Process of inducing differentiation of embryonic cell to cell expressing neural surface marker using OP9 or PA6 cells

• US 8,273,570 B2
• Filed: 05/16/2001
• Issued: 09/25/2012
• Est. Priority Date: 05/16/2000
• Status: Expired due to Fees

• Current Assignee: Riken, Yoshiki Sasai
• Original Assignee: Riken
• Inventors: Sasai, Yoshiki, Nishikawa, Shin-Ichi
• Primary Examiner(s): Leffers, Jr., Gerald
• Assistant Examiner(s): SGAGIAS, MAGDALENE K
• Application Number:
  US09/855,587
• Publication Number:
  US 20020151056A1
• Time in Patent Office: 4,150 Days
• Field of Search: 435/325, 435/363, 435/366, 435/377, 435/383, 435/368, 435/373, 435/375
• US Class Current: 435/368
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0619 Neurons

Abstract

A method for inducing differentiation of an embryonic stem cell into an ectodermal cell and an ectoderm-derived cell, which comprises culturing the embryonic stem cell under non-aggregation conditions; a medium and a medium supernatant used in the method; an agent for inducing differentiation used in the method; a stroma cell or a stroma cell-derived factor having activity of inducing differentiation in the method; an antibody which specifically recognizes the stroma cell; an antigen which recognizes the antibody; a cell induced by the method; a method for evaluating or screening a substance relating to the regulation in a differentiation step from an embryonic stem cell into an ectodermal cell or an ectoderm-derived cell by carrying out the method; and a medicament comprising the stroma cell, the stroma cell-derived cell, the antibody, the antigen or the cell.

Claims

1. A method for producing a cell expressing a neural crest marker or a neural tube marker, comprising:

culturing under serum-free conditions a mammalian embryonic stem cell in vitro in the absence of retinoic acid and in the presence of a stroma cell without forming embryoid body, wherein the stroma cell is OP9 cell or PA6 cell.

4. The method according to any one of claim 1, 2 or 3, wherein the stroma cell is a stroma cell whose proliferation potency is deleted by a physicochemical treatment.

5. The method according to any one of claim 1, 2 or 3, wherein the stroma cell is a stroma cell whose proliferative potency is deleted by an antitumor agent, irradiation or pathologic tissue fixative.

6. The method according to claim 4, wherein the physiocochemical treatment is an antitumor agent selected from the group consisting of mitomycin C, 5-fluorouracil, adriamycin and methotrexate.

7. The method according to any one of claim 1, 2 or 3, wherein the stroma cell is a stroma cell whose proliferative potency is deleted by a microwave fixation, a rapid freeze-substitution fixation, a glutaraldehyde fixation, a p-formaldehyde fixation, a formalin fixation, an acetone fixation, a Van fixation, a periodic acid fixation, a methanol fixation or an osmic acid fixation.

8. The method according to any one of claim 1, 2 or 3, wherein the stroma cell is an M-CSF deficient mouse calvaria-derived OP9 cell.

9. The method according to claim 8, wherein the stroma cell is recognized by a monoclonal antibody produced by hybridoma FERM BP-7573.

10. The production method according to claim 1, wherein the method produces a cell expressing said neural crest marker.

11. The production method according to claim 1, wherein the method produces a cell expressing said neural tube marker.

16. The method according to any one of claims 1, 2 or 3, wherein the stroma cell is a mouse calvaria-derived MC3T3-G2/PA6 cell.

17. The method according to claim 8, wherein the stroma cell is recognized by a monoclonal antibody produced by hybridoma FERM BP-7573.

18. The method according to claim 16, wherein the stroma cell is recognized by a monoclonal antibody produced by hybridoma FERM BP-7573.

2. A method for producing a dopaminergic neuron, an acetylcholinergic neuron, a γ

-aminobutyratergic neuron or a serotonergic neuron, comprising;

culturing under serum-free conditions a mammalian embryonic stem cell in vitro in the absence of retinoic acid and in the presence of a stroma cell without forming embryoid body, wherein the stroma cell is OP9 cell or PA6 cell.

12. The production method according to claim 2, wherein the method produces said dopaminergic neuron.

13. The production method according to claim 2, wherein the method produces said acetylcholinergic neuron.

14. The production method according to claim 2, wherein the method produces said γ

-aminobutyratergic neuron.

15. The production method according to claim 2, wherein the method produces said serotonergic neuron.

3. A method for producing a neural stem cell which is stained by an anti-nestin antibody comprising:

culturing under serum-free conditions a mammalian embryonic stem cell in vitro in the absence of retinoic acid and in the presence of a stroma cell without forming embryoid body, wherein the stroma cell is OP9 cell or PA6 cell.