Modified laminin and use thereof

Patent metadata

Field	Value
Publication number	US-9758765-B2
Application number	US-201214110063-A
Country	US
Kind code	B2
Filing date	Apr 9, 2012
Priority date	Apr 8, 2011
Publication date	Sep 12, 2017
Grant date	Sep 12, 2017

Abstract

Provided are a modified laminin having a cell-growth regulatory molecule bound to at least one site selected from the α chain N-terminus, the α chain C-terminus, the β chain N-terminus and the γ chain N-terminus of laminin or a heterotrimeric laminin fragment, a method for culturing cells in the presence of the modified laminin, a method for establishing iPS cells in the presence of the modified laminin, and a culture substrate coated with the modified laminin. Human stem cells cultured in a xeno-free environment with the use of the modified laminin of the present invention can be provided as highly safe human stem cells applicable to regenerative medicine.

First claim

The invention claimed is:

1. A modified laminin having a cell-growth regulatory molecule bound to at least one site selected from the α chain N-terminus, the α chain C-terminus, the β chain N-terminus and the γ chain N-terminus of a heterotrimeric laminin fragment,
wherein the cell-growth regulatory molecule is a growth factor binding molecule,
wherein the laminin fragment is a laminin E8 fragment and has integrin binding activity,
wherein the growth factor binding molecule is one or more kinds selected from
(a) perlecan or a fragment having a perlecan growth factor binding domain thereof,
(b) agrin or a fragment having an agrin growth factor binding domain thereof,
(c) XVIII type collagen or a fragment having a XVIII type collagen growth factor binding domain thereof,
(d) syndecan or a fragment having a syndecan growth factor binding domain thereof,
(e) glypican or a fragment having a glypican growth factor binding domain thereof, and
(f) latent TGF-β binding protein or a fragment having a latent TGF-β binding protein growth factor binding domain thereof,
wherein the modified laminin is a chimeric molecule comprising the growth factor binding molecule and the laminin E8 fragment.

2. The modified laminin according to claim 1 , wherein the laminin consists of one kind of α chain selected from α1 to α5, one kind of β chain selected from β1 to β3, and one kind of γ chain selected from γ1 to γ3.

3. The modified laminin according to claim 2 , wherein the laminin is laminin α5β1γ1 or laminin α3β3γ2.

4. The modified laminin according to claim 1 , which is of human origin.

5. A method for culturing mammalian cells, characterized by culturing the cells in the presence of the modified human laminin according to claim 1 .

6. The method according to claim 5 , wherein the mammalian cells are ES cells, iPS cells or somatic stem cells.

7. The method according to claim 5 , wherein no feeder cells are used.

8. A culture substrate coated with the modified human laminin according to claim 1 .

9. The culture substrate according to claim 8 , wherein the coating concentration of the modified human laminin is 0.03 to 25 μg/cm 2 .

10. A modified laminin having a cell-growth regulatory molecule bound to at least one site selected from the α chain N-terminus, the α chain C-terminus, the β chain N-terminus and the γ chain N-terminus of a heterotrimeric laminin fragment,
wherein the cell-growth regulatory molecule is a growth factor binding molecule,
wherein the laminin fragment is a laminin E8 fragment and has integrin binding activity,
wherein the growth factor binding molecule is one or more kinds selected from
(a) perlecan or a fragment having a perlecan growth factor binding domain thereof,
(b) agrin or a fragment having an agrin growth factor binding domain thereof,
(c) XVIII type collagen or a fragment having a XVIII type collagen growth factor binding domain thereof,
(d) syndecan or a fragment having a syndecan growth factor binding domain thereof,
(e) glypican or a fragment having a glypican growth factor binding domain thereof, and
(f) latent TGF-β binding protein or a fragment having a latent TGF-β binding protein growth factor binding domain thereof,
wherein the modified laminin is a chimeric molecule comprising the growth factor binding molecule and the laminin E8 fragment, and wherein the chimeric molecule is a fusion protein.

Assignees

• Sekiguchi Kiyotoshi
• Taniguchi Yukimasa
• Nakagawa Masato
• Univ Osaka
• Univ Kyoto

Inventors

• Sekiguchi Kiyotoshi
• Taniguchi Yukimasa
• Nakagawa Masato

Classifications

• C07K2319/42

  containing a HA(hemagglutinin)-tag · CPC title

• C12N5/0696 Primary

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• C07K14/78 Primary

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C07K2319/43

  containing a FLAG-tag · CPC title

• C07K2319/21

  containing a His-tag · CPC title