluripotent stem cells, optionally induced pluripotent stem cells or embryonic stem cells, and optionally, the mesoprimitive streak cells are differentiated from pluripotent stem cells by contacting the pluripotent stem cells with a TGF-beta signaling pathway activator, a Wnt signaling pathway activator, an FGF signaling pathway activator, a BMP signaling pathway activator, and a PI3K signaling pathway inhibitor. 52. The method of any one of alternatives 1-51, wherein the TGF-beta signaling pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542. 53. The method of any one of alternatives 1-52, wherein the TGF-beta signaling pathway inhibitor is A83-01. 54. The method of any one of alternatives 1-53, wherein the TGF-beta signaling pathway inhibitor is contacted at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the foregoing concentrations, including 0.1-2 μM, 0.1-1 μM, 0.5-1.5 μM, or 1-2 μM. 55. The method of any one of alternatives 1 to 54, wherein the TGF-beta signaling pathway inhibitor is contacted at a concentration of 1 μM or about 1 μM. 56. The method of any one of alternatives 1 to 55, wherein the Wnt signaling pathway inhibitor is selected from the group consisting of Wnt-C59 (C59), PNU 74654, KY-02111, PRI-724, FH-535, DIF-1, and XAV939. 57. The method of any one of alternatives 1 to 56, wherein the Wnt signaling pathway inhibitor is C59. 58. The method of any one of alternatives 1 to 57, wherein the Wnt signaling pathway inhibitor is contacted at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the above concentrations, including 0.1-2 μM, 0.1-1 μM, 0.5-1.5 μM, or 1-2 μM. 59. The method of any one of alternatives 1 to 58, wherein the Wnt signaling pathway inhibitor is contacted at a concentration of 1 μM or about 1 μM. 60. The method of any one of alternatives 1-59, wherein the BMP signaling pathway activator is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, and IDE2. 61. The method of any one of alternatives 1-60, wherein the BMP signaling pathway activator is BMP4. 62. A BMP signaling pathway activator is administered at 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, or 45 ng / mL, or at about 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, or 45 ng / mL, or any concentration within a range defined by any two of the above concentrations, including 15-45 ng / mL, 15-30 ng / mL, 30-45 ng / mL, 20-40 ng / mL, or 25-35 ng / mL. 63. The method of any one of alternatives 1 to 62, wherein the BMP signaling pathway activator is contacted at a concentration of 30 ng / mL or about 30 ng / mL. 64. The method of any one of alternatives 1-63, wherein the FGF signaling pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. 65. The method of any one of alternatives 1-64, wherein the FGF signaling pathway activator is FGF2. 66. The FGF signaling pathway activator 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, or 35 ng / mL, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 66. The method of any one of alternatives 1 to 65, wherein the contacting is performed at a concentration of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, including 5 to 35 ng / mL, 10 to 30 ng / mL, 5 to 20 ng / mL, or 20 to 35 ng / mL. 67. The method of any one of alternatives 1 to 66, wherein the FGF signaling pathway activator is contacted at a concentration of 20 ng / mL or about 20 ng / mL. 68. The method of any one of alternatives 1-67, wherein the RA signaling pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580. 69. The method of any one of alternatives 1-68, wherein the RA signaling pathway activator is RA. 70. The method of any one of alternatives 1-69, wherein the RA signaling pathway activator is contacted at a concentration of 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 μM, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, including 1-3 μM, 1-2 μM, 2-3 μM, or 1.5-2.5 μM. 71. The method of any one of alternatives 1 to 70, wherein the RA signaling pathway activator is contacted at a concentration of 2 μM or about 2 μM. 72. The method of any one of alternatives 1-71, wherein the Wnt signaling pathway activator is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpolone, kenpaullone, lithium chloride, TDZD 8, and TWS119. 73. The method of any one of alternatives 1 to 72, wherein the Wnt signaling pathway activator is CHIR99021. 74. The method of any one of alternatives 1-73, wherein the Wnt signaling pathway activator is contacted at a concentration of 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, including 0.01-20 μM, 0.01-10 μM, 1-10 μM, or 5-15 μM. 75. The method of any one of alternatives 1-74, wherein the Wnt signaling pathway activator is contacted at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or any concentration within a range defined by any two of the above concentrations, including 1-10 μM, 1-8 μM, 4-8 μM, 5-7 μM, 5-10 μM, or 6-10 μM. 76. The method of any one of alternatives 1-75, wherein the HH signaling pathway activator is selected from the group consisting of SHH, IHH, DHH, PMA, GSA 10, and SAG. 77. The method of any one of alternatives 1-76, wherein the HH signaling pathway activator is PMA. 78. The method of any one of alternatives 1-77, wherein the HH signaling pathway activator is contacted at a concentration of 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, including 1-3 μM, 1-2 μM, 2-3 μM, or 1.5-2.5 μM. 79. The method of any one of alternatives 1 to 78, wherein the HH signaling pathway activator is contacted at a concentration of 1 μM, about 1 μM, 2 μM, or about 2 μM. 80. The method of any one of alternatives 1-79, wherein the BMP signaling pathway inhibitor is selected from the group consisting of Noggin, RepSox, LY364947, LDN193189, and SB431542. 81. The method of any one of alternatives 1 to 80, wherein the BMP signaling pathway inhibitor is Noggin. 82. The method of any one of alternatives 1 to 81, wherein the BMP signaling pathway inhibitor is contacted at a concentration of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 ng / mL, or any concentration within a range defined by any two of the above concentrations, including 50-250 ng / mL, 50-200 ng / mL, 100-200 ng / mL, 100-250 ng / mL, or 200-250 ng / mL. 83. The method of any one of alternatives 1 to 82, wherein the BMP signaling pathway inhibitor is contacted at a concentration of 200 ng / mL or about 200 ng / mL. 84. Retinoic acid-responsive lateral plate mesoderm cells (RA-LPM) produced by the method of any one of alternatives 1 to 4, or 1 to 4 and 50 to 83. 85. Retinoic acid-responsive visceral mesoderm cells (RA-SpM) produced by the method of any one of alternatives 5 to 9, or 5 to 9 and 50 to 83. 86.RA-SpM, a) lack of expression of pluripotency markers OCT3 / 4 and SOX2, the middle primitive streak marker TBXT, and / or the endoderm markers FOXA2 and CDH1; b) expression of VIM, optionally decreased expression of VIM compared to cardiogenic splanchnic mesoderm (CG-SpM) and / or increased expression of VIM compared to HH-SpM; c) expression of FOXF1, optionally decreased expression of FOXF1 compared to HH-SpM and / or increased expression of FOXF1 compared to CG-SpM; d) Absence of expression of cardiac markers NKX2-5 and ISL1 or decreased expression of NKX2-5 and ISL1 compared to CG-SpM; e) expression of retinoic acid responsive markers HOXA5 and CYP26A1, and / or f) RA-SpM according to alternative example 85, characterized by one or more of the following: reduced expression of the hedgehog responsive markers GLI1 and PTCH1 compared to HH-SPm. 87. Septum transversum (STM) and mesothelial cells produced by the method of any one of alternatives 10-14, or 10-14 and 50-83. 88. The STM and mesothelial cells of alternative example 87, wherein the STM and mesothelial cells are characterized by expression of WT1, TBX18, LHX2, GATA4, UPK1B, or UPK3B, or any combination thereof, and optionally, expression of WT1, TBX18, LHX2, GATA4, UPK1B, or UPK3B is increased compared to cardiac mesoderm, liver fibroblasts, gastric mesoderm, respiratory mesoderm, esophageal mesoderm, or any combination thereof. 89. Liver fibroblasts (LF) produced by the method of any one of alternatives 15-19, or 15-19 and 50-83. 90. The liver fibroblast of alternative example 89, wherein the liver fibroblast is characterized by expression of PITX1, MSX1, MSX2, TBX5, or WNT2, or any combination thereof, and optionally, expression of PITX1, MSX1, MSX2, TBX5, or WNT2, or any combination thereof, is increased compared to cardiac mesoderm, septum transversum, gastric mesoderm, respiratory mesoderm, or esophageal mesoderm, or any combination thereof. 91. Gastric mesenchymal cells (GM) produced by the method of any one of alternatives 20-26, or 20-26 and 50-83. 92. The gastric mesenchymal cells of alternative example 91, characterized by expression of BARX1, NKX3-2, or FOXF1, or any combination thereof, and optionally, expression of BARX1, NKX3-2, or FOXF1, or any combination thereof, is increased compared to cardiac mesoderm, septum transversus, liver fibroblasts, respiratory mesoderm, or esophageal mesoderm, or any combination thereof. 93. Hedgehog-responsive lateral plate mesoderm cells (HH-LPM) produced by the method of any one of alternatives 27-30, or 27-30 and 50-83. 94. Hedgehog-responsive visceral mesoderm cells (HH-SpM) produced by the method of any one of alternatives 31-35, or 31-35 and 50-83. 95.HH-SpM, a) lack of expression of pluripotency markers OCT3 / 4 and SOX2, the middle primitive streak marker TBXT, and / or the endoderm markers FOXA2 and CDH1; b) expression of VIM, optionally reduced expression of VIM compared to cardiac visceral mesoderm (CG-SpM) and RA-SpM; c) expression of FOXF1, optionally increased expression of FOXF1 compared to CG-SpM and RA-SpM; d) Absence of expression of cardiac markers NKX2-5 and ISL1 or decreased expression of NKX2-5 and ISL1 compared to CG-SpM; e) expression of retinoic acid responsive markers HOXA5 and CYP26A1, and / or f) HH-SpM according to alternative example 94, characterized by one or more of the following: expression of hedgehog responsive markers GLI1 and PTCH1, optionally increased expression of GLI1 and PTCH1 compared to CG-SpM and RA-SpM. 96. An esophageal mesenchymal cell (EM) produced by the method of any one of alternatives 36-42, or 36-42 and 50-83. 97. The esophageal mesenchymal cells of alternative example 96, wherein the esophageal mesenchymal cells are characterized by expression of MSC, WNT4, or FOXF1, or any combination thereof, and optionally, expression of MSC, WNT4, or FOXF1, or any combination thereof, is increased compared to cardiac mesoderm, septum transversum, liver fibroblasts, gastric mesoderm, or respiratory mesoderm, or any combination thereof. 98. A respiratory mesenchymal cell (RM) produced by the method of any one of alternatives 42-49 or 42-83. 99. The respiratory mesenchymal cells of alternative example 98, wherein the respiratory mesenchymal cells are characterized by expression of TBX5, NKX6-1, WNT2, or FOXF1, or any combination thereof, and optionally, expression of TBX5, NKX6-1, WNT2, or FOXF1, or any combination thereof, is increased compared to cardiac mesoderm, septum transversus, liver fibroblasts, gastric mesoderm, or esophageal mesoderm, or any combination thereof. 100. The respiratory mesenchymal cells of alternative examples 98 or 99, wherein the respiratory mesenchymal cells are characteristic of medial respiratory mesenchyme but not ventral respiratory mesenchyme. 101.Respiratory system mesenchymal cells a) increased expression of NKX6-1 and TBX5, and / or b) characterized by one or more of reduced expression of TBX4 and WNT2; The respiratory mesenchymal cells of any one of alternative examples 98 to 100, wherein the respiratory mesenchymal cells are differentiated from HH-SpM, and a) and b) are compared to respiratory mesenchymal cells differentiated from visceral mesoderm that are differentiated from lateral plate mesoderm without the use of an HH signaling pathway activator. [Brief explanation of the drawings]

