Postmortem stem cells

• US 20020098584A1
• Filed: 11/06/2001
• Published: 07/25/2002
• Est. Priority Date: 11/06/2000
• Status: Abandoned Application

• Current Assignee: Children's Hospital of Orange County, Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Palmer, Theo D., Taupin, Philippe, Schwartz, Philip H., Gage, Fred H.
• Application Number:
  US10/012,885
• Publication Number:
  US 20020098584A1
• Time in Patent Office: Days
• US Class Current: 435/366
• 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 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2502/99 genetically modified cells
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

Disclosed are optimized methodologies for isolating and propagating stem cells from biopsies and postmortem tissues. Specifically disclosed are methods of culturing neural stem cells in the presence of a cocktail of trophic factors/co-factors for enhanced propagation.

Claims

1. A tissue culture medium for propagating postmortem stem cells comprising:

a base culture medium and a stem cell differentiating concentration of at least one trophic factors.

2. The tissue culture medium of claim 1, wherein the base culture medium is a defined medium.

3. The tissue culture medium of claim 2, wherein the defined medium is selected from the group consisting of Minimum Essential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free), F10(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification), Basal Medium Eagle (BME-with the addition of Earle'"'"'s salt base), Dulbecco'"'"'s Modified Eagle Medium (DMEM-without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy'"'"'s 5A Medium, Medium M199 (M199E-with Earle'"'"'s sale base), Medium M199 (M199H-with Hank'"'"'s salt base), Minimum Essential Medium Eagle (MEM-E-with Earle'"'"'s salt base), Minimum Essential Medium Eagle (MEM-H-with Hank'"'"'s salt base) and Minimum Essential Medium Eagle (MEM-NAA with non essential amino acids), medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams'"'"' G, Neuman &

Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, and MDBC 153.

4. The tissue culture medium of claim 2, wherein the defined medium is Dulbecc/Vogt modified Eagle'"'"'s minimal essential medium (DMEM):

F12 medium at a 1;

1 ratio.

5. The tissue culture medium of claim 1, wherein said trophic factor is a growth factor.

6. The tissue culture medium of claim 5, wherein said growth factor is selected from the group consisting of epidermal growth factor (EGF), platelet derived growth factor (PDGF), and Fibroblast Growth Factor-2 (FGF-2).

7. The tissue culture medium of claim 1, wherein the trophic factor comprises a trophic co-factor.

9. The tissue culture medium of claim 1, wherein said trophic factor comprises a combination of Fibroblast Growth Factor-2 (FGF-2), platelet derived growth factor (PDGF), epidermal growth factor (EGF), and glycosylated cystatin C.

18. A postmortem stem cell, wherein the postmortem stem cell is derived by growing a postmortem cell sample in the culture medium of claim 1.

8. The tissue culture medium of claim 8, further comprising glycosylated cystatin C.

10. A postmortem tissue culture medium kit comprising in a suitable container:

base culture medium reagents in suitable quantities to formulate a base culture medium; and

at least one trophic factors in a suitable quantity to formulate a stem cell differentiating concentration of said at least one trophic factors.

11. The postmortem tissue culture medium kit of claim 10, wherein the base culture medium is a defined medium.

12. The postmortem tissue culture medium kit of claim 11, wherein the defined medium is selected from the group consisting of Minimum Essential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free), F10(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ. Medium (with and without Fitton-Jackson Modification), Basal Medium Eagle (BME-with the addition of Earle'"'"'s salt base), Dulbecco'"'"'s Modified Eagle Medium (DMEM-without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy'"'"'s 5A Medium, Medium M199 (M199E-with Earle'"'"'s sale base), Medium M199 (M199H-with Hank'"'"'s salt base), Minimum Essential Medium Eagle (MEM-E-with Earle'"'"'s salt base), Minimum Essential Medium Eagle (MEM-H-with Hank'"'"'s salt base) and Minimum Essential Medium Eagle (MEM-NAA with non essential amino acids), medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams'"'"' G, Neuman &

Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, and MDBC 153.

13. The postmortem tissue culture medium kit of claim 11, wherein the defined medium is Dulbecco/Vogt modified Eagle'"'"'s minimal essential medium (DMEM):

F12 medium at a 1;

1 ratio.

14. The postmortem tissue culture medium kit of claim 10, wherein said trophic factor is a growth factor.

15. The postmortem tissue culture medium kit of claim 14, wherein said growth factor is is selected from the group consisting of epidermal growth factor (EGF), platelet derived growth factor (PDGF), and Fibroblast Growth Factor-2 (FGF-2).

16. The postmortem tissue culture medium kit of claim 10, further comprising glycosylated cystatin C.

17. The postmortem tissue culture medium kit of claim 16, wherein said trophic factor comprises a combination of Fibroblast Growth Factor-2 (FGF-2), platelet derived growth factor (PDGF) and epidermal growth factor (EGF).

19. The postmortem stem cell of claim 18, wherein said postmortem cell sample comprises brain cells.

20. The postmortem stem cell of claim 19, wherein the brain cells are derived from the temporal cortex, hippocampus or the ventricular zone of the brain.

21. The postmortem stem cell of claim 18, wherein said postmortem cell sample comprise neural precursor cells.

22. The postmortem stem cell of claim 18, wherein said postmortem cell sample is cultured in the presence of conditioned media taken from stem cells that produce recombinant glycosylated cystatin C.

23. The postmortem stem cell of claim 18, wherein said postmortem cell sample is grown for greater than about 70 population doublings.

24. The postmortem stem cell of claim 18, wherein said postmortem cell sample is taken from the host more than about 20 hours following death.

26. The method of claim 25, wherein said postmortem cells are brain cells.

27. The method of claim 27, wherein the brain cells are derived from the temporal cortex, hippocampus or the ventricular zone of the brain.

28. The method of claim 25, wherein said postmortem cells are neural precursor cells.

29. The method of claim 25, comprising culturing said postmortem cells in the presence of conditioned media taken from stem cells that produce recombinant glycosylated cystatin C.

30. The method of claim 25, wherein said trophic factor is selected from the group consisting of epidermal growth factor (EGF), platelet derived growth factor (PDGF), and Fibroblast Growth Factor-2 (FGF-2).

31. The method of claim 25, wherein said trophic factor comprises a combination of Fibroblast Growth Factor-2 (FGF-2), platelet derived growth factor (PDGF), epidermal growth factor (EGF), and glycosylated cystatin C.

32. The method of claim 25, wherein said postmortem cells are grown for greater than about 70 population doublings.

33. The method of claim 25, wherein the postmortem cells are taken more than about 20 hours following death.

25. A method of growing postmortem cells in culture, comprising:

providing postmortem cells; and

culturing said postmortem cells in the presence of a trophic factor and glycosylated cystatin C.