WO2011073521A1

WO2011073521A1 - Methods for enriching adult-derived endothelial progenitor cells and uses thereof

Info

Publication number: WO2011073521A1
Application number: PCT/FI2010/051036
Authority: WO
Inventors: Petri Salven
Original assignee: Petri Salven
Priority date: 2009-12-15
Filing date: 2010-12-15
Publication date: 2011-06-23

Abstract

The present invention generally relates to the discovery of new and enhanced methods for isolating and enriching for adult-derived endothelial stem cell or adult-derived endothelial progenitor cell comprising cell populations, where a large proportion of the isolated cells are capable of undergoing clonal expansion. In particular, the inventors have discovered that the use of a specific and unique combination of the cell-surface markers significantly enriches for adult endothelial progenitor cells. The methods described herein provide significant and surprising improvements in the ability to select for and isolate adult-derived endothelial progenitor cells, compared to methods where only cells positive for the markers CD31 and CD 105 are used. In addition, the invention relates methods for ex vivo culture and expansion of clonal endothelial cells originating from a single endothelial progenitor cell and use of such cell populations.

Description

METHODS FOR ENRICHING ADULT-DERIVED ENDOTHELIAL

PROGENITOR CELLS AND USES THEREOF

FIELD OF THE INVENTION

[0001] The present invention relates to stem cells, in particular methods for identifying and enriching for adult-derived endothelial progenitor cells, the use of adult-derived endothelial progenitor cells in regenerative medicine, and targeting adult-derived endothelial progenitor cells to prevent angiogenesis.

BACKGROUND OF THE INVENTION

[0002] The development of the vascular system is an essential event during the embryonic development of many animal species. During this process, local mesodermal precursors differentiate into vascular and endothelial cells (ECs) to form a primary vascular plexus, a process referred to as vasculogenesis (Timmermans et al, J. Cell. Mol. Med. (2009) 13(1): 87-102).

[0003] Until recently, it was thought that the process of vasculogenesis occurs only during embryonic development, but not in postnatal life, and that endothelial regeneration and angiogenesis occurs only via proliferation of pre-existing resident vessel wall endothelial cells. While it is now more accepted that post-natal vasculogenesis may occur due to differentiation of a subset of bone marrow-derived cells into endothelial progenitor cells (EPCs), their exact identity has remained elusive. However, conflicting results have been reported in the field, and the identification, characterization, and exact role of EPCs in vascular biology is still a subject of much discussion. In fact, some reports still suggest that such EPCs do not exist (Timmermans et al, J. Cell. Mol. Med. (2009) 13(1): 87-102).

[0004] Current methods and techniques for isolating endothelial cell progenitor or stem cell populations have not been effective due to controversies in the field relating to the identification, characterization, and exact role of endothelial stem cells in vascular biology. Specifically, the ability to isolate and identify endothelial cell progenitors has been complicated by the lack of unique endothelial stem cell marker(s), the paucity of endothelial stem cells in the circulation, and the phenotypical and functional overlap between endothelial cell progenitors, haematopoetic cells, and mature endothelial cells (Timmermans et al, J. Cell. Mol. Med. (2009) 13(1): 87-102).

[0005] Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute an admission as to the correctness of the dates or contents of these documents.

SUMMARY OF THE INVENTION

[0006] The present invention generally relates to the discovery of new and enhanced methods for isolating and enriching for adult-derived endothelial stem cell

(hESC) or adult-derived endothelial progenitor cell (hEPC) comprising cell populations, where a large proportion of the isolated cells are capable of undergoing clonal expansion. In particular, the inventors have discovered that the use of a specific and unique combination of the cell-surface markers CD31 (PECAM-1), CD 105 (endoglin), and CD117 (c-kit) significantly enriches for adult endothelial progenitor cells. In some embodiments of this method, the unique combination of cell-surface markers also includes CD44 and Sca-1. The methods described herein provide significant and surprising improvements in the ability to select for and isolate adult-derived endothelial progenitor cells, compared to methods where only cells positive for the markers CD31 and CD 105 are used. The inventors have further discovered that the novel combination of cell surface markers can also be used for isolating adult-derived endothelial progenitor cells from an adult differentiated tissue, such as the lung. In addition, the inventors have discovered methods for ex vivo culture and expansion of clonal endothelial cells originating from a single endothelial progenitor cell. [0007] Accordingly, the present invention relates to the discovery of enhanced methods for selectively isolating adult-derived endothelial progenitor cells. Described herein are methods that comprise isolating and enriching adult-derived endothelial progenitor cells from adult-derived cell samples, where such adult-derived endothelial progenitor cells are identified by being positive for the cell-surface markers CD31

(PECAM- 1 ), CD 105 (endoglin) and CD 117(c-kit). The methods provided herein can, in some embodiments, further comprise that the isolated adult-derived endothelial progenitor cells are positive for CD44 and Sca-1, and negative for at least one lineage marker selected from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a. The methods described herein can be performed, for example, using immunomagnetic selection techniques, fluorescence activated cell sorting, or a combination therein. The methods can further comprise the use of at least one antibody specific for at least one of the cell-surface markers described herein. The methods described herein are also useful for the isolation of endothelial progenitor cells from solid tissue, such as the lung, and from vascular endothelium, such as lung vascular endothelium. The methods can further comprise in vitro culturing following the selective isolation using, for example, a low-cell density adherent semi- so lid matrix colony assay.

