Progenitor cell materials and methods
View Patent ↗Provided are purified and isolated VEGF-C polypeptides capable of binding to at least one of KDR receptor tyrosine kinase (VEGFR-2) and Flt4 receptor tyrosine kinase (VEGFR-3); analogs of such peptides that have VEGF-C-like or VEGF-like biological activities or that are VEGF or VEGF-C inhibitors; polynucleotides encoding the polypeptides; vectors and host cells that embody the polynucleotides; pharmaceutical compositions and diagnostic reagents comprising the polypeptides; and methods of making and using the polypeptides.
1. A method of isolating cells that co-express CD34 and VEGFR-3, comprising steps of:
obtaining a biological sample from a human subject that contains cells; and
separating cells that express both CD34 (CD34 + ) and VEGFR-3 (VEGFR-3 + ) from cells that do not co-express CD34 and VEGFR-3 in the sample, thereby isolating CD34 + /VEGFR-3 + cells from the sample.
2. A method according to claim 1 , wherein the separating step comprises isolating CD34 + cells from the sample, and then isolating a VEGFR-3 + fraction of the CD34 + cells.
3. A method according to claim 2 , wherein the VEGFR-3 + fraction of the CD34 + cells is isolated via fluorescence-activated cell sorting of the CD34 + cells.
4. A method according to claim 1 , wherein the CD34 + /VEGFR-3 + cells are cultured with a VEGFR-3 ligand to support selective survival of a VEGFR-3+ fraction of the CD34 + cells, wherein the VEGFR-3 ligand comprises a polypeptide selected from the group consisting of
(a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, or comprising a portion thereof effective to bind human VEGFR-3; and
(b) a VEGF-C ΔCysis 156 polypeptide having an amino acid sequence comprising a portion of SEQ ID NO: 8 effective to permit binding to human VEGFR-3, wherein the cysteine residue at position 156 of SEQ ID NO: 8 has been deleted or replaced by another amino acid.
5. A method according to claim 1 , wherein the biological sample comprises human bone marrow.
6. A method according to claim 1 , wherein the biological sample comprises human blood.
7. A method according to claim 6 , wherein the biological sample comprises umbilical cord blood.
8. A method according to claim 1 , wherein the separating step comprises fluorescence-activated cell sorting of cells of the biological sample.
9. A composition comprising CD 34 + /VEGFR-3 + cells isolated according to the method of claim 2 .
10. Isolated cells that express both CD34 (CD34 + ) and VEGFR-3 (VEGFR-34 + ), wherein the cells are isolated from a biological sample from a human according to the method of claim 1 .
11. Isolated cells according to claim 10 , wherein the cells have been cultured with a VEGFR-3 ligand that comprises a polypeptide selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, or comprising a portion thereof effective to bind human VEGFR-3; and
(b) a VEGF-C ΔCys 156 polypeptide having an amino acid sequence comprising a portion of SEQ ID NO: 8 effective to permit binding to human VEGFR-3, wherein the cysteine residue at position 156 of SEQ ID NO: 8 has been deleted or replaced by another amino acid.
12. Isolated cells that express both CD34 (CD34 + ) and VEGFR-3 (VEGFR-3 + ), wherein the cells are isolated from a biological sample that comprises human bone marrow, according to the method of claim 5 .
13. Isolated cells according to claim 12 , wherein the cells have been cultured with a VEGFR-3 ligand that comprises a polypeptide selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, or comprising a portion thereof effective to bind human VEGFR-3; and
(b) a VEGF-C ΔCys 156 polypeptide having an amino acid sequence comprising a portion of SEQ ID NO: 8 effective to permit binding to human VEGFR-3, wherein the cysteine residue at position 156 of SEQ ID NO: 8 has been deleted or replaced by another amino acid.
14. Isolated cells that express both CD34 (CD34 + ) and VEGFR-3 (VEGFR-3 + ), wherein the cells are isolated from a biological sample that comprises human blood, according to the method of claim 6 .
15. Isolated cells according to claim 14 , wherein the cells have been cultured with a VEGFR-3 ligand that comprises a polypeptide selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, or comprising a portion thereof effective to bind human VEGFR-3; and
(b) a VEGF-C ΔCys 156 polypeptide having an amino acid sequence comprising a portion of SEQ ID NO: 8 effective to permit binding to human VEGFR-3, wherein the cysteine residue at position 156 of SEQ ID NO: 8 has been deleted or replaced by another amino acid.
16. Isolated cells that express both CD34 (CD34 + ) and VEGFR-3 (VEGFR-3 + ), wherein the cells are isolated from a biological sample that comprises human umbilical cord blood, according to the method of claim 7 .
17. Isolated cells according to claim 16 , wherein the cells have been cultured with a VEGFR-3 ligand that comprises a polypeptide selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, or comprising a portion thereof effective to bind human VEGFR-3; and
(b) a VEGF-C ΔCys 156 polypeptide having an amino acid sequence comprising a portion of SEQ ID NO: 8 effective to permit binding to human VEGFR-3, wherein the cysteine residue at position 156 of SEQ ID NO: 8 has been deleted or replaced by another amino acid.