Methods and compositions related to differentiated lung cells
Described herein are methods and compositions related to induced alveolar epithelial type 2 cells (iAEC2s), e.g., artificially-produced epithelial type 2 cells.
1. A method of making induced alveolar epithelial type 2 cells (iAEC2s) which produce surfactant and are capable of self-renewal in the absence of a mesenchymal cell, the method comprising:
culturing in vitro a NKX2-1+ lung epithelial progenitor cell without mesenchymal co-culture, in the simultaneous presence of:
an agonist of Wnt/beta-catenin signaling;
a corticosteroid; and
an agonist of cyclicAMP or the cyclicAMP pathway.
2. The method of claim 1 , wherein the culturing step is continued for at least 3 days.
3. The method of claim 1 , wherein the culturing step is continued for at least 15 days.
4. The method of claim 1 , further comprising a second culturing step after the first culturing step, wherein the cells are cultured without being contacted with an agonist of Wnt/beta-catenin signaling.
5. The method of claim 4 , wherein the second culturing step is continued for at least 5 days.
6. The method of claim 4 , wherein the second culturing step is continued for at least 10 days.
7. The method of claim 4 , wherein
the first culturing step is continued for a period of about 2 weeks;
the second culturing step is continued for a period of about 1 week; and
further comprising a third culturing step of culturing the cells resulting from the second culturing step in the presence of:
an agonist of Wnt/beta-catenin signaling;
a corticosteroid; and
an agonist of cyclicAMP or the cyclicAMP pathway
for a period of about 1 week.
8. The method of claim 1 , wherein the agonist of Wnt/beta catenin signaling is selected from the group consisting of:
CHIR99021; a recombinant Wnt polypeptide; a Wnt polypeptide; an exogenous Wnt polypeptide; BIO; WAY-316606; a (hetero) arylpyrimidine; IQ1; QS11; SB-216763; DCA; R-spondin; and an inhibitor of Axin2 and/or APC.
9. The method of claim 1 , wherein the corticosteroid is selected from the group consisting of:
dexamethasone; hydrocortisone; cortisone; prednisone; prednisolone; methylprednisolone; triamncinolone; betamethasone; fludrocortisone acetate; and deoxycorticosterone acetate.
10. The method of claim 1 , wherein the agonist of cyclicAMP or the cyclicAMP pathway is selected from the group consisting of:
cyclicAMP; IBMX; cholera toxin; forskolin; caffeine; theophylline; bucladesine; and pertussis toxin.
11. The method of claim 1 , wherein the NKX2-1+ lung epithelial progenitor cell is further contacted or cultured with an agonist of FGF signaling.
12. The method of claim 11 , wherein the agonist of FGF signaling is a polypeptide selected from the group consisting of:
KGF; a FGF receptor ligand; FGF1; FGF2; FGF3; FGF4; FGF6; FGF8; FGF9; FGF10; FGF17; FGF18; FGF22; and a small molecule agonist of FGF signaling.
13. The method of claim 11 , wherein the agonist of FGF signaling is KGF polypeptide.
14. The method of claim 1 , wherein the culturing further comprises culturing or contacting the cell with 3-isobutyl-1-methylxanthine (IMBX).
15. The method of claim 1 , wherein the NKX2-1+ lung epithelial progenitor cell is a CD47hi/CD2610 cell.
16. The method of claim 1 , wherein the iAEC2 cell is a NKX2-1+/SFTPC+ cell.
17. An isolated, in vitro induced alveolar epithelial type 2 cell (iAEC2) a) which is not in the presence of a mesenchymal cell, b) produces surfactant and, c) is capable of self-renewal in vitro in the absence of a mesenchymal cell.
18. The method of claim 1 , wherein the iAEC2s are capable of being maintained in the absence of a mesenchymal cell for at least 1 month.
19. The method of claim 1 , wherein the iAEC2s are capable of being maintained in the absence of a mesenchymal cell for at least 3 months.
20. The method of claim 1 , wherein the iAEC2s are capable of being maintained in the absence of a mesenchymal cell for multiple passages.
21. The iAEC2 of claim 17 , which is capable of being maintained in the absence of a mesenchymal cell for at least 1 month.
22. The iAEC2 of claim 17 , which is capable of being maintained in the absence of a mesenchymal cell for at least 3 months.
23. The iAEC2 of claim 17 , which is capable of being maintained in the absence of a mesenchymal cell for multiple passages.
24. The method of claim 1 , wherein the producing of surfactant is surfactant secretion.
25. The method of claim 1 , wherein the surfactant comprises protein and phospholipid.