Multi-Lineage Progenitor Cells

• US 20100028851A1
• Filed: 10/05/2009
• Published: 02/04/2010
• Est. Priority Date: 04/23/2004
• Status: Active Grant

• Current Assignee: BioE LLC
• Original Assignee: BioE, Inc.
• Inventors: Tigges, Barbara M., Sprague, Stacey L., Collins, Daniel P.
• Granted Patent: US 8,163,275 B2
• Time in Patent Office: Days
• US Class Current: 435/1.300
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

Abstract

Fetal blood multi-lineage progenitor cells that are capable of a wide spectrum of transdifferentiation are described.

Claims

1. A purified population of human fetal blood multi-lineage progenitor cells (MLPC), wherein said MLPC are positive for CD9 and CD45.

13. A method for cryopreserving MLPC, said method comprising:

a) contacting said purified population of MLPC of claim 1, said purified population of MLPC of claim 2, or a clonal line of MLPC with a cryopreservative; and

b) freezing said purified population of MLPC or said clonal line.

14. The method of claim 13, wherein said cryopreservative is DMSO.

15. The method of claim 13, wherein said cryopreservative is fetal bovine serum, human serum, or human serum albumin in combination with one or more of the following:

DMSO, trehalose, and dextran.

16. The method of claim 15, wherein said cryopreservative is human serum, DMSO, and trehalose.

17. The method of claim 15, wherein said cryopreservative is fetal bovine serum and DMSO.

18. The method of claim 13, wherein said purified population of MLPC or said clonal line is suspended in said cryopreservative at a concentration between 1×

10⁵ and 5×

10⁷ cells/mL.

19. The method of claim 13, wherein said purified population of MLPC or said clonal line is frozen at a controlled rate.

20. The method of claim 19, wherein the freezing rate is controlled electronically.

21. The method of claim 13, wherein said purified population of MLPC or said clonal line is frozen by placement in an ethanol bath in the vapor phase of a liquid nitrogen cryogenic storage tank.

22. A method of producing a population of differentiated cells, said method comprising culturing the purified population of MLPC of claim 1, the purified population of MLPC of claim 2, or a clonal line of MLPC with an agent effective to induce differentiation of said MLPC.

23. The method of claim 22, wherein said agent comprises insulin, glutamine, dexamethasone, indomethacin, and 3-isobutyl-1-methyl-xanthine.

24. The method of claim 22, wherein said agent comprises dexamethasone, glutamine, ascorbate, and β

-glycerophosphate.

25. The method of claim 22, wherein said agent comprises epithelial growth factor, insulin, fetuin, dexamethasone, and fibroblast growth factor-basic.

26. The method of claim 22, wherein said agent comprises fibroblast growth factor-basic, epidermal growth factor, NSF-1, and retinoic acid.

27. The method of claim 22, wherein said agent comprises heparin, bovine brain extract, epithelial growth factor, and hydrocortisone.

28. The method of claim 22, wherein said agent comprises ascorbic acid, hydrocortisone, transferrin, insulin, epidermal growth factor, hepatocyte growth factor, fibroblast growth factor-basic, fibroblast growth factor-4, and stem cell factor.

2. A purified population of human fetal blood MLPC, wherein said MLPC are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4.

3. A method for purifying a population of MLPC from human fetal blood, said method comprising:

a) contacting a human fetal blood sample with a composition, said composition comprising;

i) dextran;

ii) anti-glycophorin A antibody;

iii) anti-CD15 antibody; and

iv) anti-CD9 antibody;

b) allowing said sample to partition into an agglutinate and a supernatant phase;

c) recovering cells from said supernatant phase; and

d) purifying MLPC from the recovered cells by adherence to a solid substrate, wherein said MLPC are positive for CD9 and positive for CD45.

4. The method of claim 3, wherein said MLPC are further positive for CD34, CD133, CD41, CD44, CD105, CD29, CD73, CD90, stem cell factor, SSEA-3, SSEA-4, and CD13.

5. The method of claim 3, wherein said MLPC are negative for CD15, CD38, glycophorin-A, CD2, CD3, CD8, CD19, CD20, CD22, CD5, CD7, CD10, CD14, CD4, HLA-DR, CD16, CD33, and CD61.

6. The method of claim 3, wherein said fetal blood sample is cord blood.

7. The method of claim 3, wherein said solid substrate is a plastic substrate.

8. The method of claim 3, said method further comprising culturing said MLPC such that said MLPC obtain a fibroblast morphology, wherein said MLPC, after obtaining said fibroblast morphology, are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4.

9. The method of claim 8, wherein said MLPC, after obtaining said fibroblast morphology, are further positive for CD13, CD29, CD44, CD73, CD90, and CD105.

10. The method of claim 8, wherein said MLPC, after obtaining said fibroblast morphology, are further negative for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD41, CD61, CD62E, CD133, glycophorin-A, stem cell factor, SSEA-3, and HLA-DR.

11. The method of claim 8, further comprising testing said MLPC for CD9.

12. The method of claim 11, further comprising testing said MLPC for CD29, CD45, CD73, and CD90.