Pericyte progenitors from peripheral blood

Patent metadata

Field	Value
Publication number	US-9809798-B2
Application number	US-201214004349-A
Country	US
Kind code	B2
Filing date	Mar 12, 2012
Priority date	Mar 11, 2011
Publication date	Nov 7, 2017
Grant date	Nov 7, 2017

Abstract

Thus, provided herein are pericyte progenitor cells (e.g., isolated pericyte progenitor cells), methods for generating pericyte progenitors in clinically relevant numbers for various applications applying macromolecular crowding during cell culture, and methods of using the pericyte progenitor cells.

First claim

What is claimed is:

1. A method of obtaining pericyte progenitor cells comprising:
a) culturing blood cells with a matrix to which cells can attach, and one or more carbohydrate-based hydrophilic macromolecules, each of the one or more of the macromolecules has a molecular weight from about 50 kDa to about 1000 kDa, and wherein the one or more macromolecules are present at a concentration from about 2.5 mg/ml to about 100 mg/ml, thereby producing a cell culture; and
b) maintaining the cell culture under conditions in which pericyte progenitor cells are generated in the cell culture, wherein the pericyte progenitor cells express platelet-derived growth factor receptor-β (PDGFR-β) and neuron-glial antigen 2 (NG2),
c) thereby obtaining pericyte progenitor cells.

2. The method of claim 1 wherein the blood cells are peripheral blood mononuclear cells (PBMCs).

3. The method of claim 2 wherein about 100,000 pericyte progenitor cell per 1 ml of blood is obtained.

4. The method of claim 1 wherein the matrix comprises all or a portion of an extracellular matrix.

5. The method of claim 4 wherein the matrix comprises fibronectin.

6. The method of claim 1 wherein the culture containing the carbohydrate-based hydrophilic macromolecule has a viscosity of less than about 2 mPas.

7. The method of claim 1 wherein the carbohydrate-based hydrophilic macromolecule has a radius of about 2 to about 50 nm.

8. The method of claim 1 wherein the carbohydrate-based hydrophilic macromolecule is a polymer of glucose, sucrose or a combination thereof.

9. The method of claim 8 wherein the polymer is Ficoll™70, Ficoll™400, polyvinyl pyrrolidone (PVP), dextran, dextran sulfate, polystyrene sulfonate, pullulan, chondroitan sulfate, heparin, heparan sulfate, dermatan sulfate or a combination thereof.

10. The method of claim 1 wherein the blood cells are contacted with a mixture of carbohydrate-based hydrophilic macromolecules comprising Ficoll™70 and Ficoll™400.

11. The method of claim 10 wherein the Ficoll™70 is at a concentration range of from about 7.5 mg/ml to about 100 mg/ml, and the Ficoll™400 is at a concentration range of from about 2.5 mg/ml to about 100 mg/ml.

12. The method of claim 11 wherein the Ficoll™70 is at a concentration of about 37.5 mg/ml and the Ficoll™400 is at a concentration of about 25 mg/ml.

13. The method of claim 1 further comprising detecting pericyte progenitor cells in the cell culture.

14. The method of claim 13 wherein the detection further comprises detecting cells which adhere to the matrix and express platelet-derived growth factor receptor β (PDGFR-β), neuron-glial antigen 2 (NG2), Tie-2, vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, CD206 or a combination thereof.

15. The method of claim 13 wherein the detection further comprises detecting cells which do not express human leukocyte antigen-DR (HLA-DR), collagen I or a combination thereof.

16. The method of claim 1 wherein the blood cells are contacted with the matrix and one or more carbohydrate-based hydrophilic macromolecules at a seeding concentration of about 10,000 cells/cm 2 to about 1,000,000 cells/cm 2 .

17. The method of claim 1 wherein the conditions under which the cell culture is maintained comprise maintaining the cell culture at about 37° C. in an atmosphere of about 5% CO 2 /95% air.

18. The method of claim 17 wherein the conditions are maintained for about 1 day to about 14 days.

19. The method of claim 1 wherein the blood cells are human cells.

20. The method of claim 1 further comprising isolating the pericyte progenitor cells from the cell culture.

21. The method of claim 20 wherein the pericyte progenitor cells are isolated by harvesting cells which adhere to the matrix and express platelet-derived growth factor receptor β (PDGFRβ), neuron-glial antigen 2 (NG2), Tie-2, vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, CD206 or a combination thereof.

Assignees

• Raghunath Michael
• Blocki Anna
• Nat Univ Singapore

Inventors

• Raghunath Michael
• Blocki Anna

Classifications

• C12N2533/52

  Fibronectin; Laminin · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C12N2533/70

  Polysaccharides · CPC title

• C12N2502/28

  Vascular endothelial cells · CPC title

• C12N2500/38

  Vitamins · CPC title