[0017] In addition to the features described herein, additional features and modifications will be readily apparent from the following drawings and description of exemplary embodiments, it being understood that these drawings depict embodiments and are not intended to be limiting in scope. [Figure 1A] An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1B]An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1C]An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1D]An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1E]An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1F]An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1G]An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1H]An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1I]An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1J]An embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages is shown. Figure 1A shows representative images of mouse embryos at three developmental stages, showing the foregut region (dashed line) microscopically dissected (inset) to generate single cells. At E9.5, the anterior foregut (a.fg) and posterior foregut (p.fg) were separately isolated. E, embryonic stage; s, somite number; n, cell number. Scale bar: 1 mm. Figure 1B shows a schematic of the RNA-seq workflow. Figure 1C shows UMAP visualization of 31,268 cells isolated from pooled samples of all three stages. Cells are shaded based on their major cell lineage. Figure 1D shows whole-mount immunostaining of E9.5 mouse foregut, revealing Cdh1+ endoderm and surrounding Foxf1+ visceral mesoderm. Figures 1E and 1F show t-SNE plots of in silico isolated E9.5 endoderm (1E) and visceral mesoderm (1F) cells. Figures 1G and 1H show the pseudo-spatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show a schematic diagram of the predicted locations of E9.5 cell types mapped onto the endoderm (1I) and mesoderm (1J) of the embryonic mouse foregut. def, embryonic body; meso, mesoderm; lg, lung; eso, esophagus; lv, liver; splanch, visceral; stm, septum transversum mesenchyme; sto, stomach; pha, pharynx. [Figure 1K] Figure 1 shows one embodiment of major cell lineage definitions. UMAP of single cells from every stage with major lineages annotated by known marker genes (Panel A). UMAP of all cells from every stage with computationally assigned clusters based on transcriptome similarity (Panel B). UMAP of all cells from every stage shaded by stage and region (Panel C). t-SNE maps of single cells from each stage annotated by major lineage at E8.5 (Panel D), E9.0 (Panel E), and E9.5 (Panel F). Gene expression heatmap of selected markers in individual cells across different lineages and stages (Panel G). [Figure 1L]Figure 1 shows an embodiment of annotation of E8.5 and E9.0 DE and SM lineages. t-SNE plots of E8.5 DE (Panel A), E8.5 SM (Panel B), E9.0 DE (Panel C), and E9.0 SM cell (Panel D) annotations. E8.5 clusters are labeled "a," E9.0 "b," and E9.5 "c." Pseudo-spatial ordering of E8.5 DE (Panel E), E8.5 SM (Panel F), E9.0 DE (Panel G), and E9.0 SM cell (Panel H) along the anterior-posterior (AP) axis of the gut. Schematic diagram of the mouse embryonic foregut showing the predicted locations of E8.5 DE (Panel I), E8.5 SM (Panel J), E9.0 DE (Panel K), and E9.0 SM (Panel L) cell types mapped onto the endoderm and mesoderm. Heatmap of selected marker gene expression in individual cells across different clusters at E8.5 DE (Panel M), E8.5 SM (Panel N), E9.0 DE (Panel O), and E9.0 SM (Panel P). [Figure 1M] Figure 1 shows one embodiment of an integrated analysis of DE and SM cells. t-SNE and UMAP visualization of all SM cells from all stages annotated by major lineage (Panels A, B) and stage (Panels C, D). t-SNE and UMAP visualization of all DE cells from all stages annotated by major lineage (Panels E, F) and stage (Panels G, H). Stage-specific annotations that contribute significantly to each integrated cluster are shown in parentheses. E8.5 cells = a_cluster, E9.0 cells = b_cluster, and E9.5 cells = c_cluster. [Figure 2A]We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2B] We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2C]We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2D] We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2E]We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2F] We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2G]We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2H] We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2I]We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2J] We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2K]We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2L] We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2M]We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2N] We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2O]We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2P] We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2Q]We demonstrate one embodiment of lineage-restricted gene expression in different SM cell types. Figure 2A shows a schematic diagram of the E9.5 foregut showing the level of sectioning. Figure 2B shows dot plots depicting scRNA-seq expression of marker genes in different E9.5 SM cell clusters. Figure 2C shows whole-mount immunostaining of dissected E9.5 foregut tissue. Figures 2D-G show in situ hybridization of dissected E9.5 foregut tissue. The scale bar is 100 μm. Figures 2H-2Q show RNA-scope in situ detection in transverse E9.5 mouse embryo sections (i-iv show the AP level of the section in Figure 2A). The scale bar is 50 μm. duo, duodenum; dp, dorsal pancreas; eso, esophagus; ht, heart; lg, lung; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, venous sinus; vp, ventral pancreas. [Figure 2R] Figure 1 shows one embodiment of validation of liver mesenchymal subtypes. Schematic of mouse embryonic foregut at E9.5 (Panel A). RNA-scope in situ detection of mesodermal markers in fixed frozen sagittal sections from E9.5 mouse embryos (Panels B-F). Scale bar is 50 μm. Insets show merged and individual channels. [Figure 2S] Figure 1 shows one embodiment of coaxial Hox gene expression and transcription factor coding. Heatmap of average Hox gene expression across different DE and SM clusters arranged along the AP axis. Annotation is as follows: E8.5 = a_cluster, E9.0 = b_cluster, and E9.5 = c_cluster (Panel A). Putative location of cell clusters in foregut endoderm and mesoderm (Panel B). Transcription factor coding heatmap showing average expression of the top five differentially expressed transcription factors across DE and SM populations at E9.5 (Panel C). a, anterior; fg, foregut; post, posterior; v, ventral; stm, transverse septum mesenchyme. [Figure 3A]One embodiment of coordinated endoderm and mesoderm cell trajectories is shown. Figures 3A and 3B show SPRING visualizations of mechanically modeled cell trajectories of visceral mesoderm (3A, n = 10,097) and definitive endoderm (3B, n = 4,448). Cells are shaded by developmental stage. White arrows indicate cell lineage progression. Figures 3C and 3D show confusion matrices summarizing "parent-child" single-cell votes for SM (3C) and DE (3D) cells used to construct cell-state trees. Based on transcriptome similarity (KNN), each cell at a later time point (y-axis) voted for its most similar cell at an earlier time point (x-axis). All votes for a given cluster are tabulated, normalized to cluster size, and represented as a percentage of the vote in the heatmap. The E8.5, E9.0, and R9.5 clusters are labeled "a," "b," and "c," respectively. Figures 3E and 3F show cell state trees for the SM (3E) and DE (3F) lineages predicted by single-cell voting. The top choices linking cell states at successive time points are solid lines, while prominent secondary choices are dashed lines. Nodes are shaded by stage and annotated with cluster number. [Figure 3B]One embodiment of coordinated endoderm and mesoderm cell trajectories is shown. Figures 3A and 3B show SPRING visualizations of mechanically modeled cell trajectories of visceral mesoderm (3A, n = 10,097) and definitive endoderm (3B, n = 4,448). Cells are shaded by developmental stage. White arrows indicate cell lineage progression. Figures 3C and 3D show confusion matrices summarizing "parent-child" single-cell votes for SM (3C) and DE (3D) cells used to construct cell-state trees. Based on transcriptome similarity (KNN), each cell at a later time point (y-axis) voted for its most similar cell at an earlier time point (x-axis). All votes for a given cluster are tabulated, normalized to cluster size, and represented as a percentage of the vote in the heatmap. The E8.5, E9.0, and R9.5 clusters are labeled "a," "b," and "c," respectively. Figures 3E and 3F show cell state trees for the SM (3E) and DE (3F) lineages predicted by single-cell voting. The top choices linking cell states at successive time points are solid lines, while prominent secondary choices are dashed lines. Nodes are shaded by stage and annotated with cluster number. [Figure 3C]One embodiment of coordinated endoderm and mesoderm cell trajectories is shown. Figures 3A and 3B show SPRING visualizations of mechanically modeled cell trajectories of visceral mesoderm (3A, n = 10,097) and definitive endoderm (3B, n = 4,448). Cells are shaded by developmental stage. White arrows indicate cell lineage progression. Figures 3C and 3D show confusion matrices summarizing "parent-child" single-cell votes for SM (3C) and DE (3D) cells used to construct cell-state trees. Based on transcriptome similarity (KNN), each cell at a later time point (y-axis) voted for its most similar cell at an earlier time point (x-axis). All votes for a given cluster are tabulated, normalized to cluster size, and represented as a percentage of the vote in the heatmap. The E8.5, E9.0, and R9.5 clusters are labeled "a," "b," and "c," respectively. Figures 3E and 3F show cell state trees for the SM (3E) and DE (3F) lineages predicted by single-cell voting. The top choices linking cell states at successive time points are solid lines, while prominent secondary choices are dashed lines. Nodes are shaded by stage and annotated with cluster number. [Figure 3D]One embodiment of coordinated endoderm and mesoderm cell trajectories is shown. Figures 3A and 3B show SPRING visualizations of mechanically modeled cell trajectories of visceral mesoderm (3A, n = 10,097) and definitive endoderm (3B, n = 4,448). Cells are shaded by developmental stage. White arrows indicate cell lineage progression. Figures 3C and 3D show confusion matrices summarizing "parent-child" single-cell votes for SM (3C) and DE (3D) cells used to construct cell-state trees. Based on transcriptome similarity (KNN), each cell at a later time point (y-axis) voted for its most similar cell at an earlier time point (x-axis). All votes for a given cluster are tabulated, normalized to cluster size, and represented as a percentage of the vote in the heatmap. The E8.5, E9.0, and R9.5 clusters are labeled "a," "b," and "c," respectively. Figures 3E and 3F show cell state trees for the SM (3E) and DE (3F) lineages predicted by single-cell voting. The top choices linking cell states at successive time points are solid lines, while prominent secondary choices are dashed lines. Nodes are shaded by stage and annotated with cluster number. [Figure 3E]One embodiment of coordinated endoderm and mesoderm cell trajectories is shown. Figures 3A and 3B show SPRING visualizations of mechanically modeled cell trajectories of visceral mesoderm (3A, n = 10,097) and definitive endoderm (3B, n = 4,448). Cells are shaded by developmental stage. White arrows indicate cell lineage progression. Figures 3C and 3D show confusion matrices summarizing "parent-child" single-cell votes for SM (3C) and DE (3D) cells used to construct cell-state trees. Based on transcriptome similarity (KNN), each cell at a later time point (y-axis) voted for its most similar cell at an earlier time point (x-axis). All votes for a given cluster are tabulated, normalized to cluster size, and represented as a percentage of the vote in the heatmap. The E8.5, E9.0, and R9.5 clusters are labeled "a," "b," and "c," respectively. Figures 3E and 3F show cell state trees for the SM (3E) and DE (3F) lineages predicted by single-cell voting. The top choices linking cell states at successive time points are solid lines, while prominent secondary choices are dashed lines. Nodes are shaded by stage and annotated with cluster number. [Figure 3F]One embodiment of coordinated endoderm and mesoderm cell trajectories is shown. Figures 3A and 3B show SPRING visualizations of mechanically modeled cell trajectories of visceral mesoderm (3A, n = 10,097) and definitive endoderm (3B, n = 4,448). Cells are shaded by developmental stage. White arrows indicate cell lineage progression. Figures 3C and 3D show confusion matrices summarizing "parent-child" single-cell votes for SM (3C) and DE (3D) cells used to construct cell-state trees. Based on transcriptome similarity (KNN), each cell at a later time point (y-axis) voted for its most similar cell at an earlier time point (x-axis). All votes for a given cluster are tabulated, normalized to cluster size, and represented as a percentage of the vote in the heatmap. The E8.5, E9.0, and R9.5 clusters are labeled "a," "b," and "c," respectively. Figures 3E and 3F show cell state trees for the SM (3E) and DE (3F) lineages predicted by single-cell voting. The top choices linking cell states at successive time points are solid lines, while prominent secondary choices are dashed lines. Nodes are shaded by stage and annotated with cluster number. [Figure 3G] Figure 1 shows one embodiment of a SPRING plot of DE and SM cell trajectories. SPRING plot of all SM cells (n=10,097) shaded by stage-specific lineage annotation (Panel A) and expression of key marker genes (Panel B). SPRING plot of all DE cells (n=4,448) shaded by stage-specific lineage annotation (Panel C) and expression of key marker genes (Panel D). [Figure 3H]Figure 1 shows one embodiment of hepatic endoderm development. Cell state tree of the hepatic endoderm lineage with key marker genes indicated for each cell state (Panel A). Pseudotime analysis of the hepatic DE lineage using Monocle_v3 suggests that at E9.0, the e_b2 cluster (early hepatoblasts) is a common progenitor for e_b5 (late hepatoblasts) and e_b7 (hepatopancreatic duct progenitors) (Panel B). SPRING plot with hepatic endoderm clusters shaded by stage-specific lineage annotation (Panel C) and expression of key marker genes (Panels D-I). [Figure 4A] We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4B]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4C]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4D]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4E]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4F]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4G]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4H]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4I]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4J]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4K]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 4L]We demonstrate one embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B show schematic diagrams of the esophageal-respiratory-gastric cell state trajectories in the SM (4A) and DE (4B) using key marker genes. This suggests coordinated development of Osr1+ multilineage progenitor cells. Figures 4C and 4D show SPRING plots of the SM (4C) and DE (4E) projecting the expression of key genes. Figure 4E shows in situ hybridization of Osr1 in the dissected foregut, demonstrating that Osr1 is expressed in the respiratory, esophageal, and gastric regions. Figures 4F and 4G show in situ hybridization of Osr1 in sections spanning the respiratory and gastric regions within the foregut, demonstrating that Osr1 is expressed in both endodermal and mesenchymal cells. Figure 4H shows a SPRING plot of the esophageal-respiratory lineage in the DE. Figure 4I shows Nkx2-1 and Sox2 expression projected onto a SPRING plot, demonstrating coexpression at the esophagus-tracheal boundary. Figure 4J shows whole-mount immunostaining of Sox2 and Nkx2-1 in the mouse foregut at E9.5. Figure 4K shows immunostaining of Sox2, Nkx2-1, and Foxf1 in a transverse E9.5 foregut section, confirming a rare population of cells coexpressing Sox2 / Nkx2-1. Figure 4L shows a higher magnification of the boxed area in Figure 4K. [Figure 5A]We present one embodiment of computationally inferred receptor-ligand interactions that predict the signaling roadmap of foregut organogenesis. Figures 5A and 5B show E9.5 foregut immunostaining for Cdh1 (epithelial) and Foxf1 (mesenchymal) in whole mounts (same images as Figure 5A and Figure 1D) and sections (Figure 5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circles). Figure 5C shows predicted receptor-ligand interactions between adjacent foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. E9.5 DE and SM cell clusters are ordered along the anterior-to-posterior axis based on their in vivo location, with spatially adjacent DE and SM cell types facing each other. Shaded circles indicate relative pathway response—metagene expression levels—predicting the likelihood that a given cell population is responding to a growth factor signal. Thin vertical lines next to the clusters indicate various spatially adjacent cell populations that are all responding to a particular signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto SPRING plots in the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut cross sections, demonstrating its expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut cross sections, demonstrating BMP signaling responses in the DE and SM of the respiratory system and liver. Figures 5H and 5I show signaling roadmaps summarizing the predicted signaling states of all six pathways projected onto the DE (5H) and SM (5I) cell state trees, suggesting the combined signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signaling responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5B]We present one embodiment of computationally inferred receptor-ligand interactions that predict the signaling roadmap of foregut organogenesis. Figures 5A and 5B show E9.5 foregut immunostaining for Cdh1 (epithelial) and Foxf1 (mesenchymal) in whole mounts (same images as Figure 5A and Figure 1D) and sections (Figure 5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circles). Figure 5C shows predicted receptor-ligand interactions between adjacent foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. E9.5 DE and SM cell clusters are ordered along the anterior-to-posterior axis based on their in vivo location, with spatially adjacent DE and SM cell types facing each other. Shaded circles indicate relative pathway response—metagene expression levels—predicting the likelihood that a given cell population is responding to a growth factor signal. Thin vertical lines next to the clusters indicate various spatially adjacent cell populations that are all responding to a particular signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto SPRING plots in the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut cross sections, demonstrating its expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut cross sections, demonstrating BMP signaling responses in the DE and SM of the respiratory system and liver. Figures 5H and 5I show signaling roadmaps summarizing the predicted signaling states of all six pathways projected onto the DE (5H) and SM (5I) cell state trees, suggesting the combined signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signaling responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5C]We present one embodiment of computationally inferred receptor-ligand interactions that predict the signaling roadmap of foregut organogenesis. Figures 5A and 5B show E9.5 foregut immunostaining for Cdh1 (epithelial) and Foxf1 (mesenchymal) in whole mounts (same images as Figure 5A and Figure 1D) and sections (Figure 5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circles). Figure 5C shows predicted receptor-ligand interactions between adjacent foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. E9.5 DE and SM cell clusters are ordered along the anterior-to-posterior axis based on their in vivo location, with spatially adjacent DE and SM cell types facing each other. Shaded circles indicate relative pathway response—metagene expression levels—predicting the likelihood that a given cell population is responding to a growth factor signal. Thin vertical lines next to the clusters indicate various spatially adjacent cell populations that are all responding to a particular signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto SPRING plots in the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut cross sections, demonstrating its expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut cross sections, demonstrating BMP signaling responses in the DE and SM of the respiratory system and liver. Figures 5H and 5I show signaling roadmaps summarizing the predicted signaling states of all six pathways projected onto the DE (5H) and SM (5I) cell state trees, suggesting the combined signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signaling responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5D]We present one embodiment of computationally inferred receptor-ligand interactions that predict the signaling roadmap of foregut organogenesis. Figures 5A and 5B show E9.5 foregut immunostaining for Cdh1 (epithelial) and Foxf1 (mesenchymal) in whole mounts (same images as Figure 5A and Figure 1D) and sections (Figure 5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circles). Figure 5C shows predicted receptor-ligand interactions between adjacent foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. E9.5 DE and SM cell clusters are ordered along the anterior-to-posterior axis based on their in vivo location, with spatially adjacent DE and SM cell types facing each other. Shaded circles indicate relative pathway response—metagene expression levels—predicting the likelihood that a given cell population is responding to a growth factor signal. Thin vertical lines next to the clusters indicate various spatially adjacent cell populations that are all responding to a particular signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto SPRING plots in the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut cross sections, demonstrating its expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut cross sections, demonstrating BMP signaling responses in the DE and SM of the respiratory system and liver. Figures 5H and 5I show signaling roadmaps summarizing the predicted signaling states of all six pathways projected onto the DE (5H) and SM (5I) cell state trees, suggesting the combined signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signaling responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5E]We present one embodiment of computationally inferred receptor-ligand interactions that predict the signaling roadmap of foregut organogenesis. Figures 5A and 5B show E9.5 foregut immunostaining for Cdh1 (epithelial) and Foxf1 (mesenchymal) in whole mounts (same images as Figure 5A and Figure 1D) and sections (Figure 5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circles). Figure 5C shows predicted receptor-ligand interactions between adjacent foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. E9.5 DE and SM cell clusters are ordered along the anterior-to-posterior axis based on their in vivo location, with spatially adjacent DE and SM cell types facing each other. Shaded circles indicate relative pathway response—metagene expression levels—predicting the likelihood that a given cell population is responding to a growth factor signal. Thin vertical lines next to the clusters indicate various spatially adjacent cell populations that are all responding to a particular signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto SPRING plots in the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut cross sections, demonstrating its expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut cross sections, demonstrating BMP signaling responses in the DE and SM of the respiratory system and liver. Figures 5H and 5I show signaling roadmaps summarizing the predicted signaling states of all six pathways projected onto the DE (5H) and SM (5I) cell state trees, suggesting the combined signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signaling responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5F]We present one embodiment of computationally inferred receptor-ligand interactions that predict the signaling roadmap of foregut organogenesis. Figures 5A and 5B show E9.5 foregut immunostaining for Cdh1 (epithelial) and Foxf1 (mesenchymal) in whole mounts (same images as Figure 5A and Figure 1D) and sections (Figure 5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circles). Figure 5C shows predicted receptor-ligand interactions between adjacent foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. E9.5 DE and SM cell clusters are ordered along the anterior-to-posterior axis based on their in vivo location, with spatially adjacent DE and SM cell types facing each other. Shaded circles indicate relative pathway response—metagene expression levels—predicting the likelihood that a given cell population is responding to a growth factor signal. Thin vertical lines next to the clusters indicate various spatially adjacent cell populations that are all responding to a particular signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto SPRING plots in the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut cross sections, demonstrating its expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut cross sections, demonstrating BMP signaling responses in the DE and SM of the respiratory system and liver. Figures 5H and 5I show signaling roadmaps summarizing the predicted signaling states of all six pathways projected onto the DE (5H) and SM (5I) cell state trees, suggesting the combined signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signaling responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5G]We present one embodiment of computationally inferred receptor-ligand interactions that predict the signaling roadmap of foregut organogenesis. Figures 5A and 5B show E9.5 foregut immunostaining for Cdh1 (epithelial) and Foxf1 (mesenchymal) in whole mounts (same images as Figure 5A and Figure 1D) and sections (Figure 5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circles). Figure 5C shows predicted receptor-ligand interactions between adjacent foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. E9.5 DE and SM cell clusters are ordered along the anterior-to-posterior axis based on their in vivo location, with spatially adjacent DE and SM cell types facing each other. Shaded circles indicate relative pathway response—metagene expression levels—predicting the likelihood that a given cell population is responding to a growth factor signal. Thin vertical lines next to the clusters indicate various spatially adjacent cell populations that are all responding to a particular signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto SPRING plots in the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut cross sections, demonstrating its expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut cross sections, demonstrating BMP signaling responses in the DE and SM of the respiratory system and liver. Figures 5H and 5I show signaling roadmaps summarizing the predicted signaling states of all six pathways projected onto the DE (5H) and SM (5I) cell state trees, suggesting the combined signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signaling responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5H]We present one embodiment of computationally inferred receptor-ligand interactions that predict the signaling roadmap of foregut organogenesis. Figures 5A and 5B show E9.5 foregut immunostaining for Cdh1 (epithelial) and Foxf1 (mesenchymal) in whole mounts (same images as Figure 5A and Figure 1D) and sections (Figure 5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circles). Figure 5C shows predicted receptor-ligand interactions between adjacent foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. E9.5 DE and SM cell clusters are ordered along the anterior-to-posterior axis based on their in vivo location, with spatially adjacent DE and SM cell types facing each other. Shaded circles indicate relative pathway response—metagene expression levels—predicting the likelihood that a given cell population is responding to a growth factor signal. Thin vertical lines next to the clusters indicate various spatially adjacent cell populations that are all responding to a particular signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto SPRING plots in the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut cross sections, demonstrating its expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut cross sections, demonstrating BMP signaling responses in the DE and SM of the respiratory system and liver. Figures 5H and 5I show signaling roadmaps summarizing the predicted signaling states of all six pathways projected onto the DE (5H) and SM (5I) cell state trees, suggesting the combined signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signaling responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5I]We present one embodiment of computationally inferred receptor-ligand interactions that predict the signaling roadmap of foregut organogenesis. Figures 5A and 5B show E9.5 foregut immunostaining for Cdh1 (epithelial) and Foxf1 (mesenchymal) in whole mounts (same images as Figure 5A and Figure 1D) and sections (Figure 5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circles). Figure 5C shows predicted receptor-ligand interactions between adjacent foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. E9.5 DE and SM cell clusters are ordered along the anterior-to-posterior axis based on their in vivo location, with spatially adjacent DE and SM cell types facing each other. Shaded circles indicate relative pathway response—metagene expression levels—predicting the likelihood that a given cell population is responding to a growth factor signal. Thin vertical lines next to the clusters indicate various spatially adjacent cell populations that are all responding to a particular signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto SPRING plots in the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut cross sections, demonstrating its expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut cross sections, demonstrating BMP signaling responses in the DE and SM of the respiratory system and liver. Figures 5H and 5I show signaling roadmaps summarizing the predicted signaling states of all six pathways projected onto the DE (5H) and SM (5I) cell state trees, suggesting the combined signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signaling responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5J]One embodiment of metagene expression of all ligand-, receptor-, and context-independent response genes is shown. Dot plots showing the average scaled expression (2 to -2) of metagenes (X-axis) in each DE and SM cluster (Y-axis). For each cell signaling pathway (BMP, FGF, HH, Notch, RA, and canonical Wnt), the "ligand metagene," "receptor metagene," and "response metagene" were calculated by averaging the normalized expression of each individual gene for each pathway in each cell and cluster (e.g., Wnt ligand metagene = ΣWnt1 + Wnt2 + Wnt2b + Wnt3 · · · Wnt10b expression / n). The shading and size of each dot represent the metagene expression level for each cluster. [Figure 5K] One embodiment of computationally predicted receptor-ligand interactions between different foregut cell populations is shown. Schematics depict paracrine signaling between the DE and SM for six major pathways. Below the schematic, DE and SM cell clusters at each stage are aligned along the AP axis, consistent with their location in vivo. Spatially adjacent DE and SM cell types face each other. Shaded circles in each cluster indicate the likelihood that the cell population is responding to a signal based on pathway-responsive meta-gene expression levels. Arrows represent predicted sources of ligands, indicating paracrine and autocrine receptor-ligand pairs inferred from meta-gene expression profiles. Receptor-ligand pairings (arrows) were restricted to spatially adjacent cell populations. Thin vertical lines next to groups of clusters indicate various spatially adjacent cell populations that are all responding in the same way. [Figure 5L]One embodiment of the predicted temporal and spatial dynamics of signaling responses is shown. Expression levels of pathway response metagenes projected onto DE and SM SPRING plots, as well as cell state trees for the BMP (Panels A-B), FGF (Panels C-D), HH (Panels E-F), Notch (Panels G-H), RA (Panels I-J), and canonical Wnt (Panels K-L) pathways. This shows how coordinated spatial domains of signaling activity corresponding to cell lineages are predicted to change over a 24-hour period from E8.5 to E9.5. [Figure 6A] One embodiment of a gene study of the signaling roadmap reveals that HH promotes intestinal versus liver mesenchyme. Figures 6A and 6B show SPRING visualization of HH ligand metagene expression in DE cells (6A) and HH-responsive metagene expression in SM cells (6B). Figure 6C shows HH-responsive metagene expression projected onto the SM cell state tree, indicating low HH activity in the liver and pharyngeal SM but high activity in the intestinal mesenchyme. Figure 6D shows that Shh is expressed in the intestinal epithelium but not in the liver epithelium (outlined). The HH-responsive transgene, Gli1-lacZ, is active in the intestinal mesenchyme but not in the liver mesenchyme. Figure 6E shows differentially expressed genes between the foregut of Gli2- / -Gli3- / - and Gli2+ / -Gli3+ / - mice at E9.5 by bulk RNA sequencing (log2 FC>1, FDR<5%). Figure 6F shows a heatmap depicting the average expression of HH / Gli-regulated genes (from Figure 6E) in single-cell clusters of the DE and SM at E9.5. Figure 6G shows gene set enrichment analysis (GSEA) revealing specific cell-type enrichment of HH / Gli-regulated genes. Figure 6H shows a schematic representation of HH activity in the foregut. [Figure 6B]One embodiment of a gene study of the signaling roadmap reveals that HH promotes intestinal versus liver mesenchyme. Figures 6A and 6B show SPRING visualization of HH ligand metagene expression in DE cells (6A) and HH-responsive metagene expression in SM cells (6B). Figure 6C shows HH-responsive metagene expression projected onto the SM cell state tree, indicating low HH activity in the liver and pharyngeal SM but high activity in the intestinal mesenchyme. Figure 6D shows that Shh is expressed in the intestinal epithelium but not in the liver epithelium (outlined). The HH-responsive transgene, Gli1-lacZ, is active in the intestinal mesenchyme but not in the liver mesenchyme. Figure 6E shows differentially expressed genes between the foregut of Gli2- / -Gli3- / - and Gli2+ / -Gli3+ / - mice at E9.5 by bulk RNA sequencing (log2 FC>1, FDR<5%). Figure 6F shows a heatmap depicting the average expression of HH / Gli-regulated genes (from Figure 6E) in single-cell clusters of the DE and SM at E9.5. Figure 6G shows gene set enrichment analysis (GSEA) revealing specific cell-type enrichment of HH / Gli-regulated genes. Figure 6H shows a schematic representation of HH activity in the foregut. [Figure 6C]One embodiment of a gene study of the signaling roadmap reveals that HH promotes intestinal versus liver mesenchyme. Figures 6A and 6B show SPRING visualization of HH ligand metagene expression in DE cells (6A) and HH-responsive metagene expression in SM cells (6B). Figure 6C shows HH-responsive metagene expression projected onto the SM cell state tree, indicating low HH activity in the liver and pharyngeal SM but high activity in the intestinal mesenchyme. Figure 6D shows that Shh is expressed in the intestinal epithelium but not in the liver epithelium (outlined). The HH-responsive transgene, Gli1-lacZ, is active in the intestinal mesenchyme but not in the liver mesenchyme. Figure 6E shows differentially expressed genes between the foregut of Gli2- / -Gli3- / - and Gli2+ / -Gli3+ / - mice at E9.5 by bulk RNA sequencing (log2 FC>1, FDR<5%). Figure 6F shows a heatmap depicting the average expression of HH / Gli-regulated genes (from Figure 6E) in single-cell clusters of the DE and SM at E9.5. Figure 6G shows gene set enrichment analysis (GSEA) revealing specific cell-type enrichment of HH / Gli-regulated genes. Figure 6H shows a schematic representation of HH activity in the foregut. [Figure 6D]One embodiment of a gene study of the signaling roadmap reveals that HH promotes intestinal versus liver mesenchyme. Figures 6A and 6B show SPRING visualization of HH ligand metagene expression in DE cells (6A) and HH-responsive metagene expression in SM cells (6B). Figure 6C shows HH-responsive metagene expression projected onto the SM cell state tree, indicating low HH activity in the liver and pharyngeal SM but high activity in the intestinal mesenchyme. Figure 6D shows that Shh is expressed in the intestinal epithelium but not in the liver epithelium (outlined). The HH-responsive transgene, Gli1-lacZ, is active in the intestinal mesenchyme but not in the liver mesenchyme. Figure 6E shows differentially expressed genes between the foregut of Gli2- / -Gli3- / - and Gli2+ / -Gli3+ / - mice at E9.5 by bulk RNA sequencing (log2 FC>1, FDR<5%). Figure 6F shows a heatmap depicting the average expression of HH / Gli-regulated genes (from Figure 6E) in single-cell clusters of the DE and SM at E9.5. Figure 6G shows gene set enrichment analysis (GSEA) revealing specific cell-type enrichment of HH / Gli-regulated genes. Figure 6H shows a schematic representation of HH activity in the foregut. [Figure 6E]One embodiment of a gene study of the signaling roadmap reveals that HH promotes intestinal versus liver mesenchyme. Figures 6A and 6B show SPRING visualization of HH ligand metagene expression in DE cells (6A) and HH-responsive metagene expression in SM cells (6B). Figure 6C shows HH-responsive metagene expression projected onto the SM cell state tree, indicating low HH activity in the liver and pharyngeal SM but high activity in the intestinal mesenchyme. Figure 6D shows that Shh is expressed in the intestinal epithelium but not in the liver epithelium (outlined). The HH-responsive transgene, Gli1-lacZ, is active in the intestinal mesenchyme but not in the liver mesenchyme. Figure 6E shows differentially expressed genes between the foregut of Gli2- / -Gli3- / - and Gli2+ / -Gli3+ / - mice at E9.5 by bulk RNA sequencing (log2 FC>1, FDR<5%). Figure 6F shows a heatmap depicting the average expression of HH / Gli-regulated genes (from Figure 6E) in single-cell clusters of the DE and SM at E9.5. Figure 6G shows gene set enrichment analysis (GSEA) revealing specific cell-type enrichment of HH / Gli-regulated genes. Figure 6H shows a schematic representation of HH activity in the foregut. [Figure 6F]One embodiment of a gene study of the signaling roadmap reveals that HH promotes intestinal versus liver mesenchyme. Figures 6A and 6B show SPRING visualization of HH ligand metagene expression in DE cells (6A) and HH-responsive metagene expression in SM cells (6B). Figure 6C shows HH-responsive metagene expression projected onto the SM cell state tree, indicating low HH activity in the liver and pharyngeal SM but high activity in the intestinal mesenchyme. Figure 6D shows that Shh is expressed in the intestinal epithelium but not in the liver epithelium (outlined). The HH-responsive transgene, Gli1-lacZ, is active in the intestinal mesenchyme but not in the liver mesenchyme. Figure 6E shows differentially expressed genes between the foregut of Gli2- / -Gli3- / - and Gli2+ / -Gli3+ / - mice at E9.5 by bulk RNA sequencing (log2 FC>1, FDR<5%). Figure 6F shows a heatmap depicting the average expression of HH / Gli-regulated genes (from Figure 6E) in single-cell clusters of the DE and SM at E9.5. Figure 6G shows gene set enrichment analysis (GSEA) revealing specific cell-type enrichment of HH / Gli-regulated genes. Figure 6H shows a schematic representation of HH activity in the foregut. [Figure 6G]One embodiment of a gene study of the signaling roadmap reveals that HH promotes intestinal versus liver mesenchyme. Figures 6A and 6B show SPRING visualization of HH ligand metagene expression in DE cells (6A) and HH-responsive metagene expression in SM cells (6B). Figure 6C shows HH-responsive metagene expression projected onto the SM cell state tree, indicating low HH activity in the liver and pharyngeal SM but high activity in the intestinal mesenchyme. Figure 6D shows that Shh is expressed in the intestinal epithelium but not in the liver epithelium (outlined). The HH-responsive transgene, Gli1-lacZ, is active in the intestinal mesenchyme but not in the liver mesenchyme. Figure 6E shows differentially expressed genes between the foregut of Gli2- / -Gli3- / - and Gli2+ / -Gli3+ / - mice at E9.5 by bulk RNA sequencing (log2 FC>1, FDR<5%). Figure 6F shows a heatmap depicting the average expression of HH / Gli-regulated genes (from Figure 6E) in single-cell clusters of the DE and SM at E9.5. Figure 6G shows gene set enrichment analysis (GSEA) revealing specific cell-type enrichment of HH / Gli-regulated genes. Figure 6H shows a schematic representation of HH activity in the foregut. [Figure 6H]One embodiment of a gene study of the signaling roadmap reveals that HH promotes intestinal versus liver mesenchyme. Figures 6A and 6B show SPRING visualization of HH ligand metagene expression in DE cells (6A) and HH-responsive metagene expression in SM cells (6B). Figure 6C shows HH-responsive metagene expression projected onto the SM cell state tree, indicating low HH activity in the liver and pharyngeal SM but high activity in the intestinal mesenchyme. Figure 6D shows that Shh is expressed in the intestinal epithelium but not in the liver epithelium (outlined). The HH-responsive transgene, Gli1-lacZ, is active in the intestinal mesenchyme but not in the liver mesenchyme. Figure 6E shows differentially expressed genes between the foregut of Gli2- / -Gli3- / - and Gli2+ / -Gli3+ / - mice at E9.5 by bulk RNA sequencing (log2 FC>1, FDR<5%). Figure 6F shows a heatmap depicting the average expression of HH / Gli-regulated genes (from Figure 6E) in single-cell clusters of the DE and SM at E9.5. Figure 6G shows gene set enrichment analysis (GSEA) revealing specific cell-type enrichment of HH / Gli-regulated genes. Figure 6H shows a schematic representation of HH activity in the foregut. [Figure 7A]One embodiment for generating visceral mesoderm-like progenitor cells from human PSCs is shown. Figure 7A shows a schematic of the protocol for differentiating hPSCs into SM subtypes. Enumeration was predicted from the mouse single-cell signaling roadmap. Figure 7B shows RT-PCR of markers enriched in specific SM subtypes based on mouse single-cell data: cardiac (NKX2-5), early SM (FOXF1, HOXA1), liver-stm / mesothelial (WT1, UKP1B), liver fibroblast (MSX1), respiratory SM (NKX6-1+, MSC-), and esophagus / stomach (MSC, BARX1). Vertical bars represent mean ± SD. Tukey's test, *p<0.05, **p<0.005, ***p<0.0005. Figure 7C shows immunostaining of cell cultures at day 7. Scale bars are 50 μm (upper panel) and 10 μm (lower panel). Figure 7D shows quantification of the % of cells positive for the indicated immunostaining or RNAscope in situ hybridization. Vertical bars represent mean ± SD (n = 3). Tukey's test, *p < 0.05, **p < 0.005, ***p < 0.0005. [Figure 7B]One embodiment for generating visceral mesoderm-like progenitor cells from human PSCs is shown. Figure 7A shows a schematic of the protocol for differentiating hPSCs into SM subtypes. Enumeration was predicted from the mouse single-cell signaling roadmap. Figure 7B shows RT-PCR of markers enriched in specific SM subtypes based on mouse single-cell data: cardiac (NKX2-5), early SM (FOXF1, HOXA1), liver-stm / mesothelial (WT1, UKP1B), liver fibroblasts (MSX1), respiratory SM (NKX6-1+, MSC-), and esophagus / stomach (MSC, BARX1). Vertical bars represent mean ± SD. Tukey's test, *p<0.05, **p<0.005, ***p<0.0005. Figure 7C shows immunostaining of cell cultures at day 7. Scale bars are 50 μm (upper panel) and 10 μm (lower panel). Figure 7D shows quantification of the % of cells positive for the indicated immunostaining or RNAscope in situ hybridization. Vertical bars represent mean ± SD (n = 3). Tukey's test, *p < 0.05, **p < 0.005, ***p < 0.0005. [Figure 7C]One embodiment for generating visceral mesoderm-like progenitor cells from human PSCs is shown. Figure 7A shows a schematic of the protocol for differentiating hPSCs into SM subtypes. Enumeration was predicted from the mouse single-cell signaling roadmap. Figure 7B shows RT-PCR of markers enriched in specific SM subtypes based on mouse single-cell data: cardiac (NKX2-5), early SM (FOXF1, HOXA1), liver-stm / mesothelial (WT1, UKP1B), liver fibroblast (MSX1), respiratory SM (NKX6-1+, MSC-), and esophagus / stomach (MSC, BARX1). Vertical bars represent mean ± SD. Tukey's test, *p<0.05, **p<0.005, ***p<0.0005. Figure 7C shows immunostaining of cell cultures at day 7. Scale bars are 50 μm (upper panel) and 10 μm (lower panel). Figure 7D shows quantification of the % of cells positive for the indicated immunostaining or RNAscope in situ hybridization. Vertical bars represent mean ± SD (n = 3). Tukey's test, *p < 0.05, **p < 0.005, ***p < 0.0005. [Figure 7D]One embodiment for generating visceral mesoderm-like progenitor cells from human PSCs is shown. Figure 7A shows a schematic of the protocol for differentiating hPSCs into SM subtypes. Enumeration was predicted from the mouse single-cell signaling roadmap. Figure 7B shows RT-PCR of markers enriched in specific SM subtypes based on mouse single-cell data: cardiac (NKX2-5), early SM (FOXF1, HOXA1), liver-stm / mesothelial (WT1, UKP1B), liver fibroblasts (MSX1), respiratory SM (NKX6-1+, MSC-), and esophagus / stomach (MSC, BARX1). Vertical bars represent mean ± SD. Tukey's test, *p<0.05, **p<0.005, ***p<0.0005. Figure 7C shows immunostaining of cell cultures at day 7. Scale bars are 50 μm (upper panel) and 10 μm (lower panel). Figure 7D shows quantification of the % of cells positive for the indicated immunostaining or RNAscope in situ hybridization. Vertical bars represent mean ± SD (n = 3). Tukey's test, *p < 0.05, **p < 0.005, ***p < 0.0005. [Figure 7E] Figure 1 shows an embodiment of data demonstrating that RA suppresses cardiac mesoderm and promotes visceral mesoderm progenitors. Staining of RARE-lacZ transgenic mouse embryos confirms the single-cell RNA-seq prediction that RA activity is higher in visceral mesenchyme than cardiac mesenchyme at E8.5 (Panel A). Immunostaining of transverse sections of RARE-lacZ transgenic mouse embryos (Panel B). Day 4 PSC-derived SM cultures assayed by RT-PCR for paraxial mesoderm (PAX3), limb bud (PRRX1), cardiac mesoderm (NKX2.5, ISL1), endothelial (CD31), and SM (HOXA1, HOXA5, WNT2) markers. Scale bar is 50 μm (Panel C). Quantification of NKX2-5+ cells (Panel D). fg, foregut; hg, hindgut; ht, heart; SC, stem cell; MPS, middle primitive streak; CM, cardiac mesoderm; SM, visceral mesoderm. Vertical bars represent mean ± SD (n = 3). Tukey's test, *p < 0.05, **p < 0.005, ***p < 0.0005. [Figure 7F]Figure 7 shows an embodiment of additional analysis of day 7 SM-like PSC cultures. RNAscope in situ analysis of SM-like cultures at different days. Scale bars are 50 μm for the upper panel and 10 μm for the lower panel. Quantification of Figure 7D (Panels A-C). RT-PCR analysis of mesoderm subtype markers based on mouse scRNA-seq data. Cardiac SM (ACTC1, TBX20, TNNT2), early SM (PDE5A, HOXA5); liver-stm / mesothelial (TBX18, LHX2, UPK3B), liver fibroblasts (MSX2, HAND1), and esophageal / gastric (WNT4, NKX3-2) (Panel D). SC, stem cell; MPS, intermediate primitive streak; CM, cardiac mesoderm; SM, visceral mesoderm; STM, septum transversum mesenchyme; LF, liver fibroblasts; RM, respiratory mesenchyme; EM / GM, esophageal / gastric mesenchyme. Vertical bars indicate mean ± SD (n = 3). Tukey's test, *p<0.05, **p<0.005, ***p<0.0005. [Figure 8]

