Organoid mesoderm lineage diversification

Abstract

Disclosed herein is the in vitro method of producing visceral mesoderm cell type and its subtype from pluripotent cell.These methods can be used to produce the improved organoids from foregut and hindgut that contain enriched mesenchyme, and promote the survival rate, growth and maturation of organoids both in vitro culture and in vivo transplantation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 892,781, filed August 28, 2019, which is hereby expressly incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under P01HD093363 and P30 DK078392 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] 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 days (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 the surrounding visceral mesoderm (SM) gradually pattern the naive foregut tube into distinct progenitor domains. These domains further develop into distinct visceral organs, including the trachea, lungs, 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, that surround 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. For example, applications in genetics, drug screening, personalized breeding, and transplantation currently require a deeper understanding of mesoderm differentiation during embryogenesis and improved methods for differentiating mesoderm in vitro using pluripotent stem cells (PSCs), including patient-derived PSCs. The prior art documents relevant to the invention of this application are as follows (including documents cited in the international phase after the international filing date and documents cited when the invention entered the national phase in other countries). (Prior art document) (Patent document) (Patent Document 1) U.S. Patent Application Publication No. 2016 / 0186140 (Patent Document 2) U.S. Patent Application Publication No. 2016 / 0244724 Summary of the Invention

[0005] Disclosed herein are methods for producing visceral mesoderm cells. The methods include contacting lateral plate mesoderm cells with a TGF-β signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, and a retinoic acid (RA) signaling pathway activator, thereby differentiating the lateral plate mesoderm cells into visceral mesoderm cells. In some embodiments, the visceral mesoderm cells are human visceral mesoderm cells. In some embodiments, the lateral plate mesoderm cells are differentiated from intermediate primitive stream cells. In some embodiments, the lateral plate mesoderm cells are differentiated from intermediate primitive streak cells by contacting the intermediate primitive streak cells with a TGF-β signaling pathway inhibitor, a Wnt signaling pathway inhibitor, and a BMP signaling pathway activator. In some embodiments, the intermediate primitive streak cells are differentiated from pluripotent stem cells. In some embodiments, intermediate primitive streak cells are differentiated from pluripotent stem cells by contacting the pluripotent stem cells with a TGF-β 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, lateral plate mesoderm cells are contacted with A8301, BMP4, C59, FGF2, RA, or any combination thereof. In some embodiments, the lateral plate mesoderm cells are contacted for a time sufficient to differentiate the lateral plate mesoderm cells into visceral mesoderm cells, and / or for a time that is or 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 foregoing times. In some embodiments, the lateral plate mesoderm cells are contacted for a period of time that is at or about 48 hours.In some embodiments, visceral mesoderm cells exhibit increased expression of FOXF1, HOXA1, HOXA5, or WNT2, or any combination thereof, and decreased expression of NKX2-5, ISL1, or TBX2, or any combination thereof, compared to cardiac mesoderm cells. In some embodiments, visceral mesoderm cells exhibit decreased expression of PAX3 or PRRX1, or both, compared to intermediate primitive streak cells, and / or decreased expression of CD31 compared to cardiac mesoderm cells.

[0006] Also disclosed herein is a method for producing transverse septum cells. The method includes contacting visceral mesoderm cells with a retinoic acid signaling pathway activator and a BMP signaling pathway activator. In some embodiments, the visceral mesoderm cells are any of the visceral mesoderm cells disclosed herein. In some embodiments, the visceral mesoderm cells are contacted with RA, BMP4, or both. In some embodiments, the visceral mesoderm cells are contacted for a period of time that is or 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 period within a range defined by any two of the aforementioned periods. In some embodiments, the visceral mesoderm cells are contacted for a period of time that is or is about 72 hours. In some embodiments, 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, 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, transverse septal cells exhibit decreased expression of HOXA1 or TBX5, or both, compared to visceral mesoderm cells. In some embodiments, transverse septal cells exhibit decreased expression of NKX6.1 or HOXA5, or both, compared to respiratory mesenchymal cells. In some embodiments, 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, septum transversum cells account for about 60%, 65%, 70%, 75%, 80%, 85%, or 90% of all cells differentiated from visceral mesoderm cells.

[0007] Also disclosed herein is a method for producing fibroblasts. The method includes contacting visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and a Wnt signaling pathway activator. In some embodiments, the visceral mesoderm cells are any of the visceral mesoderm cells disclosed herein. In some embodiments, the visceral mesoderm cells are contacted with RA, BMP4, CHIR99021, or any combination thereof. In some embodiments, the fibroblasts are liver fibroblasts. In some embodiments, the visceral mesoderm cells are contacted for a period of time that is or 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 period within a range defined by any two of the aforementioned periods. In some embodiments, the visceral mesoderm cells are contacted for a period of 72 hours or about 72 hours. In some embodiments, the fibroblasts exhibit increased expression of MSX1, MSX2, or HAND1, or any combination thereof, compared to visceral mesoderm cells or septum transversum cells, or both. In some embodiments, the fibroblasts exhibit decreased expression of WT1, TBX18, LHX2, or UPK1B, or any combination thereof, compared to septum transversum cells. In some embodiments, the fibroblasts exhibit decreased expression of NKX6.1, HOXA5, or LHX2, or any combination thereof, compared to respiratory mesenchymal cells. In some embodiments, the fibroblasts exhibit decreased expression of NKX3.2, MSC, BARX1, WNT4, or HOXA5, or any combination thereof, compared to esophageal / gastric mesenchymal cells.

[0008] Also disclosed herein is a method for producing respiratory mesenchymal cells. The method includes: a) contacting visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, a Hedgehog (HH) signaling pathway activator, and a Wnt signaling pathway activator. In some embodiments, the visceral mesoderm cells are contacted for a period of 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 within a range defined by any two of the aforementioned periods. In some embodiments, the visceral mesoderm cells are contacted for a period of 72 hours or about 72 hours. In some embodiments, step a) is a second step and further comprises a first step of contacting visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and an HH signaling pathway activator prior to the second step. In some embodiments, the visceral mesoderm cells are contacted for a period of 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 period within a range defined by any two of the aforementioned periods in the first step. In some embodiments, the visceral mesoderm cells are contacted for a period of 48 hours or about 48 hours in the first step. In some embodiments, the visceral mesoderm cells are contacted for a period of time that is or 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 period of time within a range defined by any two of the aforementioned periods of the second step. In some embodiments, the visceral mesoderm cells are contacted for a period of time that is or is about 24 hours of the second step. In some embodiments, the visceral mesoderm cells are any of the visceral mesoderm cells disclosed herein.In some embodiments, the visceral mesoderm cells are contacted with RA, BMP4, PMA, CHIR99021, or any combination thereof. In some embodiments, the respiratory mesenchymal cells exhibit increased expression of NKX6-1, TBX5, HOXA1, HOXA5, FOXF1, LHX2, or WNT2, or any combination thereof, compared to cardiac endoderm cells, visceral mesoderm cells, or esophageal / gastric mesenchymal cells, or any combination thereof. In some embodiments, the respiratory mesenchymal cells exhibit decreased expression of WNT2, WT1, TBX18, LHX2, or UPK1B, or any combination thereof, compared to transverse septum cells. In some embodiments, the respiratory mesenchymal cells exhibit decreased expression of WNT2, MSX1, or MSX2, or any combination thereof, compared to fibroblasts.

[0009] Also disclosed herein is a method for producing esophageal / gastric mesenchymal cells. The method includes: a) contacting visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway inhibitor, and an HH signaling pathway activator. In some embodiments, the visceral mesoderm cells are contacted for a period of 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 within a range defined by any two of the aforementioned periods. In some embodiments, the visceral mesoderm cells are contacted for a period of about 72 hours. In some embodiments, step a) is a second step, and further includes a first step of contacting visceral mesoderm cells with a retinoic acid signaling pathway activator and an HH signaling pathway activator before the second step. In some embodiments, the visceral mesoderm cells are contacted for a period of time that is or 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 period of time within a range defined by any two of the aforementioned periods in the first step. In some embodiments, the visceral mesoderm cells are contacted for a period of time that is or is about 48 hours in the first step. In some embodiments, the visceral mesoderm cells are contacted for a period of time that is or 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 period of time within a range defined by any two of the aforementioned times in the second step. In some embodiments, the visceral mesoderm cells are contacted for a period of time that is or is about 24 hours in the second step. In some embodiments, the visceral mesoderm cells are any of the visceral mesoderm cells disclosed herein. In some embodiments, the visceral mesoderm cells are contacted with RA, Noggin, PMA, or any combination thereof.In some embodiments, the esophageal / gastric mesenchymal cells exhibit increased expression of MSC, BARX1, WNT4, HOXA1, FOXF1, or NKX3-2, or any combination thereof, compared to cardiac endoderm cells, visceral mesoderm cells, or respiratory mesenchymal cells, or any combination thereof. In some embodiments, the esophageal / gastric mesenchymal cells exhibit decreased expression of WNT2, TBX5, MSX1, MSX2, or LHX2, or any combination thereof, compared to septum transversum cells, fibroblasts, or respiratory mesenchymal cells, or any combination thereof.

[0010] In any of the embodiments, the TGF-β signaling pathway inhibitor is selected from the group consisting of A8301, RepSox, LY365947, and SB431542. In any of the embodiments, the TGF-β signaling pathway inhibitor is A8301. In any of the embodiments, the TGF-β 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. In any of the embodiments, the TGF-β signaling pathway inhibitor is contacted at a concentration of 1 μM or about 1 μM.

[0011] In any of the 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 any of the embodiments, the Wnt signaling pathway inhibitor is C59. In any of the embodiments, 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 foregoing concentrations. In any of the embodiments, the Wnt signaling pathway inhibitor is contacted at a concentration of 1 μM or about 1 μM.

[0012] In any of the 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 any of the embodiments, the BMP signaling pathway activator is BMP4. In any of the embodiments, the BMP signaling pathway activator is contacted 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, or any concentration within a range defined by any two of the foregoing concentrations. In any of the embodiments, the BMP signaling pathway activator is contacted at a concentration of at or about 30 ng / mL.

[0013] In any of the 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 any of the embodiments, the FGF signaling pathway activator is FGF2. In any of the embodiments, the FGF signaling pathway activator is contacted at a concentration of 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 any concentration within a range defined by any two of the foregoing concentrations. In any of the embodiments, the FGF signaling pathway activator is contacted at a concentration of 20 ng / mL or about 20 ng / mL.

[0014] In any of the 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 any of the embodiments, the RA signaling pathway activator is RA. In any of the embodiments, 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 at 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 at any concentration within a range defined by any two of the foregoing concentrations. In any of the embodiments, the RA signaling pathway activator is contacted at a concentration of 2 μM or about 2 μM.

[0015] In any embodiment, 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 any embodiment, the Wnt signaling pathway activator is CHIR99021. In any of the embodiments, 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. In any of the embodiments, 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 foregoing concentrations.

[0016] In any of the embodiments, the HH signaling pathway activator is selected from the group consisting of SHH, IHH, DHH, PMA, GSA10, and SAG. In any of the embodiments, the HH signaling pathway activator is PMA. In any of the embodiments, 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 at 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 at any concentration within a range defined by any two of the foregoing concentrations. In any of the embodiments, the HH signaling pathway activator is contacted at a concentration of 2 μM or about 2 μM.

[0017] In any embodiment, the BMP signaling pathway inhibitor is selected from the group consisting of Noggin, RepSox, LY364947, LDN193189 and SB431542.In any embodiment, the BMP signaling pathway inhibitor is Noggin.In any embodiment, the BMP signaling pathway inhibitor is contacted at a concentration of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150ng / mL, or about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150ng / mL, or any concentration within the range defined by any two of the aforementioned concentrations.In any embodiment, the BMP signaling pathway inhibitor is contacted at a concentration of 100ng / mL or about 100ng / mL.

[0018] Also disclosed herein are visceral mesoderm cells, septum transversum cells, fibroblasts, respiratory mesenchymal cells, and esophageal / gastric mesenchymal cells produced by any of the methods disclosed herein.

[0019] The embodiments of the present disclosure provided herein are described by the following numbered alternatives:

[0020] 1. A method for producing visceral mesoderm cells, comprising: A method comprising contacting lateral plate mesoderm cells with a TGF-β signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, and a retinoic acid (RA) signaling pathway activator.

[0021] 2. The method of alternative 1, wherein the visceral mesoderm cells are human visceral mesoderm cells.

[0022] 3. The method according to alternatives 1-2, wherein the lateral plate mesoderm cells are differentiated from middle primitive stream cells.

[0023] 4. The method of alternative 3, wherein the lateral plate mesoderm cells are differentiated from intermediate primitive streak cells by contacting the intermediate primitive streak cells with a TGF-β signaling pathway inhibitor, a Wnt signaling pathway inhibitor, and a BMP signaling pathway activator.

[0024] 5. The method of alternative 3 or 4, wherein the intermediate primitive streak cells are differentiated from pluripotent stem cells.

[0025] 6. The method of alternative 5, wherein the intermediate primitive streak cells are differentiated from pluripotent stem cells by contacting the pluripotent stem cells with a TGF-β signaling pathway activator, a Wnt signaling pathway activator, an FGF signaling pathway activator, a BMP signaling pathway activator, and a PI3K signaling pathway inhibitor.

[0026] 7. The method of any one of alternatives 1-6, wherein the lateral plate mesoderm cells are contacted with A8301, BMP4, C59, FGF2, RA, or any combination thereof.

[0027] 8. The method of any one of alternatives 1-7, wherein the lateral plate mesoderm cells are contacted for a time sufficient to differentiate the lateral plate mesoderm cells into visceral mesoderm cells, and / or for a time that is or 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 aforesaid times.

[0028] 9. The method of any one of alternatives 1-8, wherein the lateral plate mesoderm cells are contacted for a period of time that is at or about 48 hours.

[0029] 10. The method of any one of alternatives 1-9, wherein the visceral mesoderm cells exhibit increased expression of FOXF1, HOXA1, HOXA5, or WNT2, or any combination thereof, and decreased expression of NKX2-5, ISL1, or TBX2, or any combination thereof, compared to cardiac mesoderm cells.

[0030] 11. The method of any one of alternatives 1-10, wherein the visceral mesoderm cells exhibit decreased expression of PAX3 or PRRX1, or both, compared to intermediate primitive streak cells, and / or decreased expression of CD31 compared to cardiac mesoderm cells.

[0031] 12. A method for producing transverse septum cells, comprising contacting visceral mesoderm cells with a retinoic acid signaling pathway activator and a BMP signaling pathway activator.

[0032] 13. The method of alternative 12, wherein the visceral mesoderm cell is a visceral mesoderm cell according to any one of alternatives 1 to 11.

[0033] 14. The method of alternatives 12 or 13, wherein the visceral mesoderm cells are contacted with RA, BMP4, or both.

[0034] 15. The method of any one of alternatives 12-14, wherein the visceral mesoderm cells are contacted for a time sufficient to differentiate the visceral mesoderm cells into transverse septum cells, and / or for a time that is or 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 period within a range defined by any two of the foregoing times.

[0035] 16. The method of any one of alternatives 12-15, wherein the visceral mesoderm cells are contacted for a period of time that is at or about 72 hours.

[0036] 17. The method of any one of alternatives 12-16, wherein the transverse septum 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.

[0037] 18. The method of any one of alternatives 12-17, wherein the transverse septum cells exhibit decreased expression of MSX1, MSX2, or HAND1, or any combination thereof, compared to cardiac mesoderm cells or fibroblasts, or both.

[0038] 19. The method of any one of alternatives 12-18, wherein the transverse septum cells exhibit decreased expression of HOXA1 or TBX5, or both, compared to visceral mesoderm cells.

[0039] 20. The method of any one of alternatives 12-19, wherein transverse septum cells exhibit decreased expression of NKX6.1 or HOXA5, or both, compared to respiratory mesenchymal cells.

[0040] 21. The method of any one of alternatives 12-20, wherein the transverse septum cells exhibit decreased expression of NKX3.2, MSC, BARX1, WNT4, or HOXA5, or any combination thereof, compared to esophageal / gastric mesenchymal cells.

[0041] 22. The method of any one of alternatives 12-21, wherein the transverse septum cells account for approximately 60%, 65%, 70%, 75%, 80%, 85%, or 90% of all cells differentiated from visceral mesoderm cells.

[0042] 23. A method for producing fibroblasts, comprising contacting visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and a Wnt signaling pathway activator.

