Therapeutic reprogramming of germ line stem cells

• US 20070020759A1
• Filed: 07/17/2006
• Published: 01/25/2007
• Est. Priority Date: 07/15/2004
• Status: Abandoned Application

• Current Assignee: PrimeGen Biotech LLC
• Original Assignee: PrimeGen Biotech LLC
• Inventors: Silva, Francisco, Sayre, Chauncey, Pau, Kwok-Yuen
• Application Number:
  US11/488,362
• Publication Number:
  US 20070020759A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/38 Vitamins
• 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 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 5/0611 Primordial germ cells, e.g. embryonic germ cells [EG]

Abstract

Pluripotent therapeutically programmed cells and methods for making such cells are provided. The pluripotent therapeutically programmed cells are post-natal stem cells which have been matured such that they represent either a more differentiated state or a less differentiated state after contact with stimulatory factors. The pluripotent therapeutically reprogrammed cells are suitable for cellular regenerative therapy and have the potential to differentiate into more committed cell lineages. Also disclosed are culture media for therapeutically reprogramming post-natal stem cells.

Claims

1. A therapeutic reprogramming method comprising:

isolating a human post-natal stem cell;

contacting said human post-natal stem cell with a medium comprising stimulatory factors which induce development of said human post-natal stem cell into a therapeutically reprogrammed cell wherein said human post-natal stem cell is therapeutically reprogrammed to a pluripotent human stem cell.

2. The therapeutic reprogramming method of claim 1 further comprising the step of differentiating said pluripotent human stem cells to yield a tissue specific cell.

3. The therapeutic reprogramming method of claim 1 wherein said human post-natal stem cell is a primordial sex cell.

4. The therapeutic reprogramming method of claim 3 wherein said primordial sex cell is a diploid germ cell isolated from a male or female post-natal source.

5. The therapeutic reprogramming method of claim 3 wherein said primordial sex cell is a spermatogonial stem cell.

6. The therapeutic reprogramming method of claim 1 wherein said medium comprises PM-10 medium.

7. The therapeutic reprogramming method of claim 1 wherein said tissue specific cell is selected from the group consisting of cardiac myocytes, glia, neurons, hepatocytes, osteocytes and chondrocytes.

34. The pluripotent therapeutic composition of claim 30 wherein said therapeutically reprogrammed human post-natal stem cell is produced according to the therapeutic reprogramming method of claim 1.

8. A cell culture medium for therapeutically reprogramming post-natal stem cells wherein said cell culture medium comprises:

a cell culture growth medium base;

a plurality of vitamins and minerals; and

a plurality of cell growth and maturation factors,

9. The cell culture medium of claim 8 wherein said post-natal stem cell is a human stem cell.

10. The cell culture medium of claim 8 wherein said medium does not contain serum.

11. The cell culture medium of claim 8 wherein said plurality of cell growth and maturation factors comprise factors selected from the group consisting of recombinant human epidermal growth factor (rhEGF), recombinant human fibroblast growth factor 2 (rhFGF-2), recombinant human glial cell derived neurotrophic factor (rhGDNF) and human leukemia inhibitory factor (LIF), platelet-derived growth factor BB (PDGF-BB), transforming growth factor-β

3 (TGF-β

3), fibroblast growth factor 8 (FGF-8) and sonic hedgehog (SHH).

12. The cell culture medium of claim 11 wherein said recombinant human epidermal growth factor is present at a concentration of between 10 ng/mL and 40 ng/mL.

13. The cell culture medium of claim 12 wherein said recombinant human epidermal growth factor is present at a concentration of 20 ng/mL.

14. The cell culture medium of claim 11 wherein said recombinant human fibroblast growth factor 2 is present at a concentration of between 1 ng/mL and 120 ng/mL.

15. The cell culture medium of claim 14 wherein said recombinant human fibroblast growth factor 2 is present at a concentration of between 1 ng/mL and 2 ng/mL.

16. The cell culture medium of claim 14 wherein said recombinant human fibroblast growth factor 2 is present at a concentration of between 10 ng/mL and 20 ng/mL.

17. The cell culture medium of claim 14 wherein said recombinant human fibroblast growth factor 2 is present at a concentration of 110 ng/mL.