[0039] Figure 1 shows one embodiment of a schematic diagram of a protocol for generating organ-specific mesoderm from hPSCs. This protocol can be implemented by sequentially changing growth media containing growth factors and chemical components according to the stepwise process of organogenesis. A-LPM: anterior lateral plate mesoderm; CG-SpM: cardiac visceral mesoderm; CM: cardiac mesoderm; EM: esophageal mesoderm; GM: gastric mesoderm; HH-LPM: hedgehog-responsive lateral plate mesoderm; HH-SpM: hedgehog-responsive visceral mesoderm; LF: liver fibroblasts; LPM: lateral plate mesoderm; Mid PS: intermediate region of the primitive streak; RA-LPM: retinoic acid-responsive lateral plate mesoderm; RA-SpM: retinoic acid-responsive visceral mesoderm; RM: respiratory mesoderm; PSC: pluripotent stem cell; SpM: visceral mesoderm; STM: septum transverseis. [Figure 9A]Figure 9 shows an embodiment of hPSC differentiation toward the visceral mesoderm subtype. Figure 9A shows brightfield images of cells differentiating from hPSCs from days 0 to 4. Figure 9B shows the relative mRNA expression of VIM and FOXF1 by quantitative RT-PCR from days 0 to 4. Each column represents the mean from three independent wells with the standard deviation. Figure 9C shows immunostaining for the pan-mesodermal marker VIM and the epithelial marker CDH1. Figure 9D shows the relative mRNA expression of cardiac genes (ISL1 and NKX2-5), RA-responsive genes (HOXA5 and CYP26A1), and HH-responsive genes (GLI1 and PTCH1) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 9E shows immunostaining for the visceral marker FOXF1 and the cardiac mesoderm marker ISL1. Figure 9F shows quantification of differentiated cells on day 4. Each vertical bar represents the mean from three independent fields with standard deviation. [Figure 9B] Figure 9 shows an embodiment of hPSC differentiation toward the visceral mesoderm subtype. Figure 9A shows brightfield images of cells differentiating from hPSCs from days 0 to 4. Figure 9B shows the relative mRNA expression of VIM and FOXF1 by quantitative RT-PCR from days 0 to 4. Each column represents the mean from three independent wells with the standard deviation. Figure 9C shows immunostaining for the pan-mesodermal marker VIM and the epithelial marker CDH1. Figure 9D shows the relative mRNA expression of cardiac genes (ISL1 and NKX2-5), RA-responsive genes (HOXA5 and CYP26A1), and HH-responsive genes (GLI1 and PTCH1) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 9E shows immunostaining for the visceral marker FOXF1 and the cardiac mesoderm marker ISL1. Figure 9F shows quantification of differentiated cells on day 4. Each vertical bar represents the mean from three independent fields with standard deviation. [Figure 9C]Figure 9 shows an embodiment of hPSC differentiation toward the visceral mesoderm subtype. Figure 9A shows brightfield images of cells differentiating from hPSCs from days 0 to 4. Figure 9B shows the relative mRNA expression of VIM and FOXF1 by quantitative RT-PCR from days 0 to 4. Each column represents the mean from three independent wells with the standard deviation. Figure 9C shows immunostaining for the pan-mesodermal marker VIM and the epithelial marker CDH1. Figure 9D shows the relative mRNA expression of cardiac genes (ISL1 and NKX2-5), RA-responsive genes (HOXA5 and CYP26A1), and HH-responsive genes (GLI1 and PTCH1) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 9E shows immunostaining for the visceral marker FOXF1 and the cardiac mesoderm marker ISL1. Figure 9F shows quantification of differentiated cells on day 4. Each vertical bar represents the mean from three independent fields with standard deviation. [Figure 9D] Figure 9 shows an embodiment of hPSC differentiation toward the visceral mesoderm subtype. Figure 9A shows brightfield images of cells differentiating from hPSCs from days 0 to 4. Figure 9B shows the relative mRNA expression of VIM and FOXF1 by quantitative RT-PCR from days 0 to 4. Each column represents the mean from three independent wells with the standard deviation. Figure 9C shows immunostaining for the pan-mesodermal marker VIM and the epithelial marker CDH1. Figure 9D shows the relative mRNA expression of cardiac genes (ISL1 and NKX2-5), RA-responsive genes (HOXA5 and CYP26A1), and HH-responsive genes (GLI1 and PTCH1) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 9E shows immunostaining for the visceral marker FOXF1 and the cardiac mesoderm marker ISL1. Figure 9F shows quantification of differentiated cells on day 4. Each vertical bar represents the mean from three independent fields with standard deviation. [Figure 9E]Figure 9 shows an embodiment of hPSC differentiation toward the visceral mesoderm subtype. Figure 9A shows brightfield images of cells differentiating from hPSCs from days 0 to 4. Figure 9B shows the relative mRNA expression of VIM and FOXF1 by quantitative RT-PCR from days 0 to 4. Each column represents the mean from three independent wells with the standard deviation. Figure 9C shows immunostaining for the pan-mesodermal marker VIM and the epithelial marker CDH1. Figure 9D shows the relative mRNA expression of cardiac genes (ISL1 and NKX2-5), RA-responsive genes (HOXA5 and CYP26A1), and HH-responsive genes (GLI1 and PTCH1) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 9E shows immunostaining for the visceral marker FOXF1 and the cardiac mesoderm marker ISL1. Figure 9F shows quantification of differentiated cells on day 4. Each vertical bar represents the mean from three independent fields with standard deviation. [Figure 9F] Figure 9 shows an embodiment of hPSC differentiation toward the visceral mesoderm subtype. Figure 9A shows brightfield images of cells differentiating from hPSCs from days 0 to 4. Figure 9B shows the relative mRNA expression of VIM and FOXF1 by quantitative RT-PCR from days 0 to 4. Each column represents the mean from three independent wells with the standard deviation. Figure 9C shows immunostaining for the pan-mesodermal marker VIM and the epithelial marker CDH1. Figure 9D shows the relative mRNA expression of cardiac genes (ISL1 and NKX2-5), RA-responsive genes (HOXA5 and CYP26A1), and HH-responsive genes (GLI1 and PTCH1) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 9E shows immunostaining for the visceral marker FOXF1 and the cardiac mesoderm marker ISL1. Figure 9F shows quantification of differentiated cells on day 4. Each vertical bar represents the mean from three independent fields with standard deviation. [Figure 10A]Figure 10 shows an embodiment of characterization of organ-specific mesoderm on day 7. Figure 10A shows a brightfield image of differentiated organ-specific mesoderm on day 7. Figure 10B shows immunostaining for CTNNB1. Figure 10C shows the relative mRNA expression of cardiac genes (NKX2-5 and TBX20), liver septum transversum gene (WT1), liver mesothelial gene (UPK1B), liver fibroblast genes (PITX1, MSX2, and TBX5), gastric mesoderm genes (FOXF1, BARX1, and NKX3-2), respiratory mesoderm genes (FOXF1, NKX6-1, and TBX5), and esophageal mesoderm (FOXF1 and MSC) by quantitative RT-PCR. Each vertical bar represents the average from three independent cells with the standard deviation. Figure 10D shows immunostaining for FOXF1, NKX6.1, and WT1. Figure 10E shows immunostaining for PITX1 and TBX5. [Figure 10B] Figure 10 shows an embodiment of characterization of organ-specific mesoderm on day 7. Figure 10A shows a brightfield image of differentiated organ-specific mesoderm on day 7. Figure 10B shows immunostaining for CTNNB1. Figure 10C shows the relative mRNA expression of cardiac genes (NKX2-5 and TBX20), liver septum transversum gene (WT1), liver mesothelial gene (UPK1B), liver fibroblast genes (PITX1, MSX2, and TBX5), gastric mesoderm genes (FOXF1, BARX1, and NKX3-2), respiratory mesoderm genes (FOXF1, NKX6-1, and TBX5), and esophageal mesoderm (FOXF1 and MSC) by quantitative RT-PCR. Each vertical bar represents the average from three independent cells with the standard deviation. Figure 10D shows immunostaining for FOXF1, NKX6.1, and WT1. Figure 10E shows immunostaining for PITX1 and TBX5. [Figure 10C]Figure 10 shows an embodiment of characterization of organ-specific mesoderm on day 7. Figure 10A shows a brightfield image of differentiated organ-specific mesoderm on day 7. Figure 10B shows immunostaining for CTNNB1. Figure 10C shows the relative mRNA expression of cardiac genes (NKX2-5 and TBX20), liver septum transversum gene (WT1), liver mesothelial gene (UPK1B), liver fibroblast genes (PITX1, MSX2, and TBX5), gastric mesoderm genes (FOXF1, BARX1, and NKX3-2), respiratory mesoderm genes (FOXF1, NKX6-1, and TBX5), and esophageal mesoderm (FOXF1 and MSC) by quantitative RT-PCR. Each vertical bar represents the average from three independent cells with the standard deviation. Figure 10D shows immunostaining for FOXF1, NKX6.1, and WT1. Figure 10E shows immunostaining for PITX1 and TBX5. [Figure 10D] Figure 10 shows an embodiment of characterization of organ-specific mesoderm on day 7. Figure 10A shows a brightfield image of differentiated organ-specific mesoderm on day 7. Figure 10B shows immunostaining for CTNNB1. Figure 10C shows the relative mRNA expression of cardiac genes (NKX2-5 and TBX20), liver septum transversum gene (WT1), liver mesothelial gene (UPK1B), liver fibroblast genes (PITX1, MSX2, and TBX5), gastric mesoderm genes (FOXF1, BARX1, and NKX3-2), respiratory mesoderm genes (FOXF1, NKX6-1, and TBX5), and esophageal mesoderm (FOXF1 and MSC) by quantitative RT-PCR. Each vertical bar represents the average from three independent cells with the standard deviation. Figure 10D shows immunostaining for FOXF1, NKX6.1, and WT1. Figure 10E shows immunostaining for PITX1 and TBX5. [Figure 10E]Figure 10 shows an embodiment of characterization of organ-specific mesoderm on day 7. Figure 10A shows a brightfield image of differentiated organ-specific mesoderm on day 7. Figure 10B shows immunostaining for CTNNB1. Figure 10C shows the relative mRNA expression of cardiac genes (NKX2-5 and TBX20), liver septum transversum gene (WT1), liver mesothelial gene (UPK1B), liver fibroblast genes (PITX1, MSX2, and TBX5), gastric mesoderm genes (FOXF1, BARX1, and NKX3-2), respiratory mesoderm genes (FOXF1, NKX6-1, and TBX5), and esophageal mesoderm (FOXF1 and MSC) by quantitative RT-PCR. Each vertical bar represents the average from three independent cells with the standard deviation. Figure 10D shows immunostaining for FOXF1, NKX6.1, and WT1. Figure 10E shows immunostaining for PITX1 and TBX5. [Figure 11A] Figure 11A shows a comparison of protocols for generating hepatic mesoderm. Figure 11A shows an overview of three protocols (the one provided herein, Coll et al. 2018, and Takebe et al. 2017). Figure 11B shows a heatmap based on relative mRNA expression of hepatic mesenchymal genes from quantitative RT-PCR. Each column represents the average from three independent wells. Figure 11C shows immunostaining for GATA4, WT1, and KRT19. Figure 11D shows immunostaining for PITX1 and TBX5. References:Coll,M.et al.Generation of Hepatic Stellate Cells from Human Pluripotent Stem Cells Enables In Vitro Modeling of Liver Fibrosis.Cell Stem Cell 23,101-113.e107(2018);Takebe,T.et al.Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.Cell Rep 21,2661-2670(2017). [Figure 11B]Figure 11A shows a comparison of protocols for generating hepatic mesoderm. Figure 11A shows an overview of three protocols (the one provided herein, Coll et al. 2018, and Takebe et al. 2017). Figure 11B shows a heatmap based on relative mRNA expression of hepatic mesenchymal genes from quantitative RT-PCR. Each column represents the average from three independent wells. Figure 11C shows immunostaining for GATA4, WT1, and KRT19. Figure 11D shows immunostaining for PITX1 and TBX5. References:Coll,M.et al.Generation of Hepatic Stellate Cells from Human Pluripotent Stem Cells Enables In Vitro Modeling of Liver Fibrosis.Cell Stem Cell 23,101-113.e107(2018);Takebe,T.et al.Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.Cell Rep 21,2661-2670(2017). [Figure 11C]Figure 11A shows a comparison of protocols for generating hepatic mesoderm. Figure 11A shows an overview of three protocols (the one provided herein, Coll et al. 2018, and Takebe et al. 2017). Figure 11B shows a heatmap based on relative mRNA expression of hepatic mesenchymal genes from quantitative RT-PCR. Each column represents the average from three independent wells. Figure 11C shows immunostaining for GATA4, WT1, and KRT19. Figure 11D shows immunostaining for PITX1 and TBX5. References:Coll,M.et al.Generation of Hepatic Stellate Cells from Human Pluripotent Stem Cells Enables In Vitro Modeling of Liver Fibrosis.Cell Stem Cell 23,101-113.e107(2018);Takebe,T.et al.Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.Cell Rep 21,2661-2670(2017). [Figure 11D]Figure 11A shows a comparison of protocols for generating hepatic mesoderm. Figure 11A shows an overview of three protocols (the one provided herein, Coll et al. 2018, and Takebe et al. 2017). Figure 11B shows a heatmap based on relative mRNA expression of hepatic mesenchymal genes from quantitative RT-PCR. Each column represents the average from three independent wells. Figure 11C shows immunostaining for GATA4, WT1, and KRT19. Figure 11D shows immunostaining for PITX1 and TBX5. References:Coll,M.et al.Generation of Hepatic Stellate Cells from Human Pluripotent Stem Cells Enables In Vitro Modeling of Liver Fibrosis.Cell Stem Cell 23,101-113.e107(2018);Takebe,T.et al.Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.Cell Rep 21,2661-2670(2017). [Figure 12A]Figure 12A shows a comparison of protocols for generating tracheal / pulmonary mesoderm. Figure 12A shows an overview of two protocols (provided herein and Kishimoto et al. 2020). Figure 12B shows the relative mRNA expression of tracheal / pulmonary mesoderm genes (FOXF1, NKX6-1, TBX5, TBX4, and WNT2) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 12C shows immunostaining for FOXF1 and NKX6.1 on day 7. Figure 12D shows immunostaining for TBX5 on day 7. Figure 12E shows immunostaining for NKX6.1 and WNT2 in E9.5 mouse foregut tissue, demonstrating the delineation between ventral and medial respiratory mesenchyme, which is suspected to be Wnt-dependent. Figure 12F shows immunostaining for SOX9 and SMA on day 12. Figure 12G shows the relative mRNA expression of the chondrocyte progenitor gene SOX9 and the smooth muscle gene ACTA2 by quantitative RT-PCR. Each vertical bar represents the average from three independent wells with the standard deviation. Reference: Kishimoto, K. et al. Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells. Nat Commun 11, 4159 (2020), which is also described in WO 2021 / 041443, the entire contents of which are expressly incorporated herein by reference. [Figure 12B]Figure 12A shows a comparison of protocols for generating tracheal / pulmonary mesoderm. Figure 12A shows an overview of two protocols (provided herein and Kishimoto et al. 2020). Figure 12B shows the relative mRNA expression of tracheal / pulmonary mesoderm genes (FOXF1, NKX6-1, TBX5, TBX4, and WNT2) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 12C shows immunostaining for FOXF1 and NKX6.1 on day 7. Figure 12D shows immunostaining for TBX5 on day 7. Figure 12E shows immunostaining for NKX6.1 and WNT2 in E9.5 mouse foregut tissue, demonstrating the delineation between ventral and medial respiratory mesenchyme, which is suspected to be Wnt-dependent. Figure 12F shows immunostaining for SOX9 and SMA on day 12. Figure 12G shows the relative mRNA expression of the chondrocyte progenitor gene SOX9 and the smooth muscle gene ACTA2 by quantitative RT-PCR. Each vertical bar represents the average from three independent wells with the standard deviation. Reference: Kishimoto, K. et al. Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells. Nat Commun 11, 4159 (2020), which is also described in WO 2021 / 041443, the entire contents of which are expressly incorporated herein by reference. [Figure 12C]Figure 12A shows a comparison of protocols for generating tracheal / pulmonary mesoderm. Figure 12A shows an overview of two protocols (provided herein and Kishimoto et al. 2020). Figure 12B shows the relative mRNA expression of tracheal / pulmonary mesoderm genes (FOXF1, NKX6-1, TBX5, TBX4, and WNT2) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 12C shows immunostaining for FOXF1 and NKX6.1 on day 7. Figure 12D shows immunostaining for TBX5 on day 7. Figure 12E shows immunostaining for NKX6.1 and WNT2 in E9.5 mouse foregut tissue, demonstrating the delineation between ventral and medial respiratory mesenchyme, which is suspected to be Wnt-dependent. Figure 12F shows immunostaining for SOX9 and SMA on day 12. Figure 12G shows the relative mRNA expression of the chondrocyte progenitor gene SOX9 and the smooth muscle gene ACTA2 by quantitative RT-PCR. Each vertical bar represents the average from three independent wells with the standard deviation. Reference: Kishimoto, K. et al. Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells. Nat Commun 11, 4159 (2020), which is also described in WO 2021 / 041443, the entire contents of which are expressly incorporated herein by reference. [Figure 12D]Figure 12A shows a comparison of protocols for generating tracheal / pulmonary mesoderm. Figure 12A shows an overview of two protocols (provided herein and Kishimoto et al. 2020). Figure 12B shows the relative mRNA expression of tracheal / pulmonary mesoderm genes (FOXF1, NKX6-1, TBX5, TBX4, and WNT2) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 12C shows immunostaining for FOXF1 and NKX6.1 on day 7. Figure 12D shows immunostaining for TBX5 on day 7. Figure 12E shows immunostaining for NKX6.1 and WNT2 in E9.5 mouse foregut tissue, demonstrating the delineation between ventral and medial respiratory mesenchyme, which is suspected to be Wnt-dependent. Figure 12F shows immunostaining for SOX9 and SMA on day 12. Figure 12G shows the relative mRNA expression of the chondrocyte progenitor gene SOX9 and the smooth muscle gene ACTA2 by quantitative RT-PCR. Each vertical bar represents the average from three independent wells with the standard deviation. Reference: Kishimoto, K. et al. Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells. Nat Commun 11, 4159 (2020), which is also described in WO 2021 / 041443, the entire contents of which are expressly incorporated herein by reference. [Figure 12E]Figure 12A shows a comparison of protocols for generating tracheal / pulmonary mesoderm. Figure 12A shows an overview of two protocols (provided herein and Kishimoto et al. 2020). Figure 12B shows the relative mRNA expression of tracheal / pulmonary mesoderm genes (FOXF1, NKX6-1, TBX5, TBX4, and WNT2) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 12C shows immunostaining for FOXF1 and NKX6.1 on day 7. Figure 12D shows immunostaining for TBX5 on day 7. Figure 12E shows immunostaining for NKX6.1 and WNT2 in E9.5 mouse foregut tissue, demonstrating the delineation between ventral and medial respiratory mesenchyme, which is suspected to be Wnt-dependent. Figure 12F shows immunostaining for SOX9 and SMA on day 12. Figure 12G shows the relative mRNA expression of the chondrocyte progenitor gene SOX9 and the smooth muscle gene ACTA2 by quantitative RT-PCR. Each vertical bar represents the average from three independent wells with the standard deviation. Reference: Kishimoto, K. et al. Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells. Nat Commun 11, 4159 (2020), which is also described in WO 2021 / 041443, the entire contents of which are expressly incorporated herein by reference. [Figure 12F]Figure 12A shows a comparison of protocols for generating tracheal / pulmonary mesoderm. Figure 12A shows an overview of two protocols (provided herein and Kishimoto et al. 2020). Figure 12B shows the relative mRNA expression of tracheal / pulmonary mesoderm genes (FOXF1, NKX6-1, TBX5, TBX4, and WNT2) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 12C shows immunostaining for FOXF1 and NKX6.1 on day 7. Figure 12D shows immunostaining for TBX5 on day 7. Figure 12E shows immunostaining for NKX6.1 and WNT2 in E9.5 mouse foregut tissue, demonstrating the delineation between ventral and medial respiratory mesenchyme, which is suspected to be Wnt-dependent. Figure 12F shows immunostaining for SOX9 and SMA on day 12. Figure 12G shows the relative mRNA expression of the chondrocyte progenitor gene SOX9 and the smooth muscle gene ACTA2 by quantitative RT-PCR. Each vertical bar represents the average from three independent wells with the standard deviation. Reference: Kishimoto, K. et al. Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells. Nat Commun 11, 4159 (2020), which is also described in WO 2021 / 041443, the entire contents of which are expressly incorporated herein by reference. [Figure 12G]Figure 12A shows a comparison of protocols for generating tracheal / pulmonary mesoderm. Figure 12A shows an overview of two protocols (provided herein and Kishimoto et al. 2020). Figure 12B shows the relative mRNA expression of tracheal / pulmonary mesoderm genes (FOXF1, NKX6-1, TBX5, TBX4, and WNT2) by quantitative RT-PCR. Each column represents the mean from three independent wells with the standard deviation. Figure 12C shows immunostaining for FOXF1 and NKX6.1 on day 7. Figure 12D shows immunostaining for TBX5 on day 7. Figure 12E shows immunostaining for NKX6.1 and WNT2 in E9.5 mouse foregut tissue, demonstrating the delineation between ventral and medial respiratory mesenchyme, which is suspected to be Wnt-dependent. Figure 12F shows immunostaining for SOX9 and SMA on day 12. Figure 12G shows the relative mRNA expression of the chondrocyte progenitor gene SOX9 and the smooth muscle gene ACTA2 by quantitative RT-PCR. Each vertical bar represents the average from three independent wells with the standard deviation. Reference: Kishimoto, K. et al. Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells. Nat Commun 11, 4159 (2020), which is also described in WO 2021 / 041443, the entire contents of which are expressly incorporated herein by reference. [Figure 13A]