[0043] 24. The method of alternative 23, wherein the visceral mesoderm cells are visceral mesoderm cells according to any one of alternatives 1 to 11.

[0044] 25. The method of alternative 23 or 24, wherein the visceral mesoderm cells are contacted with RA, BMP4, CHIR99021, or any combination thereof.

[0045] 26. The method of any one of alternatives 23-25, wherein the fibroblasts are liver fibroblasts.

[0046] 27. The method of any one of alternatives 23-26, wherein the visceral mesoderm cells are contacted for a time sufficient to differentiate the visceral mesoderm cells into fibroblasts, and / or for a time that is or 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 period within a range defined by any two of the foregoing times.

[0047] 28. The method of any one of alternatives 23-27, wherein the visceral mesoderm cells are contacted for a period of time that is at or about 72 hours.

[0048] 29. The method of any one of alternatives 23-28, wherein the fibroblasts exhibit increased expression of MSX1, MSX2, or HAND1, or any combination thereof, compared to visceral mesoderm cells or transverse septum cells, or both.

[0049] 30. The method of any one of alternatives 23-29, wherein the fibroblasts exhibit decreased expression of WT1, TBX18, LHX2, or UPK1B, or any combination thereof, compared to transverse septal cells.

[0050] 31. The method of any one of alternatives 23-30, wherein the fibroblasts exhibit decreased expression of NKX6.1, HOXA5, or LHX2, or any combination thereof, compared to respiratory mesenchymal cells.

[0051] 32. The method of any one of alternatives 23-31, wherein the fibroblasts exhibit decreased expression of NKX3.2, MSC, BARX1, WNT4, or HOXA5, or any combination thereof, compared to esophageal / gastric mesenchymal cells.

[0052] 33. A method for producing respiratory mesenchymal cells, comprising: a) contacting visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, a Hedgehog (HH) signaling pathway activator, and a Wnt signaling pathway activator.

[0053] 34. The method of alternative 33, wherein the visceral mesoderm cells are contacted for a time sufficient to differentiate the visceral mesoderm cells into respiratory mesenchymal cells, and / or for a time that is or 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 period within a range defined by any two of the foregoing times.

[0054] 35. The method of alternative 33 or 34, wherein the visceral mesoderm cells are contacted for a period of time that is at or about 72 hours.

[0055] 36. The method of alternative 33, wherein step a) is a second step and further comprises a first step of contacting visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and an HH signaling pathway activator prior to the second step.

[0056] 37. The method of alternative 36, wherein the visceral mesodermal cells are contacted for a time sufficient to differentiate the visceral mesodermal cells into respiratory mesenchymal cells, and / or for a time that is or 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 period within a range defined by any two of the aforementioned times, for the first step.

[0057] 38. The method of alternative 36 or 37, wherein the visceral mesoderm cells are contacted for a period of time that is at or about 48 hours for the first step.

[0058] 39. The method of any one of alternatives 36-38, wherein the visceral mesodermal cells are contacted for a time sufficient to differentiate the visceral mesodermal cells into respiratory mesenchymal cells for the second step, and / or for a time that is or 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 period within a range defined by any two of the foregoing times.

[0059] 40. The method of any one of alternatives 36-39, wherein the visceral mesoderm cells are contacted for a period of time that is at or about 24 hours for the second step.

[0060] 41. The method of any one of alternatives 33-40, wherein the visceral mesoderm cells are visceral mesoderm cells according to any one of alternatives 1-11.

[0061] 42. The method of any one of alternatives 33-41, wherein the visceral mesoderm cells are contacted with RA, BMP4, PMA, CHIR99021, or any combination thereof.

[0062] 43. The method of any one of alternatives 33-42, wherein the respiratory mesenchymal cells exhibit increased expression of NKX6-1, TBX5, HOXA1, HOXA5, FOXF1, LHX2, or WNT2, or any combination thereof, compared to cardiac endoderm cells, visceral mesoderm cells, or esophageal / gastric mesenchymal cells, or any combination thereof.

[0063] 44. The method of any one of alternatives 33-43, wherein respiratory mesenchymal cells exhibit decreased expression of WNT2, WT1, TBX18, LHX2, or UPK1B, or any combination thereof, compared to transverse septal cells.

[0064] 45. The method of any one of alternatives 33-44, wherein respiratory mesenchymal cells exhibit decreased expression of WNT2, MSX1, or MSX2, or any combination thereof, compared to fibroblasts.

[0065] 46. ​​A method for producing esophageal / gastric mesenchymal cells, comprising: a) contacting visceral mesodermal cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and an HH signaling pathway activator.

[0066] 47. The method of alternative 46, wherein the visceral mesodermal cells are contacted for a time sufficient to differentiate the visceral mesodermal cells into esophageal / gastric mesenchymal cells, and / or for a time that is or 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 period within a range defined by any two of the foregoing times.

[0067] 48. The method of alternative 46 or 47, wherein the visceral mesoderm cells are contacted for a period of time that is at or about 72 hours.

[0068] 49. The method described in alternative 46, wherein step a) is a second step and further comprises a first step of contacting visceral mesoderm cells with a retinoic acid signaling pathway activator and an HH signaling pathway activator prior to the second step.

[0069] 50. The method of alternative 49, wherein the visceral mesodermal cells are contacted for a time sufficient to differentiate the visceral mesodermal cells into esophageal / gastric mesenchymal cells for the first step, and / or for a time that is or 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 period within a range defined by any two of the aforementioned times.

[0070] 51. The method of alternative 49 or 50, wherein the visceral mesoderm cells are contacted for a period of time that is, or is about, 48 hours for the first step.

[0071] 52. The method of any one of alternatives 49-51, wherein the visceral mesodermal cells are contacted for a time sufficient to differentiate the visceral mesodermal cells into esophageal / gastric mesenchymal cells for the second step, and / or for a time that is or 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 period within a range defined by any two of the aforementioned times.

[0072] 53. The method of any one of alternatives 49-52, wherein the visceral mesoderm cells are contacted for a period of time that is at or about 24 hours for the second step.

[0073] 54. The method of any one of alternatives 46-53, wherein the visceral mesoderm cells are visceral mesoderm cells of any one of alternatives 1-11.

[0074] 55. The method of any one of alternatives 46-54, wherein the visceral mesoderm cells are contacted with RA, noggin, PMA, or any combination thereof.

[0075] 56. The method of any one of alternatives 46-55, wherein the esophageal / gastric mesenchymal cells exhibit increased expression of MSC, BARX1, WNT4, HOXA1, FOXF1, or NKX3-2, or any combination thereof, compared to cardiac endodermal cells, visceral mesodermal cells, or respiratory mesenchymal cells, or any combination thereof.

[0076] 57. The method of any one of alternatives 46-56, wherein the esophageal / gastric mesenchymal cells exhibit decreased expression of WNT2, TBX5, MSX1, MSX2, or LHX2, or any combination thereof, compared to transverse septum cells, fibroblasts, or respiratory mesenchymal cells, or any combination thereof.

[0077] 58. The method of any one of alternatives 1-57, wherein the TGF-β signaling pathway inhibitor is selected from the group consisting of A8301, RepSox, LY365947, and SB431542.

[0078] 59. The method of any one of alternatives 1 to 58, wherein the TGF-β signaling pathway inhibitor is A8301.

[0079] 60. The method of any one of alternatives 1-59, wherein the TGF-β 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.

[0080] 61. The method of any one of alternatives 1-60, wherein the TGF-β signaling pathway inhibitor is contacted at a concentration of 1 μM or about 1 μM.

[0081] 62. The method of any one of alternatives 1-61, wherein the Wnt signaling pathway inhibitor is selected from the group consisting of C59, PNU74654, KY-02111, PRI-724, FH-535, DIF-1, and XAV939.

[0082] 63. The method of any one of alternatives 1 to 62, wherein the Wnt signaling pathway inhibitor is C59.

[0083] 64. The method of any one of alternatives 1-63, 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 foregoing concentrations.

[0084] 65. The method of any one of alternatives 1 to 64, wherein the Wnt signaling pathway inhibitor is contacted at a concentration of 1 μM or about 1 μM.

[0085] 66. The method of any one of alternatives 1-65, 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.

[0086] 67. The method of any one of alternatives 1 to 66, wherein the BMP signaling pathway activator is BMP4.

[0087] 68. The method of any one of alternatives 1-67, wherein the BMP signaling pathway activator is contacted 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, or any concentration within a range defined by any two of the foregoing concentrations.

[0088] 69. The method of any one of alternatives 1-68, wherein the BMP signaling pathway activator is contacted at a concentration of 30 ng / mL or about 30 ng / mL.

[0089] 70. The method of any one of alternatives 1-69, 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.

[0090] 71. The method of any one of alternatives 1-70, wherein the FGF signaling pathway activator is FGF2.

[0091] 72. The method of any one of alternatives 1-71, wherein the FGF signaling pathway activator is contacted at a concentration of 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 any concentration within a range defined by any two of the foregoing concentrations.

[0092] 73. The method of any one of alternatives 1-72, wherein the FGF signaling pathway activator is contacted at a concentration of 20 ng / mL or about 20 ng / mL.

[0093] 74. The method of any one of alternatives 1-73, 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, BS493, TTNPB, and AM580.

[0094] 75. The method of any one of alternatives 1-74, wherein the RA signaling pathway activator is RA.

[0095] 76. The method of any one of alternatives 1-75, 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.

[0096] 77. The method of any one of alternatives 1-76, wherein the RA signaling pathway activator is contacted at a concentration of 2 μM or about 2 μM.

[0097] 78. The method of any one of alternatives 1-77, 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, BML284, IQ-1, WAY262611, CHIR99021, CHIR98014, AZD2858, BIO, AR-A014418, SB216763, SB415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD8, and TWS119.

[0098] 79. The method of any one of alternatives 1 to 78, wherein the Wnt signaling pathway activator is CHIR99021.

[0099] 80. The method of any one of alternatives 1-79, 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.

[0100] 81. The method of any one of alternatives 1-80, 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 foregoing concentrations.

[0101] 82. The method of any one of alternatives 1-81, wherein the HH signaling pathway activator is selected from the group consisting of SHH, IHH, DHH, PMA, GSA10, and SAG.

[0102] 83. The method of any one of alternatives 1 to 82, wherein the HH signaling pathway activator is PMA.

[0103] 84. The method of any one of alternatives 1-83, 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.

[0104] 85. The method of any one of alternatives 1-84, wherein the HH signaling pathway activator is contacted at a concentration of 2 μM or about 2 μM.

[0105] 86. The method of any one of alternatives 1-85, wherein the BMP signaling pathway inhibitor is selected from the group consisting of noggin, RepSox, LY364947, LDN193189, and SB431542.

[0106] 87. The method of any one of alternatives 1-86, wherein the BMP signaling pathway inhibitor is noggin.

[0107] 88. The method of any one of alternatives 1-87, wherein the BMP signaling pathway inhibitor is contacted at 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within the range defined by any two of the foregoing concentrations.

[0108] 89. The method of any one of alternatives 1-88, wherein the BMP signaling pathway inhibitor is contacted at a concentration of 100 ng / mL or about 100 ng / mL.

[0109] 90. Visceral mesoderm cells produced by the method described in any one of alternatives 1 to 11.

[0110] 91. A transverse septum cell produced by the method described in any one of alternatives 12 to 22.

[0111] 92. A fibroblast produced by the method according to any one of alternatives 23 to 32.

[0112] 93. Respiratory mesenchymal cells produced by the method described in any one of alternatives 33 to 45.

[0113] 94. Esophageal / gastric mesenchymal cells produced by the method described in any one of alternatives 46 to 57. [Brief explanation of the drawings]

[0114] 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 limit the scope.

[0115] [Figure 1A-J] We demonstrate an embodiment of single-cell analysis of mouse foregut endoderm and mesoderm lineages. 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 pseudospatial ordering of E9.5 endoderm (1G) and mesoderm (1H) cells along the anterior-posterior (AP) axis. Figures 1I and 1J show schematic diagrams of the predicted locations of E9.5 cell types mapped to the embryonic mouse foregut endoderm (1I) and mesoderm (1J). 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 all stages with major lineages annotated by known marker genes (Panel A). UMAP of all cells from all stages with computationally assigned clusters based on transcriptome similarity (Panel B). UMAP of all cells from all stages shaded by stage and region (Panel C). t-SNE maps of single cells from each stage annotated by major lineage for E8.5 (Panel D), E9.0 (Panel E), and E9.5 (Panel F). Gene expression heatmaps of selected markers in individual cells for 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 annotations of E8.5 DE (Panel A), E8.5 SM (Panel B), E9.0 DE (Panel C), and E9.0 SM cells (Panel D). 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 cells (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 for different clusters: DE at E8.5 (panel M), SM at E8.5 (panel N), DE at E9.0 (panel O), and SM at E9.0 (panel P). [Figure 1M]Figure 1 shows an 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 significantly contribute 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-Q] We demonstrate an 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 various 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 RNAscope 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, embryo; liv, liver; oft, outflow tract; pha, pharynx; res, respiratory system; stm, transverse septum mesenchyme; sto, stomach; sv, sinus venosus; vp, ventral pancreas. [Figure 2R] Figure 1 shows an embodiment of validation of liver mesenchymal subtypes. Schematic of mouse embryonic foregut at E9.5 (Panel A). RNAscope 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 an embodiment of coaxial Hox gene expression and transcription factor coding. Heatmap of average Hox gene expression for various DE and SM clusters aligned along the AP axis. Annotation is as follows: E8.5 = a_cluster, E9.0 = b_cluster, and E9.5 = c_cluster (Panel A). Presumed 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 for DE and SM populations at E9.5 (Panel C). a, anterior; fg, foregut; post, posterior; v, ventral; stm, transverse septum mesenchyme. [Figure 3A-F] An example of coordinated cell trajectories of endoderm and mesoderm is shown. Figures 3A and 3B show force-directed SPRING visualization of the cell differentiation pathways 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 the cell-state tree. Based on transcriptome similarity (KNN), each cell at a later time point (y-axis) voted for the most similar cell at the previous time point (x-axis). All votes for a given cluster are tabulated, normalized to cluster size, and represented as a percentage of decisions in a heatmap. 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 numbers. [Figure 3G]1 shows embodiments of SPRING plots of DE and SM cell differentiation pathways. 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 illustrates an embodiment of hepatic endoderm development. Cell state tree of the hepatic endoderm lineage (Panel A) with key marker genes indicated for each cell state. 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 4]Figures 4A-I and 4K-L show an embodiment of coordinated development of multipotent progenitor cells. Figures 4A and 4B illustrate the differentiation pathways of the esophageal-respiratory-gastric cell states 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 co-expression at the esophagus-tracheal boundary. Figure 4K shows Sox2 and Nkx2-1 whole-mount immunostaining of the mouse foregut at E9.5. Figure 4L shows Sox2, Nkx2-1, and Foxf1 immunostaining of a transverse E9.5 foregut section, confirming a rare population of cells co-expressing Sox2 / Nkx2-1. L' shows a higher magnification of the box in Figure 4L. [Figure 5A-I]We present an example 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 (5A; same image as in 1D) and sections (5B), illustrating the epithelial-mesenchymal tissue microenvironment (dashed circle). 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 specific signaling pathway. Arrows represent predicted paracrine and autocrine receptor-ligand interactions. Figure 5D shows BMP-responsive metagene expression levels projected onto a SPRING plot of the DE and SM. Figure 5E shows in situ hybridization of Bmp4 in foregut transverse sections, demonstrating expression in the respiratory mesenchyme and stm. Figures 5F and 5G show pSmad1 immunostaining in foregut transverse sections, demonstrating BMP signaling responses in the DE and SM of the respiratory and hepatic organs. 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 combinatorial signals predicted to control lineage diversification. Letters indicate predicted signals at each step, with larger font indicating stronger signal responses. a, anterior; p, posterior; hp, hepatopancreas; stm, transverse septum mesenchyme. [Figure 5J]Figure 1 shows metagene expression of all ligand-, receptor-, and context-independent response genes. 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), "ligand metagenes," "receptor metagenes," and "response metagenes" were calculated by averaging the normalized expression of each individual gene for each pathway in cells and clusters lacking Wnt1 (e.g., Wnt-ligand metagene = ΣWnt1 + Wnt2 + Wnt2b + Wnt3 · · · Wnt10b expression / n). The shading and size of each dot represent the metagene expression level in each cluster. [Figure 5K] This figure shows examples of computationally predicted receptor-ligand interactions between different foregut cell populations. Schematics depict paracrine signaling between the DE and SM for six major pathways. Below the schematic, DE and SM cell clusters for each stage are aligned along the AP axis, consistent with their in vivo locations. 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]Figure 1 shows an example of predicted temporal and spatial dynamics of signaling responses. Pathway responses projected onto SPRING plots of DE and SM—metagene expression levels and 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-H] We demonstrate an embodiment of a gene study of the signaling roadmap, revealing that HH promotes intestinal versus liver mesenchyme signaling. Figures 6A and 6B show SPRING visualization of HH ligand-metagene expression in DE cells (6A) and HH response-metagene expression in SM cells (6B). Figure 6C shows HH response-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 response 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-D]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). Columns 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 (top panel), 10 μm (bottom panel). (Lower panel). Figure 7D shows quantification of the percentage of cells positive for the indicated immunostaining or RNAscope in situ hybridization. Columns show mean values ​​± 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, intermediate primitive streak; CM, cardiac mesoderm; SM visceral mesoderm. Columns show mean values ​​± 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 various d7SM-like cultures. Scale bars are 50 μm in the upper panel and 10 μm in the lower panel. Quantification is in Figure 7D (panels A-C). RT-PCR analysis of mesoderm subtype markers based on mouse scRNA-seq data: cardiac (ACTC1, TBX20, TNNT2), early SM (PDE5A, HOXA5); liver-stm / mesothelial (TBX18, LHX2, UPK3B), liver-fibroblast (MSX2, HAND1), and esophagus / stomach (WNT4, NKX3-2) (panel D). SC, stem cell; MPS, intermediate primitive streak; CM, cardiac mesoderm; SM, visceral mesoderm; STM, septum transversum mesenchyme; LF, liver fibroblast; RM, respiratory mesenchyme; EM / GM, esophagus / stomach mesenchyme. Columns represent mean values ​​± SD (n=3). Tukey's test, *p<0.05, **p<0.005, ***p<0.0005. DETAILED DESCRIPTION OF THE INVENTION

[0116] 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, previously undefined SM subtypes were generated from human pluripotent stem cells (hPSCs).

