Methods for Differentiating AT2 Cells
AssigneeLung Biotechnology PBC
InventorsAdrian COOPER, Andre DHARMAWAN, Elena FEDERZONI, Kurt WONG
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Abstract
The present disclosure relates to methods of differentiating pluripotent stem cells or lung progenitor cells into alveolar type 2 (AT2) cells. The present disclosure also relates to AT2 cells made by such methods, organoids containing such AT2 cells and methods of using the same. The present disclosure also relates to differentiation media for use in the same.
Claims34
A method for differentiating lung progenitor cells (LPC) into alveolar type 2 (AT2) cells, comprising:
- culturing LPC in a base culture medium comprising a glycogen synthase kinase 3 (GSK3) inhibitor, keratinocyte growth factor (KGF), fibroblast growth factor 10 (FGF10), and a gamma secretase inhibitor;
- (ii) passaging the cells of (i) in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cyclic adenosine monophosphate (cAMP), an inhibitor of cyclic nucleotide phosphodiesterases, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor for about 24 hours to about 48 hours;
- (iii) culturing the cells of (ii) in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cyclic adenosine monophosphate (cAMP), and an inhibitor of cyclic nucleotide phosphodiesterases for about 7 days;
- (iv) separating the cells of (iii) having expression of epithelial cellular adhesion molecule (EpCAM) and/or carboxypeptidase M (CPM);
- (v) passaging the cells of (iv) having expression of EpCAM and/or CPM in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterases, and a ROCK inhibitor;
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- (vi) separating the cells of (v) having expression of surfactant protein C (SFTPC) to form AT2 cells.
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A method for differentiating LPC into AT2 cells, comprising:
- (i) culturing LPC in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, and a gamma secretase inhibitor;
- (ii) separating the cells of (i) having expression of EpCAM and/or CPM;
- (iii) passaging the cells of (ii) having expression of EpCAM and/or CPM in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterases, and a ROCK inhibitor for about 24 hours to about 48 hours;
- (iv) culturing the cells of (iii) in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, and an inhibitor of cyclic nucleotide phosphodiesterases for about 7 days, wherein the base culture medium does not comprise or is essentially free of a ROCK inhibitor;
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- (v) separating the cells of (iv) having expression of SFTPC to form AT2 cells.
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A method for differentiating LPC into AT2 cells, comprising:
- (i) culturing LPC in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, and a gamma secretase inhibitor;
- (ii) passaging the cells of (i) in a base culture medium comprising a GSK3 inhibitor, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterases, and a ROCK inhibitor;
- (iii) separating the cells of (ii) having expression of EpCAM and/or CPM;
- (iv) passaging the cells of (iii) having expression of EpCAM and/or CPM in a base culture medium comprising KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterases, and a ROCK inhibitor;
- (v) culturing the cells of (iv) in a base culture medium comprising KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterases, and a GSK3 inhibitor, wherein the base culture medium does not comprise or is essentially free of a ROCK inhibitor;
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- (vi) separating the cells of (v) having expression of SFTPC to form AT2 cells.
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A differentiation medium comprising a base culture medium, KGF, FGF10, dexamethasone, cAMP, an inhibitor of cyclic nucleotide phosphodiesterases, and a ROCK inhibitor.
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Dependencies are inferred from the claim text.