Method for inducing erythropoietin-producing cell

• US 9,334,475 B2
• Filed: 04/04/2013
• Issued: 05/10/2016
• Est. Priority Date: 04/06/2012
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Hitomi, Hirofumi, Osafune, Kenji
• Primary Examiner(s): LI, QIAN JANICE
• Application Number:
  US14/390,853
• Publication Number:
  US 20150087060A1
• Time in Patent Office: 1,132 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/62 DMSO
• CPC Class Codes: C12N 2501/065 Modulators of histone acetylation
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• CPC Class Codes: C12N 5/0686 Kidney cells

Abstract

An object of the present invention is to provide a method for inducing the differentiation of erythropoietin-producing cells from human pluripotent stem cells. The above object is achieved by providing a method for inducing the differentiation of erythropoietin-producing cells from human pluripotent stem cells using a medium containing a specific growth factor and a specific compound.

Claims

1. A method for producing erythropoietin-producing cells from human pluripotent stem cells, comprising the steps of:

(i) culturing human pluripotent stem cells in a first medium comprising Activin and a GSK-3β

inhibitor; and

then

(ii) culturing the human pluripotent stem cells in a second medium comprising an IGF family gene product.

2. The method according to claim 1, wherein the GSK-3β

inhibitor is 6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile (CHIR99021).

3. The method according to claim 1, wherein the IGF family gene product is IGF-1.

4. The method according to claim 1, wherein the human pluripotent stem cells are human iPS cells or human ES cells.

5. The method according to claim 1, wherein the first medium further comprises a histone deacetylase (HDAC) inhibitor.

6. The method according to claim 5, wherein the HDAC inhibitor is NaB.

7. The method according to claim 1, wherein the second medium further comprises dimethyl sulfoxide.

8. The method according to claim 1, wherein the step (i) comprises dissociating the human pluripotent stem cells into single cells.

9. The method according to claim 1, wherein a culture period of the step (ii) is 8 to 12 days.

10. The method according to claim 9, wherein a culture period of the step (i) is 6 days and the culture period of the step (ii) is 10 days.

11. The method according to claim 1, wherein the step (ii) is performed under low oxygen conditions.

12. The method according to claim 11, wherein an oxygen concentration is 5% under the low oxygen conditions.