[0008] Accordingly, in some aspects, provided herein is an isolated cell population comprising adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for the cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44 and Sca-1, and are negative for at least one cell- surface marker from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a. Such a cell population can be a naturally isolated cell population or a genetically modified cell population. In other aspects, described herein is a pharmaceutical composition comprising an isolated adult-derived vascular endothelial progenitor cell population, where the cells of the endothelial progenitor cell population are positive for the cell surface markers CD31 (PECAM- 1 ), CD 105 (endoglin), CD 117(c-kit), CD44 and Sca-1 , and are negative for at least one cell-surface marker from the group comprising CD2, CD3, CD 14, CD 16, CD 19, CD56, and CD235a.

[0009] Accordingly, in some aspects, provided herein is an isolated cell population comprising adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for the cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit) and Sca-1, and are negative for at least one cell- surface marker from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a. Such a cell population can be a naturally isolated cell population or a genetically modified cell population. In other aspects, described herein is a pharmaceutical composition comprising an isolated adult-derived vascular endothelial progenitor cell population, where the cells of the endothelial progenitor cell population are positive for the cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit) and Sca-1, and are negative for at least one cell-surface marker from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

[00010]

[00011] The isolated endothelial progenitor cells and the methods to isolate such cells described herein are useful in a variety of therapeutic applications where

neoangiogenesis and restoration of tissue vascularization are required, such as in regenerative medicine following ischemic events and for wound healing, and for use in tissue engineering.

[0010] Accordingly, described herein are methods of inducing vascularization in a tissue comprising administering to a tissue in need of vascularization an isolated cell population comprising adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for the cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44, and Sca-1, and are negative for at least one cell- surface marker from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a. Such a cell population can be a naturally isolated cell population or a genetically modified cell population. In other aspects, a pharmaceutical composition for use in inducing vascularization is provided, where the composition comprises isolated adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44 and Sca-1, and are negative for at least cell- surface marker from the group CD2, CD3, CD 14, CD 16, CD 19, CD56, and CD235a. The isolated adult vascular endothelial progenitor cells can be autologous or allogenic cells. Also described herein are uses of such isolated adult vascular endothelial progenitor cells in inducing vascularization in tissues.

[0011] Described herein are also methods of inducing vascularization in a tissue comprising administering to a tissue in need of vascularization an isolated cell population comprising adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for the cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit) and Sca-1, and are negative for at least one cell- surface marker from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a. Such a cell population can be a naturally isolated cell population or a genetically modified cell population. In other aspects, a pharmaceutical composition for use in inducing vascularization is provided, where the composition comprises isolated adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit) and Sca-1, and are negative for at least cell- surface marker from the group CD2, CD3, CD 14, CD 16, CD 19, CD56, and CD235a. The isolated adult vascular endothelial progenitor cells can be autologous or allogenic cells. Also described herein are uses of such isolated adult vascular endothelial progenitor cells in inducing vascularization in tissues.

[0012]

[0013] Further, the ability to specifically identify endothelial stem cell-enriched cell population is useful in targeting and blocking angiogenesis, such as in tumors.

Accordingly, also provided herein are methods for blocking angiogenesis comprising targeting an angiogenesis inhibitor to a cell that is positive for CD31 (PECAM-1), CD 105 (endoglin), CDl 17(c-kit), CD44 and Sca-1, and that is negative for at least one of the cell- surface markers comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a. Such targeting can be performed using an antibody or a mixture of antibodies specific for CD31 (PECAM-1), CD105 (endoglin), CDl 17(c-kit), CD44 and Sca-1.

[0014] It is also provided herein are methods for blocking angiogenesis comprising targeting an angiogenesis inhibitor to a cell that is positive for CD31 (PECAM-1), CD 105 (endoglin), CDl 17(c-kit) and Sca-1, and that is negative for at least one of the cell- surface markers comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a. Such targeting can be performed using an antibody or a mixture of antibodies specific for CD31

(PECAM-1), CD105 (endoglin), CD117(c-kit) and Sca-1.

[0015]

BRIEF DESCRIPTION OF THE DRAWINGS [0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fees. [0017] Figure 1 shows rare endothelial Lin^"CD31 CD 105 colony- forming cells

(CFCs) can be isolated from adult mouse tissues. Figure 1 A shows in vivo BrdU labeling (green, arrows) of cellular proliferation in the adult C57BL/6 mouse lung vasculature ECs stained for vWF (red) and DAPI (blue). Figure IB shows that after one month an average of 5.8% (SD ±0.29) of all blood vessel ECs in the lungs are by a replaced by a newly formed EC. The mean percentage (±SD) of BrdU-positive ECs of total ECs in the lungs (n=4) at 30, 60, and 90 days are shown. Figure 1C shows colony forming abilities of Lin^" CD31 CD105 cells isolated from the mouse lungs. Freshly isolated cells from 4 mice were assayed in duplicate. Figure ID shows GFP-expressing CD31⁺CD105⁺ seeded on 2D EC cultures one CFU per dish together with 20 CFUs of wild-type ECs form one clonal, circular GFP⁺ batch within the wild-type monolayers when cultured until confluent. Bright field and GFP-channels are shown (right, scale bar, 100 μιη).