18. The cell culture medium of claim 11 wherein said recombinant human glial cell derived neurotrophic factor is present at a concentration of between 2 ng/mL and 40 ng/mL.

19. The cell culture medium of claim 18 wherein said recombinant human glial cell derived neurotrophic factor is present at a concentration of 2 ng/mL.

20. The cell culture medium of claim 18 wherein said recombinant human glial cell derived neurotrophic factor is present at a concentration of between 10 ng/mL and 20 ng/mL.

21. The cell culture medium of claim 11 wherein said human leukemia inhibitory factor is present at a concentration of between 1,000 units/mL and 10,000 units/mL.

22. The cell culture medium of claim 21 wherein said human leukemia inhibitory factor is present at a concentration of 1,000 units/mL.

23. A cell culture media for therapeutic reprogramming of human post-natal stem cells designated PM-10 consisting essentially of Stem Cell Basal Medium, 2 mM L-glutamine, MEM vitamin solution, 0.1 mM MEM non-essential amino acids, 10⁻

4 M ascorbic acid, 10 μ

g/mL d-biotin, 80 ng/mL β

-estradiol, 60 ng/mL progesterone, 5 mg/mL bovine albumin, 1 μ

L/mL DL-lactic acid, 30 μ

g/mL pyruvic acid, 6 mg/mL D-(+)-glucose, 30 nM sodium selenite, 60 μ

M putrescine, 100 μ

g/mL transferring, 25 μ

g/mL insulin, 1×

penicillin/streptomycin, 10×

10<sup>−

5</sup> M β

-mercaptoethanol, the improvement comprising 1% fetal bovine serum, 20 ng/mL rhEGF, 1 ng/mL rhFGF-2, 10 ng/mL rhGDNF and 1,000 units human LIF.

24. A cell culture media for therapeutic reprogramming of human post-natal stem cells designated PM-20 consisting essentially of Stem Cell Basal Medium, 2 mM L-glutamine, MEM vitamin solution, 0.1 mM MEM non-essential amino acids, 10⁻

4 M ascorbic acid, 10 μ

g/mL d-biotin, 80 ng/mL β

-estradiol, 60 ng/mL progesterone, 5 mg/mL bovine albumin, 1 μ

L/mL DL-lactic acid, 30 μ

g/mL pyruvic acid, 6 mg/mL D-(+)-glucose, 30 nM sodium selenite, 60 μ

M putrescine, 100 μ

g/mL transferring, 25 μ

g/mL insulin, 1×

penicillin/streptomycin, 10×

10<sup>−

5</sup> M β

-mercaptoethanol, the improvement comprising 1% fetal bovine serum, 20 ng/mL rhEGF, 110 ng/mL rhFGF-2, 20 ng/mL rhGDNF and 1,000 units human LIF.

25. A cell culture media for therapeutic reprogramming of human post-natal stem cells designated PM-100 consisting essentially of Stem Cell Basal Medium, 2 mM L-glutamine, MEM vitamin solution, 0.1 mM MEM non-essential amino acids, 10⁻

4 M ascorbic acid, 10 μ

g/mL d-biotin, 80 ng/mL β

-estradiol, 60 ng/mL progesterone, 5 mg/mL bovine albumin, 1 μ

L/mLDL-lactic acid, 30 μ

g/mL pyruvic acid, 6 mg/mL D-(+)-glucose, 30 nM sodium selenite, 60 μ

M putrescine, 100 μ

g/mL transferring, 25 μ

g/mL insulin, 1×

penicillin/streptomycin, 10×

10<sup>−

5</sup> M β

-mercaptoethanol, the improvement comprising 1% fetal bovine serum, 20 ng/mL rhEGF, 110 ng/mL rhFGF-2, 10 ng/mL rhGDNF and 1,000 units human LIF.