[0049] Figure 13 shows an embodiment for evaluating pluripotency genes during mesoderm differentiation. Figure 13A shows the relative mRNA expression of pluripotency marker genes OCT3 / 4 and SOX2 by quantitative RT-PCR from day 0 to day 4. Figure 13B shows the relative mRNA expression of TBXT as an early mesoderm marker by quantitative RT-PCR from day 0 to day 4. Figure 13C shows immunostaining for OCT3 / 4 and SOX2. Figure 13D shows immunostaining for TBXT. Figure 13E shows the relative mRNA expression of FOXA2 as an endoderm marker by quantitative RT-PCR from day 0 to day 4. [Figure 13B]

[0049] Figure 13 shows an embodiment for evaluating pluripotency genes during mesoderm differentiation. Figure 13A shows the relative mRNA expression of pluripotency marker genes OCT3 / 4 and SOX2 by quantitative RT-PCR from day 0 to day 4. Figure 13B shows the relative mRNA expression of TBXT as an early mesoderm marker by quantitative RT-PCR from day 0 to day 4. Figure 13C shows immunostaining for OCT3 / 4 and SOX2. Figure 13D shows immunostaining for TBXT. Figure 13E shows the relative mRNA expression of FOXA2 as an endoderm marker by quantitative RT-PCR from day 0 to day 4. [Figure 13C]

[0049] Figure 13 shows an embodiment for evaluating pluripotency genes during mesoderm differentiation. Figure 13A shows the relative mRNA expression of pluripotency marker genes OCT3 / 4 and SOX2 by quantitative RT-PCR from day 0 to day 4. Figure 13B shows the relative mRNA expression of TBXT as an early mesoderm marker by quantitative RT-PCR from day 0 to day 4. Figure 13C shows immunostaining for OCT3 / 4 and SOX2. Figure 13D shows immunostaining for TBXT. Figure 13E shows the relative mRNA expression of FOXA2 as an endoderm marker by quantitative RT-PCR from day 0 to day 4. [Figure 13D]

[0049] Figure 13 shows an embodiment for evaluating pluripotency genes during mesoderm differentiation. Figure 13A shows the relative mRNA expression of pluripotency marker genes OCT3 / 4 and SOX2 by quantitative RT-PCR from day 0 to day 4. Figure 13B shows the relative mRNA expression of TBXT as an early mesoderm marker by quantitative RT-PCR from day 0 to day 4. Figure 13C shows immunostaining for OCT3 / 4 and SOX2. Figure 13D shows immunostaining for TBXT. Figure 13E shows the relative mRNA expression of FOXA2 as an endoderm marker by quantitative RT-PCR from day 0 to day 4. [Figure 13E]

[0049] Figure 13 shows an embodiment for evaluating pluripotency genes during mesoderm differentiation. Figure 13A shows the relative mRNA expression of pluripotency marker genes OCT3 / 4 and SOX2 by quantitative RT-PCR from day 0 to day 4. Figure 13B shows the relative mRNA expression of TBXT as an early mesoderm marker by quantitative RT-PCR from day 0 to day 4. Figure 13C shows immunostaining for OCT3 / 4 and SOX2. Figure 13D shows immunostaining for TBXT. Figure 13E shows the relative mRNA expression of FOXA2 as an endoderm marker by quantitative RT-PCR from day 0 to day 4. DETAILED DESCRIPTION OF THE INVENTION

[0018] Internal organs, such as the lungs, stomach, liver, and pancreas, originate in the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding visceral mesoderm (SM). While DE lineage patterning has been fairly well studied, the paracrine signaling that controls SM regionalization and how this coordinates with epithelial identity during organogenesis remains unclear. Disclosed herein is single-cell transcriptomics to generate a high-resolution cell state map of the embryonic mouse foregut. This revealed an unexpected diversity of SM cells that arise in close concert with organ-specific epithelia. These data inferred a spatiotemporal signaling roadmap of combinatorial endoderm-mesoderm interactions that orchestrate foregut organogenesis. Key predictions were validated with mouse genetics, demonstrating the importance of endoderm-derived signals in mesoderm patterning. Leveraging this signaling roadmap, we generated diverse SM subtypes from previously undefined human pluripotent stem cells (hPSCs).

[0019] The crucial inductive role of mesenchyme in gut organogenesis was first established in the 1960s, when it was shown that SMs transplanted from different anterior-posterior (AP) regions of the embryo can instruct adjacent epithelia to adopt organ identities consistent with the original SM location. Since then, mesoderm-derived paracrine signals in endodermal organogenesis have been investigated, but most of these studies have focused on individual organ lineages or individual signaling pathways and thus lack a comprehensive understanding of the temporally dynamic combinatorial signaling in the foregut microenvironment that orchestrates organogenesis. Furthermore, several fundamental questions about the mesoderm remain unanswered for decades: How many SMs are there, and does each fetal organ primordium have its own unique mesenchyme? How are SM and DE lineages regulated during organogenesis? What role does endoderm play in mesoderm regionalization?

[0020] The initial specification and patterning of embryonic mesoderm and endoderm occurs during gastrulation in mice, from E6.25 to E8.0, when these germ layers gradually emerge from the primitive streak. Lateral plate mesoderm emerges from the streak after the extraembryonic mesoderm, followed by intermediate, paraxial, and axial mesoderm. Concomitantly, DE cells also delaminate from the streak, migrate along the outer surface of the mesoderm, and ultimately insert into the overlying visceral endoderm. By E8.0, the anterior DE folds to form a foregut diverticulum, and as the adjacent lateral plate mesoderm containing cardiac progenitors migrates toward the ventral midline, morphogenetic processes begin to transform the two-layered sheet of endoderm and mesoderm into a tubular structure. The lateral plate mesoderm further divides into an outer somatic mesoderm layer adjacent to the ectoderm that gives rise to the limbs and body wall, and an inner visceral mesoderm layer that surrounds the epithelial gut tube. The first molecular signature of regional identity in the SM is the differential expression of Hox genes along the AP axis of the embryo. However, in contrast to cardiac development, where cellular diversification is well studied, the molecular mechanisms governing regionalization of the foregut SM remain unclear, particularly during the critical 24-h period during which the foregut DE subdivides into distinct organ primordia.

[0021] Recently, single-cell transcriptomics has begun to examine organogenesis with unprecedented resolution. However, studies of the developing intestine have primarily examined either epithelial components or the subsequent fetal organs they specify. As described herein, we used single-cell transcriptomics of the mouse embryonic foregut to infer comprehensive "cell state" ontogeny of the DE and SM lineages and discovered an unexpected diversity of SM progenitor subtypes that develop in close concordance with organ-specific epithelia. Projecting transcriptional profiles of paracrine signaling pathways onto these lineages infers a roadmap of reciprocal endoderm-mesoderm inductive interactions that coordinate organogenesis. Key predictions were validated with mouse genetics, showing that distinct Hedgehog signaling from the epithelium patterns SM into intestinal mesenchyme versus liver mesenchyme. Leveraging this signaling roadmap, we generated previously unknown subtypes of human SM from hPSCs.

[0022] As disclosed herein, single-cell transcriptomics was used to define the complexity of cell types in the DE and SM in the embryonic mouse foregut over the first 24 hours of organogenesis, as the primitive gut tube subdivides into distinct organ domains. Herein, an unexpected diversity of distinct cell types in the foregut mesenchyme, defined by a combinatorial code of novel marker genes and transcription factors, is revealed. Cell trajectories indicate tightly coordinated development into organ-specific DE and SM, suggesting a tightly regulated signaling network. A putative ligand-receptor signaling roadmap of reciprocal epithelial-mesenchymal interactions likely orchestrating lineage specification of the two tissue compartments was computationally predicted. The disclosures herein represent a valuable resource for further experimental investigation of foregut organogenesis, and the data can be searched on the World Wide Web at research.cchmc.org / ZornLab-singlecell.

[0023] Previous studies on the regional identity of the SM in the early embryo have been limited. Aside from the well-known regionalization of Hox gene expression, most studies have focused primarily on individual organs, such as the stomach or lung mesenchyme. Comparing single-cell transcriptomes across the entire foregut revealed extensive regionalization of the early SM into distinct organ-specific mesenchymal subtypes. The diverse transcriptional signatures of early SM cell types may only be utilized transiently to define their location and molecular program during fetal organogenesis. After organ fate determination, various SM cell types may converge toward similar differentiation programs, such as smooth muscle or fibroblasts common to all internal organs. However, the results of fetal SM diversification here are intriguing in light of emerging ideas about organ-specific interstitial cells in the adult, such as hepatic versus pancreatic stellate cells and lung-specific fibroblasts. For example, Tbx4 is expressed in the embryonic respiratory SM and subsequently maintained specifically in adult lung fibroblasts, but not in fibroblasts of other organs. Future integrated analyses of the data herein with other single-cell RNA sequencing (scRNA-seq) datasets from later fetal and adult organs should resolve how transcriptional programs evolve during cell differentiation, homeostasis, and pathogenesis.

[0024] One unexpected observation was that the liver bud contains a more distinct SM cellular state than any other organ primordium, with a septum transversum mesenchyme (stm), a venous sinus, two mesothelium, and a fibroblast population. This may be due to the fact that, unlike other GI organs that form by epithelial bulging, the hepatic endoderm delaminates and invades the adjacent stm, a process that may require more complex epithelial-mesenchymal interactions with the extracellular matrix. Our transcriptome analysis is consistent with lineage-tracing experiments showing that the early stm gives rise to mesothelium, hepatic stellate cells, interstitial fibroblasts, and perivascular smooth muscle. It will be important to determine whether the buds of other organs undergo similar cell type elaboration as they differentiate. Alternatively, mesothelium and fibroblasts derived from the liver may migrate into other organ buds. Indeed, mesenchymal cell movement is one of the confounding limitations of our study, and there is ample evidence that the hepatoblast mesothelium, also known as the proepicardium, migrates to surround the heart and lungs.

[0025] The foregut SM and cardiac mesoderm are closely related, both arising from the anterior lateral plate mesoderm. Preliminary cross-comparison of the data provided herein with recent single-cell RNA-seq studies of the early heart suggests that this common origin is reflected in the transcriptome. The developing heart tube is adjacent to the ventral foregut SM (also known as the secondary heart field [SHF]), with its arterial pole connecting to the pharyngeal SM and its venous pole connecting to the pulmonary / liver SM. Cell fate mapping studies indicate that the secondary heart field gives rise to cardiac tissue as well as the pharyngeal SM, respiratory SM, and pulmonary vasculature. Indeed, the single-cell transcriptomics and genetic analysis of Gli mutants provided herein demonstrate that epithelial-derived HH signals are important for the development of these cardiac-pulmonary progenitors.

[0026] We further investigated the discriminators that lead to the distinct mesodermal lineages provided herein. Supplementing lateral plate mesoderm (LPM) and splanchnic mesoderm (SM, SpM) induction media with retinoic acid (RA) converted their fate from cardiac SpM (CG-SpM) to more posterior, RA-responsive SpM (RA-SpM). Single-cell data further suggest that Hedgehog (HH) signaling subdivides RA-treated SpM into either hepatic mesoderm lineages, which give rise to esophageal and respiratory mesenchyme, in the absence of HH or HH-responsive gut mesoderm (HH-SpM). Thus, disclosed herein is a method for the combined activation or inhibition of the Wnt, BMP, RA, or HH pathways in monolayer culture from days 4 to 7 to efficiently direct SpM into five distinct mesenchymal progenitor cell populations: hepatic septum transversum / mesothelium (STM / mesothelium), liver fibroblasts (LF), gastric mesoderm (GM), esophageal mesoderm (EM), and respiratory mesoderm (RM), which are characterized based on their unique transcriptional signatures.

[0027] The signaling roadmap developed herein was used to direct hPSC development into distinct SM-like cell types. The system described herein offers a unique opportunity to model human fetal mesenchymal development and interrogate how combined signaling pathways direct parallel mesenchymal fate choices. The organ-specific mesenchymal progenitor cells investigated here can be used to investigate the molecular mechanisms underlying their differentiation into mature mesenchymal cell types, such as fibroblasts or smooth muscle. For example, as provided herein, we investigated the differentiation of RMs typical of the trachea and airways into smooth muscle and cartilage. Furthermore, coculturing hPSC-derived mesoderm with hPSC-derived endoderm provides a reductionist system for investigating the complex epithelial-mesenchymal crosstalk of human foregut organogenesis in vitro. Such in vitro culture models are particularly well suited for large-scale genomic analysis of gene regulatory networks governing SpM diversification, which is difficult to study in vivo.

[0028] These approaches can also be used to model congenital disorders of LPM in vitro. LPM plays a developmental role in foregut organogenesis, and disruption of this process can lead to life-threatening congenital defects such as esophageal atresia and tracheoesophageal fistula (EA / TEF). While ongoing patient genome sequencing is rapidly identifying candidate causative mutations, a major challenge remains determining whether these genes act in the endoderm or mesoderm and how they affect development. By using patient-derived iPSCs in the protocols provided herein, researchers can determine whether patient mutations affect mesodermal or endodermal lineage differentiation for a wide variety of different congenital abnormalities affecting the foregut organ.

[0029] Furthermore, the hPSC-derived SM-like tissues produced herein can be used for tissue engineering, drug screening, and personalized medicine. To date, most hPSC-derived foregut organoids (e.g., stomach, esophagus, lung) tend to lack mesenchyme, unlike hindgut-derived intestinal organoids. This is because the traditional differentiation protocols required to create foregut epithelium are incompatible with mesenchymal development. Therefore, the protocol disclosed herein enables recombination of DE and SM lineages, a key step for engineering complex foregut tissues for regenerative medicine, allowing for increased cellular complexity of in vitro-generated organoids.

[0030] Disclosed herein is a method for producing visceral mesoderm cells in vitro. In some embodiments, the visceral mesoderm cells are differentiated from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. These pluripotent stem cells can be derived from a subject or patient, so that the visceral mesoderm cells and any downstream cell types produced can be used in various aspects of personalized medicine. These visceral mesoderm cells are early progenitor cells during embryogenesis and can further differentiate into downstream cell types, such as the hepatic, respiratory, esophageal, and / or gastric lineages. Visceral mesoderm cells and downstream cell types also affect the production of PSC-derived organoids, which, as described herein, may lack sufficient mesenchymal cells, thereby hindering the growth and maturation of the organoids. The visceral mesoderm cells and methods for producing them can be applied to any organoids and / or enteroids (organoid-like structures derived from epithelial tissue and lacking mesenchyme) described herein or otherwise known in the art. For example, methods of producing organoids or enteroids can be found in U.S. Pat. Nos. 9,719,068 and 10,174,289, and International Publication Nos. 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 by reference in its entirety. Alternative methods for producing visceral mesoderm that may differ from the retinoic acid-responsive and hedgehog-responsive visceral mesoderm described herein have been previously explored in WO 2021 / 041443, which is expressly incorporated herein by reference in its entirety.

[0031] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like elements unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0032] term Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs when read in light of this disclosure. For purposes of this disclosure, the following terms are defined below.

[0033] The articles "a" and "an" are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0034] "About" means a quantity, level, value, number, frequency, proportion, dimension, size, amount, weight, or length that varies by as much as 10% from the referenced quantity, level, value, number, frequency, proportion, dimension, size, amount, weight, or length.

[0035] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to mean the inclusion of the stated steps or elements or group of steps or elements, but not the exclusion of any other steps or elements or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and that other elements may not be present. "Consisting essentially of" means the inclusion of all elements listed before this phrase, limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or mandatory, but that other elements are optional and may or may not be present depending on whether they have a substantial effect on the activity or action of the recited elements.

[0036] As used herein, the terms "individual," "subject," or "patient" have their common and usual meaning as understood in light of the present specification, and refer to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird, such as a chicken, as well as other vertebrates or invertebrates. The term "mammal" is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including monkeys (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, etc.

[0037] The terms "effective amount" or "effective dose," as used herein, have their common and ordinary meaning as understood in light of this specification and refer to that amount of a specified composition or compound that produces an observable effect. The actual dosage level of the active ingredients in the active compositions of the presently disclosed subject matter can be varied to administer an amount of the active composition or compound that is effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend on various factors, including, but not limited to, the activity of the composition, the formulation, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. In some embodiments, a minimum dose is administered, and in the absence of dose-limiting toxicity, the dose is increased to the minimum effective amount. Determination and adjustment of effective doses, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0038] As used herein, the terms "function" and "functional" have their common and ordinary meaning as understood in light of this specification and refer to biological, enzymatic, or therapeutic function.

[0039] The term "inhibit," as used herein, has its common and ordinary meaning as understood herein and can refer to a reduction or prevention of biological activity. The reduction can be, about, at least about, less than, or about a percentage of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount within a range defined by any two of the foregoing values, e.g., 10-100%, 10-50%, 10-30%, 30-100%, 50-100%, 80-100%, or 30-60%. The term "delay," as used herein, has its common and ordinary meaning as understood herein and refers to a delay, postponement, or postponement of a biological event to a time later than would otherwise be expected. The delay can be 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a percentage that is about, at least about, less than, or about less than, or an amount within a range defined by any two of the foregoing values, e.g., 10-100%, 10-50%, 10-30%, 30-100%, 50-100%, 80-100%, or 30-60%. The terms inhibition and delay do not necessarily indicate 100% inhibition or delay. Partial inhibition or delay can be achieved.

