Method for inducing vascular endothelial cells

• US 10,669,529 B2
• Filed: 07/14/2016
• Issued: 06/02/2020
• Est. Priority Date: 07/17/2015
• Status: Active Grant

• Current Assignee: Kyoto University, Osaka University
• Original Assignee: Kyoto University
• Inventors: Nakahata, Tatsutoshi, Saito, Megumu, Niwa, Akira, Ota, Ryo, Sekiguchi, Kiyotoshi
• Primary Examiner(s): Davis, Ruth A
• Application Number:
  US15/745,425
• Publication Number:
  US 20180208893A1
• Time in Patent Office: 1,419 Days
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61L 27/00 Materials for grafts or prostheses or for coating grafts or prostheses dental prostheses A61C13/00; shape or structure of prostheses A61F2/00; use of preparations for artificial teeth A61K6/80; artificial kidneys A61M1/14
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/069 Vascular Endothelial cells
• CPC Class Codes: C12N 5/10 Cells modified by introduction of foreign genetic material

Abstract

Provided is a method for producing vascular endothelial cells from pluripotent stem cells, the method comprising the following steps (i) to (iii): (i) a step of culturing pluripotent stem cells in a culture medium comprising a BMP, on a culture vessel coated with a first matrix, to produce mesodermal progenitor cells; (ii) a step of dissociating the resulting cells into single cells; and (iii) a step of culturing the resulting cells in a culture medium comprising VEGF, on a culture vessel coated with a second matrix selected from the group consisting of laminin-411 or a fragment thereof, laminin-511 or a fragment thereof, Matrigel, type IV collagen and fibronectin.

Claims

1. A method for producing vascular endothelial cells from pluripotent stem cells, the method comprising:

(i) culturing pluripotent stem cells in a culture medium comprising a BMP, on a culture vessel coated with a first matrix, to produce mesodermal progenitor cells;

(ii) dissociating the mesodermal progenitor cells obtained in the culturing of the pluripotent stem cells into single cells; and

(iii) culturing the cells obtained by the dissociating of the mesodermal progenitor cells in a culture medium comprising VEGF, on a culture vessel coated with a second matrix selected from the group consisting of laminin-411 or a fragment thereof having an avidity for integrin, and, laminin-511 or a fragment thereof having an avidity for integrin.

2. The method according to claim 1, wherein the second matrix used in the culturing of the cells obtained by the dissociating of the mesodermal progenitor cells is a fragment of laminin-411 having an avidity for integrin.

3. The method according to claim 1, wherein the fragment of laminin-411 is laminin-411 E8.

4. The method according to claim 1, wherein the first matrix used in the culturing of the pluripotent stem cells is Matrigel, or laminin-511 or a fragment thereof having an avidity for integrin.

5. The method according to claim 4, wherein the fragment of laminin-511 used in culturing of the pluripotent stem cells is laminin-511 E8.

6. The method according to claim 1, wherein the BMP is BMP4.

7. The method according to claim 1, wherein the culture medium used in the culturing of the pluripotent stem cells further comprises a GSK3β

inhibitor and VEGF.

8. The method according to claim 7, wherein the GSK3β

inhibitor is CHIR99021.

9. The method according to claim 1, wherein the culturing of the pluripotent stem cells is carried out for two days or three days.

10. The method according to claim 1, wherein the culturing of the cells obtained by the dissociating of the mesodermal progenitor cells is carried out for four days.