Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix

Patent metadata

Field	Value
Publication number	US-9994825-B2
Application number	US-201414777258-A
Country	US
Kind code	B2
Filing date	Mar 17, 2014
Priority date	Mar 15, 2013
Publication date	Jun 12, 2018
Grant date	Jun 12, 2018

Abstract

Described are methods for differentiating mammalian pluripotent stem cells towards a population of cells that can self-organize into vascular networks in vitro when harvested cells of the present invention are embedded into a hydrophilic matrix such as a hydrogel.

First claim

The invention claimed is:

1. A method for differentiating human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) towards a population that can self-organize into vascular networks in vitro, comprising the steps of:
plating a single-cell suspension of pluripotent stems selected from the group comprising hESC, hiPSC, or a combination thereof onto a surface;
adding culture medium;
culturing the cells for several days;
adding Vascular Endothelial Growth Factor (VEGF) and a transforming growth factor-beta (TGF-β) inhibitor to the culture medium;
culturing the cells for several days forming early vascular cells (EVCs) subtypes comprising vascular endothelial cadherin (VEcad + ), or VEcad + CD31 + , or a combination thereof;
harvesting the resulting cells; and
embedding the harvested cells in a hydrophilic matrix to form an in vitro vascular network.

2. The method for differentiating of claim 1 , wherein the hydrophilic matrix is a hydrogel selected from the group consisting of hyaluronic acid (HA) hydrogel and collagen hydrogel.

3. The method for differentiating of claim 1 , wherein the TGF-β inhibitor is SB431542.

4. The method for differentiating of claim 1 , wherein the concentration of added VEGF ranges from 1 to 50 ng/ml.

5. The method for differentiating of claim 1 , wherein the surface is type IV collagen.

6. The method for differentiating of claim 1 , wherein the media used for culturing are supplemented with serum.

7. The method for differentiating of claim 6 , wherein the media are supplemented with 10% serum.

8. The method for differentiating of claim 1 , wherein prior to the step of adding VEGF the cells are harvested from the surface and then plated onto a surface with VEGF in the culture medium.

9. The method of claim 1 wherein the VEGF is VEGF-A.

10. The method of claim 1 wherein the concentration of VEGF is 50 ng/ml.

Assignees

• Univ Johns Hopkins

Inventors

• Gerecht Sharon
• Kusuma Sravanti

Classifications

• C12N2506/02

  from embryonic cells · CPC title

• C12N2533/54

  Collagen; Gelatin · CPC title

• C12N2506/03

  from non-embryonic pluripotent stem cells · CPC title

• C12N5/0691

  Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels · CPC title

• A61K38/39

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG] · CPC title