Hemangio-colony forming cells

• US 9,938,500 B2
• Filed: 09/29/2015
• Issued: 04/10/2018
• Est. Priority Date: 04/14/2006
• Status: Active Grant

• Current Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Original Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Inventors: Lanza, Robert, Lu, Shi-Jiang
• Primary Examiner(s): Fox, Allison M
• Application Number:
  US14/869,561
• Publication Number:
  US 20160115450A1
• Time in Patent Office: 924 Days
• Field of Search: None
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/99 Serum-free medium
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/20 Cytokines; Chemokines
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

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.

Claims

1. A method, comprising:

(a) culturing human embryonic stem cells in serum free media in the presence of vascular endothelial growth factor (VEGF) and bone morphogenic protein 4 (BMP-4) in an amount sufficient to induce the differentiation of said embryonic stem cells into embryoid bodies; and

(b) adding at least two first growth factors selected from the group consisting of;

basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenic protein 4 (BMP-4), stem cell factor (SCF), thrombopoietin (TPO), Flt-3L (FL), HOX protein, and erythropoietin (EPO) to the culture of embryoid bodies

(c) disaggregating the embryoid bodies into single cells; and

(d) adding erythropoietin (EPO) to said single cell culture in an amount sufficient to expand CD34⁻

CD31⁻

human hemangio-colony forming cells in the culture;

wherein the embryonic stem cells, embryoid bodies, and CD34⁻

CD31⁻

hemangio-colony forming cells are continuously grown in serum-free media to generate CD34⁻

CD31⁻

human hemangio-colony forming cells wherein the erythropoietin (EPO) is added to the single cell culture in step (d) after 72 hours of initiating of the culturing of step (a), and optionally, added multiple times throughout step (d).

2. The method of claim 1, wherein the VEGF and BMP-4 are added to the culture of human embryonic stem cells in step (a) within 0-48 hours of cell culture, and optionally, multiple times throughout step (a).

3. The method of claim 1, wherein the at least two first growth factors comprise basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein 4 (BMP-4).

4. The method of claim 1, wherein the at least two first growth factors are added to the culture of embryoid bodies in step (b) within 48-72 hours of initiating of the culturing of step (a), and optionally, added multiple times.

5. The method of claim 1, wherein at least one additional growth factor is further added to said single cell culture in step (d), said at least one additional growth factor being 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), vascular endothelial growth factor (VEGF), bone morphogenic protein 4 (BMP-4), HOX protein, thrombopoietin (TPO), and Flt-3L (FL), or a combination of two or more thereof.

6. The method of claim 5, wherein the HOX protein is HOXB4 or an active fragment thereof, and the active fragment thereof possess the same or substantially similar properties as wild-type HOXB4.

7. The method of claim 6, wherein the functional equivalent or an active fragment thereof, comprises a fusion protein with a protein transduction domain (PTD).

8. The method of claim 7, wherein the fusion protein comprises one or more copies of SEQ ID NO:

14.

9. The method of claim 1, further comprising purifying the CD34⁻

CD31⁻

human hemangio-colony forming cells.

10. The method of claim 9, further comprising the use of an anti-CD71 antibody to purify the CD34⁻

CD31⁻

human hemangio-colony forming cells.

11. The method of claim 1, further comprising isolating the CD34⁻

CD31⁻

human hemangio-colony forming cells.

12. 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 majorhistocompatibility (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 CD34⁻

CD31⁻

human hemangio-colony forming cells that are hemizygous or homozygous for at least one majorhistocompatibility (MHC) allele present in a human population, and each member of the library of CD34⁻

CD31⁻

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.

13. The method of claim 12, wherein the embryonic stem cells are hemizygous or homozygous for all majorhistocompatibility (MHC) alleles present in a human population, thereby generating a library of CD34⁻

CD31⁻

human hemangio-colony forming cells that are hemizygous or homozygous for all majorhistocompatibility (MHC) alleles present in a human population.

14. The method of claim 1, further comprising differentiating CD34⁻

CD31⁻

human hemangio-colony forming cells into human hematopoietic cells.

15. The method of claim 1, further comprising differentiating CD34⁻

CD31⁻

human hemangio-colony forming cells into human endothelial cells.