[0018] Figure 2 shows that blood vessel walls contains CD117⁺ EC

subpopulations with a very high proliferative potential. Figure 2A shows a comparison of CFCs within the CD117 depleted and CD117 enriched subpopulations of Lin^"CD31⁺ CD105⁺ ECs in vitro in colony forming assays. The results of four independent experiments, each performed in duplicate, are shown. The horizontal lines indicate 10th, 25th, 50th (median), 75th, and 90th percentiles. Practically all CFCs are encompassed within the CD117⁺ EC population. *P<0.0001, The Mann- Whitney U test. Figure 2B shows the relative distributions and overlap of SCA-1⁺, CD117⁺, and CD44⁺

subpopulations within isolated mouse lung Lin^"CD31⁺ CD105⁺ ECs. The results (mean ±SD) of eleven FACS sortings are shown. Figure 2C shows a comparison of the distribution of CFCs within Lin^"CD31⁺ CD105⁺ ECs that were divided into fractions enriched or depleted for Sca-1, CD117, or CD44 in a single FACS sort. The highest proportion of CFUs were found in the Lin^" CD31⁺ CD105⁺Scal⁺CD117⁺CD44⁺

subpopulation. However, this subpopulation is very small in size, and does not contain most of the CD117⁺ CFCs. The results of four independent experiments, each performed in duplicate, are shown. *P=0.0002. Figure 2D demonstrates that Sca-1⁺CD117⁺ ECs reside in the lung vascular endothelium. High resolution confocal scans are shown. Figure 2E shows that Scal⁺CD117⁺ ECs can be detected also in subcutaneous tissues of the ears.

[0019] Figure 3 demonstrates that a single adult stem cell with the phenotype Lin^"

CD31⁺CD105⁺Scal⁺ CD117⁺ can generate functional, perfused blood vessels. Figure 3A shows a flow diagram of the FACS sorting procedure used to obtain Lin^" CD31 CD 105 Seal CDl 17 cells. Prior to transplantation, a single colony originating from a single GFP -tagged Lin^~CD31 CD105 Scal⁺CDl 17⁺ colony forming cell was expanded for twelve days in adherent culture to amplify the cell number. Figure 3B shows that functional, perfused GFP⁺ blood vessels generated by the transplanted descendants of a single Lin^~CD31 CD105⁺Scal⁺CD117⁺ cell (D14 after transplantation). The mouse was perfused with fluorescent 0.2 μιη microspheres (red) to visualize functional blood vessels. ECs were stained for CD31 and CD 105. A 3D orthogonal projection (x-z and y-z axes) from a 1 μιη thick confocal slice is also shown (right). Six independent experiments with similar results were performed. Figure 3C shows the evaluation of self-renewal capacity, as defining characteristic of stem cells, by inoculating mice with B16 melanomas (2 million cells per mice) together with 15 CFUs of GFP-tagged isolated CD31⁺CD105⁺ ECs. After two weeks of tumor growth, repeated isolations and serial transplantations of lineage depleted single cell suspensions containing the GFP⁺ tagged ECs and the B16 cells. The figure shows GFP⁺ blood vessels in the quaternary transplant. ECs were stained for VEGFR-2 (red), vWF (white), and CD31 and CD 105 (red). A 3D reconstitution of a GFP⁺ blood vessel in the quaternary transplant is also shown (right; a 34 μιη thick stack of 34 x-y slices from a confocal scan). Six independent experiments with similar results were performed. Figure 3D shows a schematic of the novel adult endothelial stem cell hierarchy described herein and the existence of a rare slowly cycling self-renewing CDl 17 Sca-l⁺ adult vascular endothelial stem cell (VESC) that resides at the blood vessel endothelium.

[0020] Figure 4 demonstrates that CD 117 CD44 EC are abundant in angiogenic and tumor blood vessels. Figure 4A shows that CDl 17⁺ (white) or CD44⁺ (green) ECs are infrequent in quiescent vasculature in intact subcutaneous tissues. ECs are detected against Sca-1 or CD31 (both red). Figure 4B shows that neoangiogenic vessels in subcutaneous matrigel plugs contain very large numbers of CDl 17⁺ and/or CD44⁺ ECs. Figure 4C shows that in intratumor vasculature of subcutaneous B16 melanomas, CDl 17⁺ or CD44⁺ ECs constituted the majority of all ECs, with long segments of vasculature being composed of CD117⁺CD44⁺ ECs.

[0021] Figure 5 depicts a sorting protocol utilized for isolating endothelial progenitor cell populations.