[0117] 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 types of SMs are there, and is there a unique mesenchyme in each fetal organ primordium? How are SM and DE lineages coordinated during organogenesis? What role does endoderm play in mesoderm regionalization?

[0118] 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 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 eventually insert into the overlying visceral endoderm. By E8.0, the anterior DE collapses 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 endoderm and mesoderm sheet into a tubular structure. The lateral plate mesoderm further divides into an outer somatic mesoderm layer next to the ectoderm, which gives rise to the limbs and body wall, and an inner visceral mesoderm layer, which 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.

[0119] 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 form. 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.

[0120] 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, when 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 differentiation pathways demonstrate 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.

[0121] Previous studies on the regional identity of the SM in the early embryo have been limited. Beyond 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.

[0122] One unexpected observation was that the liver bud contains a more distinct SM cellular state than any other organ bud, with a septum transversum mesenchyme (STM), sinus venosus, 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 early STMs give rise to mesothelium, hepatic stellate cells, interstitial fibroblasts, and perivascular smooth muscle. It will be important to determine whether other organ buds have a similar elaboration of cell types 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; there is ample evidence that the mesothelium of the liver bud, also known as the proepicardium, migrates to surround the heart and lungs.

[0123] 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.

[0124] The signaling roadmap developed herein was used to direct hPSC development toward various SM-like cell types. The system described herein offers a unique opportunity to model human fetal mesenchyme development and interrogate how combinatorial signaling pathways direct parallel mesenchymal fate choices. 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 the recombination of DE and SM organoids, a key step for engineering complex foregut tissues for regenerative medicine.

[0125] Disclosed herein is a method for producing visceral mesoderm cells in vitro.In some embodiments, 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 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 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, so that organoid growth and maturation are hindered.Visceral mesoderm cells and the method for producing them can be applied to any organoid and / or enteroid (organoid-like structure 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. Patent Nos. 9,719,068 and 10,174,289, and PCT Publication Nos. WO2011 / 140411, WO2015 / 183920, WO2016 / 061464, WO2017 / 192997, WO2018 / 106628, WO2018 / 200481, WO2018 / 085615, WO2018 / 085622, WO2018 / 085623, WO2018 / 226267, WO2020 / 023245, each of which is expressly incorporated herein by reference in its entirety.

[0126] 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.

[0127] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood when read in light of this disclosure by one of ordinary skill in the art to which this disclosure belongs. For purposes of this disclosure, the following terms are described below.

[0128] 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.

[0129] "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.

[0130] 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 everything that the phrase "consisting of" follows. Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may 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 listed 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.

[0131] The terms "individual," "subject," or "patient" as used herein have their common and usual meanings as understood in the context of this specification, and refer to human or non-human mammals, such as dogs, cats, mice, rats, cows, sheep, pigs, goats, non-human primates, or birds, such as chickens, 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.

[0132] The terms "effective amount" or "effective dose" as used herein have their common and ordinary meaning as understood in light of the specification and refer to that amount of a described composition or compound that produces an observable effect. The actual dosage level of the active ingredient in the active composition of the presently disclosed subject matter can be varied to administer an amount of the active composition or compound 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 the effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0133] As used herein, the terms "function" and "functional" have their plain and ordinary meaning as understood in light of this specification and refer to biological, enzymatic, or therapeutic function.

[0134] The term "inhibit" as used herein has its common and ordinary meaning as understood in light of the present specification and can refer to a reduction or prevention of biological activity. The reduction can be about, about, at least about, at least about, less than, or about a percentage of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or by an amount within a range defined by any two of the foregoing values. The term "delay" as used herein has its common and ordinary meaning as understood in light of the present specification 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 about 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a percentage that is about, at least, at least about, less than, or less than about, or an amount within a range defined by any two of the foregoing values. The terms inhibition and delay do not necessarily indicate 100% inhibition or delay. Partial inhibition or delay can be achieved.

[0135] As used herein, the term "isolated" has its common and ordinary meaning as understood in light of the specification and refers to a substance and / or entity that (1) has been separated from at least some of the components with which it was associated when originally produced (in nature and / or in an experimental setting) and / or (2) has been separated from at least some of the components with which it was associated when produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or equal to 100%, about, at least, at least about, less than, or less than (or a range including and / or spanning) the aforementioned values, of other components with which they were originally associated. In some embodiments, an isolated agent is 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or equivalent to 100% pure, is about, is at least, is at least about, is less than, or is less than (or ranges including and / or spanning) the aforementioned values. 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.

[0136] 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.

[0137] As used herein, "ex vivo" is given its common and ordinary meaning as understood in light of the specification and refers to the performance of a method outside a living body with little change in natural conditions.

[0138] As used herein, "in vitro" is given its common and ordinary meaning as understood in light of the specification and refers to the performance of a method outside biological conditions, e.g., in a petri dish or test tube.

[0139] 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 occurring naturally in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by 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. Additionally, 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 nucleic acid bases 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(s) 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 to amplify and / or express the nucleic acid(s) 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.

[0140] 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 any other sequence that is, is about, is at least, is at least about, is less than, or is less than about 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 light of the 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 light of the 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 to any other sequence, but generally not between sequences that encode functional or catalytic polypeptides, proteins, or protein domains, either directly or with extra nucleic acid, for example, between linkers, repeats, or restriction enzyme sites, or that are, 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 a range defined by any two of the foregoing lengths.

[0141] The nucleic acid described herein comprises nucleobases.The primary, standard, natural or unmodified bases are adenine, cytosine, guanine, thymine and uracil.Other nucleobases include, but are not limited to, purine, pyrimidine, modified nucleobase, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl base, dye-labeled base, fluorescent base or biotin-labeled base.

[0142] 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. Many functions of peptides, polypeptides, and proteins are known in the art, including, but not limited to, enzymatic, structural, transport, defensive, hormonal, or signal transduction functions. Peptides, polypeptides, and proteins are often, but not always, biologically produced 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 a 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 light of the specification, and refers to sequences that follow 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 light of the specification, and refers to sequences that precede the N-terminus of a subsequent sequence.

[0143] As used herein, the term "purity" of any given substance, compound, or material has its common and ordinary meaning as understood in light of specifications and refers to the actual abundance of the substance, compound, or material relative 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.

[0144] As used herein, the term "yield" of any given substance, compound, or material has its common and ordinary meaning as understood in light of specifications 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 reaction or process efficiency, undesired side reactions, decomposition, quality of input substances, compounds, or materials, or loss of desired substances, compounds, or materials during any step of production.

[0145] The term "w / w%" or "weight / weight %" as used herein has its ordinary and ordinary 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 ordinary 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.

[0146] stem cells As used herein, the term "totipotent stem cells" (also known as omnipotent stem cells) are 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.

[0147] As used herein, the term "embryonic stem cells (ESCs), commonly abbreviated as ES cells, as used herein, has its plain and ordinary meaning as understood in light of the present specification and refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For purposes of this disclosure, the term "ESCs" may be used broadly to encompass embryonic germ cells.

[0148] As used herein, the term "pluripotent stem cells (PSCs)" has its plain and ordinary meaning as understood in light of this specification and encompasses any cell that can differentiate into almost any cell type of 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 progeny 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 specific 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.

[0149] As used herein, the term "induced pluripotent stem cells (iPSCs)" has its plain and ordinary meaning as understood in light of the specification. These terms are commonly abbreviated as iPSCs and refer to a type of pluripotent stem cell artificially induced from normally non-pluripotent cells, such as adult somatic cells, by inducing the "forced" expression of specific genes. hiPSCs refer to human iPSCs. 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, retrovirus system is used to transform human fibroblasts into pluripotent stem cells using four essential genes: Oct3 / 4, Sox2, Klf4, and c-Myc.In other methods, lentivirus system is 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, E-cadherin, or any combination thereof.

[0150] As used herein, the term "progenitor cell" has its plain and ordinary meaning as understood in light of the 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 (induced or non-induced), multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells can be derived from embryos, infants, children, or adults. 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).

[0151] In some embodiments, one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, pluripotent stem cells are derived from embryonic stem cells, and these embryonic stem cells are derived from the totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of blastocysts, which are early-stage embryos. 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 can be applied to any stem cells.

[0152] Additional stem cells that can be used in embodiments according to the present disclosure include, but are not limited to, those obtained by and described in databases hosted by the National Stem Cell Bank (NSCB), 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), UC01 (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.

[0153] 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 change the fate of the original cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0154] 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 the target host, 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 a similar methodology. 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.

[0155] 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 the cell 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, electric pulses, or mild chemical fixation (e.g., 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.

[0156] Some embodiments described herein 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.

[0157] As used herein, "pharmaceutically acceptable" has its plain and ordinary meaning as understood in light of the specification and refers to a carrier, excipient, and / or stabilizer that is non-toxic or has an acceptable level of toxicity to cells or mammals exposed to the cells or mammals at the dosages and concentrations employed. As used herein, "pharmaceutically acceptable," "diluent," "excipient," and / or "carrier" has its plain and ordinary meaning as understood in light of the specification and is 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 a regulatory agency of the federal government, state government, or other regulatory body, 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 and sorbitol; salt formation inhibitors such as sodium; non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The compositions can also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, as desired. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. The formulation is typically suited to the method of administration.

[0158] Cryoprotectants are cell composition additives 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 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 about less than 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.

[0159] 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 media 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 less than 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.

[0160] The term "pharmaceutically acceptable salts," as understood in light of this specification, has its plain and ordinary meaning 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; and trihydroxymethylaminoethane.

[0161] Appropriate formulations vary depending on the selected route of administration. Techniques for the formulation and administration of 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, 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.

[0162] As used herein, "carrier" has its plain and ordinary meaning as understood in light of this 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 bodily organs.

[0163] As used herein, the term "diluent" has its plain and ordinary meaning as understood in light of the 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.

[0164] 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 completely or partially excluded.

[0165] Differentiation of PSCs into mesoderm During embryonic development, the mesoderm is one of the three primary germ layers and gives rise to a wide range of tissues, including muscle, connective tissue, bone, cartilage, skin, endothelium, mesenchyme, and blood cells. 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. Lateral plate mesoderm is further subdivided into the somatic mesoderm and visceral mesoderm layers. Visceral mesoderm develops closely with endoderm and gives rise to many downstream tissue types, such as blood vessels, cardiac muscle, and connective tissue and muscle of the gastrointestinal system. As disclosed herein, the retinoic acid signaling pathway is critical for the differentiation of lateral plate mesoderm into visceral mesoderm.

[0166] 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.

[0167] 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 differentiated cell type, with molecules (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 (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.

[0168] In some embodiments, the signal transduction pathway may include, but is not limited to, the Wnt signal transduction pathway, the Wnt / APC signal transduction pathway, the FGF signal transduction pathway, the TGF-β signal transduction pathway, the BMP signal transduction pathway, the Notch signal transduction pathway, the Hedgehog signal transduction pathway, the LKB signal transduction pathway, the PI3K signal transduction pathway, the retinoic acid signal transduction pathway, the ascorbic acid signal transduction pathway, or the Par polarity signal transduction pathway, or any combination thereof. It will be understood by those skilled in the art that differentiation can be promoted in accordance with the present disclosure by altering the concentration, expression, or function of any one of the signal transduction pathways disclosed herein. In some embodiments, cellular components associated with the signal transduction pathway, such as natural inhibitors, antagonists, activators, or agonists of the pathway, can be used to inhibit or activate the signal transduction pathway. In some embodiments, siRNA and / or shRNA targeting cellular components associated with the signal transduction pathway are used to inhibit or activate these pathways.

[0169] In some embodiments, pluripotent stem cells, lateral plate mesoderm cells, 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, BML284, IQ-1, WAY262611, 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, CHIR98014, AZD2858, BIO, AR-A014418, SB216763, SB415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD8, or TWS119, or any combination thereof. In some embodiments, the Wnt signaling pathway inhibitor comprises C59, PNU74654, KY-02111, PRI-724, FH-535, DIF-1, or XAV939, or any combination thereof. In some embodiments, the cells are not 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.

[0170] 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 proliferation and differentiation processes of a wide variety of cells and tissues. In humans, 22 members of the FGF family have been identified, all of which are structurally related signaling molecules. Members FGF1–FGF10 all bind to 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.

[0171] In some embodiments, pluripotent stem cells, lateral plate mesoderm cells, 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.

[0172] In some embodiments, pluripotent stem cells, lateral plate mesoderm cells, visceral mesoderm cells, or any differentiated cells thereof are contacted with a TGF-β signaling pathway activator or a TGF-β signaling pathway inhibitor. In some embodiments, the TGF-β family includes bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-Müllerian duct hormone, activin, and nodal pathways. In some embodiments, the TGF-β 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-β signaling pathway inhibitor includes A8301, RepSox, LY365947, SB431542, or any combination thereof. In some embodiments, the cells are not treated with a TGF-β signaling pathway activator or a TGF-β signaling pathway inhibitor. The TGF-β signaling pathway activators or TGF-β 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.

[0173] In some embodiments, pluripotent stem cells, lateral plate mesoderm cells, 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.

[0174] In some embodiments, pluripotent stem cells, lateral plate mesoderm cells, 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.

[0175] In some embodiments, pluripotent stem cells, lateral plate mesoderm cells, 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.

[0176] In some embodiments, pluripotent stem cells, lateral plate mesoderm cells, 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, or 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.

[0177] In some embodiments, pluripotent stem cells, lateral plate mesoderm cells, 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 are not 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.

[0178] In some embodiments, pluripotent stem cells, lateral plate mesoderm cells, 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.

[0179] In some embodiments, cells are contacted with either a small molecule compound, a signal transduction pathway activator, a signal transduction pathway inhibitor, or a growth factor for a time that is, is about, is at least about, is at most ...

[0180] In some embodiments, cells (e.g., pluripotent stem cells, lateral plate mesoderm cells, visceral mesoderm cells, or any differentiated cells thereof) are treated with a small molecule compound, signal transduction pathway activator, signal transduction pathway inhibitor, or growth factor at a concentration of 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, or , 2000ng / mL, 5000ng / mL, 7000ng / mL, 10000ng / mL, or 15000ng / mL, or a concentration that is about, at least about, less than, or about less than, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 10ng / mL to 15000ng / mL, 100ng / mL to 5000ng / mL, 500ng / mL to 2000ng / mL, 10ng / mL to 2000ng / mL, or 1000ng / mL to 15000ng / mL. In some embodiments, cells (e.g., pluripotent stem cells, lateral plate mesoderm cells, visceral mesoderm cells, or differentiated cells of any thereof) are contacted in culture with either a small molecule compound, signaling pathway activator, signaling pathway inhibitor, or growth factor such that the concentration of either the small molecule compound, signaling pathway activator, signaling pathway inhibitor, or growth factor 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, 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, more than one small molecule compound, activator, inhibitor, or growth factor is added. In these cases, the concentrations of the multiple small molecule compounds, activators, inhibitors, or growth factors may vary.