[0040] As used herein, the term "isolated" has its common and ordinary meaning as understood in light of the present specification and refers to a substance and / or entity that (1) is separated from at least some of the components with which it is associated when originally produced (in nature and / or in an experimental setting) and / or (2) is separated from at least some of the components with which it is associated when produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can be separated from, equal to, about, at least about, less than, or about less than 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 substantially 100% (or ranges including and / or spanning the foregoing values) of other components with which they are originally associated. In some embodiments, an isolated agent is, about, at least, at least about, less than, or about (or ranges including and / or spanning) 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. As used herein, an "isolated" material can be "pure" (e.g., substantially free from other components). As used herein, the term "isolated cell" can refer to a cell that is not contained in a multicellular organism or tissue.

[0041] As used herein, "in vivo" is given its common and ordinary meaning as understood in light of the present specification and refers to the performance of methods within living organisms, usually animals, mammals, including humans, and plants, as opposed to tissue extracts or dead organisms.

[0042] As used herein, "ex vivo" is given its common and ordinary meaning as understood in light of the present specification and refers to the performance of a method outside a living body with little change in natural conditions.

[0043] As used herein, "in vitro" is given its common and ordinary meaning as understood in light of the present specification and refers to the performance of a method outside biological conditions, for example, in a petri dish or test tube.

[0044] As used herein, the terms "nucleic acid" or "nucleic acid molecule" have their common and ordinary meaning as understood in the context of this specification and refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those naturally occurring in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have alterations in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Furthermore, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications of the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, or phosphoramidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain naturally occurring or modified nucleobases linked to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded. "Oligonucleotide" can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.The nucleic acid can be contained in a nucleic acid vector or construct (e.g., a plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)) that can be used for amplification and / or expression of the nucleic acid in various biological systems. Typically, the vector or construct will also contain elements including, but not limited to, a promoter, an enhancer, a terminator, an inducer, a ribosome binding site, a translation initiation site, a start codon, a stop codon, a polyadenylation signal, an origin of replication, a cloning site, a multiple cloning site, a restriction enzyme site, an epitope, a reporter gene, a selection marker, an antibiotic selection marker, a targeting sequence, a peptide purification tag, or an accessory gene, or any combination thereof.

[0045] A nucleic acid or nucleic acid molecule can contain one or more sequences encoding different peptides, polypeptides, or proteins, which can be adjacent within the same nucleic acid or nucleic acid molecule, or can be joined with extra nucleic acid, for example, between linkers, repeats, or restriction enzyme sites, or with any other sequence that is, about, at least, at least about, less than, or about less than 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 bases in length, or any length within a range defined by any two of the foregoing lengths. The term "downstream" as used herein with respect to a nucleic acid has its common and ordinary meaning as understood in the context of this specification and refers to the sequence after the 3' end of the preceding sequence on the strand containing the coding sequence (sense strand) when the nucleic acid is double-stranded. The term "upstream" as used herein with respect to a nucleic acid has its common and ordinary meaning as understood in the context of this specification and refers to the sequence before the 5' end of the succeeding sequence on the strand containing the coding sequence (sense strand) when the nucleic acid is double-stranded.The term "grouping" as used herein with respect to nucleic acids has its general and ordinary meaning as understood in light of the present specification and refers to two or more sequences that occur in close proximity, either directly or with extra nucleic acid, for example between linkers, repeats, or restriction enzyme sites, or with any other sequence that is, about, at least, at least about, less than, or about less than 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 bases in length, but not with intervening sequences that generally encode a functional or catalytic polypeptide, protein, or protein domain.

[0046] The nucleic acids described herein comprise nucleobases. Primary, standard, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleobases include, but are not limited to, purines, pyrimidines, modified nucleobases, 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.

[0047] As used herein, the terms "peptide," "polypeptide," and "protein" have their common and ordinary meanings as understood in light of this specification and refer to polymers composed of amino acids linked by peptide bonds. The many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymatic, structural, transport, defensive, hormonal, or signal transduction functions. Peptides, polypeptides, and proteins are often, but not always, produced biologically by ribosomal complexes using nucleic acid templates, although chemical synthesis is also available. By manipulating nucleic acid templates, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of two or more peptides, polypeptides, and proteins can be performed. These fusions of two or more peptides, polypeptides, or proteins can be adjacent in the same molecule or can be joined with extra amino acids, for example, between linkers, repeats, epitopes, or tags, or any other sequence that is, about, at least about, less than, or about less than 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 bases in length, or any length within the range defined by any two of the foregoing lengths. As used herein, the term "downstream" with respect to a polypeptide has its common and ordinary meaning as understood in the context of this specification and refers to sequences that are after the C-terminus of the preceding sequence. The term "upstream" as used herein in reference to a polypeptide has its common and ordinary meaning as understood in the context of this specification, and refers to sequences that precede the N-terminus of a subsequent sequence.

[0048] As used herein, the term "purity" of any given substance, compound, or material has its common and ordinary meaning as understood herein and refers to the actual abundance of the substance, compound, or material compared to the expected abundance. For example, the 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 decimal points therebetween. Purity may be affected by unwanted impurities, including, but not limited to, nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, or contaminants, or any combination thereof. In some embodiments, the 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, mycoplasma, pyrogens, bacterial endotoxins, and adventitious infectious agents. 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-visible spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0049] The term "yield" of any given substance, compound, or material, as used herein, has its common and ordinary meaning as understood in light of this specification, and refers to the actual total amount of the substance, compound, or material relative to the expected amount present. For example, the yield of a substance, compound, or material may be, about, at least about, less than, or about less than 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, including all decimal points therebetween. Yield may be affected by the efficiency of the reaction or process, undesired side reactions, decomposition, the quality of the input substances, compounds, or materials, or the loss of the desired substance, compound, or material during any step of production.

[0050] Some embodiments described herein may relate to pharmaceutical compositions comprising, consisting essentially of, or consisting of an effective amount of a 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.

[0051] As used herein, "pharmaceutically acceptable" has its common and ordinary meaning as understood in light of the present specification and refers to a carrier, excipient, and / or stabilizer that is nontoxic or has an acceptable level of toxicity to cells or mammals exposed at the dosages and concentrations employed. As used herein, "pharmaceutically acceptable," "diluent," "excipient," and / or "carrier" have their common and ordinary meaning as understood in light of the present specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with administration to a human, feline, canine, or other vertebrate host. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are approved by federal, state, or other regulatory agencies or are listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans and non-human mammals such as cats and dogs. The terms diluent, excipient, and / or "carrier" can refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline, and aqueous dextrose and glycerol solutions can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A non-limiting example of a physiologically acceptable carrier is a pH-buffered aqueous solution.Physiologically acceptable carriers may also include one or more of the following: antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; carbohydrates such as amino acids, glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt formation inhibitors such as sodium; non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The compositions may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, as desired. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. The formulation is typically suited to the method of administration.

[0052] Cryoprotectants are additives to cell compositions that improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryoprotectants 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. Cryoprotectants can be used as part of a cryopreservation medium that contains other components, such as nutrients (e.g., albumin, serum, bovine serum, fetal calf serum [FCS]) to enhance the post-thaw survival rate of cells. In these cryopreservation media, at least one cryoprotectant may be found at a concentration that is, about, at least about, less than, or equal to 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 any percentage within a range defined by any two of the foregoing numbers.

[0053] Additional excipients with 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 residual amounts or contaminants from the manufacturing process, including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof. The amount of excipient may be found in the composition at, about, at least, at least about, less than, or about a percentage that 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 any weight percentage within a range defined by any two of the foregoing numbers.

[0054] The term "pharmaceutically acceptable salts" has its ordinary and usual meaning as understood in light of this specification and includes relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including, but not limited to, analgesics, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of inorganic bases suitable for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts can also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, classes of such 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; trihydroxymethylaminoethane.

[0055] The appropriate formulation will vary depending on the selected route of administration. Techniques for formulating and administering the compounds described herein are known to those skilled in the art. Multiple techniques for administering compounds exist in the art, including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intraarterial, and intravenous), intraportal, intraarticular, intradermal, peritoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injection. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0056] As used herein, "carrier" has its common and ordinary meaning as understood in light of the present specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or uptake of a compound into cells, tissues, and / or body organs.

[0057] As used herein, the term "diluent" has its common and ordinary meaning as understood in light of this specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent can be used to increase the bulk of a potent drug whose mass is too small to manufacture and / or administer. It can also be a liquid for dissolving a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate-buffered saline, which mimics the composition of human blood.

[0058] The term "w / w%" or "weight / weight %" as used herein has its ordinary and usual meaning as understood in the context of the present specification and refers to a percentage expressed in terms of the weight of a component or agent relative to the total weight of the composition multiplied by 100. The term "v / v%" or "volume / volume %" as used herein has its ordinary and usual meaning as understood in the context of the present specification and refers to a percentage expressed in terms of the liquid volume of a compound, substance, component or agent relative to the total liquid volume of the composition multiplied by 100.

[0059] The disclosure herein uses affirmative language to describe many embodiments, and the disclosure also includes embodiments in which subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, is wholly or partially excluded.

[0060] stem cells As used herein, the term "totipotent stem cells" (also known as omnipotent stem cells) has its common and ordinary meaning as understood in light of this specification: stem cells that can differentiate into embryonic and extraembryonic cell types. Such cells are capable of building complete, viable organisms. These cells are produced from the fusion of egg and sperm cells. Cells produced by the first few divisions of a fertilized egg are also totipotent.

[0061] As used herein, the term "embryonic stem cell (ESC)," commonly abbreviated as ES cell, has its common and ordinary meaning as understood in light of the present specification and refers to a cell that is pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For purposes of this disclosure, the term "ESC" may be used broadly to encompass embryonic germ cells.

[0062] As used herein, the term "pluripotent stem cell (PSC)" has its common and ordinary meaning as understood in light of this specification and encompasses any cell that can differentiate into almost any cell type in the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), and ectoderm (epidermal tissue and nervous system). PSCs may be the descendants of inner cell mass cells of a preimplantation blastocyst or may be obtained by the induction of non-pluripotent stem cells, e.g., 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 human, rodent, porcine, and bovine.

[0063] As used herein, the term "induced pluripotent stem cell (iPSC)" has its common and ordinary meaning as understood in light of this specification and is commonly abbreviated as iPS cell, and refers to a type of pluripotent stem cell artificially derived from normally non-pluripotent cells, such as adult somatic cells, by inducing "forced" expression of specific genes. hiPSC refers to human iPSC. In several methods known in the art, iPSCs can be derived by transfecting specific stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes can include the master transcriptional regulators Oct-3 / 4 (POU5F1) and Sox2, although other genes can also improve 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 by 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 critical 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.

[0064] The term "progenitor cell," as used herein, has its common and ordinary meaning as understood in light of the present specification and encompasses any cell that can be used in the methods described herein, through which one or more progenitor cells acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, progenitor cells can be totipotent (or omnipotent) stem cells, pluripotent stem cells (artificial or non-artificial), multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells can be derived from an embryo, an infant, a child, or an adult. In some embodiments, progenitor cells can be somatic cells that have been subjected to treatment to confer pluripotency via genetic manipulation or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSCs).

[0065] 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 early mammalian embryos and are capable of indefinite undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of an early-stage embryo, the blastocyst. Methods for deriving embryonic stem cells from blastocysts are well known in the art. It will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells.

[0066] Additional stem cells that may be used in embodiments according to the present disclosure include, but are not limited to, those provided by or described in the National Stem Cell Bank (NSCB), the database hosted by the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF), the WISC Cell Bank at the Wi Cell Research Institute, the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC), Novocell, Inc. (San Diego, Calif.), Cellartis AB (Göteborg, Sweden), ES Cell International Pte Ltd (Singapore), the Technion at the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments according to the present 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), UCO1 (HSF1), UC06 (HSF6), WA01 (HI), WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14). Exemplary human pluripotent cell lines include, but are not limited to, 72_3, 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.

[0067] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "directed differentiation" describes the process by which less specialized cells become specific specialized target cell types. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or modify the fate of the initial cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0068] In some embodiments, adenovirus can be used to deliver the four necessary genes, resulting in iPSCs that are virtually identical to embryonic stem cells. Because adenovirus does not combine its own genes with any of the target host's, the risk of tumor formation is eliminated. In some embodiments, non-viral techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without the use of any viral transfection system at all, albeit with very low efficiency. In other embodiments, direct protein delivery is used to generate iPSCs, thus eliminating the need for viral or genetic modification. In some embodiments, mouse iPSCs can be generated using similar methodologies. Repeated treatment of cells with specific proteins delivered to the cells via polyarginine anchors 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.

[0069] The term "feeder cells," as used herein, has its common and ordinary meaning as understood in light of the present specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying them on their surface. Feeder cells are generally adherent cells and may be growth-arrested. For example, feeder cells may be growth-arrested by irradiation (e.g., gamma rays), mitomycin-C treatment, electrical pulses, or mild chemical fixation (e.g., with formaldehyde or glutaraldehyde). However, feeder cells are not necessarily growth-arrested. Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the culture medium, or synthesizing extracellular matrix proteins. In some embodiments, feeder cells are allogeneic or xenogeneic to the supported target stem cells, which may affect 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 forehead fibroblasts, human skin 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 muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, conditioned medium prepared from feeder cells is used instead of or in combination with feeder cell co-culture. In some embodiments, feeder cells are not used during the expansion of target stem cells.

[0070] Differentiation of PSCs into the mesodermal lineage During embryonic development, the mesoderm is one of the three major germ layers and gives rise to a wide range of tissues, including muscle, connective tissue, bone, cartilage, skin, endothelium, mesenchyme, and blood cells.

[0071] In mammals, mesoderm and endodermal germ layers are induced by TGF-beta, BMP, and WNT signals during gastrulation, when tissues gradually emerge through the primitive streak (PS). After gastrulation, double-layered sheets of endoderm and lateral plate mesoderm (LPM) fold into the primitive gut tube. In the foregut region, the LPM divides into a lateral parietal mesoderm layer adjacent to the ectoderm (which gives rise to the forelimbs and body wall) and a medial visceral mesoderm (SM, SpM) layer (which gives rise to cardiac mesoderm (CM) and mesoderm surrounding the gut tube).

[0072] The endoderm and mesoderm of the fetal gut are then progressively patterned into organ-specific cell types along the anterior-posterior and dorsal-ventral axes by reciprocal cell signaling between tissue layers.

[0073] Mesenchyme, derived from the mesoderm, plays an important role in supporting associated tissues, including epithelial tissues, for proper growth and development. The mesoderm is composed of paraxial mesoderm, intermediate mesoderm, and lateral plate mesoderm. The lateral plate mesoderm is further subdivided into somatic and visceral mesoderm layers. The visceral mesoderm develops closely with the endoderm and gives rise to many downstream tissue types, such as blood vessels, cardiac muscle, and connective tissue and muscle of the gastrointestinal system. While combinatorial signals directing different epithelial lineages have been investigated, the visceral mesoderm is less well studied. As disclosed herein, the retinoic acid signaling pathway is involved in the differentiation of lateral plate mesoderm into visceral mesoderm.

[0074] Any method for producing any embryonic cell type (e.g., mesoderm, endoderm, or ectoderm) from pluripotent stem cells is applicable to the methods described herein. In some embodiments, the pluripotent stem cells are derived from a morula. In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. Embryonic stem cells can be derived from the inner cell mass of an embryo or the gonadal ridges of an embryo. Embryonic stem cells or induced pluripotent stem cells can be derived from various animal species, including, but not limited to, mouse, rat, monkey, cat, dog, hamster, or human. In some embodiments, the embryonic stem cells or induced pluripotent stem cells are human. In some embodiments, PSCs are genetically modified to express exogenous nucleic acids or proteins prior to differentiation into downstream cell types.

[0075] In some embodiments, PSCs, such as ESCs and iPSCs, undergo directed differentiation into embryonic germ layer cells, organ tissue progenitor cells, and then into tissues, such as gastrointestinal tissue or any other biological tissue. In some embodiments, directed differentiation is performed in a stepwise manner to obtain each of the differentiated cell types, with molecules (e.g., growth factors, ligands, agonists, antagonists) being added sequentially as differentiation progresses. In some embodiments, directed differentiation is performed in a non-stepwise manner, with molecules (e.g., growth factors, ligands, agonists, antagonists) being added simultaneously. In some embodiments, directed differentiation is achieved by selectively activating specific signaling pathways in PSCs or any downstream cells.

[0076] In some embodiments, the signaling pathway may include, but is not limited to, the Wnt signaling pathway, the Wnt / APC signaling pathway, the FGF signaling pathway, the TGF-beta signaling pathway, the BMP signaling pathway, the Notch signaling pathway, the Hedgehog signaling pathway, the LKB signaling pathway, the PI3K signaling pathway, the retinoic acid signaling pathway, the ascorbic acid signaling pathway, or the Par polarity signaling pathway, or any combination thereof. It will be understood by those skilled in the art that altering the concentration, expression, or function of any one of the signaling pathways disclosed herein can drive differentiation according to the present disclosure. In some embodiments, cellular components associated with a signaling pathway, such as natural inhibitors, antagonists, activators, or agonists of the pathway, can be used to inhibit or activate the signaling pathway. In some embodiments, siRNA and / or shRNA targeting cellular components associated with a signaling pathway are used to inhibit or activate these pathways.

[0077] In some embodiments, pluripotent stem cells, intermediate primitive streak cells, lateral plate mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive lateral plate mesoderm cells), visceral mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive visceral mesoderm cells), or any differentiated cells thereof, are contacted with a Wnt signaling pathway activator or a Wnt signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway activator comprises a Wnt protein. In some embodiments, the Wnt protein comprises a recombinant Wnt protein. In some embodiments, the Wnt signaling pathway activator comprises Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, or any combination thereof. In some embodiments, the Wnt signaling pathway activator comprises a GSK3 signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway activator comprises CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, asterpolone, kempauron, lithium chloride, TDZD 8, or TWS119, or any combination thereof. In some embodiments, the Wnt signaling pathway inhibitor comprises C59, PNU 74654, KY-02111, PRI-724, FH-535, DIF-1, or XAV939, or any combination thereof. In some embodiments, the cells have not been treated with a Wnt signaling pathway activator or Wnt signaling pathway inhibitor. The Wnt signaling pathway activators or Wnt signaling pathway inhibitors provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0078] Fibroblast growth factors (FGFs) are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGFs are heparin-binding proteins, and their interaction with cell surface-associated heparan sulfate proteoglycans has been shown to be essential for FGF signaling. FGFs play important roles in the growth 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 fibroblast growth factor receptors (FGFRs). FGF1 is also known as acidic fibroblast growth factor, and FGF2 is also known as basic fibroblast growth factor (bFGF). Members FGF11, FGF12, FGF13, and FGF14, also known as FGF homologous factors 1–4 (FHF1–FHF4), have been shown to have distinct functional differences compared to FGFs. Although these factors share striking sequence similarity, they do not bind to FGFRs and are involved in intracellular processes independent of FGFs. This group is also known as "iFGFs." Members FGF15 through FGF23 are newer and less well characterized. FGF15 is the mouse ortholog of human FGF19 (hence, there is no human FGF15). 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.

[0079] In some embodiments, pluripotent stem cells, intermediate primitive streak cells, lateral plate mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive lateral plate mesoderm cells), visceral mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive visceral mesoderm cells), or any differentiated cells thereof, are contacted with an FGF signaling pathway activator. In some embodiments, the FGF signaling pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF signaling 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 an FGF signaling pathway activator. The FGF signaling pathway activators provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0080] In some embodiments, pluripotent stem cells, mesoderm cells, lateral plate mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive lateral plate mesoderm cells), visceral mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive visceral mesoderm cells), or any differentiated cells thereof, are contacted with a TGF-beta signaling pathway activator or a TGF-beta signaling pathway inhibitor. In some embodiments, the TGF-beta family includes bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-Müllerian hormone, activin, and the nodal pathway. In some embodiments, the TGF-beta signaling pathway activator includes TGF-beta 1, TGF-beta 2, TGF-beta 3, activin A, activin B, nodal, BMPs, IDE1, IDE2, or any combination thereof. In some embodiments, the TGF-beta signaling pathway inhibitor comprises A83-01, RepSox, LY365947, SB431542, or any combination thereof. In some embodiments, the cells are not treated with a TGF-beta signaling pathway activator or a TGF-beta signaling pathway inhibitor. The TGF-beta signaling pathway activators or TGF-beta signaling pathway inhibitors provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0081] In some embodiments, pluripotent stem cells, intermediate primitive streak cells, lateral plate mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive lateral plate mesoderm cells), visceral mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive visceral mesoderm cells), or any differentiated cells thereof, are contacted with a BMP signaling pathway activator or a BMP signaling pathway inhibitor. In some embodiments, the BMP signaling pathway activator comprises a BMP protein. In some embodiments, the BMP protein is a recombinant BMP protein. In some embodiments, the BMP signaling 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 signaling pathway inhibitor comprises Noggin, RepSox, LY364947, LDN193189, SB431542, or any combination thereof. In some embodiments, the cells are not treated with a BMP signaling pathway activator or a BMP signaling pathway inhibitor. The BMP signaling pathway activators or BMP signaling pathway inhibitors provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0082] In some embodiments, pluripotent stem cells, intermediate primitive streak cells, lateral plate mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive lateral plate mesoderm cells), visceral mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive visceral mesoderm cells), or any differentiated cells thereof, are contacted with a Notch signaling pathway activator or a Notch signaling pathway inhibitor. In some embodiments, the Notch signaling pathway activator comprises a Notch protein. In some embodiments, the Notch protein comprises a recombinant Notch protein. In some embodiments, the Notch pathway activator comprises JAG1, JAG2, Notch1, Notch2, Notch3, or Notch4, or any combination thereof. In some embodiments, the Notch pathway inhibitor comprises Compound E, LY411575, DBZ, or DAPT, or any combination thereof. In some embodiments, the cells are not treated with a Notch signaling pathway activator or a Notch signaling pathway inhibitor. The Notch signaling pathway activators or Notch signaling pathway inhibitors provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0083] In some embodiments, pluripotent stem cells, intermediate primitive streak cells, lateral plate mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive lateral plate mesoderm cells), visceral mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive visceral mesoderm cells), or any differentiated cells thereof, are contacted with a Hedgehog (HH) signaling pathway activator or an HH signaling pathway inhibitor. In some embodiments, the HH signaling pathway activator comprises an HH protein. In some embodiments, the HH protein is a recombinant HH protein. In some embodiments, the HH signaling pathway activator comprises SHH, IHH, DHH, purmorphamine (PMA), GSA10, SAG, or any combination thereof. In some embodiments, the HH signaling pathway inhibitor comprises HPI-1, cyclopamine, GANT58, or GANT61, or any combination thereof. In some embodiments, the cells are not treated with an HH signaling pathway activator or an HH signaling pathway inhibitor. The HH signaling pathway activators or HH signaling pathway inhibitors provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0084] In some embodiments, pluripotent stem cells, intermediate primitive streak cells, lateral plate mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive lateral plate mesoderm cells), visceral mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive visceral mesoderm cells), or any differentiated cells thereof, are contacted with a PI3K signaling pathway activator or a PI3K signaling pathway inhibitor. In some embodiments, the PI3K signaling pathway activator comprises 740Y-P, erucic acid, or both. In some embodiments, the PI3K signaling pathway inhibitor comprises wortmannin, LY294002, hibiscon C, PI-103, IC-87114, ZSTK474, AS-605240, PIK-75, PIK-90, PIK-294, PIK-293, AZD6482, PF-04691502, GSK1059615, quercetin, pluripotin, flurbiprofen, GDC-0941, dactolisib, pictilisib, idelalisib, buparlisib, rigosertib, copanlisib, duvelisib, alpelisib, or any combination thereof. In some embodiments, the cells are not treated with a PI3K signaling pathway activator or a PI3K signaling pathway inhibitor. The PI3K signaling pathway activators or PI3K signaling pathway inhibitors provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0085] In some embodiments, pluripotent stem cells, mesoderm cells, lateral plate mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive lateral plate mesoderm cells), visceral mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive visceral mesoderm cells), or any differentiated cells thereof, are contacted with a retinoic acid signaling pathway activator or a retinoic acid signaling pathway inhibitor. In some embodiments, the retinoic acid signaling pathway activator comprises retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, or AM580, or any combination thereof. In some embodiments, the retinoic acid signaling pathway inhibitor comprises guggulsterone. In some embodiments, the cells have not been treated with a retinoic acid signaling pathway activator or a retinoic acid signaling pathway inhibitor. The retinoic acid signaling pathway activators or retinoic acid signaling pathway inhibitors provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0086] In some embodiments, pluripotent stem cells, intermediate primitive streak cells, lateral plate mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive lateral plate mesoderm cells), visceral mesoderm cells (which may include retinoic acid-responsive or hedgehog-responsive visceral mesoderm cells), or any differentiated cells thereof, are contacted with an ascorbic acid signaling pathway activator. In some embodiments, the ascorbic acid signaling pathway activator comprises ascorbic acid or 2-phospho-ascorbic acid, or both. In some embodiments, the cells are not treated with an ascorbic acid signaling pathway activator. The ascorbic acid signaling pathway activators provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0087] In some embodiments, cells are contacted with a small molecule compound, signal transduction pathway activator, signal transduction pathway inhibitor, or growth factor for a time that is, is about, is at least, is at least about, is less than, or is less than about, or is less than about, or any time within a range defined by any two of the foregoing times, e.g., 1 hour 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.

