METHODS FOR OBTAINING STEM CELLS

• US 20130149286A1
• Filed: 06/03/2011
• Published: 06/13/2013
• Est. Priority Date: 06/04/2010
• Status: Active Grant

• Current Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Original Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Inventors: Chretien, Fabrice, Latil, Mathilde, Tajbakhsh, Shahragim
• Granted Patent: US 9,499,792 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A01N 1/021 Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
• CPC Class Codes: A01N 1/0284 Temperature processes, i.e. using a designated change in temperature over time
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/14 Blood; Artificial blood perfluorocarbons A61K31/02; umbilical cord blood A61K35/51; haemoglobin A61K38/42
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0659 Satellite cells
• CPC Class Codes: C12N 5/0668 Mesenchymal stem cells from other natural sources

Abstract

The present invention relates to methods for obtaining stem cells from mammalian cadavers, methods for obtaining or purifying stem cells from a sample likely to contain non-stem cells, methods of regeneration of injured tissues and methods of treatment.

Claims

1. A method for obtaining mammalian stem cells comprising the following steps:

a) harvesting a tissue from a mammalian cadaver stored at 1-6°

C., the harvesting being performed on a part of the cadaver'"'"'s body in which stem cells are usually present in a living counterpart; and

b) extracting mononuclear cells from the harvested tissue.

2. The method according to claim 1, wherein the method comprises an additional step b′

) following step b) consisting of culturing the extracted mononuclear cells.

4. The method according to claim 1, wherein the mammalian cadaver was stored at 1-6°

C. from 0 minute to 48 hours after death.

5. The method according to claim 1, wherein step a) is performed after a period of time following death comprised between 0 minute and 30 days.

6. The method according to claim 1, wherein the mammalian cadaver is a human cadaver.

7. The method according to claim 1, wherein:

step a) is performed on trabecular bone or peripheral blood;

the harvested tissue is bone marrow or peripheral blood; and

the stems cells obtained are hematopoietic stem cells, peripheral hematopoietic stem cells or mesenchymal stem cells.

8. The method according to claim 1, wherein:

harvesting of step a) is performed on muscle, especially skeletal or smooth muscle;

the harvested tissue is muscle sample, especially skeletal or smooth muscle sample; and

the stems cells obtained are muscle stem cells.

9. The method according to claim 1, wherein:

harvesting of step a) is performed on brain or spinal cord at least 24 hours after death;

the harvested tissue is brain, spinal cord or meninges sample; and

the stems cells obtained are neural stem cells

10. The method according to claim 1, further comprising an ultimate step following step b), step b′

) or step c) where appropriate, consisting of resuspending the extracted or cultivated mononuclear cells in a pharmaceutically acceptable carrier.

3. A method for obtaining mammalian stem cells expressing a transgene of interest, comprising the following steps:

a) harvesting a tissue from a mammalian cadaver stored at 1-6°

C., the harvesting being performed on a part of the cadaver'"'"'s body in which stem cells are usually present in a living counterpart; and

b) extracting mononuclear cells from the harvested tissue;

b′

) optionally culturing the extracted mononuclear cells of step b); and

c) transforming or transfecting with a vector, especially a vector of expression, or transducing with a virus vector, the mononuclear cells of step b), or step b′

) where appropriate, said vector comprising at least one polynucleotide sequence of interest.

11. A method for obtaining stem cells comprising the following steps:

a) maintaining a biological material which usually comprises stem cells at an oxygen concentration equal to or less than 0.1%; and

b) selecting viable cell, wherein viable cells are stem cells.

13. The method according to claim 11, wherein the biological material is obtained from a living donor, except human embryo or foetus.

14. The method according to claim 11, wherein the biological material is maintained at 1-37°

C. during a period of time of 12 hours to 60 days.

15. The method according to claim 11, wherein the biological material is maintained at 1-37°

C. in the absence of oxygen.

16. The method according claim 11, wherein the biological material is selected from the group consisting of bone marrow and circulating or peripheral blood, and wherein the selected viable stem cells are hematopoietic stem cells, circulating or peripheral hematopoietic stem cells or mesenchymal stem cells.

17. The method according to claim 11, wherein the biological material is muscle sample, especially skeletal or smooth muscle sample, and wherein the selected viable stem cells are muscle stem cells.

18. The method according to claim 11, wherein the biological material is selected from the group consisting of brain, spinal cord and meninges sample, and wherein the selected viable stem cells are neural stem cells.

19. The method according claim 11, wherein the biological material was obtained from a mammal cadaver stored at 1-6°

C. from 0 minutes to 48 hours after death.

20. The method according to claim 11, wherein the biological material was obtained from a living mammal.

21. The method according to claim 11, further comprising an ultimate step following step b), or step c) where appropriate, consisting of resuspending the extracted or cultivated mononuclear cells in a pharmaceutically acceptable carrier.

22. Stem cells obtained according to the method of claim 11, for use in the regeneration of an injured tissue.

12. A method for obtaining mammalian stem cells expressing a transgene of interest, comprising the following steps:

a) maintaining a biological material which usually comprises stem cells at an oxygen concentration equal to or less than 0.1%; and

b) selecting viable cell, wherein viable cells are stem cells.

c) transforming or transfecting with a vector, especially a vector of expression, or transducing with a virus vector, the viable cells of step b), said vector comprising at least one polynucleotide sequence of interest.

23. A method for culturing mammalian stem cells comprising using an anaerobic cell culture system for culturing mammalian stem cells at an oxygen concentration equal to or less than 0.1%.

24. The method according to claim 23, wherein mammalian stem cells are cultured at 1-37°

C. for 12 hours to 30 days.

25. The method according to claim 24, wherein the mammalian stem cells are Hematopoietic stem cells or muscle stem cells.

26. A method for selecting stem cells comprising using an anaerobic cell culture system for selecting for stem cells by maintaining at an oxygen concentration equal to or less than 0.1% a biological sample comprising stem cells and non-stem cells.

27. An isolated muscle stem cell characterized in that it does not express myogenin gene (Myogenin−

).

28. An isolated muscle stem cell according to claim 27 characterized in that it is Pax7high and/or of CD34+.

29. An isolated muscle stem cell according to claim 27, characterized in that it has a low level of mitochondrial activity.

30. An isolated muscle stem cell as defined in claim 27, for use in the regeneration of an injured tissue.