[0181] In some embodiments, the cells (pluripotent stem 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 contains fetal bovine serum (FBS). In some embodiments, the growth medium contains 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.

[0182] 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 about less than 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 aforementioned MOIs, e.g., 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.

[0183] 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, protein, polypeptide, nucleic acid, sugar, 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, PSCs are expanded in cell culture medium containing a ROCK inhibitor (e.g., Y-27632).

[0184] 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.

[0185] In some embodiments, pluripotent stem cells are first differentiated into intermediate primitive streak cells. In some embodiments, to differentiate PSCs into intermediate primitive streak cells, the pluripotent stem cells are contacted with a TGF-β 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-β signaling pathway activator is selected from the group consisting of TGF-β1, TGF-β2, TGF-β3, 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.

[0186] In some embodiments, the PSCs are contacted with a TGF-β signaling pathway activator. In some embodiments, the TGF-β signaling pathway activator is or comprises activin A. In some embodiments, PSCs are contacted with a TGF-β signaling pathway activator (e.g., activin A) at a concentration that is, about, at least about, less than, or about less than 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-β signaling pathway activator (e.g., activin A) at a concentration of, about, at least about, less than, or about less than 30 ng / mL.

[0187] In some embodiments, PSCs are contacted with a Wnt signaling pathway activator. In some embodiments, the Wnt signaling pathway activator is or comprises CHIR99021. In some embodiments, PSCs are contacted with a Wnt signaling pathway activator (e.g., CHIR99021) at a concentration that is, about, at least, at least about, less than, or about less than 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 aforementioned concentrations. 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 6 μM.

[0188] 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 of, about, at least, at least about, less than, or about less than 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. In some embodiments, PSCs are contacted with an FGF signaling pathway activator (e.g., FGF2) at a concentration of, about, at least, at least about, less than, or about less than 20 ng / mL.

[0189] In some embodiments, PSCs are contacted with a BMP signaling pathway activator. In some embodiments, the BMP signaling pathway activator is or includes BMP4. In some embodiments, PSCs are 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 less than 20, 21, 22, 23, 24, 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 aforementioned concentrations. 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.

[0190] 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 activator (e.g., PIK90) at a concentration of, about, at least, 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 aforementioned concentrations. In some embodiments, the PSCs are contacted with a PI3K signaling pathway inhibitor (e.g., PIK90) at a concentration of, about, at least, at least about, less than, or about less than 100 nM.

[0191] In some embodiments, PSCs are contacted with a TGF-β 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 intermediate primitive streak cells. In some embodiments, the PSCs are contacted for, about, at least, at least about, less than, 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 aforementioned times. In some embodiments, the PSCs are contacted for, about, at least, at least about, less than, or about 24 hours.

[0192] In some embodiments, any of the methods disclosed herein or other methods known in the art can be used to differentiate intermediate primitive streak cells into lateral plate mesoderm cells. In some embodiments, the intermediate primitive streak cells are differentiated from pluripotent stem cells. In some embodiments, to differentiate the intermediate primitive streak cells into lateral plate mesoderm cells, the intermediate primitive streak cells are contacted with a TGF-β signaling pathway inhibitor, a Wnt signaling pathway inhibitor, or a BMP signaling pathway activator, or any combination thereof. In some embodiments, the TGF-β signaling pathway inhibitor is selected from the group consisting of A8301, 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, to differentiate the intermediate primitive streak cells into lateral plate mesoderm cells, the intermediate primitive streak cells are contacted with A8301, C59, BMP4, or any combination thereof, including all three.

[0193] In some embodiments, the intermediate primitive streak cells are contacted with a TGF-β signaling pathway inhibitor. In some embodiments, the TGF-β signaling pathway inhibitor is or includes A8301. In some embodiments, the intermediate primitive streak cells are contacted with a TGF-β signaling pathway inhibitor (e.g., A8301) 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 at a concentration of about, at least, at least about, less than, or about less than 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 aforementioned concentrations. In some embodiments, intermediate primitive streak cells are contacted with a TGF-β signaling pathway inhibitor (e.g., A8301) at a concentration that is, about, at least about, less than, or about 1 μM.

[0194] In some embodiments, intermediate primitive streak cells are contacted with a Wnt signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway inhibitor is or comprises C59. In some embodiments, intermediate primitive streak cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) 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 at a concentration of about, at least, at least about, less than, or less than, or at any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, intermediate primitive 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.

[0195] In some embodiments, intermediate primitive streak cells are contacted with a BMP signaling pathway inhibitor. In some embodiments, the BMP signaling pathway activator is BMP4 or includes BMP4. In some embodiments, intermediate primitive streak cells are contacted with a BMP signaling pathway activator (e.g., BMP4) 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 at a concentration of about, at least, at least about, less than, or about less than, or at any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, intermediate primitive streak cells are contacted with a BMP signaling pathway activator (e.g., BMP4) at a concentration of, about, at least about, less than, or about 30 ng / mL.

[0196] In some embodiments, intermediate primitive streak cells are contacted with a TGF-β signaling pathway inhibitor, a Wnt signaling pathway inhibitor, and a BMP signaling pathway activator for a time sufficient to differentiate the intermediate primitive streak cells into 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 time within a range defined by any two of the foregoing times. In some embodiments, the intermediate primitive streak cells are contacted for, about, at least, at least about, less than, or about less than 24 hours.

[0197] In some embodiments, lateral plate mesoderm cells are produced from pluripotent stem cells according to the methods 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, the entire contents of which are expressly incorporated by reference herein, for the purpose of differentiating lateral plate mesoderm cells.

[0198] Differentiation into visceral mesoderm Disclosed herein are methods for producing visceral mesoderm cells from lateral plate mesoderm cells. In some embodiments, the lateral plate mesoderm cells are produced according to any one of the methods disclosed herein or other methods known in the art. The method for producing visceral mesoderm cells comprises contacting lateral plate mesoderm cells with a TGF-β 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, including at least one of each. In some embodiments, the lateral plate mesoderm cells are contacted with a TGF-β 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-β signaling pathway inhibitor is selected from the group consisting of A8301, 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-β signaling pathway inhibitor is A8301. 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, lateral plate mesoderm cells are contacted with A8301, BMP4, C59, FGF2, and RA. In some embodiments, lateral plate mesoderm cells are contacted with factors described herein, e.g., A8301, BMP4, C59, FGF2, and RA, for a period of time sufficient to differentiate the lateral plate mesoderm cells into visceral mesoderm. In some embodiments, the lateral plate mesoderm cells are 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, 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, 20, 21, 2 Contacting is for, about, at least about, less than, or about 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours, or within a range defined by any two of the foregoing times, e.g., 1 to 72 hours, 12 to 36 hours, 1 to 48 hours, or 24 to 72 hours. In some embodiments, the lateral plate mesoderm cells are contacted for, about, at least, at least about, less than, 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, e.g., 36-60 hours, 40-54 hours, 36-48 hours, or 48-60 hours.In some embodiments, the lateral plate mesoderm cells are contacted for a time that is, about, at least, at least about, less than, or about less than 48 hours.

[0199] In some embodiments, lateral plate mesoderm cells are contacted with a TGF-β signaling pathway inhibitor. In some embodiments, the TGF-β signaling pathway inhibitor is or includes A8301. In some embodiments, lateral plate mesoderm cells are contacted with a TGF-β signaling pathway inhibitor (e.g., A8301) 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, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, lateral plate mesoderm cells are contacted with a TGF-β signaling pathway inhibitor (e.g., A8301) at a concentration that is, about, at least about, less than, 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, e.g., 0.1-2 μM, 0.5-1.5 μM, 0.1-1 μM, or 1-2 μM. In some embodiments, lateral plate mesoderm cells are contacted with a TGF-β signaling pathway inhibitor (e.g., A8301) at a concentration that is, about, at least about, less than, or about less than 1 μM.

[0200] In some embodiments, lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway inhibitor is or includes C59. In some embodiments, 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 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, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration of, about, at least, at least about, less than, 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, e.g., 0.1-2 μM, 0.5-1.5 μM, 0.1-1 μM, or 1-2 μM. In some embodiments, lateral plate mesoderm cells are contacted with a Wnt signaling pathway inhibitor (e.g., C59) at a concentration of, about, at least, at least about, less than, or about 1 μM.

[0201] In some embodiments, 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, visceral 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 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, 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 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, 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.

[0202] In some embodiments, 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, visceral 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 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, 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 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, 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.

[0203] In some embodiments, 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, 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 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, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, lateral plate mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration of, about, at least, at least about, or 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 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, lateral plate mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration of, about, at least, at least about, or less than 2 μM.

[0204] In some embodiments, lateral plate mesoderm cells are contacted with a TGF-β 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, lateral plate mesoderm cells are contacted with a TGF-β 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, lateral plate mesoderm cells are contacted with A8301 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, and RA at a concentration of 0.01-20 μM. In some embodiments, lateral plate mesoderm cells are contacted with A8301 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, and RA at a concentration of 1-3 μM. In some embodiments, lateral plate mesoderm cells are contacted with A8301 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.

[0205] In some embodiments, visceral mesoderm cells 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 exhibit decreased expression of NKX2-5, ISL1, or TBX2, or any combination thereof, compared to cardiac mesoderm cells. In some embodiments, visceral mesoderm cells exhibit decreased expression of PAX3, PRRX1, or both, compared to intermediate primitive streak cells. In some embodiments, visceral mesoderm cells exhibit decreased expression of CD31 compared to cardiac mesoderm cells.

[0206] In any of the embodiments provided herein, the visceral mesoderm cells 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 a disease or is at risk of developing a disease. In some embodiments, the visceral mesoderm cells are derived from PSCs derived from the subject.

[0207] Differentiation into visceral mesoderm cell types As disclosed herein, visceral mesoderm cells produced by any of the methods herein can be further differentiated into visceral mesoderm subtypes. In some embodiments, the visceral mesoderm subtypes comprise septum transversum cells, fibroblasts, respiratory mesenchymal cells, or esophageal / gastric mesenchymal cells, or any combination thereof. In some embodiments, the septum transversum cells comprise hepatic septum transversum cells. In some embodiments, the fibroblasts comprise hepatic fibroblasts. An embodiment of differentiation of visceral mesoderm cells into visceral mesoderm subtypes is disclosed in Figure 7A.

[0208] Production of transverse septum cells In some embodiments, the method includes contacting visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, or both. In some embodiments, the visceral mesoderm cells are visceral mesoderm cells produced by any of the methods described herein. In some embodiments, the contacting causes the visceral mesoderm cells to differentiate into transverse septum cells. In some embodiments, the 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 visceral mesoderm cells are contacted with RA, BMP4, or both.

[0209] In some embodiments, visceral mesoderm cells are contacted with a retinoic acid signaling pathway activator (e.g., RA) at, 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, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM, and , 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, visceral mesoderm cells are contacted with a retinoic acid signaling pathway activator (e.g., RA) at, about, at least, at least about, or less than 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, and are contacted with a BMP signaling pathway activator (e.g., BMP4) at, about, at least, at least about, or less than about 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.In some embodiments, visceral mesoderm cells are contacted with a retinoic acid signaling pathway activator (e.g., RA) at, about, at least, at least about, or less than, or about 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, and with a BMP signaling pathway activator (e.g., BMP4) at, about, at least, at least about, or 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. In some embodiments, 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 40 ng / mL.

[0210] In some embodiments, 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, 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, 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, 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, visceral mesoderm cells are contacted with RA at a concentration of 2 μM and BMP4 at a concentration of 40 ng / mL.

[0211] In some embodiments, a retinoic acid signaling pathway activator (e.g., RA), or a BMP signaling pathway activator (e.g., BMP4), or both, are contacted at a concentration described herein for a period of time sufficient to differentiate the visceral mesoderm cells into septum transversum cells. In some embodiments, the visceral mesoderm cells are contacted with factors described herein, e.g., RA and BMP4, for a period of time sufficient to differentiate the visceral mesoderm cells into septum transversum cells. In some embodiments, the contacting 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, In some embodiments, the contact is for, about, at least, at least about, less than, or about 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 108 hours, or any period within a range defined by any two of the foregoing times. In some embodiments, the contact is for, about, at least, 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 within a range defined by any two of the foregoing times. 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.

[0212] 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, the transverse septum cells represent 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% of the total cells separated from visceral mesoderm cells, or a percentage of the total cells differentiated from visceral mesoderm cells that is about that, at least about that, less than that, or about less than that, or any percentage within a range defined by any two of the foregoing percentages, e.g., 60%-100%, 70%-90%, or 75%-85%.

[0213] Fibroblast production In some embodiments, the method includes contacting visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, a Wnt signaling pathway activator, or any combination thereof. In some embodiments, the visceral mesoderm cells are visceral mesoderm cells produced by any of the methods described herein. In some embodiments, the contacting causes the visceral mesoderm cells to differentiate into fibroblasts. In some embodiments, the 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 visceral mesoderm cells are contacted with RA, BMP4, CHIR99021, or any combination thereof, including all three.

[0214] In some embodiments, the visceral mesoderm cells are at, about, at least about, less than, 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, e.g., 0. and the antibody is contacted with an RA signaling pathway activator (e.g., RA) at a concentration of 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM, and is at, about, at least about, or less than 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, and is contacted with a Wnt signaling pathway activator (e.g., CHIR99021) at, about, at least, 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, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM.In some embodiments, the visceral mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at, about, at least, at least about, or 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, 2.8, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations, and are at, about, at least, at least about, or less than 10, 20, 30, 40, 50, 60, 70, or 80 ng / mL. and contacted with a BMP signaling pathway activator (e.g., BMP4) at or about less than, or any concentration within a range defined by any two of the foregoing concentrations, and contacted with a Wnt signaling pathway activator (e.g., CHIR99021) at, about, at least, at least about, or less than, or about less than 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. In some embodiments, visceral mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at a concentration of, or about, at least, at least about, or less than, or about 2 μM; with a BMP signaling pathway activator (e.g., BMP4) at a concentration of, or about, at least, at least about, or less than, or about 40 ng / mL; and with a Wnt signaling pathway activator (e.g., CHIR99021) at a concentration of, or about, at least, at least about, or less than, or about 6 μM.

[0215] In some embodiments, 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, 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, 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, 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, visceral mesoderm cells are contacted with RA at a concentration of 2 μM, BMP4 at a concentration of 40 ng / mL, and CHIR99021 at a concentration of 6 μM.

[0216] In some embodiments, 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) are contacted at concentrations described herein for a period of time sufficient to differentiate the visceral mesoderm cells into fibroblasts. In some embodiments, the visceral mesoderm cells are contacted with factors described herein, e.g., RA, BMP4, and CHIR99021, for a period of time sufficient to differentiate the visceral mesoderm cells into fibroblasts. In some embodiments, the contacting 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, In some embodiments, the contact is for, about, at least, at least about, less than, or about 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 108 hours, or any period within a range defined by any two of the foregoing times. In some embodiments, the contact is for, about, at least, 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 within a range defined by any two of the foregoing times. 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.

[0217] In some embodiments, the fibroblasts exhibit increased expression of MSX1, MSX2, or HAND1, or any combination thereof, compared to visceral mesoderm cells or transverse septum cells, or both. In some embodiments, the fibroblasts exhibit decreased expression of WT1, TBX18, LHX2, or UPK1B, or any combination thereof, compared to transverse septum cells. In some embodiments, the fibroblasts exhibit decreased expression of NKX6.1, HOXA5, or LHX2, or any combination thereof, compared to respiratory mesenchymal cells. In some embodiments, the fibroblasts exhibit decreased expression of NKX3.2, MSC, BARX1, WNT4, or HOXA5, or any combination thereof, compared to esophageal / gastric mesenchymal cells.