[0088] In some embodiments, cells (e.g., pluripotent stem cells, mesoderm cells, lateral plate mesoderm cells, visceral mesoderm cells, or any differentiated cells thereof) are treated with a small molecule compound, a signaling pathway activator, a signaling pathway inhibitor, or a growth factor at a concentration of 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 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, The antibody is contacted in the medium at a concentration that is, about, at least, at least about, less than, or about equal to 2000 ng / mL, 5000 ng / mL, 7000 ng / mL, 10000 ng / mL, or 15000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 5 to 15000 ng / mL, 10 ng / mL to 15000 ng / mL, 100 ng / mL to 5000 ng / mL, 500 ng / mL to 2000 ng / mL, 5 to 2000 ng / mL, 10 ng / mL to 2000 ng / mL, or 1000 ng / mL to 15000 ng / mL. In some embodiments, cells (e.g., pluripotent stem cells, lateral plate mesoderm cells, visceral mesoderm cells, or any differentiated cells thereof) are contacted with a small molecule compound, signaling pathway activator, signaling pathway inhibitor, or growth factor in a medium such that the concentration of the small molecule compound, signaling pathway activator, signaling pathway inhibitor, or growth factor is, is about, is at least about, is less than, or is less than about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM.In some embodiments, the concentration of the small molecule compound, activator, inhibitor, or growth factor is maintained at a constant level throughout treatment. In some embodiments, the concentration of the small molecule compound, activator, inhibitor, or growth factor is varied over the course of treatment. In some embodiments, two or more small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the concentrations of the two or more small molecule compounds, activators, inhibitors, or growth factors may be different.

[0089] In some embodiments, cells (e.g., pluripotent stem cells, mesoderm cells, lateral plate mesoderm cells, visceral mesoderm cells, or any differentiated cells thereof) are cultured in a growth medium that supports the growth of stem cells and their differentiated cells. In some embodiments, the growth medium is RPMI 1640, DMEM, DMEM / F12, mTeSR1, or mTeSR Plus medium. In some embodiments, the growth medium comprises fetal bovine serum (FBS). In some embodiments, the growth medium comprises FBS at a concentration that is, about, at least about, less than, or equal to 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 any percentage within a range defined by any two of the foregoing concentrations, e.g., 0%-20%, 0.2%-10%, 2%-5%, 0%-5%, or 2%-20%. In some embodiments, the growth medium does not contain xenogeneic components. In some embodiments, the growth medium comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0090] 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 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, pluripotent stem cells are prepared from PBMCs by viral transduction. In some embodiments, PBMCs are transduced with Sendai virus, lentivirus, adenovirus, or adeno-associated virus, or any combination thereof. In some embodiments, PBMCs are transduced with Sendai virus containing expression vectors for Oct3 / 4, Sox2, Klf4, or L-Myc, or any combination thereof. In some embodiments, PBMCs are transduced with one or more viruses at an MOI that is, about, at least about, less than, or equal to 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or any MOI within a range defined by any two of the foregoing MOIs, e.g., an MOI of 0-5.0, 1.0-4.0, 2.0-3.0, 0-3.0, or 1.0-5.0. In some embodiments, after transduction, the PBMCs express stem cell reprogramming factors. In some embodiments, after transduction, the PBMCs are reprogrammed into iPSCs. In some embodiments, the iPSCs are grown on a feeder cell substrate. In some embodiments, the iPSCs are grown on a MEF feeder cell substrate. In some embodiments, the iPSCs are grown on an irradiated MEF feeder cell substrate. In some embodiments, iPSCs are grown in RPMI 1640, DMEM, DMEM / F12, mTeSR1, or mTeSR Plus medium.

[0091] In some embodiments, PSCs are expanded in cell culture. In some embodiments, iPSCs are expanded in an extracellular matrix, or a mimetic or derivative thereof. In some embodiments, the extracellular matrix, or a mimetic or derivative thereof, comprises a polymer, a protein, a polypeptide, a nucleic acid, a sugar, a lipid, polylysine, polyornithine, collagen, gelatin, fibronectin, vitronectin, laminin, elastin, tenascin, heparan sulfate, entactin, nidogen, osteopontin, basement membrane, Matrigel, Geltrex, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof. In some embodiments, PSCs are expanded in Matrigel, Geltrex, or 1% gelatin, or any combination thereof. In some embodiments, iPSCs are expanded in cell culture medium containing a ROCK inhibitor (e.g., Y-27632).

[0092] Differentiation into lateral plate mesoderm Any method for producing lateral plate mesoderm cells from pluripotent stem cells disclosed herein or otherwise known in the art is applicable to the methods described herein.

[0093] In some embodiments, pluripotent stem cells are first differentiated into mesoprimitive streak cells. In some embodiments, to differentiate PSCs into mesoprimitive streak cells, the pluripotent stem cells are contacted with a TGF-beta signaling pathway activator, a Wnt signaling pathway activator, an FGF signaling pathway activator, a BMP signaling pathway activator, or a PI3K signaling pathway inhibitor, or any combination thereof. In some embodiments, the TGF-beta signaling pathway activator is selected from the group consisting of TGF-beta 1, TGF-beta 2, TGF-beta 3, activin A, activin B, Nodal, BMP, IDE1, and IDE2. In some embodiments, the Wnt signaling pathway activator is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML284, IQ-1, WAY262611, CHIR99021, CHIR98014, AZD2858, BIO, AR-A014418, SB216763, SB415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD8, and TWS119. In some embodiments, the FGF signaling pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, the BMP signaling pathway activator is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, and IDE2.In some embodiments, the PI3K signaling pathway inhibitor is selected from the group consisting of wortmannin, LY294002, hibiscon C, PI-103, IC-87114, ZSTK474, AS-605240, PIK-75, PIK-90, PIK-294, PIK-293, AZD6482, PF-04691502, GSK1059615, quercetin, pluripotin, flurbiprofen, GDC-0941, dactolisib, pictilisib, idelalisib, buparlisib, rigosertib, copanlisib, duvelisib, and alpelisib. In some embodiments, the PSCs are contacted with activin A, CHIR99021, FGF2, BMP4, or PIK90, or any combination thereof, including all five, to differentiate the PSCs into intermediate primitive streak cells. In some embodiments, the induced pluripotent stem cells are human induced pluripotent stem cells. In some embodiments, the intermediate primitive streak cells are human intermediate primitive streak cells.

[0094] In some embodiments, the PSCs are contacted with a TGF-beta signaling pathway activator. In some embodiments, the TGF-beta signaling pathway activator is or comprises activin A. In some embodiments, PSCs are contacted with a TGF-beta signaling pathway activator (e.g., activin A) at a concentration that is, about, at least about, less than, or equal to 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, or 45 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 15-45 ng / mL, 20-40 ng / mL, 15-30 ng / mL, or 30-45 ng / mL. In some embodiments, the PSCs are contacted with a TGF-beta signaling pathway activator (e.g., activin A) at a concentration that is, about, at least about, less than, or about less than 30 ng / mL.

[0095] In some embodiments, the PSCs are contacted with a Wnt signaling pathway activator. In some embodiments, the Wnt signaling pathway activator is or includes CHIR99021. In some embodiments, the PSCs are contacted with a Wnt signaling pathway activator (e.g., CHIR99021) at a concentration that is, about, at least about, less than, or equal to 1, 2, 3, 4, 5, 5.1, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 8, 9, or 10 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-10 μM, 1-8 μM, 4-8 μM, 5-7 μM, 5-10 μM, or 6-10 μM. In some embodiments, the PSCs are contacted with a Wnt signaling pathway activator (e.g., CHIR99021) at a concentration that is, about, at least about, less than, or about less than 6 μM.

[0096] In some embodiments, PSCs are contacted with an FGF signaling pathway activator. In some embodiments, the FGF signaling pathway activator is or includes FGF2. In some embodiments, PSCs are contacted with an FGF signaling pathway activator (e.g., FGF2) at a concentration that is, about, at least about, less than, or equal to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-100 ng / mL, 1-50 ng / mL, 10-40 ng / mL, 50-100 ng / mL, 75-100 ng / mL, or 5-50 ng / mL. In some embodiments, the PSCs are contacted with an FGF signaling pathway activator (e.g., FGF2) at a concentration that is, about, at least about, less than, or about less than 20 ng / mL.

[0097] In some embodiments, the PSCs are contacted with a BMP signaling pathway activator. In some embodiments, the BMP signaling pathway activator is or comprises BMP4. In some embodiments, PSCs are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or equal to 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 20-60 ng / mL, 30-50 ng / mL, 20-40 ng / mL, 40-60 ng / mL, or 25-55 ng / mL. In some embodiments, the PSCs are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or about less than 40 ng / mL.

[0098] In some embodiments, the PSCs are contacted with a PI3K signaling pathway inhibitor. In some embodiments, the PI3K signaling pathway inhibitor is or includes PIK90. In some embodiments, the PSCs are contacted with a PI3K signaling pathway inhibitor (e.g., PIK90) at a concentration that is, about, at least about, less than, or about less than 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 50-150 nM, 50-120 nM, 80-120 nM, or 100-150 nM. In some embodiments, the PSCs are contacted with a PI3K signaling pathway inhibitor (e.g., PIK90) at a concentration that is, about, at least, at least about, less than, or about less than 100 nM.

[0099] In some embodiments, PSCs are contacted with a TGF-beta signaling pathway activator, a Wnt signaling pathway activator, an FGF signaling pathway activator, a BMP signaling pathway activator, and a PI3K signaling pathway inhibitor for a time sufficient to differentiate the PSCs into mesoprimitive streak cells. In some embodiments, the PSCs are contacted for, about, at least, at least about, less than, or about less than 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any amount of time within a range defined by any two of the foregoing periods, e.g., 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours. In some embodiments, the PSCs are contacted for a time that is, about, at least, at least about, less than, or about less than 24 hours.

[0100] In some embodiments, the intermediate primitive streak cells are differentiated into retinoic acid-responsive lateral plate mesoderm cells (RA-LPM). In some embodiments, the intermediate primitive streak cells are differentiated from pluripotent stem cells. In some embodiments, the intermediate primitive streak cells are differentiated from induced pluripotent stem cells or embryonic stem cells. In some embodiments, the intermediate primitive streak cells are differentiated from pluripotent stem cells by contacting the pluripotent stem cells with a TGF-beta signaling pathway activator, a Wnt signaling pathway activator, an FGF signaling pathway activator, a BMP signaling pathway activator, and a PI3K signaling pathway inhibitor. In some embodiments, to differentiate the intermediate primitive streak cells into retinoic acid-responsive lateral plate mesoderm cells, the intermediate primitive streak cells are contacted with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, or a retinoic acid (RA) signaling pathway activator, or any combination thereof. In some embodiments, the TGF-beta signaling pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542. In some embodiments, the Wnt signaling pathway inhibitor is selected from the group consisting of C59, PNU74654, KY-02111, PRI-724, FH-535, DIF-1, and XAV939. In some embodiments, the BMP signaling pathway activator is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, and IDE2. In some embodiments, the RA signaling pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580. In some embodiments, to differentiate the intermediate primitive streak cells into retinoin-responsive lateral plate mesoderm cells, the intermediate primitive streak cells are contacted with A83-01, C59, BMP4, RA, or any combination thereof, including all four.

[0101] In some embodiments for producing RA-LPM, mesenteroprimitive streak cells are contacted with a TGF-beta signaling pathway inhibitor. In some embodiments, the TGF-beta signaling pathway inhibitor is or includes A83-01. In some embodiments, mesenteroprimitive streak cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.1-2 μM, 0.1-1 μM, 0.5-1.5 μM, or 1-2 μM. In some embodiments, the mesoprimitive streak cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or about 1 μM.

[0102] In some embodiments for producing RA-LPM, mesoprimitive streak cells are contacted with a Wnt signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway inhibitor is or includes Wnt-C59 (C59). In some embodiments, mesoprimitive streak cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.1-2 μM, 0.1-1 μM, 0.5-1.5 μM, or 1-2 μM. In some embodiments, the mesoprimitive streak cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or about 1 μM.

[0103] In some embodiments of producing RA-LPM, the mesenteroprimitive streak cells are contacted with a BMP signaling pathway activator, which in some embodiments is or includes BMP4. In some embodiments, mesoprimitive streak cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or equal to 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, or 45 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 15-45 ng / mL, 15-30 ng / mL, 30-45 ng / mL, 20-40 ng / mL, or 25-35 ng / mL. In some embodiments, the mesoprimitive streak cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or about less than 30 ng / mL.

[0104] In some embodiments for producing RA-LPM, mesoprimitive streak cells are contacted with an RA signaling pathway activator. In some embodiments, the RA signaling pathway activator is or comprises RA. In some embodiments, mesoprimitive streak cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration that is, about, at least about, less than, or equal to 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-3 μM, 1-2 μM, 2-3 μM, or 1.5-2.5 μM. In some embodiments, the mesoprimitive streak cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration that is, about, at least about, less than, or about 2 μM.

[0105] In some embodiments for producing RA-LPM, the intermediate primitive streak cells are contacted with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, and an RA signaling pathway activator for a time sufficient to differentiate the intermediate primitive streak cells into retinoic acid-responsive lateral plate mesoderm cells. In some embodiments, the intermediate primitive streak cells are contacted for, about, at least, at least about, less than, or about less than 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any amount of time within a range defined by any two of the foregoing periods, e.g., 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours. In some embodiments, the mesoprimitive streak cells are contacted for an amount of time that is, about, at least, at least about, less than, or about less than 24 hours.

[0106] In some embodiments, the intermediate primitive streak cells are differentiated into hedgehog-responsive lateral plate mesoderm cells (HH-LPM). In some embodiments, the intermediate primitive streak cells are differentiated from pluripotent stem cells. In some embodiments, the intermediate primitive streak cells are differentiated from induced pluripotent stem cells or embryonic stem cells. In some embodiments, the intermediate primitive streak cells are differentiated from pluripotent stem cells by contacting the pluripotent stem cells with a TGF-beta signaling pathway activator, a Wnt signaling pathway activator, an FGF signaling pathway activator, a BMP signaling pathway activator, and a PI3K signaling pathway inhibitor. In some embodiments, to differentiate the intermediate primitive streak cells into hedgehog-responsive lateral plate mesoderm cells, the intermediate primitive streak cells are contacted with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, a retinoic acid (RA) signaling pathway activator, or a hedgehog (HH) signaling pathway activator, or any combination thereof. In some embodiments, the TGF-beta signaling pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542. In some embodiments, the Wnt signaling pathway inhibitor is selected from the group consisting of C59, PNU74654, KY-02111, PRI-724, FH-535, DIF-1, and XAV939. In some embodiments, the BMP signaling pathway activator is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, and IDE2. In some embodiments, the RA signaling pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580. In some embodiments, the HH signaling pathway activator is selected from the group consisting of SHH, IHH, DHH, PMA, GSA10, and SAG.In some embodiments, to differentiate the intermediate primitive streak cells into hedgehog-responsive lateral plate mesoderm cells, the intermediate primitive streak cells are contacted with A83-01, C59, BMP4, RA, PMA, or any combination thereof, including all five.

[0107] In some embodiments for producing HH-LPM, mesenteroprimitive streak cells are contacted with a TGF-beta signaling pathway inhibitor. In some embodiments, the TGF-beta signaling pathway inhibitor is or includes A83-01. In some embodiments, mesenteroprimitive streak cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.1-2 μM, 0.1-1 μM, 1-2 μM, or 0.5-1.5 μM. In some embodiments, the mesoprimitive streak cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or about 1 μM.

[0108] In some embodiments for producing HH-LPM, mesoprimitive streak cells are contacted with a Wnt signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway inhibitor is or includes Wnt-C59 (C59). In some embodiments, mesoprimitive streak cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.1-2 μM, 0.1-1 μM, 1-2 μM, or 0.5-1.5 μM. In some embodiments, the mesoprimitive streak cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or about 1 μM.

[0109] In some embodiments of producing HH-LPM, the mesenteroprimitive streak cells are contacted with a BMP signaling pathway activator, hi some embodiments, the BMP signaling pathway activator is or includes BMP4. In some embodiments, mesoprimitive streak cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or equal to 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, or 45 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 15-45 ng / mL, 15-30 ng / mL, 30-45 ng / mL, 20-40 ng / mL, or 25-35 ng / mL. In some embodiments, the mesoprimitive streak cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or about less than 30 ng / mL.

[0110] In some embodiments for producing HH-LPM, mesoprimitive streak cells are contacted with an RA signaling pathway activator. In some embodiments, the RA signaling pathway activator is or includes RA. In some embodiments, mesoprimitive streak cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration that is, about, at least about, less than, or equal to 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 μM, or any concentration within a range defined by any three of the foregoing concentrations, e.g., 1-3 μM, 1-2 μM, 2-3 μM, or 1.5-2.5 μM. In some embodiments, the mesoprimitive streak cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration that is, about, at least about, less than, or about 2 μM.

[0111] In some embodiments of producing HH-LPM, the mesoprimitive streak cells are contacted with an HH signaling pathway activator. In some embodiments, the HH signaling pathway activator is or includes PMA. In some embodiments, the mesoprimitive streak cells are contacted with an HH signaling pathway activator (e.g., PMA) at a concentration that is, about, at least about, less than, or equal to 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.5-3 μM, 0.5-2 μM, 0.5-1 μM, 1-2 μM, or 1-3 μM. In some embodiments, the mesoprimitive streak cells are contacted with an HH signaling pathway activator (e.g., PMA) at a concentration that is, about, at least about, less than, or about 1 μM.

[0112] In some embodiments for producing HH-LPM, intermediate primitive streak cells are contacted with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an RA signaling pathway activator, and an HH signaling pathway activator for a time sufficient to differentiate the intermediate primitive streak cells into hedgehog-responsive lateral plate mesoderm cells. In some embodiments, the intermediate primitive streak cells are contacted for, about, at least, at least about, less than, or about less than 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any amount of time within a range defined by any two of the foregoing periods, e.g., 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours. In some embodiments, the mesoprimitive streak cells are contacted for an amount of time that is, about, at least, at least about, less than, or about less than 24 hours.

[0113] In some embodiments, lateral plate mesoderm cells are produced from pluripotent stem cells according to an alternative method found in Loh et al., "Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types," Cell. (2016) 166(2):451-467, which is expressly incorporated by reference in its entirety for purposes of differentiation into lateral plate mesoderm cells.

[0114] Differentiation into visceral mesoderm Disclosed herein are methods for producing retinoic acid-responsive visceral mesoderm cells (RA-SPm) from retinoic acid-responsive lateral plate mesoderm cells. In some embodiments, the retinoic acid-responsive lateral plate mesoderm cells are produced according to any one of the methods disclosed herein. The method for producing retinoic acid-responsive visceral mesoderm cells comprises contacting the retinoic acid-responsive lateral plate mesoderm cells with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, or a retinoic acid (RA) signaling pathway activator, or any combination thereof comprising at least one of each, thereby differentiating the RA-LPM into RA-SpM. In some embodiments, the retinoic acid-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, and an RA signaling pathway activator. In some embodiments, the TGF-beta signaling pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542. In some embodiments, the Wnt signaling pathway inhibitor is selected from the group consisting of C59, PNU74654, KY-02111, PRI-724, FH-535, DIF-1, and XAV939. In some embodiments, the BMP signaling pathway activator is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, and IDE2. In some embodiments, the FGF signaling pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.In some embodiments, the RA signaling pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580. In some embodiments, the TGF-beta signaling pathway inhibitor is A83-01. In some embodiments, the Wnt signaling pathway inhibitor is C59. In some embodiments, the BMP signaling pathway activator is BMP4. In some embodiments, the FGF signaling pathway activator is FGF2. In some embodiments, the RA signaling pathway activator is RA. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with A83-01, BMP4, C59, FGF2, and RA. In some embodiments, the retinoic acid-responsive lateral plate mesoderm cells are contacted with factors described herein, e.g., A83-01, BMP4, C59, FGF2, and RA, for a period of time sufficient to differentiate the retinoic acid-responsive lateral plate mesoderm cells into retinoic acid-responsive visceral mesoderm cells. In some embodiments, the retinoic acid responsive lateral plate mesoderm cells are selected from the group consisting of: 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, Contact for an amount of time that is, is about, is at least, is at least about, is less than, or is about less than 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours, or any amount of time within a range defined by any two of the foregoing times, for example, 1 to 72 hours, 12 to 36 hours, 1 to 48 hours, or 24 to 72 hours.In some embodiments, the retinoic acid-responsive lateral plate mesoderm cells are contacted for an amount of time that is, about, at least, at least about, less than, or about less than 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or any amount of time within a range defined by any two of the foregoing times, e.g., 36-60 hours, 40-54 hours, 36-48 hours, or 48-60 hours. In some embodiments, the retinoic acid-responsive lateral plate mesoderm cells are contacted for an amount of time that is, about, at least, at least about, less than, or about less than 48 hours. In some embodiments, RA-SpM is characterized by one or more of: a) lack of expression of pluripotency markers OCT3 / 4 and SOX2, intermediate primitive streak marker TBXT, and / or endodermal markers FOXA2 and CDH1; b) expression of VIM, which may include decreased expression of VIM compared to cardiac visceral mesoderm (CG-SpM) and / or increased expression of VIM compared to HH-SpM; c) expression of FOXF1, which may include decreased expression of FOXF1 compared to HH-SpM and / or increased expression of FOXF1 compared to CG-SpM; d) lack of expression of cardiac markers NKX2-5 and ISL1, or decreased expression of NKX2-5 and ISL1 compared to CG-SpM; e) expression of retinoic acid-responsive markers HOXA5 and CYP26A1; and / or f) decreased expression of Hedgehog-responsive markers GLI1 and PTCH1 compared to HH-SpM.

