Pluripotent cells

• US 9,834,751 B2
• Filed: 08/11/2016
• Issued: 12/05/2017
• 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): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US15/234,839
• Publication Number:
  US 20160348071A1
• Time in Patent Office: 481 Days
• Field of Search: 435325
• 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 method of reducing spontaneous behavioral epileptic seizure activity in a mammalian subject comprising:

(a) isolating mammalian cells from fetal telencephalon and/or mesencephalon, wherein the mammalian cells co-express nestin, Oct-4, and a marker selected from the group consisting of TRA-1-60, TRA-1-81 and SSEA-4,

(b) suspending the isolated mammalian cells in a culture medium that has a total calcium concentration of 0.03 to 0.15 mM,

(c) transplanting the suspended mammalian cells of step (b) into a cerebral ventricle of the subject who has had a seizure within the preceding week, and

(d) measuring spontaneous behavioral epileptic seizures in the mammalian subject treated with the transplanted cells of step (c), wherein spontaneous behavioral epileptic seizure activity is reduced following transplantation.

2. The method of claim 1, wherein the subject has had a seizure within the preceding 48 hours.

3. The method of claim 1, wherein the cells in step (a) continue to proliferate in an undifferentiated state for at least 4 months.

4. The method of claim 1, wherein the cells in step (a) have a doubling rate of less than 12 days.

5. The method of claim 1, wherein the cells in step (a) have a doubling rate of about 5 days.

6. The method of claim 1, wherein the cells in step (a) continue to proliferate for 2 years in vitro.

7. The method of claim 1, wherein the subject is human, equine, canine, feline, porcine, ovine or rodent.

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

9. The method of claim 8, wherein the total calcium concentration is about 0.05 mM.

10. The method 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

).

11. The method of claim 10, wherein the medium further comprises:

(d) about 25-150 ng/μ

l leukemia inhibiting factor (LIF).

12. The method of claim 11, wherein the LIF is about 25 ng/μ

l.

13. The method of claim 10, wherein the EGF is about 20 ng/μ

l.

14. The method of claim 10, wherein the bFGF is about 10 ng/μ

l.

15. The method of claim 10, wherein the TGFα

is about 10 ng/μ

l.

16. The method of claim 10, wherein the medium is free of a feeder layer.

17. The method of claim 10, wherein the medium is serum-free.

18. The method of claim 10, wherein the medium further comprises 0.5-2.5% B27 supplement.

19. The method of claim 10, wherein the growth factors EGF, bFGF and TGFα

are recombinant growth factors.

20. The method of claim 10, wherein the cells and the growth factors are human.

21. The method of claim 10, wherein the medium further comprises about 0.11 mg/ml sodium pyruvate.