Differentiation and expansion of endothelial cells from pluripotent stem cells and the in vitro formation of vasculature like structures

• US 10,119,122 B2
• Filed: 06/10/2014
• Issued: 11/06/2018
• Est. Priority Date: 06/10/2013
• Status: Active Grant

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: Mummery, Christine Lindsay, Orlova, Valeriya Viktorovna
• Primary Examiner(s): Epps-Smith, Janet L
• Application Number:
  US14/897,619
• Publication Number:
  US 20160115453A1
• Time in Patent Office: 1,610 Days
• Field of Search: 4353071, 435325, 435377, 506 10
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2503/06 Screening or testing on artificial skin
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/069 Vascular Endothelial cells
• CPC Class Codes: C12N 5/0691 Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
• CPC Class Codes: G01N 33/5061 Muscle cells
• CPC Class Codes: G01N 33/5064 Endothelial cells

Abstract

The disclosure is concerned among others with means and methods for obtaining endothelial cells and to means and methods for in vitro cell culture comprising endothelial cells and pericytes and/or smooth muscle cells derived from the pericytes. The endothelial cells or the pericytes and/or smooth muscle cells, or both, are preferably derived from in vitro differentiated pluripotent stem cells.

Claims

1. A method for producing an in vitro cell culture comprising endothelial cells derived from in vitro differentiated pluripotent stein cells and smooth muscle cells derived from in vitro differentiated pluripotent stem cells or derived from pericytes, the method comprising the following steps:

(a) culturing pluripotent stem cells in defined medium comprising ActivinA, BMP4, VEGF, and a canonical WNT ligand or GSK3 inhibitor, to produce a culture comprising differentiated cells;

(b) culturing the cells obtained in step (a) in defined medium containing VEGF and a TGF{beta} signaling inhibitor to produce endothelial cells; and

(c) culturing the endothelial cells obtained in step (b) with smooth muscle cells derived from in vitro differentiated pluripotent stem cells or derived from pericytes.

2. The method according to claim 1, further comprising:

collecting the cells produced in step (b) and obtaining therefrom a collection of cells that comprises more than 90% endothelial cells.

3. The method according to claim 1, further comprising:

collecting the cells produced in step (b) and obtaining therefrom a collection of cells that comprises more than 90% pericytes.

4. The method according to claim 2, wherein said collection of cells is obtained by separating the cells of step (b) on the basis of CD31 expression.

5. The method according to claim 3, further comprising:

providing the endothelial cells with a cell storage medium and storing the endothelial cells at a temperature of −

70°

C. or less.

6. The method according to claim 2, further comprising:

culturing said endothelial cells together with smooth muscle cells.

8. The method according to claim 2, wherein the collection of cells comprises more than 90% endothelial cells based on the expression of CD31, CD34 or VECadherin.

9. The method according to claim 3, wherein the collection of cells comprises more than 90% pericytes based on the expression of CD31, CD34 or VECadherin.

10. The method according to claim 4, wherein separating the cells of step (b) on the basis of CD31 expression is by means of beads comprising a CD31 binding antibody.

7. A method of producing an in vitro cell culture comprising endothelial cells and smooth muscle cells, wherein the endothelial cells are able to integrate into a vascular network in vivo, the method comprising:

(a) culturing pluripotent stem cells in a defined medium comprising ActivinA, BMP4, VEGF, and a canonical WNT ligand or GSK3 inhibitor, to produce a culture comprising differentiated cells;

(b) culturing the cells of step (a) in defined medium containing VEGF and a TGF(beta) signaling inhibitor to produce endothelial cells; and

(c) culturing the endothelial cells obtained in step (b) with smooth muscle cells.

11. A method for producing an in vitro cell culture comprising purified endothelial cells and purified smooth muscle cells, wherein the cell culture comprises a capillary network comprising the endothelial cells and the smooth muscle cells, the method comprising:

deriving endothelial cells from in vitro differentiated induced pluripotent stem cells (iPSCs) and purifying the endothelial cells,

deriving smooth muscle cells from in vitro differentiated induced pluripotent stem cells or from pericytes that are derived from in vitro differentiated induced pluripotent stem cells and purifying the smooth muscle cells, and

culturing the purified endothelial cells together with the purified smooth muscle cells.

12. The method according to claim 11, wherein the endothelial cells and the smooth muscle cells are derived from induced pluripotent stem cells (iPSCs) generated from skin biopsy fibroblasts or blood outgrowth endothelial cells.

13. The method according to claim 11, wherein the endothelial cells are able to integrate into a vascular network in vivo.

14. The method according to claim 11, wherein the endothelial cells have a genetic defect that in vivo exhibits a phenotype in vasculature.

15. The method according to claim 11, wherein the smooth muscle cells have a genetic defect that in vivo exhibits a phenotype in vasculature.

16. The method according to claim 11, further comprising:

isolating the endothelial cells and the smooth muscle cells, and

cryopreserving the endothelial cells and the smooth muscle cells.

17. The method according to claim 11, further comprising:

screening the cell culture for angiogenic and anti-angiogenic compounds.