[0115] In some embodiments for producing RA-SpM, retinoic acid-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor. In some embodiments, the TGF-beta signaling pathway inhibitor is or includes A83-01. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or equal to 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.1-2 μM, 0.5-1.5 μM, 0.1-1 μM, or 1-2 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or about 1 μM.

[0116] In some embodiments for producing RA-SpM, retinoic acid-responsive lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway inhibitor is or includes Wnt-C59 (C59). In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or equal to 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.1-2 μM, 0.5-1.5 μM, 0.1-1 μM, or 1-2 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or about 1 μM.

[0117] In some embodiments for producing RA-SpM, retinoic acid-responsive lateral plate mesoderm cells are contacted with a BMP signaling pathway activator. In some embodiments, the BMP signaling pathway activator is or includes BMP4. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or equal to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-100 ng / mL, 5-40 ng / mL, 10-80 ng / mL, 1-50 ng / mL, or 50-100 ng / mL. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or equal to 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, or 45 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 15-45 ng / mL, 20-40 ng / mL, 15-30 ng / mL, or 30-45 ng / mL. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or about less than 30 ng / mL.

[0118] In some embodiments for producing RA-SpM, retinoic acid-responsive lateral plate mesoderm cells are contacted with an FGF signaling pathway activator. In some embodiments, the FGF signaling pathway activator is or includes FGF2. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with an FGF signaling pathway activator (e.g., FGF2) at a concentration that is, about, at least about, less than, or equal to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-100 ng / mL, 5-40 ng / mL, 10-80 ng / mL, 1-50 ng / mL, or 50-100 ng / mL. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with an FGF signaling pathway activator (e.g., FGF2) at a concentration that is, about, at least about, less than, or equal to 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, or 35 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 5-35 ng / mL, 10-30 ng / mL, 5-20 ng / mL, or 20-35 ng / mL. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with an FGF signaling pathway activator (e.g., FGF2) at a concentration that is, about, at least about, less than, or about less than 20 ng / mL.

[0119] In some embodiments for producing RA-SpM, retinoic acid-responsive lateral plate mesoderm cells are contacted with a retinoic acid signaling pathway activator. In some embodiments, the retinoic acid signaling pathway activator is or comprises RA. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a retinoic acid signaling pathway activator (e.g., RA) at a concentration that is, about, at least about, less than, or equal to 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration that is, about, at least, at least about, less than, or about equal to 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-3 μM, 1.5-2.5 μM, 1-2 μM, or 2-3 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration that is, about, at least, at least about, less than, or about equal to 2 μM.

[0120] In some embodiments for producing RA-SpM, retinoic acid-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor at a concentration of 0.01-20 μM, a Wnt signaling pathway inhibitor at a concentration of 0.01-20 μM, a BMP signaling pathway activator at a concentration of 1-100 ng / mL, an FGF signaling pathway activator at a concentration of 1-100 ng / mL, and an RA signaling pathway activator at a concentration of 0.01-20 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor at a concentration of 0.1-2 μM, a Wnt signaling pathway inhibitor at a concentration of 0.1-2 μM, a BMP signaling pathway activator at a concentration of 15-45 ng / mL, an FGF signaling pathway activator at a concentration of 5-35 ng / mL, and an RA signaling pathway activator at a concentration of 1-3 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with A83-01 at a concentration of 0.01 to 20 μM, C59 at a concentration of 0.01 to 20 μM, BMP4 at a concentration of 1 to 100 ng / mL, FGF2 at a concentration of 1 to 100 ng / mL, and RA at a concentration of 0.01 to 20 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with A83-01 at a concentration of 0.1 to 2 μM, C59 at a concentration of 0.1 to 2 μM, BMP4 at a concentration of 15 to 45 ng / mL, FGF2 at a concentration of 5 to 35 ng / mL, and RA at a concentration of 1 to 3 μM. In some embodiments, retinoic acid-responsive lateral plate mesoderm cells are contacted with A83-01 at a concentration of 1 μM, C59 at a concentration of 1 μM, BMP4 at a concentration of 30 ng / mL, FGF2 at a concentration of 20 ng / mL, and RA at a concentration of 2 μM.

[0121] In some embodiments, RA-SpM is characterized by one or more of: a) lack of expression of pluripotency markers OCT3 / 4 and SOX2, intermediate primitive streak marker TBXT, and / or endodermal markers FOXA2 and CDH1; b) expression of VIM, which may include decreased expression of VIM compared to cardiac visceral mesoderm (CG-SpM) and / or increased expression of VIM compared to HH-SpM; c) expression of FOXF1, which may include decreased expression of FOXF1 compared to HH-SpM and / or increased expression of FOXF1 compared to CG-SpM; d) lack of expression of cardiac markers NKX2-5 and ISL1, or decreased expression of NKX2-5 and ISL1 compared to CG-SpM; e) expression of retinoic acid-responsive markers HOXA5 and CYP26A1; and / or f) decreased expression of Hedgehog-responsive markers GLI1 and PTCH1 compared to HH-SpM.

[0122] Also disclosed herein are methods for producing hedgehog-responsive visceral mesoderm cells (HH-SpM) from hedgehog-responsive lateral plate mesoderm cells. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are produced according to any one of the methods disclosed herein. The method for producing hedgehog-responsive visceral mesoderm cells comprises contacting hedgehog-responsive lateral plate mesoderm cells with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, a retinoic acid (RA) signaling pathway activator, or an HH signaling pathway activator, or any combination thereof comprising at least one of each, thereby differentiating the HH-LPM into HH-SpM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, an RA signaling pathway activator, and an HH signaling pathway activator. In some embodiments, the TGF-beta signaling pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542. In some embodiments, the Wnt signaling pathway inhibitor is selected from the group consisting of C59, PNU74654, KY-02111, PRI-724, FH-535, DIF-1, and XAV939. In some embodiments, the BMP signaling pathway activator is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, and IDE2. In some embodiments, the FGF signaling pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.In some embodiments, the RA signaling pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580. In some embodiments, the HH signaling pathway activator is selected from the group consisting of SHH, IHH, DHH, PMA, GSA10, and SAG. In some embodiments, the TGF-beta signaling pathway inhibitor is A83-01. In some embodiments, the Wnt signaling pathway inhibitor is C59. In some embodiments, the BMP signaling pathway activator is BMP4. In some embodiments, the FGF signaling pathway activator is FGF2. In some embodiments, the RA signaling pathway activator is RA. In some embodiments, the HH signaling pathway activator is PMA. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with A83-01, BMP4, C59, FGF2, RA, and PMA. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with factors described herein, e.g., A83-01, BMP4, C59, FGF2, RA, and PMA, for a period of time sufficient to differentiate the hedgehog-responsive lateral plate mesoderm cells into hedgehog-responsive visceral mesoderm cells. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are selected from the group consisting of: 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, Contact for an amount of time that is, is about, is at least, is at least about, is less than, or is about less than 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours, or any amount of time within a range defined by any two of the foregoing times, for example, 1 to 72 hours, 12 to 36 hours, 1 to 48 hours, or 24 to 72 hours.In some embodiments, the hedgehog-responsive lateral plate mesoderm cells are contacted for an amount of time that is, about, at least, at least about, less than, or about less than 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or any amount of time within a range defined by any two of the foregoing times, e.g., 36-60 hours, 40-54 hours, 36-48 hours, or 48-60 hours. In some embodiments, the hedgehog-responsive lateral plate mesoderm cells are contacted for an amount of time that is, about, at least, at least about, less than, or about less than 48 hours. In some embodiments, HH-SpM is characterized by one or more of: a) lack of expression of pluripotency markers OCT3 / 4 and SOX2, the intermediate primitive streak marker TBXT, and / or the endodermal markers FOXA2 and CDH1; b) expression of VIM, which may include decreased VIM expression compared to cardiac visceral mesoderm (CG-SpM) and RA-SpM; c) expression of FOXF1, which may include increased FOXF1 expression compared to CG-SpM and RA-SpM; d) lack of expression of cardiac markers NKX2-5 and ISL1, or decreased NKX2-5 and ISL1 expression compared to CG-SpM; e) expression of retinoic acid-responsive markers HOXA5 and CYP26A1; and / or f) expression of hedgehog-responsive markers GLI1 and PTCH1, which may include increased GLI1 and PTCH1 expression compared to CG-SpM and RA-SpM.

[0123] In some embodiments for producing HH-SpM, hedgehog-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor. In some embodiments, the TGF-beta signaling pathway inhibitor is or includes A83-01. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or equal to 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.1-2 μM, 0.5-1.5 μM, 0.1-1 μM, or 1-2 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor (e.g., A83-01) at a concentration that is, about, at least about, less than, or about 1 μM.

[0124] In some embodiments of producing HH-SpM, hedgehog-responsive lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway inhibitor is or includes Wnt-C59 (C59). In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or equal to 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.1-2 μM, 0.5-1.5 μM, 0.1-1 μM, or 1-2 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration that is, about, at least about, less than, or about 1 μM.

[0125] In some embodiments for producing HH-SpM, hedgehog-responsive lateral plate mesoderm cells are contacted with a BMP signaling pathway activator. In some embodiments, the BMP signaling pathway activator is or includes BMP4. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or equal to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-100 ng / mL, 5-40 ng / mL, 10-80 ng / mL, 1-50 ng / mL, or 50-100 ng / mL. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or equal to 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, or 45 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 15-45 ng / mL, 20-40 ng / mL, 15-30 ng / mL, or 30-45 ng / mL. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or about less than 30 ng / mL.

[0126] In some embodiments for producing HH-SpM, hedgehog-responsive lateral plate mesoderm cells are contacted with an FGF signaling pathway activator. In some embodiments, the FGF signaling pathway activator is or includes FGF2. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with an FGF signaling pathway activator (e.g., FGF2) at a concentration that is, about, at least about, less than, or equal to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-100 ng / mL, 5-40 ng / mL, 10-80 ng / mL, 1-50 ng / mL, or 50-100 ng / mL. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with an FGF signaling pathway activator (e.g., FGF2) at a concentration that is, about, at least about, less than, or equal to 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, or 35 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 5-35 ng / mL, 10-30 ng / mL, 5-20 ng / mL, or 20-35 ng / mL. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with an FGF signaling pathway activator (e.g., FGF2) at a concentration that is, about, at least about, less than, or about less than 20 ng / mL.

[0127] In some embodiments of producing HH-SpM, hedgehog-responsive lateral plate mesoderm cells are contacted with a retinoic acid signaling pathway activator. In some embodiments, the retinoic acid signaling pathway activator is or includes RA. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a retinoic acid signaling pathway activator (e.g., RA) at a concentration that is, about, at least about, less than, or equal to 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration that is, about, at least, at least about, less than, or about less than 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 μM, or any concentration within a range defined by any three of the foregoing concentrations, e.g., 1-3 μM, 1.5-2.5 μM, 1-2 μM, or 2-3 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration that is, about, at least, at least about, less than, or about less than 2 μM.

[0128] In some embodiments of producing HH-SpM, hedgehog-responsive lateral plate mesoderm cells are contacted with an HH signaling pathway activator. In some embodiments, the HH signaling pathway activator is or comprises PMA. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with an HH signaling pathway activator (e.g., PMA) at a concentration that is, about, at least about, less than, or equal to 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.5-3 μM, 0.5-1.5 μM, or 1-2 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with an HH signaling pathway activator (e.g., HH) at a concentration that is, about, at least about, less than, or about 1 μM.

[0129] In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor at a concentration of 0.01-20 μM, a Wnt signaling pathway inhibitor at a concentration of 0.01-20 μM, a BMP signaling pathway activator at a concentration of 1-100 ng / mL, an FGF signaling pathway activator at a concentration of 1-100 ng / mL, an RA signaling pathway activator at a concentration of 0.01-20 μM, and an HH signaling pathway activator at a concentration of 0.5-3 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with a TGF-beta signaling pathway inhibitor at a concentration of 0.1-2 μM, a Wnt signaling pathway inhibitor at a concentration of 0.1-2 μM, a BMP signaling pathway activator at a concentration of 15-45 ng / mL, an FGF signaling pathway activator at a concentration of 5-35 ng / mL, an RA signaling pathway activator at a concentration of 1-3 μM, and an HH signaling pathway activator at a concentration of 0.5-1.5 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with A83-01 at a concentration of 0.01-20 μM, C59 at a concentration of 0.01-20 μM, BMP4 at a concentration of 1-100 ng / mL, FGF2 at a concentration of 1-100 ng / mL, RA at a concentration of 0.01-20 μM, and an HH signaling pathway activator at a concentration of 0.5-3 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with A83-01 at a concentration of 0.1-2 μM, C59 at a concentration of 0.1-2 μM, BMP4 at a concentration of 15-45 ng / mL, FGF2 at a concentration of 5-35 ng / mL, RA at a concentration of 1-3 μM, and an HH signaling pathway activator at a concentration of 0.5-1.5 μM. In some embodiments, hedgehog-responsive lateral plate mesoderm cells are contacted with A83-01 at a concentration of 1 μM, C59 at a concentration of 1 μM, BMP4 at a concentration of 30 ng / mL, FGF2 at a concentration of 20 ng / mL, RA at a concentration of 2 μM, and PMA at a concentration of 1 μM.

[0130] In some embodiments, HH-SpM is characterized by one or more of: a) lack of expression of pluripotency markers OCT3 / 4 and SOX2, the intermediate primitive streak marker TBXT, and / or the endodermal markers FOXA2 and CDH1; b) expression of VIM, which may include decreased VIM expression compared to cardiac visceral mesoderm (CG-SpM) and RA-SpM; c) expression of FOXF1, which may include increased FOXF1 expression compared to CG-SpM and RA-SpM; d) lack of expression of cardiac markers NKX2-5 and ISL1, or decreased NKX2-5 and ISL1 expression compared to CG-SpM; e) expression of retinoic acid-responsive markers HOXA5 and CYP26A1; and / or f) expression of hedgehog-responsive markers GLI1 and PTCH1, which may include increased GLI1 and PTCH1 expression compared to CG-SpM and RA-SpM.

[0131] In some embodiments, visceral mesoderm cells (which may comprise RA-SpM or HH-SpM) produced according to any of the methods herein exhibit increased expression of FOXF1, HOXA1, HOXA5, or WNT2, or any combination thereof, compared to cardiac mesoderm cells. In some embodiments, visceral mesoderm cells (which may comprise RA-SpM or HH-SpM) exhibit decreased expression of NKX2-5, ISL1, or TBX2, or any combination thereof, compared to cardiac mesoderm cells. In some embodiments, visceral mesoderm cells (which may comprise RA-SpM or HH-SpM) exhibit decreased expression of PAX3, PRRX1, or both, compared to intermediate primitive streak cells. In some embodiments, visceral mesoderm cells (which may comprise RA-SpM or HH-SpM) exhibit decreased expression of CD31 compared to cardiac mesoderm cells.

[0132] In any of the embodiments provided herein, the visceral mesoderm cells (which may include RA-SpM or HH-SpM) are mammalian cells. In some embodiments, the visceral mesoderm cells are human visceral mesoderm cells. In some embodiments, the visceral mesoderm cells are derived from a subject. In some embodiments, the subject is human. In some embodiments, the subject has or is at risk for a disease. In some embodiments, the visceral mesoderm cells are derived from PSCs derived from the subject.

[0133] Differentiation into visceral mesoderm cell types As disclosed herein, visceral mesoderm cells (which may include RA-SpM or HH-SpM) produced by any of the methods herein can be further differentiated into visceral mesoderm subtypes, including subtypes that differentiate under the influence of retinoic acid and / or the Hedgehog pathway. In some embodiments, the visceral mesoderm subtypes include septum transversum and mesothelial cells, fibroblasts, gastric mesenchymal cells, respiratory mesenchymal cells, or esophageal mesenchymal cells, or any combination thereof. In some embodiments, the septum transversum and mesothelial cells include hepatic septum transversum and hepatic mesothelial cells. In some embodiments, the fibroblasts include hepatic fibroblasts. Embodiments of differentiation of visceral mesoderm cells into visceral mesoderm subtypes are disclosed in Figures 7A and 8.

[0134] Septum transversum and mesothelial cell production In some embodiments, the method comprises contacting retinoic acid-responsive visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, or both, thereby differentiating RA-SpM into septum transversum (STM) and mesothelial cells. In some embodiments, the retinoic acid-responsive visceral mesoderm cells are retinoic acid-responsive visceral mesoderm cells produced by any of the methods described herein. In some embodiments, the retinoic acid-responsive visceral mesoderm cells are contacted with a retinoic acid signaling pathway activator and a BMP signaling pathway activator. In some embodiments, the retinoic acid signaling activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580. In some embodiments, the BMP signaling pathway activator is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, and IDE2. In some embodiments, the retinoic acid signaling pathway activator is RA. In some embodiments, the BMP signaling pathway activator is BMP4. In some embodiments, the retinoic acid-responsive visceral mesoderm cells are contacted with RA, BMP4, or both. In some embodiments, the resulting septum transversum and mesothelial cells are characterized by expression of WT1, TBX18, LHX2, GATA4, UPK1B, or UPK3B, or any combination thereof. In some embodiments, expression of WT1, TBX18, LHX2, GATA4, UPK1B, or UPK3B is increased compared to cardiac mesoderm, liver fibroblasts, gastric mesoderm, respiratory mesoderm, esophageal mesoderm, or any combination thereof.

[0135] In some embodiments, retinoic acid responsive visceral mesoderm cells are treated with a retinoic acid signaling pathway activator (e.g., RA) at a concentration that is, about, at least about, less than, or equal to 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. Alternatively, the cells may be contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration of 1, 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 a concentration of about 1, 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 a range defined by any two of the aforementioned concentrations, for example, 1 to 100 ng / mL, 5 to 40 ng / mL, 10 to 80 ng / mL, 1 to 50 ng / mL, or 50 to 100 ng / mL.In some embodiments, retinoic acid responsive visceral mesoderm cells are treated with retinoic acid responsive visceral mesoderm cells at a concentration that is, about, at least about, less than, or equal to 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-3 μM, 1-2 μM, 2-3 μM, or 1.5-2.5 μM. The cells are contacted with a receptor pathway activator (e.g., RA) and with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least about, less than, or equal to 10, 20, 30, 40, 50, 60, 70, or 80 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 10-80 ng / mL, 10-30 ng / mL, 10-40 ng / mL, 30-80 ng / mL, 40-80 ng / mL, or 20-40 ng / mL. In some embodiments, retinoic acid-responsive visceral mesoderm cells are treated with a retinoic acid signaling pathway activator (e.g., RA) at a concentration of, about, at least about, less than, or equal to 1.8, 1.9, 2, 2.1, or 2.2 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1.8-2.2 μM, 1.8-2 μM, 2-2.2 μM, or 1.9-2.1 μM. and also contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least, at least about, less than, or about 20, 30, 40, 50, or 60 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 20-60 ng / mL, 20-40 ng / mL, 20-30 ng / mL, 30-60 ng / mL, or 40-60 ng / mL.In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with a retinoic acid signaling pathway activator (e.g., RA) at a concentration that is, about, at least, at least about, less than, or about 2 μM, and with a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least, at least about, less than, or about 30 ng / mL.

[0136] In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with an RA signaling pathway activator at a concentration of 0.01-20 μM and a BMP signaling pathway activator at a concentration of 1-100 ng / mL. In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with an RA signaling pathway activator at a concentration of 1-3 μM and a BMP signaling pathway activator at a concentration of 10-80 ng / mL. In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with RA at a concentration of 0.01-20 μM and BMP4 at a concentration of 1-100 ng / mL. In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with RA at a concentration of 1-3 μM and BMP4 at a concentration of 10-80 ng / mL. In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with RA at a concentration of 2 μM and BMP4 at a concentration of 30 ng / mL.

[0137] In some embodiments, the retinoic acid-responsive visceral mesoderm cells are contacted with a retinoic acid signaling pathway activator (e.g., RA), or a BMP signaling pathway activator (e.g., BMP4), or both, at concentrations described herein, for a period of time sufficient to differentiate the retinoic acid-responsive visceral mesoderm cells into transverse septum cells. In some embodiments, the retinoic acid-responsive visceral mesoderm cells are contacted with factors described herein, e.g., RA and BMP4, for a period of time sufficient to differentiate the retinoic acid-responsive visceral mesoderm cells into transverse septum cells. In some embodiments, the contact is 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 14 9, 100, 101, 102, 103, 104, 105, 106, 107, or 108 hours, or about, at least, at least about, less than, or about less than, or any period within a range defined by any two of the foregoing times, for example, 36 to 108 hours, 36 to 80 hours, 50 to 80 hours, 36 to 72 hours, 72 to 108 hours, or 68 to 76 hours. In some embodiments, the contact is for a period of time that is, about, at least, at least about, less than, or about less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 hours, or any period of time within a range defined by any two of the foregoing periods, e.g., 60-84 hours, 60-72 hours, 72-84 hours, or 70-74 hours. In some embodiments, the contact is for a period of time that is, about, at least, at least about, less than, or about less than 72 hours.

[0138] In some embodiments, the resulting septum transversum and mesothelial cells are characterized by expression of WT1, TBX18, LHX2, GATA4, UPK1B, or UPK3B, or any combination thereof, hi some embodiments, expression of WT1, TBX18, LHX2, GATA4, UPK1B, or UPK3B is increased compared to cardiac mesoderm, liver fibroblasts, gastric mesoderm, respiratory mesoderm, esophageal mesoderm, or any combination thereof.

[0139] In some embodiments, the resulting transverse septal cells exhibit increased expression of WT1, TBX18, LHX2, UPK3B, or UPK1B, or any combination thereof, compared to cardiac mesoderm cells, visceral mesoderm cells, or fibroblasts, or any combination thereof. In some embodiments, the transverse septal cells exhibit decreased expression of MSX1, MSX2, or HAND1, or any combination thereof, compared to cardiac mesoderm cells or fibroblasts, or both. In some embodiments, the transverse septal cells exhibit decreased expression of HOXA1 or TBX5, or both, compared to visceral mesoderm cells. In some embodiments, the transverse septal cells exhibit decreased expression of NKX6.1 or HOXA5, or both, compared to respiratory mesenchymal cells. In some embodiments, the transverse septal cells exhibit decreased expression of NKX3.2, MSC, BARX1, WNT4, or HOXA5, or any combination thereof, compared to esophageal / gastric mesenchymal cells. In some embodiments, transverse septum cells represent 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% of the total cells differentiated from visceral mesoderm cells, or a percentage of the total cells differentiated from visceral mesoderm cells that is about, at least about, less than, or about less than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%, or any percentage within a range defined by any two of the foregoing percentages, e.g., 60%-100%, 70%-90%, or 75%-85%.