[0218] Production of respiratory mesenchymal cells In some embodiments, the method includes contacting visceral mesoderm cells with an RA signaling pathway activator, a BMP signaling pathway activator, an HH signaling pathway activator, or a Wnt signaling pathway activator, or any combination thereof. In some embodiments, the visceral mesoderm cells are visceral mesoderm cells produced by any of the methods described herein. In some embodiments, this contacting differentiates the visceral mesoderm cells into respiratory mesenchymal cells. In some embodiments, the visceral mesoderm cells are contacted with an RA signaling pathway activator, a BMP signaling pathway activator, an HH signaling pathway activator, and a Wnt signaling pathway activator. In some embodiments, the method may further include contacting the visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and an HH signaling pathway activator before contacting the visceral mesoderm cells with an RA signaling pathway activator, a BMP signaling pathway activator, an HH signaling pathway activator, and a Wnt signaling pathway activator. In some embodiments, this two-step process enhances the differentiation of visceral mesoderm cells into respiratory mesenchymal cells.

[0219] 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 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 HH signaling pathway activator is selected from the group consisting of SHH, IHH, DHH, PMA, GSA10, and SAG. 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 RA signaling pathway activator is RA.In some embodiments, the BMP signaling pathway activator is BMP4. In some embodiments, the HH signaling pathway activator is PMA. In some embodiments, the Wnt signaling pathway activator is CHIR99021. In some embodiments, the visceral mesoderm cells are contacted with RA, BMP4, PMA, CHIR99021, or any combination thereof, including all four.

[0220] In some embodiments, the visceral mesoderm cells are irradiated with RA signaling at, 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, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. and contacting the pathway activator (e.g., RA) with a concentration of, about, at least about, less than, or 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 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, and contacted with a BMP signaling pathway activator (e.g., BMP4), at, about, at least, at least about, or less than, 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, e.g., 0.01-20 μM, 0.01-10 μM , 1-15 μM, or 10-20 μM, and optionally, at, about, at least about, less than, 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, e.g., 0.01-20 μM, 0.The cells are contacted with a Wnt signaling pathway activator (e.g., CHIR99021) at 1-10 μM, 1-15 μM, or 10-20 μM.

[0221] In some embodiments, the visceral mesoderm cells are treated with an RA signaling pathway activator at, about, 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, 2.8, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations. and a BMP signaling pathway activator (e.g., BMP4) at, about, at least about, less than, or about 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, and , 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM, or at least about, less than, or about less than, or any concentration within a range defined by any two of the foregoing concentrations, and optionally, at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 μM ... , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or at about, at least about, less than, or about less than, or any concentration within a range defined by any two of the foregoing concentrations, such as 0.1-2 μM, 0.5-1.5 μM, 0.1-1 μM, or 1-2 μM, is contacted with a Wnt signaling pathway activator (e.g., CHIR99021).

[0222] In some embodiments, 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 40 ng / mL; an HH signaling pathway activator (e.g., PMA) at a concentration that is, about, at least, at least about, or less than about 2 μM; and optionally, a Wnt signaling pathway activator (e.g., CHIR99021) at a concentration that is, about, at least, at least about, or less than about 1 μM.

[0223] In some embodiments, 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, an HH signaling pathway activator at a concentration of 0.01-20 μM, and optionally a Wnt signaling pathway activator at a concentration of 0.01-20 μM. In some embodiments, 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, an HH signaling pathway activator at a concentration of 1-3 μM, and optionally a Wnt signaling pathway activator at a concentration of 0.1-2 μM. In some embodiments, visceral mesoderm cells are contacted with RA at a concentration of 0.01-20 μM, BMP4 at a concentration of 1-100 ng / mL, PMA at a concentration of 0.01-20 μM, and optionally CHIR99021 at a concentration of 0.01-20 μM. In some embodiments, visceral mesoderm cells are contacted with RA at a concentration of 1-3 μM, BMP4 at a concentration of 10-80 ng / mL, PMA at a concentration of 1-3 μM, and optionally CHIR99021 at a concentration of 0.1-2 μM. In some embodiments, visceral mesoderm cells are contacted with RA at a concentration of 2 μM, BMP4 at a concentration of 40 ng / mL, PMA at a concentration of 2 μM, and optionally CHIR99021 at a concentration of 1 μM.

[0224] In some embodiments, visceral mesoderm cells are differentiated into respiratory mesenchymal cells in a one-step process. In these embodiments, the method includes contacting the visceral mesoderm cells with an RA signaling pathway activator (e.g., RA), a BMP signaling pathway activator (e.g., BMP4), an HH signaling pathway activator (e.g., PMA), and a Wnt signaling pathway activator (e.g., CHIR99021). In some embodiments, the RA signaling pathway activator, BMP signaling pathway activator, and Wnt signaling pathway activator of the one-step process are contacted at concentrations described herein for a period of time sufficient to differentiate the visceral mesoderm cells into respiratory mesenchymal cells. In some embodiments, the visceral mesoderm cells are contacted with factors described herein, such as RA, BMP4, PMA, and CHIR99021, for a period of time sufficient to differentiate the visceral mesoderm cells into respiratory mesenchymal cells. In some embodiments, the contacting 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, In some embodiments, the contact is for, about, at least, at least about, less than, or about 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 108 hours, or any period within a range defined by any two of the foregoing times. In some embodiments, the contact is for, about, at least, 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 within a range defined by any two of the foregoing times.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.

[0225] In some embodiments, visceral mesoderm cells differentiate into respiratory mesenchymal cells in a two-step process. In some embodiments, the method comprises a first step of contacting visceral mesoderm cells with an RA signaling pathway activator, a BMP signaling pathway activator, and an HH signaling pathway activator, before a second step of contacting the visceral mesoderm cells with an RA signaling pathway activator, a BMP signaling pathway activator, an HH signaling pathway activator, and a Wnt signaling pathway activator (e.g., CHIR99021). In some embodiments, the RA signaling pathway activator (e.g., RA), BMP signaling pathway activator (e.g., BMP4), and HH signaling pathway activator (e.g., PMA) in the first and second steps are the same. In some embodiments, the RA signaling pathway activator, BMP signaling pathway activator, and HH signaling pathway activator in the first and second steps are different. In some embodiments, the RA signaling pathway activator, BMP signaling pathway activator, and HH signaling pathway activator of the first step of the two-step process and the RA signaling pathway activator, BMP signaling pathway activator, HH signaling pathway activator, and Wnt signaling pathway activator of the second step of the two-step process are contacted at concentrations described herein for a period of time sufficient to differentiate visceral mesoderm cells into respiratory mesenchymal cells. In some embodiments, the visceral mesoderm cells are contacted with factors described herein, such as RA, BMP4, PMA, and CHIR99021, for a period of time sufficient to differentiate visceral mesoderm cells into respiratory mesenchymal cells.In some embodiments, the RA signaling pathway activator (e.g., RA), the BMP signaling pathway activator (e.g., BMP4), and the HH signaling pathway activator (e.g., PMA) in the first step are 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, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 The contact may be for a period of time that is, about, at least about, less than, or about less than 0, 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, or 84 hours, or any period of time within a range defined by any two of the foregoing periods. In some embodiments, the RA signaling pathway activator (e.g., RA), BMP signaling pathway activator (e.g., BMP4), and HH signaling pathway activator (e.g., PMA) in the first step are contacted for a period of, 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 period within a range defined by any two of the aforementioned times. In some embodiments, the RA signaling pathway activator (e.g., RA), BMP signaling pathway activator (e.g., BMP4), and HH signaling pathway activator (e.g., PMA) in the first step are contacted for a period of, about, at least, at least about, less than, or about less than 48 hours.In some embodiments, the RA signaling pathway activator (e.g., RA), BMP signaling pathway activator (e.g., BMP4), HH signaling pathway activator (e.g., PMA), and Wnt signaling pathway activator (e.g., CHIR99021) in the second step are contacted for a period of time that is, is about, is at least, is at least about, is less than, or is about less than 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, or 48 hours, or any period of time within a range defined by any two of the foregoing times. In some embodiments, the RA signaling pathway activator (e.g., RA), BMP signaling pathway activator (e.g., BMP4), HH signaling pathway activator (e.g., PMA), and Wnt signaling pathway activator (e.g., CHIR99021) in the second step are contacted for a period of, 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 period within a range defined by any two of the foregoing times. In some embodiments, the RA signaling pathway activator (e.g., RA), BMP signaling pathway activator (e.g., BMP4), HH signaling pathway activator (e.g., PMA), and Wnt signaling pathway activator (e.g., CHIR99021) in the second step are contacted for a period of time that is, about, at least, at least about, less than, or about less than 24 hours.

[0226] In some embodiments, respiratory mesenchymal cells exhibit increased expression of NKX6-1, TBX5, HOXA1, HOXA5, FOXF1, LHX2, or WNT2, or any combination thereof, compared to cardiac endoderm cells, visceral mesoderm cells, or esophageal / gastric mesenchymal cells, or any combination thereof. In some embodiments, respiratory mesenchymal cells exhibit decreased expression of WNT2, WT1, TBX18, LHX2, or UPK1B, or any combination thereof, compared to transverse septum cells. In some embodiments, respiratory mesenchymal cells exhibit decreased expression of WNT2, MSX1, or MSX2, or any combination thereof, compared to fibroblasts.

[0227] Esophageal / gastric mesenchymal cell production In some embodiments, the method comprises contacting visceral mesoderm cells with an RA signaling pathway activator, an HH signaling pathway activator, or a BMP signaling pathway activator, or any combination thereof. In some embodiments, the visceral mesoderm cells are visceral mesoderm cells produced by any of the methods described herein. In some embodiments, this contacting differentiates the visceral mesoderm cells into esophageal / gastric mesenchymal cells. In some embodiments, the visceral mesoderm cells are contacted with an RA signaling pathway activator, an HH signaling pathway activator, and a BMP signaling pathway inhibitor. In some embodiments, the method may further comprise contacting the visceral mesoderm cells with a retinoic acid signaling pathway activator and an HH signaling pathway activator before contacting the visceral mesoderm cells with a retinoic acid signaling pathway activator, an HH signaling pathway activator, and a BMP signaling pathway activator. In some embodiments, this two-step process enhances the differentiation of visceral mesoderm cells into esophageal / gastric mesenchymal cells.

[0228] 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 BMP signaling pathway inhibitor is selected from the group consisting of noggin, RepSox, LY364947, LDN193189, and SB431542. In some embodiments, the RA signaling pathway activator is RA. In some embodiments, the HH signaling pathway activator is PMA. In some embodiments, the BMP signaling pathway inhibitor is noggin. In some embodiments, the visceral mesoderm cells are contacted with RA, PMA, noggin, or any combination thereof, including all three.

[0229] In some embodiments, the visceral mesoderm cells are at, about, at least about, less than, 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, e.g., 0.01-20 μM, 0.01-10 μM, or 20 μM. or 1-15 μM, or 10-20 μM, and is at, about, at least about, or less than, or about 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 is defined by any two of the foregoing concentrations. and optionally contacting the HH signaling pathway activator (e.g., PMA) at any concentration within the range, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM, and optionally contacting the HH signaling pathway activator (e.g., PMA) at 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, or 250 ng / mL, 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., 1-250 ng / mL, 5-150 ng / mL, 10-100 ng / mL, 1-150 ng / mL, or 50-250 ng / mL.

[0230] In some embodiments, visceral mesoderm cells are contacted with an RA signaling pathway activator (e.g., RA) at, or 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, 2.8, 2.9, or 3 μM, or any concentration within a range defined by any two of the foregoing concentrations; The subject is contacted with an HH signaling pathway activator (e.g., PMA) at, about, at least, at least about, or less than, or about 3 μM, or any concentration within a range defined by any two of the aforementioned concentrations, and optionally with a BMP signaling pathway inhibitor (e.g., Noggin) at, about, at least, at least about, or less than, or about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations.

[0231] In some embodiments, 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; an HH signaling pathway activator (e.g., PMA) at a concentration that is, about, at least, at least about, or less than about 2 μM; and optionally, a BMP signaling pathway inhibitor (e.g., noggin) at a concentration that is, about, at least, at least about, or less than about 100 ng / mL.

[0232] In some embodiments, visceral mesoderm cells are contacted with an RA signaling pathway activator at a concentration of 0.01-20 μM, an HH signaling pathway activator at a concentration of 0.01-20 μM, and optionally a BMP signaling pathway inhibitor at a concentration of 1-250 ng / mL. In some embodiments, visceral mesoderm cells are contacted with an RA signaling pathway activator at a concentration of 1-3 μM, an HH signaling pathway activator at a concentration of 1-3 μM, and optionally a BMP signaling pathway inhibitor at a concentration of 50-150 ng / mL. In some embodiments, visceral mesoderm cells are contacted with RA at a concentration of 0.01-20 μM, PMA at a concentration of 0.01-20 μM, and optionally Noggin at a concentration of 1-250 ng / mL. In some embodiments, visceral mesoderm cells are contacted with RA at a concentration of 1-3 μM, PMA at a concentration of 1-3 μM, and optionally Noggin at a concentration of 50-150 ng / mL. In some embodiments, visceral mesoderm cells are contacted with RA at a concentration of 2 μM, PMA at a concentration of 2 μM, and optionally Noggin at a concentration of 100 ng / mL.

[0233] In some embodiments, visceral mesoderm cells are differentiated into esophageal / gastric mesenchymal cells in a one-step process. In these embodiments, the method includes contacting the visceral mesoderm cells with an RA signaling pathway activator (e.g., RA), an HH signaling pathway activator (e.g., PMA), and a BMP signaling pathway inhibitor (e.g., Noggin). In some embodiments, the RA signaling pathway activator, HH signaling pathway activator, and BMP signaling pathway inhibitor of the one-step process are contacted at concentrations described herein for a period of time sufficient to differentiate the visceral mesoderm cells into esophageal / gastric mesenchymal cells. In some embodiments, the visceral mesoderm cells are contacted with factors described herein, such as RA, PMA, and Noggin, for a period of time sufficient to differentiate the visceral mesoderm cells into esophageal / gastric mesenchymal cells. In some embodiments, the contacting 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, In some embodiments, the contact is for, about, at least, at least about, less than, or about 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 108 hours, or any period within a range defined by any two of the foregoing times. In some embodiments, the contact is for, about, at least, 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 within a range defined by any two of the foregoing times. 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.

[0234] In some embodiments, visceral mesoderm cells differentiate into esophageal / gastric mesenchymal cells in a two-step process. In some embodiments, the method includes a first step of contacting visceral mesoderm cells with an RA signaling pathway activator and an HH signaling pathway activator before a second step of contacting visceral mesoderm cells with an RA signaling pathway activator, an HH signaling pathway activator, and a BMP signaling pathway inhibitor (e.g., noggin). In some embodiments, the RA signaling pathway activator (e.g., RA) and the HH signaling pathway activator (e.g., PMA) in the first and second steps are the same. In some embodiments, the RA signaling pathway activator and the HH signaling pathway activator in the first and second steps are different. In some embodiments, the RA signaling pathway activator (e.g., RA) and HH signaling pathway activator (e.g., PMA) in the first step and the RA signaling pathway activator (e.g., RA), HH signaling pathway activator (e.g., PMA), and BMP signaling pathway inhibitor (e.g., Noggin) in the second step are contacted at concentrations described herein for a period of time sufficient to differentiate visceral mesoderm cells into respiratory mesenchymal cells. In some embodiments, the visceral mesoderm cells are contacted with factors described herein, such as RA, PMA, and Noggin, for a period of time sufficient to differentiate visceral mesoderm cells into esophageal / gastric mesenchymal cells.In some embodiments, the RA signaling pathway activator (e.g., RA) and the HH signaling pathway activator (e.g., PMA) in the first step are selected from the group consisting of 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, The contact may be for a period of, about, at least about, less than, or about less than 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, or 84 hours, or any period of time within a range defined by any two of the foregoing periods. In some embodiments, the RA signaling pathway activator (e.g., RA) and the HH signaling pathway activator (e.g., PMA) in the first step are contacted for a period of, 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 period within a range defined by any two of the aforementioned times. In some embodiments, the RA signaling pathway activator (e.g., RA) and the HH signaling pathway activator (e.g., PMA) in the first step are contacted for a period of, about, at least, at least about, less than, or about less than 48 hours.In some embodiments, the RA signaling pathway activator (e.g., RA), HH signaling pathway activator (e.g., PMA), and BMP signaling pathway inhibitor (e.g., noggin) in the second step are contacted for a period of time that is, is about, is at least, is at least about, is less than, or is about less than 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, or 48 hours, or any period of time within a range defined by any two of the foregoing times. In some embodiments, the RA signaling pathway activator (e.g., RA), HH signaling pathway activator (e.g., PMA), and BMP signaling pathway inhibitor (e.g., Noggin) in the second step are contacted for a period of, 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 period within a range defined by any two of the aforementioned periods. In some embodiments, the RA signaling pathway activator (e.g., RA), HH signaling pathway activator (e.g., PMA), and BMP signaling pathway inhibitor (e.g., Noggin) in the second step are contacted for a period of, about, at least, at least about, less than, or about less than 24 hours.