DETAILED DESCRIPTION OF THE INVENTION [0022] The present invention generally relates to the discovery of new and enhanced methods for isolating and enriching for adult-derived endothelial stem cell (ESC) or adult-derived endothelial progenitor cell (EPC) comprising cell populations. The invention provides, in part, a specific and unique combination of cell-surface markers that significantly enriches for adult-derived endothelial progenitor cells, thus allowing for a significant improvement in the ability to select for and isolate adult-derived endothelial progenitor cells from adult biological samples. These unique methods are also useful in the isolation of adult-derived endothelial progenitor cells from differentiated tissues. In other aspects, the invention provides compositions of, and methods for isolating, adult-derived endothelial progenitor cells for use in a variety of therapeutic applications in regenerative medicine, where neoangiogenesis and restoration of tissue vascularization, i.e., vascular regeneration, are required, including, but not limited to, following ischemic events, wound healing, and for use in tissue engineering. In other aspects, the invention provides methods for targeting and blocking tumor angiogenesis based on the ability to specifically identify adult-derived endothelial progenitor cell populations.

Isolating and Enriching for Endothelial Progenitor Cells

[0023] Described herein are methods for isolating and enriching for adult-derived endothelial progenitor cells (EPCs) from an adult-derived biological sample, such as a cell sample. Such methods comprise selecting from an adult-derived cell sample those cells that are positive for cell- surface markers comprising CD31 (PECAM-1), CD 105 (endoglin) and CD117(c-kit), thereby enriching the adult-derived cell sample for EPCs.

[0024] Accordingly, in some aspects, the invention provides methods for isolating and enriching for adult-derived endothelial progenitor cells. The methods comprise using the specific and novel combination of the cell-surface molecules, CD31 (PECAM-1), CD 105 (endoglin), and CD117 (c-kit), to enrich for adult-derived endothelial progenitor cells (EPCs), such as human adult-derived EPCs, from a variety of sources and biological samples, including differentiated tissues.

[0025] "CD31" refers to the cell-surface adhesion molecule, also known as

PECAM-1, that is expressed in large amounts on endothelial cells at intercellular junctions, on T cell subsets, and to a lesser extent, on platelets and most leukocytes. Expression of CD31 is required for the transendothelial migration of leukocytes through the intercellular spaces between vascular endothelial cells.

[0026] "CD 105," also known as "endoglin," refers to the type I membrane glycoprotein located on cell surfaces that is part of the TGF-β receptor complex. The protein consists of a homodimer of 180 kDA with disulfide links. It has been found on endothelial cells, activated macrophages, fibroblasts, and smooth muscle cells. As CD 105 has been found to be part of the TGF- βΐ receptor complex, it may be involved in the binding of TGF- βΐ, TGF- β3, activin-A, BMP-2, and BMP-7. In addition to its role in TGF- β signaling, it has been postulated that endoglin is involved in the cytoskeletal organization affecting cell morphology and migration, development of the cardiovascular system, and in vascular remodeling. Its expression is regulated during heart development. Experimental mice without the endoglin gene die due to cardiovascular abnormalities.

[0027] "CDl 17" refers to the transmembrane cytokine receptor expressed on the surface of hematopoietic stem cells, as well as other cell types, also known as C-kit receptor. c-Kit is a proto-oncogene that is a member of the receptor tyrosine kinase family and, more specifically, is closely related to the platelet derived growth factor receptor (PDGFR). c-Kit is the normal cellular homolog of the HZ4-feline sarcoma virus transforming gene (v-Kit). c-Kit regulates a variety of biological responses including chemotaxis, cell proliferation, apoptosis, and adhesion. c-Kit is also identical with the product of the W locus in mice and, as such, is integral to the development of mast cells and hematopoiesis. The ligand for the c-Kit receptor (KL) has been identified and is encoded at the murine steel (SI) locus. Kit is the human homolog of the proto-oncogene c- Kit. In various cancers, it has been found that mutations in Kit are integral for tumor growth and progression. [0028] CDl 17 is often used to identify certain types of hematopoietic (blood) progenitors in the bone marrow. It is known that hematopoietic stem cells (HSC), multipotent progenitors (MPP), and common myeloid progenitors (CMP) express high levels of CDl 17, while common lymphoid progenitors (CLP) expresses low surface levels of CDl 17. CDl 17 is also used to identify the earliest thymocyte progenitors in the thymus, specifically early T lineage progenitors (ETP/DN1) and DN2 thymocytes, which express high levels of CDl 17. Additionally mast cells, melanocytes in the skin, and interstitial cells of Cajal in the digestive tract have been shown to express CDl 17. CDl 17 is also a marker for mouse prostate stem cells. CDl 17 is the receptor for the cytokine stem cell factor (SCF), also known as "steel factor" or "c-kit ligand". SCF exists in two forms, cell surface bound SCF and soluble (or free) SCF.

[0029] In one aspect, a method to isolate a substantially pure population of adult- derived endothelial progenitor cells is provided, where a combination of agents specific for the group of cell-surface markers comprising CD31 (PECAM-1), CD 105 (endoglin), and CDl 17 (c-kit) are contacted with a biological sample to isolate a substantially pure population of adult-derived EPCs from the biological sample. In some embodiments of this aspect, the group of cell- surface markers consists essentially of CD31, CD 105, and CDl 17. In some embodiments of the aspect, the group of cell-surface markers consists of CD31 , CD 105, and CDl 17. In some embodiments of this aspect and all such aspects described herein, the substantially pure population of EPCs is positive for the expression of CD31, CD105, and CDl 17.

