METHOD FOR INDUCING DIFFERENTIATION INTO EPITHELIAL PROGENITOR CELL/STEM CELL POPULATION AND CORNEAL EPITHELIAL CELL POPULATION FROM INDUCED PLURIPOTENT STEM CELLS

• US 20120142103A1
• Filed: 05/18/2010
• Published: 06/07/2012
• Est. Priority Date: 05/18/2009
• Status: Abandoned Application

• Current Assignee: Kyoto University, Tohoku University National Universities Corporation
• Original Assignee: Kyoto University, Tohoku University National Universities Corporation
• Inventors: Yamanaka, Shinya, Okita, Keisuke, Nishida, Kohji, Hayashi, Ryuhei, Sakurai, Miharu, Kageyama, Tomofumi
• Application Number:
  US13/321,130
• Publication Number:
  US 20120142103A1
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: A61L 27/3834 Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
• CPC Class Codes: A61L 27/3869 Epithelial tissues other than skin
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• 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 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2502/1394 Bone marrow stromal cells; whole marrow
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062
• CPC Class Codes: C12N 5/0629 Keratinocytes; Whole skin

Abstract

The present invention relates to: a method for inducing differentiation into an epithelial progenitor cell/stem cell population or a corneal epithelial cell population by culturing, under particular conditions, induced pluripotent stem cells induced from mammalian somatic cells or undifferentiated stem cells; an epithelial progenitor cell/stem cell population or a corneal epithelial cell population obtained by the method; and a cell preparation for the treatment of epithelial disease and a cell sheet, which are prepared using these cell populations.

Claims

1. A method for inducing differentiation into a keratin 14-positive and p63-positive epithelial progenitor cell/stem cell population from induced pluripotent stem cells induced from mammalian somatic cells or undifferentiated stem cells, comprising:

culturing said induced pluripotent stem cells on feeder cells or a support selected from collagen, basement membrane matrix, amnion, fibronectin, and laminin using a medium for epidermal cells containing an epidermal growth factor and/or cholera toxin and serum.

2. The method according to claim 1, wherein the medium further contains one or more selected from hydrocortisone, insulin, transferrin, and selenium.

3. The method according to claim 1, wherein the feeder cells are stromal cells.

4. The method according to claim 3, wherein the stromal cells are 3T3 cells.

5. The method according to claim 1, wherein the medium further contains at least one of BMP4 (Bone Morphogenetic Protein 4) retinoic acid.

6. The method according to claim 1, wherein the differentiation into the epithelial progenitor cell/stem cell population is induced without embryoid body formation.

12. A method for inducing further differentiation into an epithelial cell population from the epithelial progenitor cell/stem cell population which has been induced by a method according to claim 1.

13. The method according to claim 12, wherein the epithelial cell population is any selected from a corneal epithelial cell population, an oral mucosal epithelial cell population, a urinary bladder epithelial cell population, a conjunctival epithelial cell population, a gastric mucosal epithelial cell population, a small intestinal epithelial cell population, a large intestinal epithelial cell population, a renal epithelial cell population, a renal tubular epithelial cell population, a gingival mucosal epithelial cell population, an esophagus epithelial cell population, a hepatic epithelial cell population, a pancreatic epithelial cell population, a pulmonary epithelial cell population, and a gallbladder epithelial cell population.

14. A method for inducing differentiation into a keratin 12-positive corneal epithelial cell population from the epithelial progenitor cell/stem cell population, comprising continuing to culture in a method according to claim 1.

15. The method according to claim 1, further comprising the step of isolating a keratin 14-positive and p63-positive cell population.

16. The method according to any one of claim 14, further comprising the step of isolating a keratin 12-positive and keratin 14-negative cell population.

17. A cell preparation for epithelial disease comprising an epithelial progenitor cell/stem cell population obtained by a method according to claim 1 and/or an epithelial cell population induced from said epithelial progenitor cell/stem cell population.

18. A cell sheet comprising layers of an epithelial progenitor cell/stem cell population obtained by a method according to claim 1 and/or an epithelial cell population induced from said epithelial progenitor cell/stem cell population.

7. A method for inducing differentiation into a keratin 14-positive and p63-positive epithelial progenitor cell/stem cell population from induced pluripotent stem cells induced from mammalian somatic cells or undifferentiated stem cells, comprising:

culturing said induced pluripotent stem cells on 3T3 cells or in the presence of a 3T3 cell-derived differentiation factor.

8. The method according to claim 7, wherein the induced pluripotent stem cells are cultured in an epithelial induction medium containing serum and/or BMP4 or in a medium for epidermal cells containing an epidermal growth factor and/or cholera toxin and serum.

9. The method according to claim 8, wherein the epithelial induction medium further contains one or more selected from retinoic acid, nonessential amino acid, β

-mercaptoethanol, and sodium pyruvate, and the medium for epidermal cells further contains one or more selected from hydrocortisone, insulin, transferrin, and selenium.

10. The method according to claim 8, wherein the induced pluripotent stem cells are cultured in a differentiation medium containing one or more selected from a serum substitute, BMP4 (Bone Morphogenetic Protein 4), and retinoic acid and then further cultured in the epithelial induction medium or the medium for epidermal cells.

11. The method according to claim 10, wherein the differentiation medium further contains one or more selected from nonessential amino acid, β

-mercaptoethanol, and sodium pyruvate.

19. (canceled)

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)