Compositions and methods for identifying, isolating and enriching germline-like stem cells from amniotic fluid

• US 20080159999A1
• Filed: 10/23/2007
• Published: 07/03/2008
• Est. Priority Date: 10/23/2006
• Status: Abandoned Application

• Current Assignee: Konstantinos Stefanidis
• Original Assignee: Konstantinos Stefanidis
• Inventors: Stefanidis, Konstantinos
• Application Number:
  US11/976,321
• Publication Number:
  US 20080159999A1
• Time in Patent Office: Days
• US Class Current: 424/93.21
• 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 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

The present invention is directed to pluripotent embryonic stem cells derived from amniotic fluid and the methods for isolating, expanding and differentiating these cells, and their therapeutic uses such as manipulating the cells by gene transfection and other means for therapeutic applications.

Claims

1. An amniotic fluid cell composition comprising:

pluripotent embryonic stem cells expressing DAZL; and

amniotic fluid.

2. The composition of claim 1, wherein said amniotic fluid, comprises 5-50% of said basal growth medium.

3. The composition of claim 1 wherein said stem cells may further express at least one marker selected from alkaline phosphatase;

SSEA-3;

SSEA-4;

TRA-1-60;

TRA-1-81;

TRA2-54;

c-kit;

Oct-4 and oxytocin receptor.

4. The composition of claim 1 wherein said germline-like stem cells may further express at least one of HLA Class I, CD13, CD44, CD49b, and CD105.

5. The composition of claim 1 wherein said germline-like stem cells may further express at least one of POU5F1, TDGF, GABRB3, FGF4 and TERT.

6. The composition of claim 1 wherein said composition further comprises an agent selected from the group consisting of forskolin ([3R-(3a, 4α

β

, 5B, 6B, 6aα

, 10α

, 10α

β

, 10bα

)]-5-(acetyloxy)-3-ethenyldodecahydro-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1H-naphtho[2,1-b]pyran-1-one), cholera toxin, isobutylmethylxanthine (IBMX), and dibutyrladenosine cyclic monophosphate (dbcAMP).

7. The composition of claim 1, wherein said growth factor is basic fibroblast growth factor (bFGF).

8. The composition of claim 7 wherein the concentration of said bFGF is in the range of about 1-10 ng/ml.

9. The composition of claim 1, wherein said basal growth medium comprises one or more of L-glutamine, essential amino acids, non-essential amino acids, antibiotics and combinations thereof.

10. A method for isolating germline-like stem cells from amniotic fluid, said method comprising:

(a) providing amniotic fluid cells on a substrate for a sufficient time to permit a portion of said amniotic fluid cells to adhere to said substrate;

(b) removing a non-adherent portion of said amniotic fluid cells;

(c) identifying from said non-adherent portion of amniotic fluid cells, those cells expressing at least one germline-like stem marker, said at least one germline-like stem marker being DAZL.

11. The method of claim 10, wherein said identifying step comprises performing flow cytometry analysis, immunocytochemical analysis, or RT-PCR or a combination thereof.

12. A cell line isolated by method of claim 10.

13. The method of claim 10 wherein said germline-like stem cells may further express at least one of alkaline phosphatase;

SSEA-3;

SSEA-4;

TRA-1-60;

TRA-1-81;

TRA2-54;

c-kit;

Oct-4 and oxytocin receptor.

14. The method of claim 10 wherein said germline-like stem cells do not express SSEA-1.

15. The method of claim 10 wherein said germline-like stem cells may further express at least one of HLA Class I, CD13, CD44, CD49b, and CD105.

16. The method of claim 10 wherein said germline-like stem cells may further express at least one of POU5F1, TDGF, GABRB3, FGF4 and TERT.

17. An isolated amniotic fluid germline-like stem cell which is pluripotent and DAZL positive.

18. The isolated germline-like stem cell of claim 17 wherein said cell further expresses at least one of alkaline phosphatase;

SSEA-3;

SSEA-4;

TRA-1-60;

TRA-1-81;

TRA2-54;

c-kit;

Oct-4 and oxytocin receptor.

19. The isolated germline-like stem cell as in claim 18 wherein said germline-like stem cells do not express SSEA-1.

20. The isolated germline-like stem cell as in claim 19 wherein said cell further expresses at least one of HLA Class I, CD13, CD44, CD49b, and CD105.

21. The isolated germline-like stem cell as in claim 17 wherein said cell further expresses at least one of POU5F1, TDGF, GABRB3, FGF4 and TERT.

22. A method of treating a disease in a human comprising administering the cell or a plurality of said cell of claim 17 into an individual in need thereof.

23. The method of claim 22 wherein said disease is selected from the group consisting of:

infertility, cirrhosis of the liver, pancreatitis, diabetes, Parkinson'"'"'s disease, spinal cord injury, stroke, burns, heart disease, certain types of cancer, osteoarthritis, rheumatoid arthritis, leukemia, lymphoma, genetic blood disorders, and Alzheimer'"'"'s disease.

24. A culture medium for the growth of germline-like stem cells isolated from amniotic fluid, said culture medium comprising aminotic fluid and basal medium, said basal medium comprising:

about 80% Dulbeco'"'"'s modified Eagle'"'"'s medium; and

serum replacement medium.

25. A culture medium of claim 24, further comprising one of more of L-glutamine, nonessential amino acids, antibiotics, a growth factor and other agent.

26. The culture medium of claim 24, wherein said growth factor is basic fibroblast growth factor (bFGF).