[0030] Also described herein are methods for isolating and enriching for adult- derived endothelial progenitor cells (EPCs) from an adult-derived biological sample, such as a cell sample. Such methods comprise selecting from an adult-derived cell sample those cells that are positive for cell- surface markers comprising CD31 (PECAM-1), CD 105 (endoglin), CDl 17(c-kit), CD44, and Sca-1, and negative for one or more lineage markers, thereby enriching the adult-derived cell sample for EPCs. [0031] Accordingly, in other embodiments of the aspects described herein, the method further comprises the use of additional cell-surface markers, such as CD44, Seal (Ly6A/E), and lineage markers to isolate the substantially pure population of adult-derived EPCs from the biological sample.

[0032] "CD44," as used herein, refers to a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. CD44 is a receptor for hyaluronic acid and can also interact with other ligands, such as osteopontin, collagens, and matrix metalloproteinases (MMPs). A specialized sialofucosylated glycoform of CD44 called HCELL is found natively on human hematopoietic stem cells, and is a highly potent E- selectin and L-selectin ligand. HCELL functions as a "bone homing receptor", directing migration of human hematopoietic stem cells and mesenchymal stem cells to bone marrow. CD44 participates in a wide variety of cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis.

[0033] As used herein, "Sca-1" refers to anl8 kDa member of the Ly-6 family of

GPI-linked surface proteins found in mice, and is expressed by mouse hematopoietic stem cells, myeloid population and peripheral T and B cells. Sca-1 is expressed at high levels upon activation regardless of the Ly-6 haplotype. The Ly-6 family is involved in regulation and function of T cell activation. Sca-1 serves as a major phenotypic marker for mouse hematopoietic progenitor/stem cell subset.

[0034] A number of different cell-surface markers have specific expression on specific differentiated cell lineages, and are not expressed by the adult-derived endothelial progenitor cells isolated by the methods described herein. Accordingly, when agents specific for these lineage cell-markers are contacted with endothelial progenitor cells, the cells will be "negative." Lineage cell-markers that are not expressed by endothelial progenitor cells contemplated for use in the invention include, but are not limited to, CD 13 and CD33 (expressed on myeloid cells); CD71 (expressed on erythroid cells); CD19 and B220 (expressed on B cells), CD61 (expressed on human megakaryocyte cells); Mac-1 (CDl lb/CD 18) (expressed on monocytes); Gr-1 (expressed on granulocytes); Terl 19 (expressed on erythroid cells); and I17Ra, CD2, CD3, CD4, CD5, CD8 (expressed on T cells); CD14, CD56, and CD235a. In some embodiments of the aspects described herein, the lineage markers used may be dependent on the species from which the adult-derived endothelial progenitor cells are being isolated, as determined by one of skill in the art. For example, when isolating human adult-derived endothelial progenitor cells the combination of lineage markers can comprise CD2, CD3, CD16, CD19, CD56, and CD235a. For example, when isolating murine adult-derived endothelial progenitor cells the combination of lineage markers can comprise CD5, CD45R (B220), CDl lb, Gr-1 (Ly-6G/C), 7-4, and Ter-119.

[0035] Accordingly, in some embodiments of this aspect, the group of cell-surface markers comprises CD31, CD105, CD117, and CD44. in some embodiments of this aspect, the group of cell- surface markers comprises CD31, CD 105, and CDl 17. In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, and CD44. In some embodiments of this aspect, the group of cell-surface markers consists of CD31, CD105, CDl 17, and CD44. In some embodiments of this aspect and all such aspects described herein, the substantially pure population of adult-derived EPCs is positive for the expression of CD31, CD105, CDl 17, and CD44.

[0036] In other embodiments of this aspect, the group of cell-surface markers comprises CD31, CD 105, CDl 17, CD44, and one or more lineage markers. In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD 105, CDl 17, CD44, and one or more lineage markers. In some embodiments of this aspect, the group of cell-surface markers consists of CD31, CD 105, CDl 17, CD44, and one or more lineage markers. In some embodiments of this aspect and all such aspects described herein, the substantially pure population of adult-derived EPCs is positive for the expression of CD31, CD105, CDl 17, and CD44, and negative for the expression of the one or more lineage markers. In some embodiments of this aspect and all such aspects described herein, the substantially pure population of adult-derived EPCs is positive for the expression of CD31 , CD 105and CD 117, and negative for the expression of the one or more lineage markers. In some embodiments, the one or more lineage markers comprise the group CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

[0037] In some embodiments of this aspect, the group of cell-surface markers comprises CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of this aspect, the group of cell- surface markers comprises CD31, CD 105, CDl 17 and Sca-1, and one or more lineage markers. In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, CD44,

Sca-1, and one or more lineage markers. In some embodiments of this aspect, the group of cell-surface markers consists of CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments, the one or more lineage markers comprise the group CD2, CD3, CD14, CD16, CD19, CD56, and CD235a. In some embodiments of this aspect and all such aspects described herein, the substantially pure population of adult- derived EPCs is positive for the expression of CD31, CD 105, CDl 17, CD44, and Sca-1 and negative for the expression of the one or more lineage markers. In some embodiments of this aspect and all such aspects described herein, the substantially pure population of adult-derived EPCs is positive for the expression of CD31, CD 105, CDl 17 and Sca-1 and negative for the expression of the one or more lineage markers.