[0235] In some embodiments, the esophageal / gastric mesenchymal cells exhibit increased expression of MSC, BARX1, WNT4, HOXA1, FOXF1, or NKX3-2, or any combination thereof, compared to cardiac endoderm cells, visceral mesoderm cells, or respiratory mesenchymal cells, or any combination thereof. In some embodiments, the esophageal / gastric mesenchymal cells exhibit decreased expression of WNT2, TBX5, MSX1, MSX2, or LHX2, or any combination thereof, compared to visceral mesoderm cells, septum transversum cells, fibroblasts, or respiratory mesenchymal cells, or any combination thereof.

[0236] Factors for differentiation of visceral mesoderm In any of the embodiments provided herein, the visceral mesoderm cells are contacted with an RA signaling pathway activator. 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, or AM580. In some embodiments, the RA signaling pathway activator is or comprises RA. In some embodiments, visceral mesoderm cells are contacted with an RA signaling pathway activator at a concentration that is, about, at least about, less than, 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, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, visceral mesoderm cells are not contacted with an RA signaling pathway activator.

[0237] In any of the embodiments provided herein, the visceral mesoderm cells are contacted with a BMP signaling pathway activator. 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 BMP signaling pathway activator is or includes BMP4. In some embodiments, the visceral mesoderm cells are contacted with a BMP signaling pathway activator at a concentration that is, about, at least about, less than, or about less than 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, the visceral mesoderm cells are not contacted with a BMP signaling pathway activator.

[0238] In any of the embodiments provided herein, visceral mesoderm cells are contacted with a Wnt signaling pathway activator.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 Wnt signaling pathway activator is or comprises CHIR99021. In some embodiments, the visceral mesoderm cells are contacted with the Wnt signaling pathway activator 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, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, the visceral mesoderm cells are not contacted with the Wnt signaling pathway activator.

[0239] In any of the embodiments provided herein, visceral mesoderm cells are contacted with an HH signaling pathway activator. 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 HH signaling pathway activator is or comprises PMA. In some embodiments, the visceral mesoderm cells are contacted with the HH signaling pathway activator 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, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM. In some embodiments, the visceral mesoderm cells are not contacted with an HH signaling pathway activator.

[0240] In any of the embodiments provided herein, visceral mesoderm cells are contacted with a BMP signaling pathway inhibitor.In some embodiments, the BMP signaling pathway inhibitor is selected from the group consisting of Noggin, RepSox, LY364947, LDN193189, and SB431542.In some embodiments, the BMP signaling pathway inhibitor is Noggin or comprises Noggin. In some embodiments, the visceral mesoderm cells are contacted with a BMP signaling pathway inhibitor at a concentration that is, about, at least about, less than, or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 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 foregoing concentrations, e.g., 1-250 ng / mL, 5-150 ng / mL, 10-100 ng / mL, 1-150 ng / mL, or 50-250 ng / mL. In some embodiments, the visceral mesoderm cells are not contacted with a BMP signaling pathway activator.

[0241] In any of the embodiments provided herein, the visceral mesoderm cells are contacted with the one or more signaling pathway activators or signaling pathway inhibitors for, about, or at least about 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, or 48 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. The visceral mesoderm cells are allowed to differentiate into visceral mesoderm subtypes for a period of time that is at least about, less than, or about less than.

[0242] Also disclosed herein are any one of the visceral mesoderm cells produced by any one of the methods provided herein. Also disclosed herein are any one of the transverse septum cells produced by any one of the methods provided herein. Also disclosed herein are any one of the fibroblast cells produced by any one of the methods provided herein. Also disclosed herein are any one of the respiratory mesenchymal cells produced by any one of the methods provided herein. Also disclosed herein are any one of the esophageal / gastric mesenchymal cells produced by any one of the methods provided herein. [Example]

[0243] Some aspects of the embodiments discussed herein are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Those skilled in the art will recognize that many other embodiments are also within the scope of the disclosure, as described herein and in the claims.

[0244] Example 1. Single-cell transcriptome defines progenitor cell diversity in the developing foregut. To comprehensively define lineage diversification during foregut organogenesis, single-cell RNA sequencing (scRNA-seq) of the mouse embryonic foregut was performed at three time points spanning the time of early patterning and lineage induction: E8.5 (5–10 mesodermal segments [s]), E9.0 (12–15 s), and E9.5 (25–30 s) (Figure 1A–B). The foregut was microscopically dissected between the posterior pharynx and midgut, and tissue was pooled from 15–20 embryos at each time point. At E9.5, the anterior and posterior regions, containing the lung / esophagus and liver / pancreas primordia, respectively, were isolated. A total of 31,268 single-cell transcriptomes passed quality control criteria, with an average read depth of 3,178 transcripts / cell. Cells were clustered based on the expression of highly variable genes across the population and visualized using uniform manifold approximation projection (UMAP) and t-SNE (t-SNE) (Figure 1C, 1K). This identified 24 cell clusters that could be grouped into nine major cell lineages based on well-known marker genes: DE, SM, cardiac, other mesoderm (parietal and paraaxial), endodermis, blood, ectoderm, neural crest, and extraembryonic (Figure 1K). The DE cluster (4,448 cells) was characterized by co-expression of Foxa1 / 2, Cdh1, and / or Epcam, whereas the SM cluster (10,097 cells) was defined by co-expression of Foxf1 (Figure 1D), Vim, and / or Pdgfra, as well as negativity for cardiac and other mesoderm-specific transcripts.

[0245] To pinpoint lineage diversification in the DE and SM, we selected these cells in silico for further analysis. We identified 11 major DE clusters consisting of 26 stage-specific subclusters (12 clusters at E9.5, 8 clusters at E9.0, and 6 clusters at E8.5) and 13 major SM clusters consisting of 36 stage-specific subclusters (17 clusters at E9.5, 12 clusters at E9.0, and 7 clusters at E8.5) (Figure 1E-F, 1L-M). The clusters were annotated by comparing their discriminant genes with the expression patterns of over 160 genes published in the Mouse Genome Informatics (MGI) database. These data provide a comprehensive single-cell resolution view of early foregut organogenesis and can be searched on the World Wide Web at research.cchmc.org / ZornLab-singlecell.

[0246] Annotation identified all major DE organ lineages at E9.5, including a Tbx1+ pharynx, two Nkx2-1 / Foxa2+ respiratory clusters, two Sox2+ esophageal clusters, two Sox2 / Osr1+ stomach clusters, two Alb / Prox1 / Afp+ liver clusters (c1 hepatocytes and c10 early hepatocytes with higher Alb / HNF4a expression), a Sox17 / Pdx1+ hepatopancreatic duct, a Pdx1 / Mnx1+ pancreas, and a Cdx2+ duodenum (Figure 1E). Consistent with the dissection, no Nkx2-1+ / Hhex+ thyroid progenitors were detected. Similar to a recent scRNA-seq analysis of E8.75 intestinal epithelium, we also annotated half a dozen distinct DE precursor states between E8.5 and E9.0 based on the restricted expression of lineage-specifying transcription factors (TFs), including Otx2+ foregut, Sox2 / Sp5-rich dorsal foregut, Osr1 / Irx1-rich foregut, Hhex+ hepatic endoderm, Nkx2-3+ ventral DE adjacent to the heart, and a small population of Cdx2+ midgut cells ( Figure 1L ).

[0247] Example 2. Verification of novel mesenchymal subtypes At all stages, the diversity of SM cell types in the foregut was much more complex than previously recognized (Figure 1F, 1L). However, unlike the DE, SM populations were typically defined by combinations of multiple transcripts, not just one or two markers (Figure 2A-B). In situ hybridization and immunostaining of E9.5 foregut and embryo sections confirmed that combinations of co-expressed transcripts define distinct organ-specific SM subtypes (Figure 2C-Q). The 17 SM cell populations at E9.5 included five Tbx1 / Prrx1+ pharyngeal clusters, Isl1 / Mtus2+ cardiac outflow tract cells, Nkx6-1 / Gata4 / Wnt2+ respiratory cells, and Nkx6-1 / Sfrp2 / Wnt4+ esophageal mesenchyme (Figure 2B-J). We annotated three Barx1 / Hlx+ gastric mesenchyme populations (one likely ventral based on Gata4 expression) and one Hand1 / Hoxc8+ duodenal mesenchyme. No organ-specific mesenchyme was identified and was suspected to be in the gastric or duodenal clusters ( Figure 2P–Q ).

[0248] Unexpectedly, the liver bud contained five distinct mesenchymal populations. MGI data mining and in situ validation allowed the annotation of Alcam / Wnt2 / Gata4-enriched stm, Tbx5 / Wnt2 / Gata4 / Vsnl1+ sinusoids, Msx1 / Wnt2 / Hand1 / Col1a1+ fibroblast populations, and two Wt1 / Gata4 / Uroplakin+ mesothelial populations (Figure 2K-N, 2R). Interestingly, restricted expression of Hand1 and Hand2 in the posterior liver bud compared to the anterior liver bud (Figure 2R, panel b) and mutually exclusive expression of Wnt2 and Msx1 from Wt1 were observed (Figure 2R, panels e-f).

[0249] Example 3. Pseudo-temporal ordering of foregut cells Various organs form at precise locations along the anterior-posterior (AP) axis of the intestine. To assess whether this is reflected in the transcriptional profiles of single cells, we employed pseudotemporal analysis, which has been used to examine cell positional information within a continuous field of embryonic tissues. To this end, DE and SM cells were analyzed at each stage using diffusion maps, a dimensionality reduction method for reconstructing developmental differentiation pathways. The most anterior pharyngeal cluster was fixed as the root, and the pseudotemporal density distribution of each cluster was plotted based on the transition probability from the root cell to all other cells in the graph. Remarkably, this ordered both DE and SM cell populations according to their appropriate AP position in the embryo, indicating that the analysis represents an unbiased proxy for pseudospace (Figure 1G-J, 1L). The data also showed that at this point in development, cells within the embryonic intestinal tract exhibit a continuum of transcriptional signatures, with spatially adjacent cell types having more similar expression profiles than distant cell types. Indeed, E9.5 clusters from the anterior incision were located in the anterior half of the pseudotemporal continuum compared to posterior tissues, confirming the robustness of the computational ordering. Finally, we examined Hox genes known to be expressed collinearly along the AP axis and observed a progressive increase in posterior Hox paralog expression, particularly in more posterior clusters within the SM ( Figure 2S ).

[0250] Combining pseudospatial analysis, MGI curation, and in situ validation, we mapped each DE and SM population to its approximate location within the intestinal tract (Figures 1I-J, 1L), revealing that SM diversity mirrors DE lineages, indicating tightly coordinated development from the beginning of organogenesis.

[0251] Example 4. Transcription factor codes in foregut endoderm and mesenchyme DE organ lineages have historically been defined by overlapping expression domains of several transcription factors (TFs). While several regionally expressed TFs have been reported in the SM, single-cell RNA-seq data allow us to define a comprehensive combinatorial code of differentially expressed TFs that distinguish different SM and DE subtypes (Figure 2S). This revealed novel lineage-restricted markers, such as the homeodomain TF Nkx6-1. Well known for its expression in pancreatic endoderm (Figure 2P), Nkx6-1 was also specifically expressed in respiratory and esophageal mesoderm at E9.5 (Figure 2B-C, H-J). This TF code facilitates lineage tracing experiments and studies examining their role in mesenchymal differentiation.

[0252] Example 5. Synchronized endodermal and mesenchymal lineage differentiation pathways The complexity of DE and SE transcriptional cell states doubled in just 24 h between E8.5 and E9.5, reflecting the formation of more specialized cell types from progenitor cells. To examine the temporal dynamics of lineage diversification, we visualized single-cell data using SPRING (Figure 3A-B), an algorithm that represents k-nearest neighbors in a force-directed graph, to facilitate analysis of developmental differentiation pathways. Both DE and SM differentiation pathways progressed from a continuum of closely related cell states at E8.5 to transcriptionally distinct cell populations at E9.5 (Figure 3A-B, 3G), consistent with the transition from multipotent progenitors to organ-specific lineages. Importantly, the cell clusters defined by tSNE were well preserved in SPRING (Figure 3G), supporting the robustness of the clustering. One striking observation evident in the structure of the SPRING plot was the apparent coordination of SM and DE lineage diversification over 24 h.

[0253] To more clearly visualize the developmental differentiation pathways associated with lineage diversification, a consensus cell state tree using single-cell voting was generated, in which each cell at a later time point votes for the most likely parent at the previous time point based on gene expression similarity. All cell votes were then tabulated by cluster (Figure 3C-D), which is represented by a simple tree manifold (Figure 3E-F). While SM migrations that bring distant cell types into specific organs cannot be ruled out, the data supported the notion of transcriptionally related cell states arising from the subdivision of a common progenitor cell population. Given that time points were generated from pooled embryos of slightly different ages, it was possible that parent-child relationships existed within specific time points. To address this and confirm the single-cell voting results, each differentiation pathway was evaluated using pseudotime analysis, which computationally predicts the progenitor state of a cell population (Monocle; Cao, J. et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature 566, 496-502 (2019)). In general, pseudotime analysis was consistent with single-cell voting. However, in the case of hepatic endoderm, Monocle predicted parent-child relationships within E9.0, where Hhex+ hindgut endoderm (cluster e_b2) gave rise to both Prox1 / Afp+ hepatoblasts (e_b5) and Prox1 / Sox17 / Pdx1+ hepatopancreatic biliary progenitors (e_b7) (Figure 3H), consistent with in vitro lineage tracing experiments.

[0254] Overall, the DE trajectories inferred by single-cell transcriptomes are consistent with experimentally determined fate maps, demonstrating the robustness of the analysis herein and suggesting that previously poorly defined SM differentiation pathways may also represent lineage relationships. That said, it should be noted that cells with similar transcriptomes are not necessarily lineage-related. Indeed, cells of different lineages, such as the ventral and dorsal pancreas, may converge on similar transcriptional profiles. Thus, the results provided here establish a theoretical framework for future experimental analyses of foregut mesenchymal development.

[0255] Example 6. Coordinated generation of multipotent progenitor cells A closer look at the differentiation pathways between the DE and SM suggests coordinated development from multipotent progenitors within adjacent endodermal and mesodermal tissue layers. For example, at E8.5, both the DE lateral foregut cells (e_a2) and the spatially adjacent SM cells (m_a0) express the TF Osr1, a trajectory predicting that these two cell populations are multipotent progenitors that give rise to the respiratory, esophageal, and gastric epithelium and mesenchyme, respectively (Figure 4A-B). As development progresses, the different cell populations appear segregated as they gradually express distinct lineage-regulating TFs and growth factors (Figure 4A-D). In situ validation confirmed that Osr1 is expressed in both the presumptive esophageal, lung, and gastric epithelium and mesenchyme at E9.5 (Figure 4E-G).

[0256] Furthermore, closer examination of the DE tracheal cluster suggested a transitional epithelial cell population that co-expresses the respiratory marker Nkx2-1 and the esophageal marker Sox2 at E9.5, when the foregut is patterning along the dorsal-ventral axis (Figure 4H-I). Immunostaining confirmed that this is indeed a rare Nkx2-1 / Sox2+ cell population at the anterior border of the tracheoesophagus, which recent studies have demonstrated is important for tracheoesophageal morphogenesis (Figure 4K-L). In summary, the differentiation pathways of foregut lineages predicted from single-cell transcriptomes represent a valuable resource for further research.

[0257] Example 7. Prediction of the signaling roadmap for organ induction We computationally predicted the paracrine signaling microenvironment of the foregut, which controls cell fate decisions (Figure 5A-B). For six major signaling pathways involved in organogenesis: BMP, FGF, Hedgehog (HH), Notch, retinoic acid (RA), and canonical Wnt, metagene expression profiles were calculated for all ligand-, receptor-, and context-independent response genes in each DE and SM cluster (Figure 5J). Using spatial maps of each cell population in the foregut (Figure 1I-J), cell populations along the AP axis were ordered such that DE and SM cell types most likely to directly contact each other in the signaling diagram were opposite each other (Figure 5C). Metagene expression levels were then used to predict potential ligand-receptor pairs and the likelihood that a particular cell population is responding to local paracrine or autocrine signals (Figure 5A-C, 5K). Metagene expression thresholds were benchmarked against experimentally validated interactions in the literature. Furthermore, potential ligand-receptor pairings were restricted to nearby cell clusters, consistent with the generally accepted view that these pathways act over a relatively short range. Overall, this analysis revealed a hypothetical combinatorial signaling network (Figures 5A-C, 5K).

