Hemangio-colony forming cells

Patent metadata

Field	Value
Publication number	US-11566228-B2
Application number	US-201815902817-A
Country	US
Kind code	B2
Filing date	Feb 22, 2018
Priority date	Apr 14, 2006
Publication date	Jan 31, 2023
Grant date	Jan 31, 2023

Abstract

Methods of generating and expanding human hemangio-colony forming cells in vitro and methods of expanding and using such cells are disclosed. The methods permit the production of large numbers of hemangio-colony forming cells as well as derivative cells, such as hematopoietic and endothelial cells. The cells obtained by the methods disclosed may be used for a variety of research, clinical, and therapeutic applications.

First claim

What is claimed is:

1. A method for generating human embryonic stem cell-derived hemangio-colony forming cells in vitro, the method comprising:
(a) culturing human embryonic stem cells in a serum-free media comprising vascular endothelial growth factor (VEGF) and bone morphogenic protein 4 (BMP—4) for promoting the formation of embryoid bodies;
(b) culturing the cells produced in step (a) in a serum-free media comprising at least two growth factors selected from the group consisting of VEGF, BMP-4 and basic fibroblast growth factor (bFGF), thereby forming embryoid bodies;
(c) disaggregating the embryoid bodies after step (b) into single cells; and
(d) culturing the single cells in a serum free media comprising at least two growth factors selected from the group consisting of insulin, transferrin, granulocyte macrophage colony—stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte colony—stimulating factor (G-CSF), stem cell factor (SCF), thrombopoietin (TPO), FLT3 (FL), vascular endothelial growth factor (VEGF), and bone morphogenic protein 4 (BMP-4), thereby generating human hemangio-colony forming cells,
wherein the human embryonic stem cell-derived hemangio-colony forming cells are capable of differentiating into hematopoidic cells and endothelial cells, and wherein the human embryonic stem cell-derived hemangio-colony forming cells express at least two markers selected from the group consisting of CD61, integrin β 1 and NF-E2.

2. The method of claim 1 , wherein the serum free media in step (b) or step (d) further comprises a HOXB4 protein.

3. The method of claim 1 , wherein the serum free media of step (b) further comprises one or more growth factors selected from the group consisting of stem cell factor (SCF), Flt-3L (FL) thrombopoietin (TPO) and erythropoietin (EPO).

4. The method of claim 1 , wherein the human embryonic stem cells are cultured in the serum free media comprising BMP-4 and VEGF in step (a) for at least the first 48 hours of cell culture.

5. The method of claim 3 , wherein step (b) is performed within 48-72 hours of culturing the human embryonic stem cells in step (a).

6. The method of claim 1 , wherein step (b) is performed within 48-72 hours of culturing the human embryonic stem cells in step (a).

7. The method of claim 1 , wherein the serum free media of step (a), step (b) and step (d) further comprise erythropoietin (EPO).

8. The method of claim 1 , further comprising purifying the human hemangio-colony forming cells.

9. The method of claim 8 , wherein the purifying of the hemangio-colony forming cells comprises the use of immunoaffinity column chromatography with an anti-CD71 antibody.

10. The method of claim 1 , further comprising isolating the human hemangio-colony forming cells.

11. The method of claim 10 , wherein the isolating of the hemangio-colony forming cells comprises isolating the hemangio-colony forming cells by size and/or by morphology.

12. The method of claim 1 , wherein step (d) is performed 72 hours after the initiation of culturing the human embryonic stem cells in step (a).

13. The method of claim 1 , wherein the culturing in step (a), step (b) and step (d) is performed under low attachment conditions.

14. The method of claim 1 , wherein the serum free media in step (d) further comprises methylcellulose.

15. The method of claim 1 , wherein in step (a) the concentration of VEGF is 25-100 ng/ml, and the concentration of BMP-4 is 25-100 ng/ml.

16. The method of claim 3 , wherein in step (b) the concentration of VEGF is 25-100 ng/ml, the concentration of BMP-4 is 25-100 ng/ml, the concentration of SCF is 10-50 ng/ml, the concentration FL is 10-50 ng/ml, and the concentration of TPO is 10-50 ng/ml.

17. The method of claim 1 , wherein in step (d) the concentration of insulin is 10 μg/ml, the concentration of transferrin is 200 μg/ml, the concentration of GM-CSF is 20 μg/ml, the concentration of IL-3 is 20 ng/ml, the concentration of IL-6 is 10 ng/ml or 20 ng/ml, the concentration of G-CSF is 20 ng/ml, the concentration of SCE is 20 ng/ml or 50 ng/ml, the concentration of TPO is 20 ng/ml or 50 ng/ml, the concentration of FL is 20 ng/ml or 50 ng/ml, the concentration of VEGF is 25-100 ng/ml, and the concentration of BMP-4 is 25-100 ng/ml.

18. The method of claim 1 , wherein the embryonic stem cells are from a library of embryonic stem cells that are hemizygous or homozygous for at least one major histocompatibility (MHC) allele present in a human population, and each member of the library is hemizygous or homozygous for a unique set of MHC alleles compared to other members of the library of embryonic stem cells, thereby generating a library of human hemangio-colony forming cells that are hemizygous or homozygous for at least one major histocompatibility (MHC) allele present in a human population, and each member of the library of human hemangio-colony forming cells is hemizygous or homozygous for a unique set of MHC alleles compared to other members of the library of human hemangio colony forming cells.

19. The method of claim 1 , further comprising differentiating the hemangio-colony forming cells into human hematopoietic cells.

20. The method of claim 1 , further comprising differentiating the hemangio-colony forming cells into human endothelial cells.

Assignees

• Astellas Inst For Regenerative Medicine

Inventors

• Lanza Robert
• Lu Shi-Jiang

Classifications

• C12N2501/20

  Cytokines; Chemokines · CPC title

• C12N2500/90

  Serum-free medium, which may still contain naturally-sourced components · CPC title

• A61K35/44

  Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells · CPC title

• C12N2500/99

  Serum-free medium · CPC title

• C12N2506/02

  from embryonic cells · CPC title