[0038] [0039] In another aspect, the invention provides a method to isolate an enriched population of adult-derived endothelial progenitor cells, where a combination of agents specific for the group of cell- surface markers comprising CD31 (PECAM-1), CD 105 (endoglin), and CDl 17 (c-kit) are contacted with an adult-derived biological sample to isolate an enriched population of adult-derived EPCs from the biological sample. In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD 105, and CDl 17. In some embodiments of the aspect, the group of cell- surface markers consists of CD31, CD 105, and CDl 17. In some embodiments of this aspect and all such aspects described herein, the enriched population of adult-derived EPCs is positive for the expression of CD31, CD105, and CDl 17.

[0040] In other embodiments, the method further comprises the use of additional cell-surface markers, such as Seal (Ly6A/E), lineage markers, and CD44, to isolate the enriched population of adult-derived EPCs from the biological sample.

[0041] In some embodiments of this aspect, the group of cell- surface markers comprises CD31, CD105, CDl 17, and CD44. In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, and CD44. In some embodiments of this aspect, the group of cell-surface markers consists of CD31 , CD105, CDl 17, and CD44. In some embodiments of this aspect and all such aspects described herein, the enriched population of adult-derived EPCs is positive for the expression of CD31, CD105, CDl 17, and CD44.

[0042] In other embodiments of this aspect, the group of cell-surface markers comprises CD31, CD 105, CDl 17, CD44, and one or more lineage markers. In other embodiments of this aspect, the group of cell- surface markers comprises CD31, CD 105, CDl 17 and one or more lineage markers. In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD 105, CDl 17, CD44, and one or more lineage markers. In some embodiments of this aspect, the group of cell-surface markers consists of CD31, CD 105, CDl 17, CD44, and one or more lineage markers. In some embodiments of this aspect and all such aspects described herein, the enriched population of adult-derived EPCs is positive for the expression of CD31, CD105, CDl 17, and CD44, and negative for the expression of the one or more lineage markers. In some embodiments, the one or more lineage markers comprise the group CD2, CD3, CD 14, CD 16, CD 19, CD56, and CD235a. [0043] In some embodiments of this aspect, the group of cell-surface markers comprises CD31, CD105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of this aspect, the group of cell- surface markers comprises CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of this aspect, the group of cell-surface markers consists of CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of this aspect and all such aspects described herein, the enriched population of adult-derived EPCs is positive for the expression of CD31, CD 105, CDl 17, CD44, and Sca-1 and negative for the expression of the one or more lineage markers. In some embodiments, the one or more lineage markers comprise the group CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

[0044] As referred to herein, an "adult-derived endothelial progenitor cell" refers to a population of adult immature progenitor cells that are capable of mediating post-natal vasculogenic activity, including the formation of new endothelial cells and blood vessels in vivo. An adult-derived endothelial progenitor cell is a cell that can be clonally and serially replated in culture and will give rise to endothelium either by differentiation in vitro or direct incorporation into the vessel wall in vivo. Exemplary methods to test whether an isolated population is an EPC population are described herein. Such EPCs, unlike the majority of adult endothelial cells found in differentiated tissues, are capable of robust clonal expansion, secondary and tertiary colony formation upon replating, and de novo blood vessel formation when transplanted into immunodeficient mice. Such cells can be isolated from a variety of sources, including, but not limited to, differentiated adult tissues, such as lung tissue, peripheral blood, and bone marrow. As referred to herein, the term "adult" refers to any stage of organism development past the fetal stage, i.e., from the moment of birth, all tissues and cells are referred to herein as "adult," unless expressly indicated otherwise. Accordingly, an adult-derived cell population or biological sample is that is obtained from an adult organism.

[0045] The terms "isolate" and "methods of isolation," as used herein, refers to a process whereby a cell or population of cells is removed from a subject or sample in which it was originally found, or a descendant of such a cell or cells. The term "isolated population" with respect to an isolated population of cells, as used herein, refers to a population of cells that has been removed and separated from a biological sample, or a mixed or heterogeneous population of cells found in such a sample. Such a mixed population includes, for example, a population of peripheral blood mononuclear cells obtained from isolated blood, or a cell suspension of a tissue sample. In some

embodiments, an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched from. In some embodiments of this aspect and all aspects described herein, the isolated population is an isolated population of endothelial progenitor cells. In other embodiments of this aspect and all aspects described herein, the isolated population comprises a substantially pure population of endothelial progenitor cells as compared to a

heterogeneous population of cells comprising various somatic cells types from which the endothelial progenitor cells were derived. In some embodiments, an isolated cell or cell population, such as a population of adult-derived endothelial progenitor cells, is further cultured in vitro, e.g., in the presence of growth factors or cytokines, to further expand the number of cells in the isolated cell population or substantially pure cell population. Such culture can be performed using any method known to one of skill in the art, for example, low-cell density adherent semi- so lid matrix colony assay, as described in the Examples section. In some embodiments, the isolated or substantially pure adult-derived cell populations obtained by the methods disclosed herein are later introduced into a second subject, or re-introduced into the subject from which the cell population was originally isolated (e.g., allogenic transplantation).

