CHEMICALLY DEFINED DIFFERENTIATION PROTOCOL FOR PERICYTE DIFFERENTIATION FROM PLURIPOTENT STEM CELLS

• US 20190316094A1
• Filed: 04/16/2019
• Published: 10/17/2019
• Est. Priority Date: 04/16/2018
• Status: Active Application

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Daly, William T., Murphy, William L., Soref, Cheryl M., Torr, Elizabeth E., Aisenbrey, Elizabeth A., Johnson, Hunter J.
• Application Number:
  US16/385,543
• Publication Number:
  US 20190316094A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0691 Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
• CPC Class Codes: C12N 5/0692 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

The present invention provides methods of differentiating pericytes from pluripotent stem cells comprising culturing steps in chemically defined culture medium. A population of exogenously derived pericytes from PSCs are also contemplated. Further uses of the exogenously cultured pericytes for an in vitro disease model or in vitro angiogenesis assay are contemplated, including an in vitro 3D model of vasculature.

Claims

1. A method of isolating pericytes from pluripotent stem cells, the method comprising:

a) culturing pluripotent stem cells on coated plates in a chemically defined culture medium supplemented with about 1-30 ng/ml BMP4, about 10-30 ng/ml activin and about 1-5 μ

M GSK-3 inhibitor for at least 2 days,

b) culturing the cultured cells of step (a) in chemically defined medium supplemented with about 5-15 μ

g/ml transferrin, about 10-30 μ

g/ml insulin, about 70-150 ng/ml fibroblast growth factor 2 (FGF2), about 25-75 ng/ml vascular endothelial growth factor-A (165) (VEGF-A (165)) and about 2-10 μ

M TGFβ

1 inhibitor sufficient to differentiate the cells into CD34−

or CD31−

cell population,

c) detecting CD34−

or CD31−

cell population at day 6 of culture and isolating the CD34+ or CD31+ cells from the population,

d) culturing the CD34−

or CD31−

cells in step (c) in EGM2 media for at least 2 days on coated plates; and

e) detecting at least one of the pericyte markers selected from the group consisting of PDGFRβ

, SM22, CD13 and Desmin in a population of cultured pericyte cells of step (d), wherein the population is at least about 95% pure.

2. The method of claim 1, wherein the chemically defined medium of step a) is E8 medium.

3. The method of claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

4. The method of claim 1, wherein the TGFβ

1 inhibitor is SB431542.

5. The method of claim 1, wherein in step (a), the chemically defined medium is supplemented with about 5 ng/ml BMP4, about 25 ng/ml activin A and about 1 μ

M GSK-3 inhibitor, and in step (b), the chemically defined media is supplemented with

about 10-11 ng/ml transferrin,

about 20 ng/ml insulin,

about 100 ng/ml FGF2,

about 50 ng/ml VEGF-A (165), and

about 5 μ

M TGFβ

1 inhibitor.

6. The method of claim 1, wherein the method does not comprise a step using feeder cells embryoid bodies.

7. The method of claim 1, wherein step (c) comprises isolating a population of at least 95% CD31−

or CD34−

cells.

8. The method of claim 1, wherein step (e) provides a population of at least 98% pericytes expressing PDGFRβ

, SM22, CD13 and Desmin.

9. The method of claim 8, wherein step (e) provides a population of at least 99% pericytes expressing PDGFRβ

, SM22, CD13 and Desmin.

10. The method of claim 1, wherein the adherent substrate of step (a) are Martigel coated plates, vitronectin coated plates, fibronectin coated plates, collagen IV coated plates or a combination thereof.

11. The method of claim 1, wherein the adherent substrate of step (d) are selected from plates coated with gelatin, collagen, fibronectin or a combination thereof.

12. The method of claim 11, wherein the coated plates are coated with a mixture of collagen and fibronectin.

13. The method of claim 1, wherein the pericytes are isolated in about 8-12 days.

14. The method of claim 1, wherein the GSK-3 inhibitor is CHIR990221.

15. The method of claim 1, wherein the CD31−

or CD34−

cells are isolated by magnetic activated cell sorting.

16. A population of exogenously derived pericytes from PSCs made by the method of claim 1, wherein at least 95% of the population express at least one of the markers PDGFRβ

, SM22, CD13 and Desmin.

17. An exogenously cultured pericyte population derived from PSCs of claim 16, wherein the pericytes are able to enwrap blood vessel endothelial cells to form blood vessels.

21. An in vitro disease model or in vitro angiogenesis system comprising the exogenously cultured pericytes of claim 16.

22. An in vitro 3D model of vasculature comprising the exogenously cultured pericytes of claim 16.

23. The exogenously cultured pericytes of claim 16, wherein the pericytes are able to expand in culture for at least three passages.

18. A method of isolating pericytes from CD34−

or CD31−

cell populations, the method comprising;

a) culturing a substantially pure population of CD34−

or CD31−

cells derived from PSCs in EGM2 media for at least 2 days on adherent substrate; and

b) detecting the pericyte markers PDGFRβ

, SM22, CD13 and Desmin on a population of cells, and isolating the population of pericytes expressing at least one of the markers PDGFRβ

, SM22, CD13 and Desmin.

19. The method of claim 18, wherein the adherent substrate are plates coated with gelatin, collagen, fibronectin or a combination thereof.

20. The method of claim 19, wherein the coated plates are coated with a mixture of collagen and fibronectin.