Modified laminin and use thereof
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.
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.