[0046] The term "substantially pure," with respect to a particular cell population, refers to a population of cells that is at least about 75%, at least about 80%, at least about 85%o, at least about 90%>, at least about 95%, at least about 98%>, or at least about 99% pure, with respect to the cells making up a total cell population. In other words, the terms "substantially pure" or "essentially purified", with regard to a population of endothelial progenitor cells isolated using the methods as disclosed herein, refers to a population of adult-derived EPCs that contain fewer than about 25%, fewer than about 20%, fewer than about 15%), fewer than about 10%>, fewer than about 9%, fewer than about 8%, fewer than about 7%), fewer than about 6%, fewer than about 5%, fewer than about 4%, fewer than about 4%, fewer than about 3%, fewer than about 2%, fewer than about 1%, or less than 1%, of cells that are not adult-derived EPCs as defined by the terms herein. Some embodiments of these aspects further encompass methods to expand a population of substantially pure or enriched adult-derived EPCs, wherein the expanded population of EPCs is also a substantially pure or enriched population of adult-derived EPCs. [0047] The terms "enriching" or "enriched" are used interchangeably herein and mean that the yield (fraction) of cells of one type, such as endothelial progenitor cells, is increased by at least 15%, by at least 20%, by at least 25%, by at least 30%>, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, or by at least 75%, over the fraction of cells of that type in the starting biological sample, culture, or preparation. A population of endothelial progenitor cells obtained by the methods described herein using a combination of agents specific for the group of cell-surface molecules comprising CD31, CD 105, CDl 17, CD44, and Sca-1 is most preferably at least 60% enriched for endothelial progenitor cells. [0048] A "marker," as used herein, describes the characteristics and/or phenotype of a cell. Markers can be used for selection of cells comprising characteristics of interest. Markers will vary with specific cells. Markers are characteristics, whether morphological, functional or biochemical (enzymatic), particular to a cell type, or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, possess an epitope for antibodies or other binding molecules available in the art. However, a marker may consist of any molecule found in a cell including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages. Markers may be detected by any method available to one of skill in the art.

[0049] Accordingly, as used herein, a "cell-surface marker" refers to any molecule that is expressed on the surface of a cell. Cell-surface expression usually requires that a molecule possesses a transmembrane domain. Some molecules that are normally not found on the cell-surface can be engineered by recombinant techniques to be expressed on the surface of a cell. Many naturally occurring cell-surface markers are termed "CD" or "cluster of differentiation" molecules. Cell-surface markers often provide antigenic determinants to which antibodies can bind to. Cell-surface markers of particular relevance to the methods described herein include CD31, CD 105, CDl 17, CD44, and Sca-1. [0050] A cell can be designated "positive" or "negative" for any of the cell-surface markers described herein, and both such designations are useful for the practice of the methods described herein. A cell is considered "positive" for a cell-surface marker if it expresses the marker on its cell-surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker, and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is bound the cell. It is to be understood that while a cell may express messenger R A for a cell-surface marker, in order to be considered positive for the methods described herein, the cell must express it on its surface. Similarly, a cell is considered "negative" for a cell-surface marker if it doe not express the marker on its cell-surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is bound the cell. In such embodiments, where agents specific for cell-surface lineage markers used, the agents can all comprise the same label or tag, such as fluorescent tag, and thus all cells positive for that label or tag can be excluded or removed in the methods to isolate endothelial progenitor cells described herein, so that the remaining adult-derived endothelial cells are "negative" for the one or more lineage markers used. [0051] Accordingly, as defined herein, an "agent specific for a cell-surface marker" refers to an agent that can selectively react with or bind to that cell-surface marker, but has little or no detectable reactivity to another cell-surface marker or antigen. For example, an agent specific for CD117 will not be specific for CD 105. Thus, agents specific for cell-surface markers recognize unique structural features of the markers. In some embodiments, an agent specific for a cell-surface marker binds to the cell-surface marker, but does not cause initiation of downstream signaling events mediated by that cell- surface marker, for example, a non-activating antibody. Agents specific for cell-surface molecules include, but are not limited to, antibodies or antigen-binding fragments thereof, natural or recombinant ligands, small molecules; nucleic acid sequence and nucleic acid analogues; intrabodies; aptamers; and other proteins or peptides.

[0052] In some embodiments of this aspect and all aspects described herein, the preferred agents specific for cell-surface markers are antibody agents that specifically bind the cell-surface markers, and can include polyclonal and monoclonal antibodies, and antigen-binding derivatives or fragments thereof. Well-known antigen binding fragments include, for example, single domain antibodies (dAbs; which consist essentially of single VL or VH antibody domains), Fv fragment, including single chain Fv fragment (scFv), Fab fragment, and F(ab')2 fragment. Methods for the construction of such antibody molecules are well known in the art. Accordingly, as used herein, the term "antibody" refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region. Antigen- binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. "Antigen-binding fragments" include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. The terms Fab, Fc, pFc', F(ab') 2 and Fv are employed with standard immunological meanings [Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford)]. Such antibodies or antigen-binding fragments specific for CD31, CD 105, CD 105, CD44, and Sca-1 are available

commercially from vendors such as R&D Systems, BD Biosciences, e-Biosciences and Miltenyi, or can be raised against these cell-surface markers by methods known to those skilled in the art.