[0258] Overall, our computational predictions, organized around known expression patterns of ligands and receptors, defined the most known signaling interactions controlling DE lineage specification. This included mesoderm-derived BMPs, FGFs, and Wnts that promote DE liver and lung fates, and autocrine Notch signaling in the DE endocrine pancreas. This suggests that previously undefined SM signaling predictions are also likely to be accurate. To test this, we examined BMP signaling as an example. Consistent with the scRNA-seq data, in situ hybridization confirmed high levels of Bmp4 ligand expression in the STM and respiratory mesenchyme, while immunostaining for phospho-Smad1 / 5 / 8, cellular effectors of BMP signaling, confirmed autocrine and paracrine signaling in the developing liver and respiratory mesenchyme and epithelium, respectively, as predicted (Figure 5E-G).

[0259] Signaling response metagene expression levels were projected onto SPRING plots and cell state trees, revealing spatiotemporally dynamic signaling domains that correlate with cell lineages (Figure 5D, 5L). In general, transcriptome data predict locally restricted interactions in which the SM is a major source of BMP, FGF, RA, and Wnt ligands, signaling both to the adjacent DE and to the SM itself (Figure 5C). In contrast, HH ligands are produced by signals to the DE and intestinal SM, but there is no evidence of autocrine activity in the DE (Figure 5C). Combining data from all six signaling pathways into cell state trees generated a comprehensive roadmap of combinatorial signals predicted to orchestrate the temporal and spatial development of each DE and SM lineage (Figure 5H-I). This analysis predicts many previously unrecognized signaling interactions and represents a hypothesis-generating resource for further experimental validation.

[0260] Example 8. Examining the role of epithelial hedgehog signaling in foregut mesenchymal patterning To genetically test the predictive value of the signaling roadmap, we examined HH activity, which has been suggested to be high in the intestinal SM (esophagus, respiratory tract, stomach, and duodenum) but low in the pharyngeal and hepatic SM by scRNA-seq (Figure 6A-C). HH ligand stimulates the activation of Gli2 and Gli3 TFs, which in turn promote the transcription of HH target genes (e.g., Gli1). Mouse embryo sections confirmed that Shh ligands are expressed in the intestinal DE and high levels of Gli1-LacZ are expressed in the adjacent SM. In contrast, the hepatic endoderm does not express Shh, and there were very few, if any, Gli1-LacZ-positive cells in the hepatic SM (Figure 6D). To define the function of HH in SM patterning, bulk RNA-seq was performed on the foregut of Gli2- / -;Gli3- / - double mutant embryos, which lack all HH activity and cannot specify a respiratory fate. By comparing homozygous mutants with heterozygous littermates, we identified 156 HH / Gli-regulated transcripts (Figure 6E). With the caveat that this bulk RNA-seq is performed in both endoderm and mesoderm, we examined the enrichment of these HH-regulated transcripts in the transcriptomes of DE and SM single-cell clusters. This revealed that most transcripts were expressed in the SM compared to the DE. Transcripts downregulated in Gli2 / 3- mutants (n = 80) were typically enriched in the intestinal SM, whereas upregulated transcripts (n = 76) were typically enriched in the liver or pharyngeal SM (Figure 6E-G). Interestingly, HH / Gli-regulated transcripts, including downregulated TFs (Osr1, Tbx4 / 5, Foxf1 / 2) and upregulated TFs (Tbx18, Lhx2, Wt1), are implicated in respiratory and liver development, respectively (Figure 6E). This genetic analysis confirmed the predicted value of the signaling roadmap, where differential HH activity promotes intestinal SM versus hepatic and pharyngeal SM, in part by regulating other lineage specifying TFs and signaling proteins ( Figure 5I ).

[0261] The data provided herein suggest a model in which a reciprocal epithelial-mesenchymal signaling network coordinates the DE and SM lineages during organogenesis. In this model, SM-derived RA signals back to the SM, inducing regionally restricted expression of Shh in the DE by E9.0, establishing the broad pharyngeal, intestinal, and hepatic domains. Other SM ligands (BMP, FGF, Notch, RA, and Wnt), with distinct localized expression in these three broad domains, then coordinately differentiate the DE and SM progenitors. This model can be tested by cell-specific genetic manipulation.

[0262] Example 9. Differentiation of visceral mesenchymal-like lineages from human PSCs. We then used the novel SM markers and signaling roadmap disclosed herein to direct the differentiation of distinct SM subtypes from human pluripotent stem cells (hPSCs), which had previously been elusive. Previous studies have established protocols for differentiating hPSCs into lateral plate mesoderm (LPM) and cardiac tissue. Although both SM and heart are derived from the LPM, single-cell data suggested that in mice, early SMs experience more RA signaling than early cardiac mesoderm. This was confirmed by the expression of the RA-responsive RARE:lacZ transgene in E8.5 embryos (Figure 7E). (d) Addition of RA to the LPM differentiation medium on days 2–4 downregulated cardiac markers NKX2-5, ISL1, and TBX20 and promoted SM markers FOXF1, HOXA1, HOXA5, and WNT2 (Figure 7B, 7E). This is consistent with mouse scRNA-seq data showing that E8.5 SM expresses Nkx2-5, Isl1, and Tbx20 at lower levels than cardiac mesoderm. Examination of PAX3, PRRX1, and CD31 confirmed that d4 SM cultures do not express significant levels of endothelial, somatic, or limb mesenchymal markers (Figure 7E).

[0263] Next, to drive organ-specific SM-like lineages based on the roadmap, we treated primitive SMs with various combinations of HH, RA, Wnt, and BMP agonists or antagonists from d4 to d7 (Figure 7A). As predicted, HH agonists promoted intestinal identity and efficiently blocked hepatic fate. In HH-treated cultures, addition of RA and BMP4 (RA / BMP4) followed by WNT from d6 to d7 promoted gene expression consistent with respiratory mesenchyme (NKX6-1, TBX5, and WNT2) with low levels of esophageal, gastric, or hepatic markers. In contrast, addition of RA and BMP4 antagonists from d6 to d7 promoted esophageal / gastric-like identity (MSC, BARX1, WNT4, and NKX3-2) (Figure 7B-C, 7F). In the absence of HH agonists, cells treated with RA / BMP4 / WNT had a gene expression profile similar to that of liver SM and mesothelial markers (WT1, TBX18, LHX2, and UPK1B), whereas RA / BMP4 / WNT-treated cells expressed liver-fibroblast markers (MSX1 / 2 and HAND1). Immunostaining and RNA microscopy confirmed the RT-PCR analysis (Figures 7C-D, 7F) showing that approximately 70-80% of cells in liver SM / mesothelial-like cultures were WT1+, MSX1-, and NKX6-1-, whereas other populations appeared to be approximately 30-40%. The remaining cells appeared undifferentiated rather than alternative lineages. These data provide evidence that the signaling roadmap inferred from mouse scRNA-seq data can be used to direct the differentiation of various organ-specific SM subtypes from hPSCs.

[0264] Example 10. Materials and Methods Embryo collection and single cell dissociation All mouse experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children's Hospital Medical Center. No statistical sample size estimation was performed prior to the experiment, and sufficient embryos were used to generate the necessary materials for the experiment. Randomization was not utilized because no specific treatments were performed in different groups. Timed matings were performed between C57BL / 6J mice, and the day a vaginal plug was detected was considered embryonic day 0.5. Stage classification was verified by counting somite numbers at E8.5 (5–10 somites [s]), E9.0 (12–15 s), and E9.5 (25–30 s) (Figure 1A–B). The foregut between the posterior pharynx and midgut was microscopically dissected, and the heart and most of the paraxial tissues were removed, excluding the thyroid gland. At E9.5, the anterior and posterior regions, containing the lung / esophagus and liver / pancreas rudiments, respectively, were isolated separately. Dissected foregut tissue was pooled from 16, 20, 18, and 15 embryos from E8.5, E9.0, E9.5 anterior, and E9.5 posterior, respectively, isolated from two to three litters.

[0265] Single-cell dissociation using a cold-activated protease protocol was performed as known in the art. Rapidly dissected C57BL / 6J mouse embryo tissue was transferred to ice-cold PBS containing 5 mM CaCl, 10 mg / ml Bacillus licheniformis (Sigma), and 125 U / ml DNAse (Qiagen) and incubated on ice with pipette mixing. After 7 minutes, single-cell dissociation was confirmed under a microscope. Cells were then transferred to a 15 mL conical tube and 3 mL of ice-cold PBS containing 10% FBS (FBS / PBS) was added. Cells were pelleted (1200 G for 5 minutes) and resuspended in 2 mL of PBS / FBS. Cells were washed three times with 5 mL of PBS / 0.01% BSA (PBS / BSA) and resuspended to a final cell concentration of 100,000 cells / mL for scRNA-seq. Single-cell suspensions from each stage were loaded into a Chromium Single Cell Controller instrument (10x Genomics) to generate single-cell gel beads in emulsion. Single-cell RNA-seq libraries for high-throughput sequencing were prepared using the Chromium Single Cell 5' Library and Gel Bead Kit (10x Genomics). All samples were multiplexed together and sequenced on an Illumina HiSeq2500. The individuals performing the RNA extraction, library preparation, and sequencing steps were blinded.

[0266] Immunofluorescence staining, in situ hybridization and RNAscope Mouse embryos were collected at the indicated stages and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. Fixed samples were washed three times for 10 minutes with PBS, and the foregut was dissected under a microscope, if necessary. Embryos or dissected foreguts were then processed for antibody staining as previously described or for in situ hybridization.

[0267] For RNAscope of mouse tissues, fixed embryos were immersed in 30% sucrose / PBS overnight, embedded in OCT, cryosectioned (12 μm) onto Superfrost Plus slides (Thermo Fisher), and stored overnight at -80°C. For RNAscope of adherent hPSC cultures, cells were differentiated on Geltrex-coated u-Slide 8-wells (ibid.) and fixed with 4% PFA for 30 minutes at room temperature. Cells were dehydrated through an ethanol gradient and stored in 100% ethanol at -20°C. RNAscope fluorescent in situ hybridization was performed using RNAscope Multiplex Fluorescent Detection Reagents V2 (Advanced Cell Diagnostics, Inc.) and Opal fluorophores (Akoya Biosciences) according to the manufacturer's instructions.

[0268] Preprocessing of 10x Genomics raw scRNA-seq data Raw scRNA-seq data were processed using CellRanger (v2.0.0, available on the World Wide Web at github.com / 10XGenomics / cellranger). Reads were aligned to the mouse genome [mm10] to calculate gene counts across barcodes. Barcodes with UMI counts below approximately 5k were not included in downstream analyses. The percentage of reads that mapped to the transcriptome was approximately 70% for each sample. The resulting data consisted of 9748 cells at E8.5, 9265 cells at E9.0, 7208 cells in the E9.5 forward sample, and 5085 cells in the E9.5 backward sample.

[0269] Quality control, dimensionality reduction, clustering, and marker prediction Subsequent QC and clustering were performed using the Seurat [v2.3.4] package in R. Basic filtering was performed if all genes were expressed in three or more cells and included all cells with at least 100 detected genes. QC was based on the nGene and percent.mito parameters to remove multiples and cells with high mitochondrial gene expression. After filtering, 9,748, 9,265, and 12,255 cells were retained for E8.5, E9.0, and E9.5 samples, respectively. Global scaling was used to normalize the counts of all cells in each sample [scale factor: 10,000], and cell cycle effects were removed by regressing the difference between S and G2M phases from the normalized data using default parameters. First, each developmental stage was clustered separately to identify major cell lineages. Approximately 1,500 highly variable genes (HVGs) were selected across each population by marking outliers from the variance vs. avgExp plot. PCA was performed using HVG, and the first 20 principal components were used for cell clustering, which was then visualized using t-distributed stochastic neighbor embedding (tSNE). Marker genes defining each cluster were identified using the "FindAllMarkers" function in Seurat (Wilcoxon rank sum test), and these were used to annotate clusters based on well-known cell type-specific genes.

[0270] Cells from all three time points were integrated with Seurat (v3.0) using diagonalized canonical correlation analysis (CCA) to reduce the dimensionality of the dataset, followed by L2 normalization of the canonical correlation vectors (CCVs). Finally, we obtained mutual neighbors (MNNs), also known as integration anchors (cell pairs) for integrating cells. First, 30 CCs (canonical correlation components) were used for clustering, and then nonlinear dimensionality reduction approaches (UMAP and tSNE) were used to reduce the dimensionality and visualize cells in two dimensions.

[0271] In silico selection and clustering for definitive endoderm and visceral mesenchyme The definitive endoderm (DE) cluster (4,448 cells) was defined by coexpression of Foxa1 / 2, Cdh1, and / or Epcam, while the visceral SM (10,097 cells) was defined by coexpression of Foxf1, Vim, and / or Pdgfra, and by negativity for cardiac, somatic, and axial mesoderm-specific transcripts. Cells from the DE and SM clusters were extracted from each time point and reclustered using Seurat [v2.3.4] to define lineage subtypes. Prior to reclustering the blood, mitochondrial, ribosomal, and lineage-dependent noncoding RNA genes were regressed from the data. Dimensionality reduction, clustering, and marker prediction steps were performed as described above for each stage. DE and SM cell subtypes were annotated through manual curation of cluster marker genes compared to over 300 publicly available expression profiles in the MGI database and independent gene expression validation. DE and SM clusters from all three time points were analyzed together using the Seurat (v3.0) integration approach described above.

[0272] Transcription factor codes from DE and SM lineages To identify TFs specifically enriched in various DE and SM cell types, we utilized the "FindAllMarkers" function in Seurat [v3.0] on a set of 1623 TFs expressed in the mouse genome [AnimalTFDB]. Raw counts of TFs were normalized and scaled in Seurat [v3.0]. Cells within a cluster served as replicates in finding marker TFs for each lineage. The Wilcoxon rank-sum test was used to identify marker TFs. The top five marker TFs were then visualized using the DimHeatmap function in Seurat (v3.0).

[0273] Pseudotemporal analysis of the spatial organization of cell populations To investigate whether pseudotime analysis can inform the spatial organization of cells within a continuous sheet of DE or SM tissue, we performed pseudotime analysis using URD [v1.0]. First, to calculate pseudotime, we used the diffusion map to calculate the transition probability between DE and SM cells at each stage. Next, we generated diffusion map components using the calcDM function and used the first eight components to calculate the transition probability between cells. Next, to calculate pseudotime, we anchored the root cell to the most anterior cluster based on manual annotation. Starting from the root cell, we performed a probabilistic breadth-first graph search using the transition probabilities until all cells in the graph were visited. Multiple simulations were performed, and the pseudotime was equal to the average iteration of visiting each cell in the graph from the root cell. We calculated pseudotime using the following functions in URD ("floodPseudotime" and "floodPsuedotimeProcess"). Finally, we used the plotDists function to plot the density distribution of pseudotimes for each cluster / cell type. Density distribution of ordered clusters in pseudotime, as well as the manually curated ordering of cell types along the AP axis.

[0274] SPRING analysis of cell differentiation pathways To explore cell differentiation pathways across three time points, we ran SPRING [v1.0] using a k-nearest neighbor (KNN) graph (5-neighborhood) to obtain a force-directed layout of cells and their neighbors. To understand transcriptional changes across cell states (lineages), the first 40 principal components (PCs) were learned from the most recent time point, E9.5, and this PC space was used to rescale the entire dataset (E8.5, E9.0, and E9.5). This transformed data was used to generate a distance matrix, and then a KNN graph was obtained using default parameters.

[0275] Inferring cell state trees by parent-child single-cell voting To visualize differentiation pathways in a simple transcriptional cell-state tree, we used a parent-child single-cell voting approach based on the KNN classification algorithm. First, we generated a normalized count matrix using discriminative marker genes from all DE or SM clusters as features for each stage. The marker genes were used as features to train KNN, during which KNN learns the distances between cells in the training set based on feature representation. Each cell was classified based on its Seurat cluster assignment. Cells at later time points vote for the most likely parent cell at the previous time point as follows: E8.5 cells were used to train KNN, and E9.0 cells were used to test E8.5 cells. KNN yielded the voting probability of each E9.0 cell for each E8.5 cluster, and then averaged the voting probabilities of each E9.0 cluster for each E8.5 cluster. This approach was repeated with E9.5 cells voting for their E9.0 parent. The average voting probability for a particular cluster was tabulated, normalized for cluster size, and expressed as a percentage of the total votes in the confusion matrix. The highest number of votes linking a later time point to an earlier time point was displayed as a solid line on the tree. Outstanding second choices with >60% of the votes were reported as dashed lines on the tree. This voting probability was also compared with the confusion matrix obtained from KNN to evaluate the transcriptional cell state tree. In >99% of cases, the two methods resulted in the same first and second choices, validating the inferred parent-child relationship.