[0140] Liver fibroblast production In some embodiments, the method comprises contacting retinoic acid-responsive visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, or a Wnt signaling pathway activator, or any combination thereof, thereby differentiating RA-SpM into liver fibroblasts (LFs). In some embodiments, the retinoic acid-responsive visceral mesoderm cells are retinoic acid-responsive visceral mesoderm cells produced by any of the methods described herein. In some embodiments, the retinoic acid-responsive visceral mesoderm cells are contacted with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and a Wnt signaling pathway activator. In some embodiments, the retinoic acid signaling pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS493, TTNPB, and AM580. In some embodiments, the BMP signaling pathway activator is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, and IDE2. In some embodiments, the Wnt signaling pathway activator is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML284, IQ-1, WAY262611, CHIR99021, CHIR98014, AZD2858, BIO, AR-A014418, SB216763, SB415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD8, and TWS119. In some embodiments, the retinoic acid signaling pathway activator is RA. In some embodiments, the BMP signaling pathway activator is BMP4. In some embodiments, the Wnt signaling pathway activator is CHIR99021.In some embodiments, the retinoic acid-responsive visceral mesoderm cells are contacted with RA, BMP4, CHIR99021, or any combination thereof, including all three. In some embodiments, the resulting liver fibroblasts are characterized by expression of PITX1, MSX1, MSX2, TBX5, or WNT2, or any combination thereof. In some embodiments, expression of PITX1, MSX1, MSX2, TBX5, or WNT2, or any combination thereof, is increased compared to cardiac mesoderm, septum transversus, gastric mesoderm, respiratory mesoderm, or esophageal mesoderm, or any combination thereof.

[0141] In some embodiments, the retinoic acid responsive visceral mesoderm cells are administered to a subject in need of treatment with retinoic acid responsive visceral mesoderm cells at a concentration that is, about, at least about, less than, or about less than 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations. and a RA signaling pathway activator (e.g., RA) at a concentration of, for example, 0.01 to 20 μM, 0.01 to 10 μM, 1 to 15 μM, or 10 to 20 μM, ...1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL. The cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration of at or about equal to or less than 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or with a Wnt signaling pathway activator (e.g., CHIR99021) at a concentration of at or about equal to or less than 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or with any concentration within a range defined by any two of the aforementioned concentrations, for example, 0.01 to 20 μM, 0.01 to 10 μM, 1 to 15 μM, or 10 to 20 μM.In some embodiments, retinoic acid responsive visceral mesoderm cells are cultured in a 500-well platelet-free environment, comprising a RA signaling pathway activator (e.g., RA) at a concentration that is at, about, at least, at least about, less than, or about equal to 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-3 μM, 1-2 μM, 2-3 μM, or 1.5-2.5 μM, and a RA signaling pathway activator (e.g., RA) at a concentration that is at, about, at least, at least about, less than, or about equal to 10, 20, 30, 40, 50, 60, 70, or 80 ng / mL. and a BMP signaling pathway activator (e.g., BMP4) at any concentration within a range defined by one of the following: 1, 2, 3, 4, 5, 5.1, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, or 10 μM, or about, or at least about, or less than, or about, or less than, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-10 μM, 1-8 μM, 4-8 μM, 5-7 μM, 5-10 μM, or 6-10 μM.In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration that is, about, at least, at least about, or less than about 2 μM; a BMP signaling pathway activator (e.g., BMP4) at a concentration that is, about, at least, at least about, or less than about 30 ng / mL; or a Wnt signaling pathway activator (e.g., CHIR99021) at a concentration that is, about, at least, at least about, or less than about 6 μM.

[0142] In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with an RA signaling pathway activator at a concentration of 0.01-20 μM, a BMP signaling pathway activator at a concentration of 1-100 ng / mL, and a Wnt signaling pathway activator at a concentration of 0.01-20 μM. In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with an RA signaling pathway activator at a concentration of 1-3 μM, a BMP signaling pathway activator at a concentration of 10-80 ng / mL, and a Wnt signaling pathway activator at a concentration of 5-7 μM. In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with RA at a concentration of 0.01-20 μM, BMP4 at a concentration of 1-100 ng / mL, and CHIR99021 at a concentration of 0.01-20 μM. In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with RA at a concentration of 1-3 μM, BMP4 at a concentration of 10-80 ng / mL, and CHIR99021 at a concentration of 5-7 μM. In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with RA at a concentration of 2 μM, BMP4 at a concentration of 30 ng / mL, and CHIR99021 at a concentration of 6 μM.

[0143] In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA), a BMP signaling pathway activator (e.g., BMP4), and a Wnt signaling pathway activator (e.g., CHIR99021) at concentrations described herein for a period of time sufficient to differentiate the retinoic acid-responsive visceral mesoderm cells into liver fibroblasts. In some embodiments, retinoic acid-responsive visceral mesoderm cells are contacted with factors described herein, e.g., RA, BMP4, and CHIR99021, for a period of time sufficient to differentiate the retinoic acid-responsive visceral mesoderm cells into liver fibroblasts. In some embodiments, the contact is 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 14 9, 100, 101, 102, 103, 104, 105, 106, 107, or 108 hours, or about, at least, at least about, less than, or about less than, or any period within a range defined by any two of the foregoing times, for example, 36 to 108 hours, 36 to 80 hours, 50 to 80 hours, 36 to 72 hours, 72 to 108 hours, or 68 to 76 hours. In some embodiments, the contact is for a period of time that is, about, at least about, less than, or about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 hours, or any period of time within a range defined by any two of the foregoing periods, e.g., 60-84 hours, 60-72 hours, 72-84 hours, or 70-74 hours.In some embodiments, the contact is for a period of time that is, about, at least, at least about, less than, or about less than 72 hours.

[0144] In some embodiments, the obtained liver fibroblasts are characterized by expression of PITX1, MSX1, MSX2, TBX5, or WNT2, or any combination thereof. In some embodiments, the expression of PITX1, MSX1, MSX2, TBX5, or WNT2, or any combination thereof, is increased compared to cardiac mesoderm, septum transversus, gastric mesoderm, respiratory mesoderm, or esophageal mes...

Claims

1. 1. A method for producing retinoic acid-responsive lateral plate mesoderm cells (RA-LPM), comprising: contacting a mesoprimitive streak cell with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, and a retinoic acid (RA) signaling pathway activator, thereby differentiating the mesoprimitive streak cell into an RA-LPM; Optionally, (a) the mesoprimitive streak cells are contacted with A83-01, Wnt-C59 (C59), BMP4, RA, or any combination thereof; and / or (b) the intermediate primitive streak cells are contacted for a time sufficient to differentiate the intermediate primitive streak cells into RA-LPM, and / or for a time that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the foregoing times, inclusive of 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours; and / or (c) the mesoprimitive streak cells are contacted for a period of time that is at or about 24 hours. method.

2. 1. A method for producing retinoic acid-responsive visceral mesoderm cells (RA-SpM), comprising: contacting RA-LPM with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, and an RA signaling pathway activator, thereby differentiating the RA-LPM into RA-SpM; Optionally, (a) the RA-LPM is RA-LPM produced by the method of claim 1; and / or (b) contacting the RA-LPM with A83-01, C59, BMP4, bFGF (FGF2), RA, or any combination thereof; and / or (c) culturing the RA-LPM for a time sufficient to differentiate the RA-LPM into RA-SpM, and / or for 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours or about 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or any time within a range defined by any two of the above times, inclusive of 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours; preferably, the RA-LPM is contacted for a time that is at or about 48 hours; method.

3. A method for producing septum transversum (STM) and mesothelial cells, comprising contacting RA-SpM with a retinoic acid signaling pathway activator and a BMP signaling pathway activator, thereby differentiating the RA-SpM into STM and mesothelial cells; Optionally, (a) the RA-SpM is the RA-SpM according to claim 2, and / or (b) contacting the visceral mesoderm cells with RA, BMP4, or both; and / or (c) contacting the RA-SpM for a time sufficient to differentiate the RA-SpM into STM and mesothelial cells, and / or for a time that is at or about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 hours, or any time within a range defined by any two of the foregoing times, inclusive: 60-84 hours, 60-72 hours, 72-84 hours, or 70-74 hours; and / or (d) contacting the RA-SpM for a period of time that is at or about 72 hours; method.

4. A method for producing liver fibroblasts (LF), comprising contacting RA-SpM with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and a Wnt signaling pathway activator, thereby differentiating the RA-SpM into liver fibroblasts; Optionally, (a) The RA-SpM is the RA-SpM according to claim 2; (b) contacting the RA-SpM with RA, BMP4, CHIR99021, or any combination thereof; and / or (c) contacting for a time sufficient to differentiate the RA-SpM into liver fibroblasts, and / or for a time that is at or about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, or 84 hours, or any time within a range defined by any two of the foregoing times, inclusive: 60-84 hours, 60-72 hours, 72-84 hours, or 70-74 hours; and / or (d) contacting the RA-SpM for a period of time that is at or about 72 hours; method.

5. 1. A method for producing gastric mesenchymal cells (GM), comprising: a) contacting RA-SpM with a retinoic acid signaling pathway activator and an HH signaling pathway activator; b) contacting the cells obtained in step a) with a retinoic acid signaling pathway activator, a BMP signaling pathway inhibitor, and an HH signaling pathway activator; Thereby, the RA-SpM are differentiated into GM, Optionally, (a) the RA-SpM is RA-SpM produced by the method of claim 2; and / or (b) contacting the RA-SpM for step a) for 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or for any time within a range defined by any two of the above times, inclusive: 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours; and / or (c) contacting the RA-SpM for a period of time that is at or about 48 hours for step a), and / or (d) contacting the cells obtained in step a) with step b) for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or for a time that is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the above times, including 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours; and / or (e) contacting the cells obtained in step a) for a period of time that is 24 hours or about 24 hours with respect to step b), and / or (f) contacting the RA-SpM with RA, Noggin, PMA, or any combination thereof; method.

6. 1. A method for producing hedgehog-responsive lateral plate mesoderm cells (HH-LPM), comprising: contacting a mesenteroprimitive streak cell with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an RA signaling pathway activator, and a Hedgehog (HH) signaling pathway activator, thereby differentiating the mesenteroprimitive streak cell into an HH-LPM; Optionally, (a) contacting the mesoprimitive streak cells with A83-01, C59, BMP4, RA, PMA, or any combination thereof; and / or (b) contacting said intermediate primitive streak cells for a time sufficient to differentiate said intermediate primitive streak cells into HH-LPM, and / or for a time that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the foregoing times, inclusive of 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours; and / or (c) contacting the mesoprimitive streak cells for a period of time that is at or about 24 hours; method.

7. 1. A method for producing hedgehog-responsive visceral mesoderm cells (HH-SpM), comprising: contacting HH-LPM with a TGF-beta signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, an RA signaling pathway activator, and an HH signaling pathway activator, thereby differentiating the HH-LPM into HH-SpM; Optionally, (a) the HH-LPM is HH-LPM produced by the method of claim 7, and / or (b) contacting the HH-LPM with A83-01, C59, BMP4, bFGF, RA, PMA, or any combination thereof; and / or (c) culturing the HH-LPM for a time sufficient to differentiate the HH-LPM into HH-SpM, and / or for 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours or about 36, 37, 38, 39, 40, 41, 42, 43, , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or any time within a range defined by any two of the foregoing times, inclusive: 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours; and / or (d) contacting the HH-LPM for a period of time that is at or about 48 hours; method.

8. 1. A method for producing esophageal mesenchymal cells (EM), comprising: a) contacting HH-SpM with a retinoic acid signaling pathway activator and an HH signaling pathway activator; b) contacting the cells obtained in step a) with a retinoic acid signaling pathway activator, a BMP signaling pathway inhibitor, and an HH signaling pathway activator; Thereby, the HH-SpM are differentiated into EMs, Optionally, (a) the HH-SpM is HH-SpM produced by the method of claim 7; and / or (b) contacting the HH-SpM for step a) for 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or for any time within a range defined by any two of the above times, inclusive: 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours; and / or (c) contacting the HH-SpM for a period of time that is at or about 48 hours for step a), and / or (d) contacting the cells obtained in step a) with step b) for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or for a time that is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the above times, including 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours; and / or (e) contacting the cells obtained in step a) for a period of time that is 24 hours or about 24 hours with respect to step b), and / or (f) contacting the HH-SpM with RA, Noggin, PMA, or any combination thereof; method.

9. 1. A method for producing respiratory mesenchymal cells (RM), comprising: a) contacting HH-SpM with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and a hedgehog (HH) signaling pathway activator; b) contacting the cells obtained in step a) with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, an HH signaling pathway activator, and a Wnt signaling pathway activator; Thereby, the HH-SpM are differentiated into RM, Optionally, (a) the HH-SpM is HH-SpM produced by the method of claim 7; and / or (b) contacting the HH-SpM for step a) for 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 hours, or for any time within a range defined by any two of the above times, inclusive: 36-60 hours, 36-54 hours, 36-48 hours, 48-54 hours, or 48-60 hours; and / or (c) contacting the HH-SpM for a period of time that is at or about 48 hours for step a), and / or (d) contacting the cells obtained in step a) with step b) for 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or for a time that is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, or any time within a range defined by any two of the above times, including 12-36 hours, 12-24 hours, 24-36 hours, or 18-30 hours; and / or (e) contacting the cells obtained in step a) for a period of time that is 24 hours or about 24 hours with respect to step b), and / or (f) contacting the HH-SpM with RA, BMP4, PMA, CHIR99021, or any combination thereof; method.

10. The method according to claim 1, wherein (a) the intermediate streak cells are human intermediate streak cells, and / or (b) the mesoprimitive streak cells have been differentiated from pluripotent stem cells, optionally induced pluripotent stem cells or embryonic stem cells, and optionally the mesoprimitive streak cells have been differentiated from pluripotent stem cells by contacting the pluripotent stem cells with a TGF-beta signaling pathway activator, a Wnt signaling pathway activator, an FGF signaling pathway activator, a BMP signaling pathway activator, and a PI3K signaling pathway inhibitor; and / or (c) the TGF-beta signaling pathway inhibitor is selected from the group consisting of A83-01, RepSox, LY365947, and SB431542, and / or (d) the TGF-beta signaling pathway inhibitor is A83-01, and / or (e) contacting the TGF-beta signaling pathway inhibitor at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the above concentrations, including 0.1-2 μM, 0.1-1 μM, 0.5-1.5 μM, or 1-2 μM; and / or (f) contacting the TGF-beta signaling pathway inhibitor at a concentration of 1 μM or about 1 μM; and / or (g) the Wnt signaling pathway inhibitor is selected from the group consisting of Wnt-C59 (C59), PNU 74654, KY-02111, PRI-724, FH-535, DIF-1, and XAV939, and / or (h) the Wnt signaling pathway inhibitor is C59, and / or (i) contacting the Wnt signaling pathway inhibitor at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the above concentrations, including 0.1-2 μM, 0.1-1 μM, 0.5-1.5 μM, or 1-2 μM; and / or (j) contacting the Wnt signaling pathway inhibitor at a concentration of 1 μM or about 1 μM; and / or (k) the BMP signaling pathway activator is selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, and IDE2; and / or (l) the BMP signaling pathway activator is BMP4, and / or (m) administering the BMP signaling pathway activator to a patient in need thereof at a concentration of 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, or 45 ng / mL or about 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, or 45 ng / mL. , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ng / mL, or any concentration within a range defined by any two of the above concentrations, including 15-45 ng / mL, 15-30 ng / mL, 30-45 ng / mL, 20-40 ng / mL, or 25-35 ng / mL; and / or (n) contacting the BMP signaling pathway activator at a concentration of 30 ng / mL or about 30 ng / mL; and / or (o) the FGF signaling pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23; and / or (p) the FGF signaling pathway activator is FGF2, and / or (q) the FGF signaling pathway activator 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, or 35 ng / mL, or about 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, or 35 ng / mL or 35 ng / mL, or any concentration within a range defined by any two of the above concentrations, including 5-35 ng / mL, 10-30 ng / mL, 5-20 ng / mL, or 20-35 ng / mL; and / or (r) contacting the FGF signaling pathway activator at a concentration of 20 ng / mL or about 20 ng / mL; and / or (s) the RA signaling pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, and AM580; and / or (t) the RA signaling pathway activator is RA, and / or (u) contacting the RA signaling pathway activator at a concentration of 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 μM, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, inclusive of 1-3 μM, 1-2 μM, 2-3 μM, or 1.5-2.5 μM; and / or (v) contacting the RA signaling pathway activator at a concentration of 2 μM or about 2 μM; and / or (w) The Wnt signaling pathway activator is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpolone, kenpaullone, lithium chloride, and TDZD. 8, and TWS119, and / or (x) the Wnt signaling pathway activator is CHIR99021, and / or (y) contacting the Wnt signaling pathway activator at a concentration of 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the above concentrations, including 0.01-20 μM, 0.01-10 μM, 1-10 μM, or 5-15 μM; and / or (z) contacting the Wnt signaling pathway activator at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM, or any concentration within a range defined by any two of the above concentrations, including 1-10 μM, 1-8 μM, 4-8 μM, 5-7 μM, 5-10 μM, or 6-10 μM; and / or (aa) the HH signaling pathway activator is selected from the group consisting of SHH, IHH, DHH, PMA, GSA 10, and SAG; and / or (ab) the HH signaling pathway activator is PMA, and / or (ac) contacting the HH signaling pathway activator at a concentration of 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM, or any concentration within a range defined by any two of the above concentrations, inclusive of 1-3 μM, 1-2 μM, 2-3 μM, or 1.5-2.5 μM; and / or (ad) contacting the HH signaling pathway activator at a concentration of 1 μM, about 1 μM, 2 μM, or about 2 μM; and / or (ae) the BMP signaling pathway inhibitor is selected from the group consisting of Noggin, RepSox, LY364947, LDN193189, and SB431542, and / or (af) the BMP signaling pathway inhibitor is Noggin, and / or (ag) contacting the BMP signaling pathway inhibitor at a concentration of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 ng / mL, or any concentration within a range defined by any two of the above concentrations, including 50-250 ng / mL, 50-200 ng / mL, 100-200 ng / mL, 100-250 ng / mL, or 200-250 ng / mL; and / or (ah) contacting the BMP signaling pathway inhibitor at a concentration of 200 ng / mL or about 200 ng / mL; method.

11. A retinoic acid-responsive lateral plate mesoderm cell (RA-LPM) produced by the method of claim 1.

12. 3. Retinoic acid-responsive visceral mesoderm cells (RA-SpM) produced by the method of claim 2, optionally comprising: The RA-SpM is a) lack of expression of the pluripotency markers OCT3 / 4 and SOX2, the middle primitive streak marker TBXT, and / or the endoderm markers FOXA2 and CDH1; and / or b) expression of VIM, optionally decreased expression of VIM compared to cardiac splanchnic mesoderm (CG-SpM) and / or increased expression of VIM compared to HH-SpM; and / or c) expression of FOXF1, optionally decreased expression of FOXF1 compared to HH-SpM and / or increased expression of FOXF1 compared to CG-SpM; and / or d) lack of expression of cardiac markers NKX2-5 and ISL1 or reduced expression of NKX2-5 and ISL1 compared to CG-SpM; and / or e) expression of the retinoic acid responsive markers HOXA5 and CYP26A1, and / or f) reduced expression of the hedgehog-responsive markers GLI1 and PTCH1 compared to HH-SPm; RA-SpM.

13. 4. Septum transversum (STM) and mesothelial cells produced by the method of claim 3, optionally characterized by expression of WT1, TBX18, LHX2, GATA4, UPK1B, or UPK3B, or any combination thereof, and preferably wherein expression of WT1, TBX18, LHX2, GATA4, UPK1B, or UPK3B is increased compared to cardiac mesoderm, liver fibroblasts, gastric mesoderm, respiratory mesoderm, esophageal mesoderm, or any combination thereof.

14. 5. A liver fibroblast (LF) produced by the method of claim 4, optionally characterized by expression of PITX1, MSX1, MSX2, TBX5, or WNT2, or any combination thereof, preferably wherein the expression of PITX1, MSX1, MSX2, TBX5, or WNT2, or any combination thereof, is increased compared to cardiac mesoderm, septum transversum, gastric mesoderm, respiratory mesoderm, or esophageal mesoderm, or any combination thereof.

15. Gastric mesenchymal cells (GM) produced by the method of claim 5, optionally comprising: The gastric mesenchymal cells are characterized by expression of BARX1, NKX3-2, or FOXF1, or any combination thereof, and preferably, the expression of BARX1, NKX3-2, or FOXF1, or any combination thereof, is increased compared to cardiac mesoderm, septum transversus, liver fibroblasts, respiratory mesoderm, or esophageal mesoderm, or any combination thereof.

16. Hedgehog-responsive lateral plate mesoderm cells (HH-LPM) produced by the method of claim 6.

17. 8. Hedgehog-responsive visceral mesoderm cells (HH-SpM) produced by the method of claim 7, optionally comprising: The HH-SpM a) lack of expression of the pluripotency markers OCT3 / 4 and SOX2, the middle primitive streak marker TBXT, and / or the endoderm markers FOXA2 and CDH1; and / or b) expression of VIM, optionally reduced expression of VIM compared to cardiac visceral mesoderm (CG-SpM) and RA-SpM; and / or c) expression of FOXF1, optionally increased expression of FOXF1 compared to CG-SpM and RA-SpM; and / or d) lack of expression of cardiac markers NKX2-5 and ISL1 or reduced expression of NKX2-5 and ISL1 compared to CG-SpM; and / or e) expression of the retinoic acid responsive markers HOXA5 and CYP26A1, and / or f) expression of the hedgehog responsive markers GLI1 and PTCH1, optionally increased expression of GLI1 and PTCH1 compared to CG-SpM and RA-SpM; HH-SpM.

18. 9. An esophageal mesenchymal cell (EM) produced by the method of claim 8, optionally characterized by expression of MSC, WNT4, or FOXF1, or any combination thereof, preferably wherein expression of MSC, WNT4, or FOXF1, or any combination thereof, is increased compared to cardiac mesoderm, septum transversus, liver fibroblasts, gastric mesoderm, or respiratory mesoderm, or any combination thereof.

19. 10. Respiratory mesenchymal cells (RM) produced by the method of claim 9, optionally comprising: (a) the respiratory mesenchymal cells are characterized by expression of TBX5, NKX6-1, WNT2, or FOXF1, or any combination thereof, preferably with increased expression of TBX5, NKX6-1, WNT2, or FOXF1, or any combination thereof, compared to cardiac mesoderm, septum transversus, liver fibroblasts, gastric mesoderm, or esophageal mesoderm, or any combination thereof; and / or (b) the respiratory mesenchymal cells are characteristic of medial respiratory mesenchyme but not ventral respiratory mesenchyme; and / or (c) the respiratory mesenchymal cells are a) increased expression of NKX6-1 and TBX5, and / or b) characterized by one or more of reduced expression of TBX4 and WNT2; The respiratory mesenchymal cells are differentiated from HH-SpM, and a) and b) are compared with respiratory mesenchymal cells differentiated from visceral mesoderm differentiated from lateral plate mesoderm without the use of an HH signaling pathway activator. Respiratory mesenchymal cells.

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