Method for inducing differentiation mesodermal stem cells, es cells or immortalized cells into nervous system cells

• US 20040259254A1
• Filed: 08/03/2004
• Published: 12/23/2004
• Est. Priority Date: 10/30/2001
• Status: Abandoned Application

• Current Assignee: ASKA Pharmaceutical Company Limited, NC Medical Research Inc. (e-solutions, Inc. /Japan/), Mitsui Sumitomo Insurance Care Network Co., Ltd. (Ms&Ad Insurance Group Holdings Incorporated)
• Original Assignee: NC Medical Research Inc. (e-solutions, Inc. /Japan/)
• Inventors: Hamada, Hirofumi, Honmou, Osamu
• Application Number:
  US10/493,964
• Publication Number:
  US 20040259254A1
• Time in Patent Office: Days
• US Class Current: 435/456
• 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: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/1353 from bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 2506/1369 from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
• CPC Class Codes: C12N 5/0618 Cells of the nervous system

Abstract

Mesodermal stem cells or ES cells, prepared from the mononuclear cell fraction isolated from bone marrow fluid or umbilical blood, were found to differentiate into neural stem cells, neurons, or glial cells when cultured in a basal culture medium. In addition, the differentiation of the mesodermal stem cells or ES cells into neural cells was promoted through the addition of an ischemic brain extract to the above-mentioned basal culture medium. Furthermore, the neural cells obtained using the above-described method for inducing differentiation were revealed to have neural regeneration potency in a brain infarction model, a dementia model, a spinal cord injury model and a demyelination model.

In addition, according to the present invention, mesodermal stem cells can be differentiated into neural cells by immortalizing the mesodermal stem cells by highly expressing or activating an immortalization gene in the mesodermal stem cells and culturing the cells under an appropriate condition. The methods of the present invention are very useful in the medical field of neural regeneration.

Claims

1. A method for inducing differentiation of a mesodermal stem cell or ES cell derived from a mononuclear cell fraction isolated from bone marrow fluid or umbilical blood collected from a vertebrate into a neural cell by incubating the cell in a basal culture medium at 33°

C. to 38°

C.

11. The method according to claim 1, wherein the mononuclear cell fraction can be prepared by subjecting the bone marrow fluid or umbilical blood collected from a vertebrate to density-gradient centrifugation in a solution at 2000 rpm for a sufficient time ensuring separation depending on the specific gravity, and then recovering a cell fraction within the range of specific gravity 1.07 g/ml to 1.1 g/ml.

12. The method according to claim 1, wherein the mesodermal stem cell has the characteristics of SH2(+), SH3(+), SH4(+), CD29(+), CD44(+), CD14(−

), CD34(−

), and CD45(−

).

14. The method according to claim 1, which further comprises adding bFGF, EGF, or ischemic brain extract to a basal culture medium.

15. The method according to claim 1 wherein the neural cell is selected from the group consisting of neural stem cell, neural progenitor cell, neuron, and glial cell.

16. A cell obtained by the method according to claim 1.

17. A composition for treating a neurological disease, which comprises the cell according to claim 16.

18. The composition according to claim 17, wherein the neurological disease is selected from the group consisting of degenerative diseases and demyelinating diseases in the central and peripheral nervous systems, cerebral apoplexy, brain tumor, higher brain dysfunction, psychiatric diseases, epilepsy, traumatic neurological diseases, inflammatory diseases, infectious diseases and spinal cord infarction.

19. A method for treating a neurological disease, which comprises transplanting the cell or the composition according to claim 17 into a recipient.

20. The method according to claim 19, wherein the neurological disease is selected from the group consisting of degenerative diseases and demyelinating diseases in the central and peripheral nervous systems, cerebral apoplexy, brain tumor, higher brain dysfunction, psychiatric diseases, epilepsy, traumatic neurological diseases, inflammatory diseases, infectious diseases and spinal cord infarction.

21. The method according to claim 19, wherein the cell for transplantation is derived from the recipient.

2. A method for inducing differentiation of a mesodermal stem cell into a neural cell, which comprises the steps of:

(a) providing an immortalized mesodermal stem cell by highly expressing or activating an immortalization gene; and

(b) inducing the differentiation of the immortalized mesodermal stem cell of step (a) into a neural cell by culturing the mesodermal stem cell in a basal culture medium.

3. The method according to claim 2, wherein the immortalization gene is highly expressed or activated through the introduction of the immortalization gene into the mesodermal stem cell.

4. The method according to claim 3, wherein the mesodermal stem cell is immortalized by introducing the immortalization gene into the mesodermal stem cell using a retroviral vector.

5. The method according to claim 4, wherein the retroviral vector is pBabe.

6. The method according to claim 2, wherein the immortalization gene of the neural cell can be or has been removed by a gene-removing treatment.

7. The method according to claim 6, wherein the gene-removing treatment of the immortalization gene is performed by sandwiching the immortalization gene within a pair of loxP sequences or loxP-like sequences and then treating with a recombinase.

8. The method according to claim 2, wherein the immortalization gene is any one selected from the group of telomerase gene, genes derived from the telomerase, and genes which regulate the expression or activity of the telomerase.

9. The method according to claim 2, wherein the mesodermal stem cell is derived from bone marrow fluid, umbilical blood, peripheral blood, skin, hair root cells of skin, muscle tissue, ES cells or cells derived from ES cells.

10. The method according to claim 2, wherein the mesodermal stem cell is contained in a mononuclear cell fraction isolated from bone marrow fluid or umbilical blood collected from a vertebrate.

13. The method according to claim 2, wherein the culture is carried out at 33°

C. to 38°

C.