Compositions and methods for obtaining 3-dimensional lung-like epithelium and related uses thereof
The invention disclosed herein generally relates to methods and systems for growing, expanding and/or obtaining 3-dimensional lung-like epithelium comprising cells having SOX9 protein activity and SOX2+ protein activity. In particular, the invention disclosed herein relates to methods and systems for growing human cells having SOX9 protein activity and SOX2+ protein activity in vitro, and for promoting pluripotent stem cell derived ventral-anterior foregut spheroid tissue into 3-dimensional lung-like epithelium comprising cells having SOX9 protein activity and SOX2+ protein activity.
1. A method, comprising:
culturing in vitro for approximately forty days cells from NKX2.1+ ventral-anterior foregut spheroid tissue derived from pluripotent stem cells, wherein the culturing results in differentiation of the cells from the NKX2.1+ ventral-anterior foregut spheroid tissue derived from pluripotent stem cells into tissue comprising 3-dimensional lung-like epithelium, wherein the culturing comprises activating the FGF signaling pathway, the retinoic acid signaling pathway, and the Wnt signaling pathway; and
obtaining 3-dimensional lung tissue from the cultured tissue comprising 3-dimensional lung-like epithelium, wherein the obtained 3-dimensional lung tissue comprises a 3-dimensional lung tissue peripheral region and a 3-dimenstional lung tissue internal region,
wherein the 3-dimensional lung tissue peripheral region comprises cells expressing SOX9 protein and SOX2 protein,
wherein the 3-dimensional lung tissue internal region comprises cells expressing SOX2 protein, SCGB1A1 protein, and MUC5AC, wherein the 3-dimensional lung tissue internal region does not comprise cells expressing SOX9 protein.
2. The method of claim 1 ,
wherein activating the Wnt signaling pathway comprises culturing the with a small molecule or agonist that activates the Wnt signaling pathway,
wherein activating the FGF signaling pathway comprises culturing the cells with a small molecule or agonist that activates the FGF signaling pathway,
wherein activating the RA signaling pathway comprises culturing the cells with a small molecule or agonist that activates the RA signaling pathway.
3. The method of claim 2 ,
wherein the small molecule or agonist that activates the Wnt signaling pathway is CHIR99021,
wherein the small molecule or agonist that activates the FGF signaling pathway is selected from FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23,
wherein the small molecule or agonist that activates the RA signaling pathway is selected from the group consisting of all-trans retinoic acid, AC 261066, AC 55649, adapalene, AM 580, AM 80, BMS 753, BMS 961, CD 1530, CD 2314, CD 437, Ch 55, isotretinoin, tazarotene, and TTNPB.
4. The method of claim 1 , wherein the NKX2.1+ ventral-anterior foregut spheroid tissue is derived from definitive endoderm cells, wherein the definitive endoderm cells are derived from pluripotent stem cells, wherein the pluripotent stem cells are embryonic stem cells and/or induced pluripotent stem cells and/or or cells obtained through somatic cell nuclear transfer.
5. A method of treating a mammalian subject having a damaged lung tissue with reduced function, comprising:
a) culturing in vitro for approximately forty days cells from NKX2.1+ ventral-anterior foregut spheroid tissue derived from pluripotent stem cells, wherein the culturing results in differentiation of the obtained NKX2.1+ ventral-anterior foregut spheroid tissue into tissue comprising 3-dimensional lung-like epithelium, wherein the culturing comprises activating the FGF signaling pathway, the retinoic acid signaling pathway, and the Wnt signaling pathway;
b) obtaining 3-dimensional lung tissue from the cultured tissue comprising 3-dimensional lung-like epithelium, wherein the obtained 3-dimensional lung tissue comprises a 3-dimensional lung tissue peripheral region and a 3-dimensional lung tissue internal region,
wherein the 3-dimensional lung tissue peripheral region comprises cells expressing SOX9 protein and SOX2 protein,
wherein the 3-dimensional lung tissue internal region comprises cells expressing SOX2 protein, SCGB1A1 protein, and MUC5AC, wherein the 3-dimensional lung tissue internal region does not have cells expressing SOX9 protein;
c) engrafting the obtained 3-dimensional lung tissue at the site of injury, wherein engrafted 3-dimensional lung tissue engraft at the site of injury and repopulate at least a portion of the site with the engrafted cells, wherein the repopulated cells supplement the function, thereby treating the subject.
6. The method of claim 5 , wherein the damaged lung tissue is associated with a condition caused by one or more of
an injury that results in a loss of epithelial function,
a post-lung transplant complication, and/or
a genetic disorder.
7. The method of claim 6 ,
wherein the injury that results in loss of epithelial function is bronchiolitis obliterans;
wherein the post-lung transplant complication is bronchiolitis obliterans;
wherein the genetic disorder is cystic fibrosis.
8. The method of claim 5 ,
wherein activating the Wnt signaling pathway comprises culturing the NKX2.1+ ventral-anterior foregut spheroid tissue with a small molecule or agonist that activates the Wnt signaling pathway,
wherein activating the FGF signaling pathway comprises culturing the NKX2.1+ ventral-anterior foregut spheroid tissue with a small molecule or agonist that activates the FGF signaling pathway,
wherein activating the RA signaling pathway comprises culturing the NKX2.1+ ventral-anterior foregut spheroid tissue with a small molecule or agonist that activates the RA signaling pathway.
9. The method of claim 8 ,
wherein the small molecule or agonist that activates the Wnt signaling pathway is CHIR99021,
wherein the small molecule or agonist that activates the FGF signaling pathway is selected from FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23,
wherein the small molecule or agonist that activates the RA signaling pathway is selected from the group consisting of all-trans retinoic acid, AC 261066, AC 55649, adapalene, AM 580, AM 80, BMS 753, BMS 961, CD 1530, CD 2314, CD 437, Ch 55, isotretinoin, tazarotene, and TTNPB.
10. The method of claim 5 , comprising at least one of the following:
wherein the activating one or more signaling pathways within the NKX2.1+ ventral-anterior foregut spheroid tissue occurs over a specified temporal period,
wherein the activating one or more signaling pathways within the NKX2.1+ ventral-anterior foregut spheroid tissue occurs comprises activating two or more signaling pathways.
11. The method of claim 5 , wherein the NKX2.1+ ventral-anterior foregut spheroid tissue is derived from definitive endoderm cells, wherein the definitive endoderm cells are derived from pluripotent stem cells, wherein the pluripotent stem cells are embryonic stem cells and/or induced pluripotent stem cells and/or or cells obtained through somatic cell nuclear transfer.