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1. WO2021058956 - PROCÉDÉ D'OBTENTION DE CELLULES DE TYPE BASAL DES VOIES RESPIRATOIRES DÉRIVÉES DE CELLULES SOUCHES PLURIPOTENTES ET MODÈLE D'ÉPITHÉLIUM DES VOIES RESPIRATOIRES

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CLAIMS

1. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells comprising the steps:

differentiating a population of pluripotent stem cells to obtain a heterogeneous population of pluripotent stem cell-derived lung progenitor cells;

culturing the pluripotent stem cell-derived lung progenitor cells in the presence of feeder cells and a rho-kinase inhibitor to obtain a population of pluripotent stem cell-derived airway basal-like cells; and

culturing the pluripotent stem cell-derived lung progenitor cells and feeder cells in a serum-free medium to obtain a substantially pure population of pluripotent stem cell-derived airway basal-like cells.

2. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in Claim 1, wherein the population of pluripotent stem cells are induced pluripotent stem cells (iPSCs).

3. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in Claim 2, wherein the induced pluripotent stem cells are derived from a patient without any known genetic disorder or respiratory disease.

4. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in Claim 2, wherein the induced pluripotent stem cells are derived from a patient with a known genetic disorder or respiratory disease.

5. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of the previous Claims, wherein the obtained substantially pure population of pluripotent stem-cell derived airway basal-like cells comprises cells expressing one or more airway basal cell markers.

6. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in Claim 5, wherein at least 70% of the obtained substantially pure population of pluripotent stem-cell derived airway basal-like cells express one or more airway basal cell markers, preferably at least 90% of the obtained substantially pure population of pluripotent stem-cell derived airway basal- like cells express one or more airway basal cell markers.

7. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of Claims 5 to 6, wherein the airway basal cell markers are DNR63, NGFR, cytokeratin 14 and integrin a6.

8. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of the previous Claims, wherein the obtained substantially pure population of pluripotent stem-cell derived airway basal-like cells contains cells having a cuboidal morphology.

9. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of the previous Claims, wherein the obtained substantially pure population of pluripotent stem-cell derived airway basal-like cells contains cells having enlarged nuclei.

10. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of the previous Claims, wherein the obtained pluripotent stem cell-derived lung progenitor cells are plated at a 1:1 ratio with the feeder cells.

11. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of the previous Claims, wherein the feeder cells are mouse fibroblast cells.

12. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of the previous Claims, wherein the feeder cells are 3T3-J2 cells.

13. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of the previous Claims, wherein the feeder cells are mitotically inactivated.

14. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of the previous Claims, wherein the feeder cells and pluripotent stem cell-derived lung progenitor cells are cultured in a serum- free medium.

15. A method for obtaining a substantially pure population of pluripotent stem cell- derived airway basal-like cells as in any of the previous Claims, wherein the rho- kinase inhibitor is used at a concentration of between 5 mM and 30 pM, most preferably the rho-kinase inhibitor is used at a concentration of around 10 pM.

16. A substantially pure population of induced pluripotent stem cell-derived airway basal-like cells wherein at least 50% of the cells express NGFR and at least 70% of the cells express Integrin a6; preferably at least 60% of the cells express NGFR and at least 80% of the cells express Integrin a6; more preferably at least 70% of the cells express NGFR and at least 90% of the cells express Integrin a6 and optionally, at least 50%, more preferably 70%, of the cells express cytokeratin 14.

17. A substantially pure population of induced pluripotent stem cell-derived airway basal-like cells as in Claim 16 wherein the cells have a cuboidal morphology and/or enlarged nuclei.

18. A method of treating an individual having respiratory disease, comprising implanting a pluripotent stem-cell derived airway basal-like cells obtained by the methods of claims 1 to 15 or the cells of Claims 16 or 17.

19. Use of the substantially pure population of pluripotent stem cell-derived airway basal-like cells obtained by the methods of claims 1 to 15 or the cells of Claims 16 or 17 in a drug discovery screen; toxicity assay; inhalation assay; research of differentiation pathways; research of disease aetiology.

20. A method for obtaining an in vitro pluripotent stem cell-derived airway epithelium model or lung model comprising the steps of:

obtaining a substantially pure population of pluripotent stem cell-derived airway basal-like cells by the methods of claims 1 to 15 or obtaining the cells of Claims 16 or 17; and

culturing the population of pluripotent stem cell-derived airway basal-like cells on an air-liquid interface to obtain an in vitro pluripotent stem cell-derived airway epithelium model.