26. A cell culture media for therapeutic reprogramming of human post-natal stem cells designated PM-1 consisting essentially of Stem Cell Basal Medium, 2 mM L-glutamine, MEM vitamin solution, 0.1 mM MEM non-essential amino acids, 10⁻

4 M ascorbic acid, 10 μ

g/mL d-biotin, 80 ng/mL β

-estradiol, 60 ng/mL progesterone, 5 mg/mL bovine albumin, 1 μ

L/mLDL-lactic acid, 30 μ

g/mL pyruvic acid, 6 mg/mL D-(+)-glucose, 30 nM sodium selenite, 60 μ

M putrescine, 100 μ

g/mL transferring, 25 μ

g/mL insulin, 1×

penicillin/streptomycin, 10×

10<sup>−

5</sup> M β

-mercaptoethanol, the improvement comprising 1% fetal bovine serum, 20 ng/mL rhEGF, 10 ng/mL rhFGF-2, 2 ng/mL rhGDNF and 1,000 units human LIF.

27. A cell culture media for therapeutic reprogramming of human post-natal stem cells designated PM-3 consisting essentially of Stem Cell Basal Medium, 2 mM L-glutamine, MEM vitamin solution, 0.1 mM MEM non-essential amino acids, 10⁻

4 M ascorbic acid, 10 μ

g/mL d-biotin, 80 ng/mL β

-estradiol, 60 ng/mL progesterone, 5 mg/mL bovine albumin, 1 μ

L/mLDL-lactic acid, 30 μ

g/mL pyruvic acid, 6 mg/mL D-(+)-glucose, 30 nM sodium selenite, 60 μ

M putrescine, 100 μ

g/mL transferring, 25 μ

g/mL insulin, 1×

penicillin/streptomycin, 10×

10<sup>−

5</sup> M β

-mercaptoethanol, the improvement comprising 12% fetal bovine serum, 20 ng/mL rhEGF, 10 ng/mL rhFGF-2, 2 ng/mL rhGDNF and 1,000 units human LIF.

28. A cell culture media for therapeutic reprogramming of human post-natal stem cells designated PM-5 consisting essentially of Stem Cell Basal Medium, 2 mM L-glutamine, MEM vitamin solution, 0.1 mM MEM non-essential amino acids, 10⁻

4 M ascorbic acid, 10 μ

g/mL d-biotin, 80 ng/mL β

-estradiol, 60 ng/mL progesterone, 5 mg/mL bovine albumin, 1 μ

L/mLDL-lactic acid, 30 μ

g/mL pyruvic acid, 6 mg/mL D-(+)-glucose, 30 nM sodium selenite, 60 μ

M putrescine, 100 μ

g/mL transferring, 25 μ

g/mL insulin, 1×

penicillin/streptomycin, 10×

10<sup>−

5</sup> M β

-mercaptoethanol, the improvement comprising 1% fetal bovine serum, 20 ng/mL rhEGF, 2 ng/mL rhFGF-2, 20 ng/mL rhGDNF and 1,000 units human LIF.

29. A cell culture media for therapeutic reprogramming of human post-natal stem cells designated PM-101 consisting essentially of Stem Cell Basal Medium, 2 mM L-glutamine, MEM vitamin solution, 0.1 mM MEM non-essential amino acids, 10⁻

4 M ascorbic acid, 10 μ

g/mL d-biotin, 80 ng/mL β

-estradiol, 60 ng/mL progesterone, 5 mg/mL bovine albumin, 1 μ

L/mLDL-lactic acid, 30 μ

g/mL pyruvic acid, 6 mg/mL D-(+)-glucose, 30 nM sodium selenite, 60 μ

M putrescine, 100 μ

g/mL transferring, 25 μ

g/mL insulin, 1×

penicillin/streptomycin, 10×

10<sup>−

5</sup> M β

-mercaptoethanol, the improvement comprising 1% fetal bovine serum, 20 ng/mL rhEGF, 10 ng/mL rhFGF-2, 10 ng/mL rhGDNF and 1,000 units human LIF.

30. A pluripotent therapeutic composition comprising a therapeutically reprogrammed human post-natal stem cell.

31. The pluripotent therapeutic composition of claim 30 wherein said human post-natal stem cell is a primordial sex cell.

32. The therapeutic reprogramming method of claim 31 wherein said primordial sex cell is a diploid germ cell isolated from a male or female post-natal source.

33. The pluripotent therapeutic composition of claim 31 wherein said primordial sex cell is a spermatogonial stem cell.