Hemangio colony forming cells and non-engrafting hemangio cells
Methods of generating and expanding human hemangio-colony forming cells and non-engrafting hemangio 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, non-engrafting hemangio 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. Human non-engrafting hemangio cells are a novel progenitor cell population that is related to but distinct from the hemangioblast and human hemangio-colony forming cells. The invention also provides compositions, preparations, and solutions comprising hemangio-colony forming cells, non-engrafting hemangio cells or cells differentiated therefrom. The compositions, preparations, and solutions include cryopreserved preparations and substantially purified preparations, as well as mixed compositions formulated in combination with related hemangioblast progenitor cell types that can engraft into the bone marrow.
1. A method for expanding hemangioblasts comprising growing mammalian hemangioblasts in serum-free media comprising BMP4 protein and VEGF protein or functional equivalents or active fragments thereof in an amount sufficient to support the proliferation of said hemangioblasts.
2. The method according to claim 1 , wherein said hemangioblasts are enriched, purified, or isolated from cord blood, peripheral blood, or bone marrow.
3. The method according to claim 1 , wherein said hemangioblasts are human hemangioblasts.
4. The method according to claim 1 , wherein said hemangioblasts are grown in vitro to achieve at least 1×10 6 hemangioblasts.
5. The method according to claim 1 , wherein said serum-free media further comprises a homeobox protein, a functional equivalent or an active fragment thereof.
6. The method according to claim 5 , wherein the homeobox protein, functional equivalent or active fragment thereof is a HOXB4 protein or a functional equivalent or an active fragment thereof.
7. The method according to claim 6 , wherein said HOXB4 is mouse or human HOXB4.
8. The method according to claim 7 , wherein said HOXB4 is human HOXB4.
9. The method according to claim 6 , wherein said HOXB4 comprises a full-length protein.
10. The method according to claim 6 , wherein said HOXB4 comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
11. The method according to claim 1 , wherein said serum-free media further comprises a fusion protein comprising HOXB4 and a protein transduction domain (PTD).
12. The method according to claim 11 , wherein said protein transduction domain (PTD) is TAT protein, a functional variant or an active fragment thereof.
13. The method according to claim 12 , wherein said TAT protein comprises the amino acid sequence of SEQ ID NO: 14.
14. The method according to claim 11 , wherein said PTD comprises one or more copies of a TAT polypeptide with the amino acid sequence of SEQ ID NO: 14.