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

Patent metadata

Field	Value
Publication number	US-9506037-B2
Application number	US-201313844313-A
Country	US
Kind code	B2
Filing date	Mar 15, 2013
Priority date	Mar 15, 2013
Publication date	Nov 29, 2016
Grant date	Nov 29, 2016

Abstract

A bicellular vascular population derived from human pluripotent stem cells (hPSCs) undergoes morphogenesis and assembly in a synthetic hydrogel. It is shown that hPSCs can be induced to co-differentiate into early vascular cells (EVCs) in a clinically-relevant strategy dependent upon Notch activation. These EVCs mature into ECs and pericytes, and self-organize to form vascular networks in an engineered matrix. Upon in vivo implantation, multicellular human vascular networks are functionally perfused. Thus, a derived bicellular population is exploited for its intrinsic self-assembly capability to create functional microvasculature in a deliverable matrix.

First claim

What is claimed is:

1. A method for differentiating human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into early vascular cells (EVCs) in vitro, comprising the steps of:
plating a single-cell suspension of hESCs or hiPSCs onto a surface coated with suitable materials selected from the group consisting of type I collagen, fibronectin, and type IV collagen;
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; and
harvesting the resulting EVCs,
wherein said EVCs express CD73 and one or more of CD105 and CD146.

2. The method for differentiating of claim 1 , wherein the TGF βinhibitor is SB431542.

3. The method for differentiating of claim 1 , wherein the concentration of VEGF is between about 1 and 50 ng/ml.

4. The method for differentiating of claim 1 , wherein the suitable material is type IV collagen.

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

6. The method for differentiating of claim 5 , wherein the media are supplemented with 10% serum.

7. 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 coated with the suitable materials with VEGF in the culture medium.

8. The method for differentiating of claim 1 , further comprising a step of embedding the harvested resulting EVC cells in a matrix following the step of harvesting.

9. The method for differentiating of claim 8 , wherein after embedding the harvested resulting EVC cells in a matrix, the cells comprise a population that can self-organize into vascular networks.

10. The method for differentiating of claim 9 , wherein the matrix is a hydrophilic matrix.

11. The method for differentiating of claim 10 , wherein the hydrophilic matrix is a hydrogel selected from the group consisting of hyaluronic acid (HA) hydrogel and collagen hydrogel.

12. The method for differentiating of claim 8 , wherein the matrix is a hydrophilic matrix.

13. The method for differentiating of claim 12 , wherein the hydrophilic matrix is a hydrogel selected from the group consisting of hyaluronic acid (HA) hydrogel and collagen hydrogel.

14. The method for differentiating of claim 1 , further comprising the step of sub-culturing the EVCs in a medium containing serum on a tissue-culture treated surface and removing unattached cells after 4 hours.

15. The method for differentiating of claim 14 , wherein the medium for sub-culturing EVCs also contains Angiopoietin 1 or TGFb-1.

Assignees

• Univ Johns Hopkins

Inventors

• Gerecht Sharon
• Kusuma Sravanti

Classifications

• A61K38/39

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

• A61K31/00

  Medicinal preparations containing organic active ingredients · CPC title

• C12N2533/54

  Collagen; Gelatin · CPC title

• C12N2501/998

  Proteins not provided for elsewhere · CPC title

• C12N2501/905

  Hyaluronic acid · CPC title