Pluripotent cells

• US 8,367,406 B2
• Filed: 07/20/2009
• Issued: 02/05/2013
• Est. Priority Date: 12/02/2003
• Status: Active Grant

• Current Assignee: Celavie Biosciences, LLC
• Original Assignee: Celavie Biosciences, LLC
• Inventors: Kopyov, Oleg V.
• Primary Examiner(s): FALK, ANNE MARIE
• Application Number:
  US12/506,128
• Publication Number:
  US 20090280097A1
• Time in Patent Office: 1,296 Days
• Field of Search: None
• US Class Current: 435/325
• CPC Class Codes: A01N 1/0205 Chemical aspects
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• 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: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: C12N 2500/14 Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• 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 2501/148 Transforming growth factor alpha [TGF-a]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

Pluripotent cells that are immunopositive for both the neural progenitor marker nestin and a pluripotent cell marker are provided. The cells exhibit rapid doubling times and can be maintained in vitro for extended periods. Also provided are cell cultures containing the pluripotent cells, a method of transplanting human pluripotent cells to a host, and a method of reducing seizure activity in a subject. These pluripotent cells, when transplanted into the ventricle of a host animal, migrate to the site of damage and adopt a suitably corrective phenotype, resulting in both structural and functional restoration.

Claims

1. A composition comprising a plurality of isolated mammalian cells that co-express nestin and a marker of pluripotent cells, and a culture medium that has a total calcium concentration of 0.03 to 0.15 mM, wherein the cells are suspended in the culture medium and wherein the cells continue to proliferate in an undifferentiated state for at least 4 months.

2. The composition of claim 1, wherein the marker of pluripotent cells is selected from the group consisting of TRA-1-60, TRA-1-81 and SSEA-4.

3. The composition of claim 1, wherein the cells have a doubling rate of less than 12 days.

4. The composition of claim 1, wherein the cells have a doubling rate of about 5 days.

5. The composition of claim 1, wherein the cells continue to proliferate for 2 years in vitro.

6. The composition of claim 1, wherein the cells are obtained from fetal forebrain.

7. The composition of claim 1, wherein the medium has a total calcium concentration of less than 0.1 mM.

8. The composition of claim 7, wherein the total calcium concentration is about 0.05 mM.

9. The composition of claim 1, wherein the medium further comprises:

(a) about 15-100 ng/μ

l epidermal growth factor (EGF);

(b) about 10-150 ng/μ

l basic fibroblast growth factor (bFGF);

(c) about 10-75 ng/μ

l transforming growth factor-alpha (TGFα

).

10. The composition of claim 9, further comprising:

(d) about 25-150 ng/μ

l leukemia inhibiting factor (LIF).

11. The composition of claim 10, wherein the LIF is about 25 ng/μ

l.

12. The composition of claim 9, wherein the EGF is about 20 ng/μ

l.

13. The composition of claim 9, wherein the bFGF is about 10 ng/μ

l.

14. The composition of claim 9, wherein the TGFα

is about 10 ng/μ

l.

15. The composition of claim 9, wherein the culture medium is free of a feeder layer.

16. The composition of claim 9, wherein the culture medium is serum-free.

17. The composition of claim 9, further comprising 0.5-2.5% B27 supplement.

18. The composition of claim 9, wherein the growth factors EGF, bFGF and TGFα

are recombinant growth factors.

19. The composition of claim 9, wherein the cells and the growth factors are human.

20. The composition of claim 9, wherein the culture medium further comprises about 0.11 mg/ml sodium pyruvate.

21. A method of propagating pluripotent cells, comprising culturing the composition of claim 1.

22. The method of claim 21, wherein the culturing is in the absence of a feeder layer.

23. A plurality of isolated mammalian cells that co-express nestin and a marker of pluripotent cells, wherein the cells are immunopositive for nestin and for a marker of pluripotent cells, the marker selected from the group consisting of TRA-1-60, TRA-1-81 and SSEA-4, and a culture medium, wherein the cells are suspended in the culture medium, wherein the culture medium has a total calcium concentration of 0.03 to 0.09 mM, and wherein the cells continue to proliferate in an undifferentiated state for at least 4 months.