Culture substrate for human pluripotent stem cells and use thereof

• US 8,877,493 B2
• Filed: 10/07/2010
• Issued: 11/04/2014
• Est. Priority Date: 10/08/2009
• Status: Active Grant

• Current Assignee: Kyoto University, Osaka University
• Original Assignee: Kyoto University, Osaka University
• Inventors: Sekiguchi, Kiyotoshi, Futaki, Sugiko, Taniguchi, Yukimasa, Hayashi, Maria, Nakatsuji, Norio, Miyazaki, Takamichi, Kawase, Eihachiro, Suemori, Hirofumi
• Primary Examiner(s): Lankford, Blaine
• Assistant Examiner(s): Visone, Thomas J
• Application Number:
  US13/500,452
• Publication Number:
  US 20120220031A1
• Time in Patent Office: 1,489 Days
• Field of Search: None
• US Class Current: 435/366
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention provides a culture substrate which enables maintenance culture of human pluripotent stem cells in a pluripotent state under a feeder-free culture environment, and a culture method of human pluripotent stem cells using the culture substrate. By seeding human pluripotent stem cells dissociated into single cells at a cell density of 4×10⁴ to 10×10⁴ cells/cm² onto a culture substrate coated with human laminin α5β1γ1 E8 fragment or human laminin α3β3γ2 E8 fragment preferably at a concentration of 0.5 to 25 μg/cm², the human pluripotent stem cells can be rapidly expanded in a pluripotent state.

Claims

1. A culture method of human pluripotent stem cells dissociated into single cells in feeder-free culture conditions, comprising the steps of:

dissociating human pluripotent stem cells into single cells, and

culturing the human pluripotent stem cells dissociated into single cells using a culture substrate, the culture substrate being coated with human laminin β

5β

2γ

1 E8 fragment or human laminin α

3β

3γ

2 E8 fragment.

2. The culture method according to claim 1, wherein the coating concentration of human laminin α

5β

1γ

1 E8 fragment or human laminin α

3β

3γ

2 E8 fragment is 0.5 to 25 μ

g/cm².

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

4. The culture method according to claim 1, wherein the human pluripotent stem cells dissociated into single cells are seeded at a cell density of 2×

10⁴ to 20×

10⁴ cells/cm².

5. The culture method according to claim 1, further comprising forming single-cell-derived colonies through culturing the human pluripotent stem cells dissociated into single cells.

6. The culture method according to claim 1, further comprising maintaining the human pluripotent stem cells in a single-cell state.

7. The culture method according to claim 1, further comprising using a synthetic medium.