Method for preparing skin-derived pluripotent precursor cells

Patent metadata

Field	Value
Publication number	US-2022340868-A1
Application number	US-202017633082-A
Country	US
Kind code	A1
Filing date	Aug 4, 2020
Priority date	Aug 6, 2019
Publication date	Oct 27, 2022
Grant date	—

Abstract

The present invention relates to improvement of the yield of skin-derived pluripotent precursor cells in induction of differentiation of stem cells to skin-derived pluripotent precursor cells. The present invention provides a method for preparing skin-derived pluripotent precursor cell comprising culturing a neural crest stem cells in the presence of at least one selected from the group consisting of laminin and a fragment thereof to differentiate the cells to skin-derived pluripotent precursor cells, wherein the laminin is at least one selected from the group consisting of laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, laminin 521, and a variant thereof.

First claim

1 . A method for preparing skin-derived pluripotent precursor cells, the method comprising
culturing neural crest stem cells in the presence of at least one selected from the group consisting of laminin and a fragment thereof to differentiate the cells to skin-derived pluripotent precursor cells, wherein the laminin is at least one selected from the group consisting of laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, laminin 521, and a variant thereof.

2 . The method according to claim 1 , further comprising preparing the neural crest stem cells,
wherein the preparation comprises culturing pluripotent stem cells in the presence of at least one selected from the group consisting of laminin and a fragment thereof to differentiate the cells to neural crest stem cells, wherein the laminin is at least one selected from the group consisting of laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, laminin 521, and a variant thereof.

3 . The method according to claim 1 , wherein the laminin fragment is a laminin fragment having an integrin binding activity.

4 . The method according to claim 1 , wherein the laminin fragment is a laminin fragment comprising laminin E8 fragment.

5 . The method according to claim 1 , wherein the at least one selected from the group consisting of the laminin and the fragment thereof is the laminin or the fragment thereof bound to perlecan or a fragment comprising a growth factor binding site thereof.

6 . The method according to claim 1 , wherein the culturing the neural crest stem cells in the presence of the at least one selected from the group consisting of the laminin and the fragment thereof comprises culturing the cells on a culture substrate comprising the at least one selected from the group consisting of the laminin and the fragment thereof or culturing the cells in a medium comprising the at least one selected from the group consisting of the laminin and the fragment thereof.

7 . The method according to claim 2 , wherein the culturing the pluripotent stem cells in the presence of the at least one selected from the group consisting of the laminin and the fragment thereof comprises culturing the cells on a culture substrate comprising the at least one selected from the group consisting of the laminin and the fragment thereof or culturing the cells in a medium comprising the at least one selected from the group consisting of the laminin and the fragment thereof.

8 . The method according to claim 1 , wherein the culturing neural crest stem cells comprises culturing the cells in a differentiation medium comprising a Wnt signaling agonist.

9 . The method according to claim 2 , wherein the culturing pluripotent stem cells comprises culturing the cells in a differentiation medium comprising at least one selected from the group consisting of TGFβ signal inhibitor and BMP signal inhibitor.

10 . The method according to claim 1 , wherein the skin-derived pluripotent precursor cells co-expresses nestin and fibronectin.

11 . The method according to claim 1 , wherein the neural crest stem cells are human-derived neural crest stem cells.

12 . A method for preparing neural crest stem cells, the method comprising
culturing pluripotent stem cells in the presence of at least one selected from the group consisting of laminin and a fragment thereof bound to perlecan or a fragment comprising a growth factor binding site thereof to differentiate the cells to neural crest stem cells, wherein the laminin is at least one selected from the group consisting of laminin 111, laminin 121, laminin 332, laminin 421, laminin 511, laminin 521, and a variant thereof.

13 . The method according to claim 12 , wherein the laminin fragment is a laminin fragment having an integrin binding activity.

14 . The method according to claim 12 , wherein the laminin fragment is a laminin fragment comprising a laminin E8 fragment.

15 . The method according to claim 12 , wherein the culturing the cells in the presence of the at least one selected from the group consisting of the laminin and the fragment thereof bound to perlecan or a fragment comprising a growth factor binding site thereof comprises culturing the cells on a culture substrate comprising the at least one selected from the group consisting of the laminin and the fragment thereof bound to perlecan or a fragment comprising a growth factor binding site thereof or culturing the cells in a medium comprising the at least one selected from the group consisting of the laminin and the fragment thereof bound to perlecan or a fragment comprising a growth factor binding site thereof.

16 . The method according to claim 12 , wherein the culturing the pluripotent stem cells comprises culturing the cells in a differentiation medium comprising at least one selected from the group consisting of TGFβ signal inhibitor and BMP signal inhibitor.

17 . The method according to claim 12 , wherein the pluripotent stem cells are human-derived pluripotent stem cells.

18 . The method according to claim 1 , wherein the culturing the cells is performed without using feeder cells.

19 . The method according to claim 1 , wherein the culturing the cells is performed in the absence of a xenogeneic component.

20 - 27 . (canceled)

Assignees

• Kao Corp
• Univ Osaka

Inventors

• Nakagiri Yoriko
• Sekiguchi Kiyotoshi

Classifications

• C12N2501/115

  Basic fibroblast growth factor (bFGF, FGF-2) · CPC title

• C07K14/78

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

• C12N2501/415

  Wnt; Frizzeled · CPC title

• A61K35/36

  Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells (islets of Langerhans A61K35/39) · CPC title

• C12N2501/15

  Transforming growth factor beta (TGF-β) · CPC title