Compositions and methods of preparing airway cells
AssigneeYale University
InventorsMahboobe Ghaedi, Laura Niklason
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Abstract
The present invention provides compositions and methods of preparing airway cells. In one aspect, an epithelial airway cell derived from an induced pluripotent stem (iPS) cell characterized by expression of airway cell surface markers and an ability to proliferate is described. In another aspect, methods of differentiating an iPS into an epithelial airway cell is provided. Engineered lungs, methods of making such engineered lungs comprising the epithelial airway cells and treating respiratory disorders are also disclosed.
Claims22
A method of differentiating an induced pluripotent stem (iPS) cell into an airway epithelial cell, the method comprising:
- culturing the iPS cell in the absence of serum to induce differentiation into a definitive endoderm (DE) cell;
- culturing the DE cell in the presence of serum to induce differentiation into an anterior foregut endoderm (AFE) cell;
- and
- culturing the AFE cell in the presence of serum, a cytokine cocktail that inhibits the WNT pathway, and at least 0.5 μM retinoic acid to induce differentiation of the AFE cell into the airway epithelial cell;
- wherein the airway epithelial cell is characterized by:
- expression of airway cell surface markers, wherein the airway cell surface markers comprise Clara cell secretory protein (CCSP), cytokeratin 5 (KRT5), and FOXJ1;
- and
- an ability to proliferate in culture for at least about 30 days without loss of the airway cell surface markers.
An engineered three-dimensional lung tissue comprising airway epithelial cells derived from induced pluripotent stem (iPS) cells and a three-dimensional scaffold, wherein the airway epithelial cells are characterized by:
- expression of airway cell surface markers, wherein the airway cell surface markers comprise Clara cell secretory protein (CCSP), cytokeratin 5 (KRT5), and FOXJ1;
- and
- an ability to proliferate in culture for at least about 30 days without loss of the airway cell surface markers;
- wherein the iPS cells are cultured in the absence of serum to induce differentiation into definitive endoderm (DE) cells;
- wherein the DE cells are cultured in the presence of serum to induce differentiation into anterior foregut endoderm (AFE) cells;
- and
- wherein the AFE cells are cultured in the presence of serum, a cytokine cocktail that inhibits the WNT pathway, and at least 0.5 μM retinoic acid to induce differentiation of the AFE cells into the airway epithelial cells.
A method of engineering a three-dimensional lung tissue, the method comprising:
- (i) differentiating a population of induced pluripotent stem (iPS) into airway epithelial cells, wherein the airway epithelial cells express airway cell surface markers comprising Clara cell secretory protein (CCSP), cytokeratin 5 (KRT5), and FOXJ1;
- and
- are capable of proliferation in culture for at least about 30 days without loss of the airway cell markers;
- wherein the iPS cells are cultured in the absence of serum to induce differentiation into definitive endoderm (DE) cells;
- wherein the DE cells are cultured in the presence of serum to induce differentiation into anterior foregut endoderm (AFE) cells;
- and
- wherein the AFE cells are cultured in the presence of serum, a cytokine cocktail that inhibits the WNT pathway, and at least 0.5 μM retinoic acid to induce differentiation of the AFE cells into the airway epithelial cells;
- and
- (ii) seeding the airway epithelial cells onto a three-dimensional scaffold.
A method of improving, treating or relieving a symptom of a respiratory disorder in a subject in need thereof, the method comprising:
- (i) differentiating induced pluripotent stem (iPS) cells into airway epithelial cells, wherein the airway epithelial cells express airway cell surface markers comprising Clara cell secretory protein (CCSP), cytokeratin 5 (KRT5), and FOXJ1, and are capable of proliferation in culture for at least about 30 days without loss of the airway cell markers;
- wherein the iPS cells are cultured in the absence of serum to induce differentiation into definitive endoderm (DE) cells;
- wherein the DE cells are cultured in the presence of serum to induce differentiation into anterior foregut endoderm (AFE) cells;
- and
- wherein the AFE cells are cultured in the presence of serum, a cytokine cocktail that inhibits the WNT pathway, and at least 0.5 μM retinoic acid to induce differentiation of the AFE cells into the airway epithelial cells;
- and
- (ii) administering the airway epithelial cells in an amount effective to treat the respiratory disorder in the subject.
Dependencies are inferred from the claim text.