Generation of airway basal stem cells from human pluripotent stem cells
Described herein is a method of generating in-vitro differentiated airway basal cells and compositions thereof. Also described herein is a method of treating a pulmonary disease comprising administering the in-vitro differentiated airway basal cells and compositions thereof. In another aspect, described herein is a disease model comprising patient-derived or genetically modified in-vitro differentiated airway basal cells and compositions thereof.
1. A method comprising:
(a) culturing a population of Nkx2-1 + lung progenitor cells in a first culture medium and contacting the Nkx2-1 + lung progenitor cells with, one or more of: at least one Wnt agonist; at least one retinoic acid or a derivative thereof; and at least one bone morphogenic protein (BMP) agonist;
(b) re-suspending the Nkx2-1 + lung progenitor cells in a hydrogel to form epithelial spheres;
(c) culturing the epithelial spheres in a second culture medium and contacting the epithelial spheres with one or more of: at least one fibroblast growth factor (FGF) agonist; at least one steroid; 3′,5′-cyclic monophosphate sodium salt; 3-isobutyl-1-methyxanthine (IBMX); and at least one Rho kinase (ROCK) inhibitor, thereby differentiating the epithelial spheres into airway progenitor cells;
(d) passaging and re-suspending the airway progenitor cells in the second culture medium, thereby expanding the airway progenitor cells; and
(e) culturing the airway progenitor cells from step (d) in a third culture medium and contacting the airway progenitor cells from step (d) with at least one inhibitor of SMAD; at least one inhibitor of transforming growth factor β (TGF-β); at least one bone morphogenetic protein (BMP) inhibitor; and at least one ROCK inhibitor; thereby differentiating the airway progenitor cells into airway basal cells (BCs).
2. The method of claim 1 , further comprising a step of sorting and/or isolating airway BCs that express one or more markers selected from the group consisting of: Nkx2-1; tumor protein 63 (TP63); cytoskeletal protein keratin 5 (KRT5); and nerve growth factor receptor (NGFR).
3. The method of claim 1 , wherein the hydrogel comprises one or more extracellular matrix components.
4. The method of claim 1 , wherein the Wnt agonist is an inhibitor of GSK-3.
5. The method of claim 4 , wherein the Wnt agonist is CHIR99021.
6. The method of claim 1 , wherein the BMP agonist is BMP4.
7. The method of claim 1 , wherein the fibroblast growth factors are FGF2 and FGF10.
8. The method of claim 1 , wherein the ROCK inhibitor is Y-27632.
9. The method of claim 1 , wherein the inhibitor of SMAD is A83-01.
10. The method of claim 1 , wherein the inhibitor of transforming growth factor β (TGF-β) is selected from the group consisting of: ALK5 inhibitor II, SB431542, LY364947, and A83-01.
11. The method of claim 1 , wherein the inhibitor of BMP is selected from the group consisting of: dorsomorphin and DMH1.
12. The method of claim 1 , wherein step (a) comprises contacting the cells with CHIR99021, retinoic acid, and BMP4.
13. The method of claim 1 , wherein step (c) comprises contacting the cells with FGF2, FGF10, dexamethasone, IBMX, 3′,5′-cyclic monophosphate sodium salt, and Y-27632.
14. The method of claim 1 , wherein step (e) comprises contacting the cells with A83-01 and DMH1.
15. The method of claim 1 , wherein the cells in steps (b) and/or (d) are cultured in a 3-dimensional microenvironment.
16. The method of claim 1 , wherein the Nkx2-1 + lung progenitor cells are generated by a method comprising:
(a) culturing a population of pluripotent stem cells in a serum-free medium;
(b) culturing the population of pluripotent stem cells in a culture medium wherein the culture medium comprises or the stem cells are contacted with a Wnt agonist or a Wnt polypeptide;
(c) culturing the population of pluripotent stem cells in a culture medium and contacting the pluripotent stem cells with a bone morphogenic protein (BMP) and a Wnt agonist, thereby inducing the differentiation of the pluripotent stem cells into a population of lung progenitor cells; and
(d) sorting or isolating a population of Nkx2-1 + lung progenitor cells from the population of lung progenitor cells generated in (c).
17. The method of claim 16 , wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.
18. The method of claim 16 , further comprising, during or following step (d), sorting the population of Nkx2-1+ lung progenitor cells for F3+/EGFR+ lung/airway progenitor cells.
19. The method of claim 1 , further comprising a step of cryopreserving the airway BCs.
20. The method of claim 16 , wherein the pluripotent stem cells are derived from a healthy subject.
21. The method of claim 16 , wherein the pluripotent stem cells are derived from a subject with a pulmonary disease.
22. The method of claim 21 , wherein the pulmonary disease is selected from the group consisting of: asthma, cystic fibrosis, primary ciliary dyskinesia, interstitial lung disease, and genetic lung malformations.
23. The method of claim 1 , further comprising administering the airway basal cells to the trachea of a subject with a pulmonary disease.
24. The method of claim 23 , wherein the pulmonary disease is selected from the group consisting of: asthma, cystic fibrosis, primary ciliary dyskinesia interstitial lung disease, and genetic lung malformations.
25. The method of claim 1 , wherein the airway basal cell expresses NKX2-1, TP63; KRT5; and NGFR.
26. The method of claim 2 , the one or more markers is nerve growth factor receptor (NGFR).