METHODS OF GENERATING EPITHELIAL LINEAGE CELLS FROM EMBRYOID BODIES AND PLURIPOTENT CELLS

• US 20090075374A1
• Filed: 07/24/2008
• Published: 03/19/2009
• Est. Priority Date: 07/24/2007
• Status: Abandoned Application

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Ji, Lin, Metallo, Christian M., Palecek, Sean P., De Pablo, Juan J.
• Application Number:
  US12/179,462
• Publication Number:
  US 20090075374A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0629 Keratinocytes; Whole skin

Abstract

Methods of generating p63-positive cells from embryoid bodies and pluripotent cells by culturing the cells in the presence of a retinoid and optionally a bone morphogenetic protein, such that the cells express at least p63. The p63-positive cells can be further cultured without the retinoid and optional bone morphogenetic protein to K14-positive cells. The K14-positive cells can be further cultured into various terminally differentiated cell types of the epithelial lineage.

Claims

1. A method of generating p63-positive cells, the method comprising the step of:

culturing embryoid bodies in a medium comprising a retinoid and optionally a bone morphogenetic protein for about two days to about nine days, each in an amount sufficient to generate p63-positive cells.

2. The method of claim 1, wherein the embryoid bodies are human embryoid bodies.

3. The method of claim 1, wherein the retinoid is selected from the group consisting of tretinoin, alitretinoin and isotretinoin.

4. The method of claim 1, wherein the bone morphogenetic protein is bone morphogenetic protein-4.

5. The method of claim 1, wherein the retinoid is provided at a concentration between about 0.1 μ

M to about 10 μ

M.

6. The method of claim 1, wherein the bone morphogenetic protein is provided at a concentration between about 5 ng/ml to about 50 ng/ml.

7. The method of claim 1, wherein the cells are also K18 positive.

8. The method of claim 1, wherein the medium is a defined medium.

9. A method of generating p63 positive cells, the method comprising the step of:

culturing pluripotent cells in a medium comprising a retinoid and optionally a bone morphogenetic protein for about five days to about seven days, each in an amount sufficient to generate p63 positive cells.

10. The method of claim 9, wherein the pluripotent cells are selected from the group consisting of human embryonic stem cells and induced pluripotent stem cells.

11. The method of claim 9, wherein the retinoid is selected from the group consisting of tretinoin, alitretinoin and isotretinoin.

12. The method of claim 9, wherein the bone morphogenetic protein is bone morphogenetic protein-4.

13. The method of claim 9, wherein the retinoid is at a concentration between about 0.1 μ

M to about 10 μ

M.

14. The method of claim 9, wherein the bone morphogenetic protein is at a concentration between about 5 ng/ml to about 50 ng/ml.

15. The method of claim 9, wherein the cells are also K18 positive.

16. The method of claim 9, wherein the medium is a defined medium.

17. A method of generating K14-positive cells, the method comprising the step of:

culturing p63-positive cells to confluence in a defined serum-free medium to obtain K14-positive cells, wherein the cells are on an adherent surface, and wherein the medium does not comprise a retinoid and bone morphogenetic protein.

18. The method of claim 17, wherein the adherent surface comprises a material selected from the group consisting of collagen, fibronectin, gelatin, glycosaminoglycans, laminin, Matrigel®

, osteocalcin, osteonectin and combinations thereof.

19. A method of generating filaggrin- and involucrin-positive cells, the method comprising the step of:

culturing K14-positive cells to confluence in a defined serum-free medium to obtain filaggrin- and involucrin-positive cells, wherein the cells are on an adherent surface.

20. The method of claim 19, wherein the adherent surface comprises a material selected from the group consisting of collagen, fibronectin, gelatin, glycosaminoglycans, laminin, Matrigel®

, osteocalcin, osteonectin and combinations thereof.

21. The method of claim 19, wherein the cells are also K10 positive.

22. A method of generating K3/K12-positive cells, the method comprising the step of:

culturing K14-positive cells to confluence in a defined serum-free medium to obtain K3/K12-positive cells, wherein the cells are on an adherent surface.

23. The method of claim 22, wherein the adherent surface comprises a material selected from the group consisting of collagen, fibronectin, gelatin, glycosaminoglycans, laminin, Matrigel®

, osteocalcin, osteonectin and combinations thereof.

24. A population of cultured K14-positive cells, wherein at least about 85% of the cells express K14.

25. The population of cultured cells of claim 24, wherein the K14-positive cells are produced from cells selected from the group consisting of embryoid bodies and pluripotent cells.

26. A population of cultured filaggrin- and involucrin-positive cells, wherein at least about 85% of the cells express filaggrin and involucrin.

27. The population of cultured cells of claim 26, wherein the cells also express K10.