21. A method for obtaining an in vitro pluripotent stem cell-derived airway epithelium or lung model as in Claim 20, wherein the obtained in vitro pluripotent stem cell- derived airway epithelium model comprises cells expressing one or more airway epithelial cell markers.

22. A method for obtaining an in vitro pluripotent stem cell-derived airway epithelium or lung model as in Claim 21, wherein the airway epithelial cell markers are; Club Cell Protein 10, Mucin 1, DNR63 and Acetylated Tubulin.

23. A method for obtaining an in vitro pluripotent stem cell-derived airway epithelium model as in any of Claims 20 to 22, wherein the in vitro pluripotent stem cell-derived airway epithelium model has a substantially layered structure which resembles a

naturally occurring airway epithelium and comprises a plurality of cell types selected from basal cells, ciliated cells, goblet cells and club cells.

24. A method for obtaining an in vitro pluripotent stem cell-derived airway epithelium model as in any of Claims 20 to 23, wherein the air-liquid interface is provided by culturing the pluripotent stem cell-derived airway basal-like cells on an insert placed in a cell culture vessel, preferably the cells are cultured on the top of the insert in the cell culture vessel and cell culture medium is added beneath the insert such that the cells on the top of the insert are exposed to the atmosphere.

25. A method for obtaining an in vitro pluripotent stem cell-derived airway epithelium model as in any of Claims 20 to 24, wherein the air-liquid interface culture is allowed to mature for 5 or more days, more preferably the air-liquid interface culture is allowed to mature for 14 or more days, most preferably the air-liquid interface culture is allowed to mature for 21 or more days.

26. An in vitro pluripotent stem cell-derived airway epithelium or lung model which expresses one or more airway epithelial cell markers selected from Club Cell Protein 10, Mucin 1, DNR63 and Acetylated Tubulin; and which has a substantially layered structure which resembles a naturally occurring airway epithelium and comprises a plurality of cell types selected from basal cells, ciliated cells, goblet cells and club cells.

27. Use of the in vitro pluripotent stem cell-derived airway epithelium model obtained by the methods of Claims 20 to 25 or of Claim 26, used in a drug discovery screen; toxicity assay; inhalation assay; research of differentiation pathways; pharmacokinetic studies of a compound; pharmacodynamic studies of a compound; studies of disease aetiology.

28. A method of making a COVID-19 lung model comprising infecting the in vitro pluripotent stem cell-derived airway epithelium model obtained by the methods of Claims 20 to 25, or infecting the model of Claim 26, with coronavirus SARS-CoV-2.

29. A COVID 19 lung model, comprising an in vitro pluripotent stem cell-derived airway epithelium model obtained by the methods of Claims 20 to 25, or the model of Claim 26, which has been infected with coronavirus SARS-Cov-2.

30. An in vitro pluripotent stem cell-derived airway epithelium or lung model as in Claim 26, or a COVID-19 lung model as in Claim 29, which comprises cilia which have a cilia beat frequency of 11 ± 1 Hz to 14 ± 1 Hz, or a cilia beat frequency 12.4 ± 1.6 Hz to 13.6 ± 2.0 Hz, or a cilia beat frequency of 12.4 ± 1.6 Hz, or a cilia beat frequency of 13.6 ± 2.0 Hz.

31. A COVID-19 lung model as in either of Claim 29 or 30 wherein the cells show increased secretion of IL-6, preferably increased secretion of IL-6 IL-10 and TNFa, after infection with coronavirus SARS-Cov-2 compared to a model where cells are not infected with coronavirus SARS-Cov-2.

32. A COVID-19 lung model as in any of Claims 29 to 31 wherein IL-6 secretion equating to greater than 2-fold increase, more preferably a greater than 2.5 fold increase, most preferably a greater than 2.9 fold increase, and optionally a four-fold increase above that of the uninfected control is seen within 48 hours post infection.

33. An in vitro method of screening for prophylactic and therapeutic agents for COVID- 19, comprising the steps of;

Obtaining the COVID-19 lung model of Claims 29 to 32;

bringing a test agent into contact with the COVID-19 lung model; and

detecting or measuring a response in the infected model.

34. An in vitro method of screening for prophylactic and therapeutic agents for COVID- 19, comprising the steps of;

infecting an in vitro pluripotent stem cell-derived airway epithelium model obtained by the methods of Claims 20 to 25, or infecting the model of Claim 26, with coronavirus SARS-CoV-2 to give an infected model;

bringing a test agent into contact with the infected model; and

detecting or measuring a response in the infected model.