BRAIN-DERIVED STEM CELLS FOR REPAIR OF MUSCULOSKELETAL SYSTEM IN VERTEBRATE SUBJECTS

• US 20100209399A1
• Filed: 10/13/2009
• Published: 08/19/2010
• Est. Priority Date: 02/13/2009
• Status: Abandoned Application

• Current Assignee: Celavie Biosciences, LLC
• Original Assignee: Celavie Biosciences, LLC
• Inventors: Kopyov, Oleg V.
• Application Number:
  US12/578,263
• Publication Number:
  US 20100209399A1
• Time in Patent Office: Days
• US Class Current: 424/93.700
• 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 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system

Abstract

The stem cells that can be propagated and maintained for extended periods of time in culture in the absence of a feeder layer, and can be used to repair tissue damage. These cells are derived from fetal tissues and are able to repair different types of damage in musculoskeletal system, with significantly greater efficacy than stem cells derived from adult tissues. These cells are hypoimmunogenic and can be used for allogeneic transplantation to vertebrate hosts having disease and/or damage in musculoskeletal and other tissues. The cells can be administered by direct injection to the site in need of repair or by systemic (e.g., intravenous) administration. The stem cells of the invention are capable of migrating to the sites in need of repair, and of adopting a phenotype most appropriate to the nature of the damage, injury or disease.

Claims

1. A method of ameliorating connective tissue damage in a mammalian subject, the method comprising introducing into a site of connective tissue damage in the subject about 1 to about 10 million stem cells, wherein the stem cells are derived from fetal mammalian brain.

2. The method of claim 1, wherein the stem cells do not express major histocompatibility complex (MHC).

3. The method of claim 1, wherein the stem cells are allogeneic.

4. The method of claim 1, wherein the stem cells are cultured for about 30 days prior to the introducing.

5. The method of claim 4 wherein the stem cells are immunopositive for telomerase, Oct-4, SSEA-4 and Nanog, and do not express major histocompatibility complex (MHC) or p53.

6. The method of claim 4 wherein the stem cells are cultured in a medium having a total calcium concentration of 0.03 to 0.15 mM and comprising:

(a) about 15-100 ng/ml epidermal growth factor (EGF);

(b) about 10-150 ng/ml basic fibroblast growth factor (bFGF);

(c) about 10-75 ng/ml transforming growth factor-alpha (TGFα

); and

(d) about 30-50 ng/ml insulin-like growth factor (IGF).

7. The method of claim 6, wherein the medium further comprises:

(e) about 1-3% by volume B27;

(f) about 40-60 ng/ml leukemia inhibitory factor (LIF);

(g) about 0.05-0.2 mM GLUTAMAX; and

(h) about 0.5-2% by volume N2 supplement.

8. The method of claim 7, wherein the medium is Eagle'"'"'s minimum essential medium (EMEM) and comprises:

(a) about 40 ng/ml epidermal growth factor (EGF);

(b) about 40 ng/ml basic fibroblast growth factor (bFGF); and

(c) about 40 ng/ml transforming growth factor-alpha (TGFα

).

(d) about 40 ng/ml insulin-like growth factor (IGF);

(e) about 50 ng/ml leukemia inhibitory factor (LIF);

(f) about 2% by volume B27;

(g) about 0.05-0.2 mM GLUTAMAX;

(h) about 0.5-2% by volume N2 supplement; and

(i) about 0.05 mM calcium chloride.

9. The method of claim 1, wherein the introducing comprises injection into the site of connective tissue damage.

10. The method of claim 9, wherein the injection is performed under ultrasound guidance.

11. The method of claim 1, wherein the cells are derived from equine fetal tissue.

12. The method of claim 1, wherein the mammalian subject is equine.

13. The method of claim 1, wherein the mammalian subject is canine or feline.

14. The method of claim 1, wherein the mammalian subject is human.

15. The method of claim 1, wherein the connective tissue damage comprises ligament damage, osteochondrosis, tendonitis, navicular syndrome, cartilage damage, laminitis or arthritis.

16. The method of claim 1, wherein the introducing comprises intravenous injection.

17. The method of claim 1, wherein the introducing comprises distal end perfusion.

18. A kit comprising:

(a) a container comprising about 1 to about 10 million stem cells, wherein the stem cells are derived from fetal mammalian brain;

(b) a label that indicates use of the cells for implantation into a site of connective tissue damage.

19. The kit of claim 18, further comprising:

(c) a needle suitable for transcutaneous injection.

20. The kit of claim 18, wherein the container comprises a syringe.

21. The kit of claim 18, wherein the fetal mammalian brain is equine.

22. The kit of claim 18, wherein the cells do not express MHC.

23. The kit of claim 18, further comprising a second container, the second container comprising platelet-rich plasma.