[0276] To validate the cell-state tree assertions using pseudotime analysis, Monocle [v3.0.0] was deployed on individual lineages / cell states. tSNE was used for dimensionality reduction, and a principle graph was trained using SimplePPT. All other parameters were set to default.

[0277] Calculation of meta-gene profiles For the six major paracrine signaling pathways involved in foregut organogenesis (BMP, FGF, HH, Notch, RA, and classical Wnt), we curated lists of all established ligand, receptor, and context-independent pathway response genes encoded in the mouse genome. Ligand-metagene, receptor-metagene, and response-metagene profiles were then calculated by summing the normalized expression of individual genes of each pathway in each cell and cluster, as follows (e.g., Wnt-ligand metagene = Σ(Wnt1 + Wnt2 + Wnt2b + Wnt3 / / / Wnt10b expression)).

[0278] Assume there are x genes and n cells in a gene set: Gene1 has (a1, a2...an) counts, Gene2 has (b1, b2...bn) counts, etc.

[0279] Step 1: The counts for each gene were normalized using the maximum count of the gene across all DE and SM cells (n = 14,545 cells): Gene1_norm = (a1, a2···an) / max(a1, a2···an).

[0280] Step 2: The normalized gene counts were summed per cell to generate meta-gene_v1, which contains the counts for the cell: meta-gene_v1 = Gene1_norm + Gene2_norm + + Genex_norm. The estimated total counts are m1, m2, mn.

[0281] Step 3: The total count of metagene_v1 was normalized by the maximum count of metagene_v1 to create a metagene profile for each cell: MetaGene = (m1, m2 ··· mn) / max(m1, m2 ··· mn). The average metagene expression profile of the ligand, receptor, and response genes for each DE and SM cluster was then calculated in Seurat [v3.0] using the "AverageExpression" function. The average metagene expression profiles for all DE and SM clusters were visualized as dot plots using Seurat. The average expression of the metagene expression profiles was scaled from -2 to 2 for dot plot visualization.

[0282] Prediction of receptor-ligand interactions A given cell type was scored as expressing sufficient ligand to signal or sufficient receptor to respond to the ligand if the mean ligand-metagene or receptor-metagene expression level was ≥ -1 and expressed in ≥ 25% of cells (except for the Notch ligand-metagene, for which an expression threshold of ≥ -1.5 was used due to low overall expression in all cells). These thresholds were empirically conservatively set and benchmarked against experimentally validated signaling interactions in DE liver, lung, and pancreas. Additionally, we determined the likelihood that a particular cell population was responding based on a context-independent pathway response-metagene expression level of ≥ -1 and expressed in ≥ 25% of cells. Context-independent response genes are genes known in the art to be directly transcribed in most cell types in response to ligand-receptor activation.

[0283] The DE and SM clusters at each stage are ordered along the AP axis, and the diagram uses spatially adjacent DE and SM cell types to match the location of the tracheal primordium in vivo. To assign receptor-ligand interactions to each cell cluster, we determined whether a particular cluster was responsive based on thresholds for response metagene and receptor metagene levels ≥ −1. If a responsive cluster also expressed ligand-metagene levels ≥ −1, autocrine signaling was established. For paracrine signaling, adjacent cell populations within the same tissue layer and from adjacent layers that expressed ligand-metagenes above the threshold were identified, establishing a receptor-ligand interaction. Signal intensity was calculated as the sum of the ligand-metagene and response-metagene values. If this value was ≥ 1, the signal was considered "strong."

[0284] Comparison of bulk RNA-seq vs. scRNA-seq Foregut tissue was dissected from E9.5 double mutant Gli2- / -;Gli3- / - mice (n = 3) and Gli2+ / -;Gli3+ / - heterozygous littermate controls (n = 3). Each dissected foregut was used separately for RNA extraction, library preparation, and bulk RNA-seq. These mice were of mixed strain, and embryo sex was unknown. Using the CSBB [v3.0] (available on the World Wide Web at github.com / csbbcompbio / CSBB-v3.0) pipeline, the genome was aligned to the mouse genome [mm110]. Differentially expressed transcripts between the two genotypes were obtained using RUVSeq (LogFC ≥ |1| and FDR ≤ 0.1). Differentially expressed genes were clustered using hierarchical clustering and visualized for the samples using Morpheus (available on the World Wide Web at software.broadinstitute.org / morpheus).

[0285] To compare the bulk analysis with scRNA-seq, the expression of differentially expressed genes relative to cells was visualized in scClusters using the "DoHeatmap" function in Seurat. Cells were arranged according to their anterior / posterior axis position in each cluster, and genes were reordered by reverting from the clustering order obtained above. Gene set enrichment analysis (GSEA) [v3.0] was also performed to examine the statistical enrichment of differentially expressed genes in the intestinal SM (respiratory, esophagus, stomach, and duodenum), pharynx, and liver SM clusters. The normalized counts of genes relative to cells and up- and down-regulated genes from bulk RNA-seq were used as custom gene sets to perform the GSEA analysis.

[0286] Maintaining PSCs This study used two hPSC lines: 1) WA01-H1 human embryonic stem cells purchased from WiCell (NIH approval numbers, NIHhESC-10-0043 and NIHhESC-10-0062) and 2) human iPSC72_3, generated by the CCHMC Pluripotent Stem Cell Facility. Both cell lines have been authenticated for: i) cell identity by STR profiling by the Genetica DNA Laboratory; ii) genetic stability by standard mitotic spread and G-banded karyotype analysis in the CCHMC Cytogenetics Laboratory; and iii) functional pluripotency; cells were subjected to analysis of functional pluripotency by teratoma assay and demonstrate the ability to differentiate into each of the three germ layers. Both cell lines were routinely tested negative for mycoplasma contamination. hPSC lines were maintained in feeder-free conditions in mTeSR1 medium (StemCell Technologies) on Geltrex-coated 6-well Nunclon surface plates (Thermo Fisher) at 37°C with 5% CO2. Cells were checked daily, and differentiated cells were manually removed. Cells were passaged every 4 days using dispase solution (Thermo Fisher).

[0287] Mesenchymal differentiation of PSCs Differentiation of hPSCs into lateral plate mesoderm was induced using a previously described method with modifications. Briefly, partially confluent hPSCs were dissociated into very fine clumps with Accutase (Invitrogen) and passaged at 1:18 onto new Geltrex-coated 24-well plates for immunocytochemistry and 12-well plates for RNA preparation in mTeSR1 containing 1 μM thiazovivin (Tocris) (Day 1). The following day, after a brief wash with DMEM / F12, the cells were incubated for 24 hours in Day 0 medium (30 ng / mL activin A (Cell Guidance Systems), 40 ng / mL BMP4 (R&D Systems), 6 μM CHIR99021 (Tocris), 20 ng / mL FGF2 (Thermo Fisher), 100 nM PIK90 (EMD Millipore)). Basal medium consisting of Advanced DMEM / F12, N2, B27, 15 mM HEPES, 2 mM L-glutathione, and penicillin-streptomycin was used for this day 0 culture and all subsequent differentiations. On day 1, cells were briefly washed with DMEM / F12 followed by 24 hours of Day 1 culture medium (1 μM A8301 (Tocris), 30 ng / mL BMP4, 1 μM C59 (Cellagen Technology)). For cardiac mesoderm generation, cells were cultured in 1 μM A8301, 30 ng / mL BMP4, 1 μM C59, and 20 ng / mL FGF2 from days 2 to 4 (medium was changed daily). From day 4, cells were cultured in 200 μg / mL 2-phospho-ascorbic acid (Sigma), 1 μM XAV939 (Sigma), and 30 ng / mL BMP4 for 3 days. For visceral mesoderm generation, cells were cultured in 1 μM A8301, 30 ng / mL BMP4, 1 μM C59, 20 ng / mL FGF2, and 2 μM RA (Sigma) from day 2 to day 4 (medium was changed daily).To further direct local visceral mesoderm, we performed one of the following: (1) 2 μM RA and 40 ng / mL BMP4 to promote STM fate for 3 days; (2) 2 μM RA and 2 μM purmorphamine (PMA) (Tocris) for 2 days, followed by 2 μM RA, 2 μM PMA, and 100 ng / mL Noggin (R&D Systems) for the final day to promote esophageal / gastric mesenchymal fate; or (3) 2 μM RA, 40 ng / mL BMP4, and 2 μM PMA for 2 days, followed by 2 μM RA, 40 ng / mL BMP4, 2 μM PMA, and 1 μM CHIR99021 for the final day to promote respiratory mesenchymal fate. The medium was changed daily. Similar results were obtained with WA-01 hESCs and human iPSC 72_3.

[0288] Quantitative RT-PCR Total RNA was prepared from differentiating human ES cells using a Nucleospin kit according to the manufacturer's protocol. Reverse transcription PCR was performed using the Superscript VILO cDNA synthesis kit. QuantStudio 5 and 6 were used for qPCR analysis. Statistics were performed using PRISM8 (GraphPad Software). Significance was determined by one-way analysis of variance followed by Tukey's test.

[0289] immunocytochemistry Cells were fixed with 4% PFA / PBS for 30 minutes at room temperature. After perforation with 0.5% Triton X-100 / PBS for 10 minutes, the cells were incubated with 5% normal donkey serum for 2 hours. The cells were incubated with primary antibodies overnight at 4°C. The next day, the cells were washed with PBS and then incubated with secondary antibodies for 1 hour at room temperature.

[0290] Data and Code Availability The scRNA-seq and bulk RNA-seq data (including BAM, raw counts, and cell annotations) are available on the Gene Expression Omnibus (GEO): GSE136689 and GSE136687. All code (scripts, R packages, and software) and documentation have been uploaded to the World Wide Web at github.com / ZornLab / Single-cell-transcriptomics-reveals-a-signaling-roadmap-coordinating-endoderm-and-mesoderm-lineage. All deposited code is available under GPLv3.0. The scRNA-seq data can be explored on the World Wide Web at research.cchmc.org / ZornLab-singlecell.p

[0291] In at least some of the foregoing embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment, except where such substitution is not technically feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be included within the scope of the subject matter, as defined by the appended claims.

[0292] With respect to the use of substantially all plural and / or singular terms herein, those of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein.

[0293] With respect to the use of "eg," it is understood to mean "for example," and thus is a non-limiting example.

[0294] Those skilled in the art will understand that, generally, the terms used in this specification, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" is typically interpreted as "including, but not limited to," the term "having" is typically interpreted as "having at least," the term "include" is typically interpreted as "including, but not limited to," etc.). Those skilled in the art will further understand that if a specific number of introduced claim recitations is intended, such intention will be expressly recited in the claim, and that in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the appended claims below may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases is typically interpreted as meaning that introducing a claim recitation with the indefinite article "a" or "an" means limiting any particular claim that includes the claim recitation so introduced to embodiments that include only one such recitation, even if the same claim also includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"), and the same applies to the use of definite articles used to introduce claim recitations. In addition, those skilled in the art will recognize that when a specific number of introduced claim recitations is explicitly recited, such recitations are typically interpreted to mean at least the recited number (e.g., the mere recitation of "two recitations" without any other modifier means at least two recitations or more than two recitations).Furthermore, when a convention similar to "such as at least one of A, B, and C" is used, such syntax is typically intended to mean how one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "such as at least one of A, B, or C" is used, such syntax is typically intended to mean how one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, will typically be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0295] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0296] As will be understood by those skilled in the art, for all purposes, including in terms of written description, all ranges disclosed herein encompass all possible subranges and combinations of these subranges. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least 2, 3, 4, 5, 10, etc. divisions. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As will be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," and "less than" refer to ranges that are inclusive of the recited numbers and that can subsequently be broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5, etc. items, and so on.

[0297] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0298] All references cited herein, including, but not limited to, references to published and unpublished applications, patents, and literature, are incorporated herein by reference in their entirety and any specific disclosures referenced herein, and are hereby made a part of this specification. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, the specification supersedes and / or is intended to take precedence over such conflicting material.

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Claims

1. 1. A method for producing visceral mesoderm cells, comprising: A method comprising contacting lateral plate mesoderm cells with a TGF-β signaling pathway inhibitor, a Wnt signaling pathway inhibitor, a BMP signaling pathway activator, an FGF signaling pathway activator, and a retinoic acid (RA) signaling pathway activator for 36 to 60 hours to differentiate the lateral plate mesoderm cells into visceral mesoderm cells, wherein the contacting inhibits the TGF-β signaling pathway and the Wnt signaling pathway in the lateral plate mesoderm cells and activates the BMP signaling pathway, FGF signaling pathway, and retinoic acid (RA) signaling pathway in the lateral plate mesoderm cells.

2. The method of claim 1 , wherein the visceral mesoderm cells are human visceral mesoderm cells.

3. 3. The method of claim 1 or 2, wherein the lateral plate mesoderm cells are differentiated from intermediate primitive streak cells.

4. 4. The method of claim 3, wherein the lateral plate mesoderm cells are differentiated from intermediate primitive streak cells by contacting the intermediate primitive streak cells with a TGF-β signaling pathway inhibitor, a Wnt signaling pathway inhibitor, and a BMP signaling pathway activator, wherein the contacting inhibits the TGF-β signaling pathway and the Wnt signaling pathway in the intermediate primitive streak cells and activates the BMP signaling pathway in the intermediate primitive streak cells.

5. The method of claim 3 or 4, wherein the intermediate primitive streak cells are differentiated from pluripotent stem cells.

6. 6. The method of claim 5, wherein the intermediate primitive streak cells are differentiated from pluripotent stem cells by contacting the pluripotent stem cells with a TGF-β signaling pathway activator, a Wnt signaling pathway activator, an FGF signaling pathway activator, a BMP signaling pathway activator, and a PI3K signaling pathway inhibitor, wherein the contacting activates the TGF-β signaling pathway, the Wnt signaling pathway, the FGF signaling pathway, the BMP signaling pathway, and inhibits the PI3K signaling pathway of the pluripotent stem cells.

7. The method of any one of claims 1 to 6, wherein the lateral plate mesoderm cells are contacted with A8301, BMP4, C59, FGF2, RA, or any combination thereof.

8. 8. The method of any one of claims 1 to 7, wherein the visceral mesoderm cells exhibit increased expression of FOXF1, HOXA1, HOXA5, or WNT2, or any combination thereof, and decreased expression of NKX2-5, ISL1, or TBX2, or any combination thereof, compared to cardiac mesoderm cells.

9. 1. A method for producing septum transversum cells, comprising: Producing visceral mesoderm cells according to the method of any one of claims 1 to 8; contacting the visceral mesoderm cells with a retinoic acid signaling pathway activator and a BMP signaling pathway activator for 60 to 84 hours to differentiate the visceral mesoderm cells into septum transverse cells, wherein the contacting activates the retinoic acid signaling pathway and the BMP signaling pathway in the visceral mesoderm cells. A method comprising:

10. 10. The method of claim 9, wherein the visceral mesoderm cells are contacted with RA, BMP4, or both.

11. 1. A method for producing respiratory mesenchymal cells, comprising: a) producing visceral mesoderm cells according to the method of any one of claims 1 to 8; b) contacting the visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, a Hedgehog (HH) signaling pathway activator, and a Wnt signaling pathway activator for 60 to 84 hours to differentiate the visceral mesoderm cells into respiratory mesenchymal cells, wherein the contacting activates the retinoic acid signaling pathway, the BMP signaling pathway, the Hedgehog (HH) signaling pathway, and the Wnt signaling pathway in the visceral mesoderm cells. A method comprising:

12. The method of claim 11, wherein step b) is the third step and further comprises a second step of contacting the visceral mesoderm cells with a retinoic acid signaling pathway activator, a BMP signaling pathway activator, and an HH signaling pathway activator prior to the third step.

13. 13. The method of any one of claims 11 to 12, wherein the visceral mesoderm cells are contacted with RA, BMP4, PMA, CHIR99021, or any combination thereof.

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