[0053] In some embodiments of the aspects described herein, an agent specific for a cell-surface molecule, such as an antibody or antigen-binding fragment, is labeled with a tag to facilitate the isolation of the adult-derived endothelial cell population. The terms "label" or "tag", as used herein, refer to a composition capable of producing a detectable signal indicative of the presence of a target, such as, the presence of a specific cell-surface marker in a biological sample. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bio luminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods to isolate and enrich endothelial cell progenitor cells. [0054] The terms "labeled antibody" or "tagged antibody", as used herein, includes antibodies that are labeled by detectable means and include, but are not limited to, antibodies that are fluorescently, enzymatically, radioactively, and chemiluminescently labeled. Antibodies can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS, which can be detected using an antibody specific to the tag, for example, an anti-c-Myc antibody. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Non-limiting examples of fluorescent labels or tags for labeling the antibodies for use in the methods of invention include Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester,

Methoxycoumarin, Succinimidyl ester, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7.

[0055] In some embodiments of these aspects and all such aspects described herein, a variety of methods to isolate a substantially pure or enriched population of adult-derived endothelial progenitor cells are available to a skilled artisan, including immunoselection techniques, such as high-throughput cell sorting using flow cytometric methods, affinity methods with antibodies labeled to magnetic beads, biodegradable beads, nonbiodegradable beads, and antibodies panned to surfaces including dishes and combination of such methods.

[0056] As defined herein, "flow cytometry" refers to a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them through an electronic detection apparatus. Flow cytometry allows simultaneous multiparametric analysis of the physical and/or chemical parameters of up to thousands of particles per second, such as fiuorescent parameters. Modern flow cytometric instruments usually have multiple lasers and fluorescence detectors. Increasing the number of lasers and detectors allows for labeling by multiple antibodies, and can more precisely identify a target population by their phenotypic markers. Certain flow cytometric instruments can even take digital images of individual cells, allowing for the analysis of fluorescent signal location within or on the surface of cells.

[0057] A common variation of flow cytometric techniques is to physically sort particles based on their properties, so as to purify populations of interest, using

"fluorescence-activated cell sorting" As defined herein, "fluorescence-activated cell sorting" refers to a flow cytometric method for sorting a heterogeneous mixture of cells from a biological sample into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell and provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell per droplet. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately-prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some flow cytometric systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off. Accordingly, in those embodiments when the agents specific for the cell-surface markers CD31, CD 105, CD117, CD44, and Sca-1 are antibodies labeled with tags that can be detected by flow cytometers, fluorescence- activated cell sorting can be used in and with the methods, assays, and kits described herein to isolate and enrich for populations of adult-derived endothelial progenitor cells.

[0058] In other embodiments of these aspects and all aspects described herein, isolation and enrichment for populations of endothelial progenitor cells can be performed using bead based sorting mechanisms, such as magnetic beads. In such methods, the sample of cells to be isolated or enriched is contacted with magnetic beads coated with antibodies against one or more specific cell- surface antigens, such as CD31, CD 105, CD117, CD44, and Sca-1. This causes the cells expressing this antigen to attach to the magnetic beads. Afterwards the contacted cell solution is transferred to a strong magnetic field, such as a column or rack having a magnet. The cells attached to the beads

(expressing the cell-surface markers) stay on the column or sample tube, while other cells (not expressing the antigen) flow through or remain in solution. Using this method, cells can be separated positively or negatively with respect to the particular cell-surface markers. As defined herein, "positive selection" refers to techniques that result in the isolation or enrichment of cells expressing specific cell-surface markers, while "negative selection" refers techniques that result in the isolation or enrichment of cells not expressing specific cell-surface markers. In some embodiments, beads can be coated with antibodies by a skilled artisan using standard techniques known in the art, such as commercial bead conjugation kits. In some embodiments, a negative selection step is performed to remove cells expressing one or more lineage markers, followed by fluorescence activated cell sorting to positively select adult-derived endothelial progenitor cells expressing CD31 , CD105, CD117, CD44, and Sca-1. For example, a cell sample is first contacted with labeled antibodies specific for cell- surface markers of interest, such as CD2, CD3, CD 14, CD 16, CD 19, CD56, and CD235a and the sample is then contacted with beads that are specific for the labels of the antibodies, and the cells expressing any of the markers CD2, CD3, CD14, CD16, CD19, CD56, and CD235a are removed using immunomagnetic lineage depletion.

Expansion and Characterization of EPCs

[0059] Also described herein are methods to further expand propagate the isolated or enriched populations of adult-derived endothelial progenitor cells for functional identification of such cells, or for use in other aspects and embodiments of the invention.

[0060] In some embodiments, populations of adult-derived EPCs isolated or enriched using a combination of agents specific for the cell- surface markers CD31, CD 105, CD117, CD44, and Sca-1 are used in colony- forming assays to expand and further characterize the adult-derived EPCs. In some embodiments, the isolated adult-derived

EPCs are expanded in culture using methods known to one of skill in the art prior to use in a subject in need, for example. Such expansion methods can comprise the use of low- density adherent semi- so lid matrix colony assays as described herein in the Example section. Isolated adult-derived EPCs can be cultured under primary culture conditions resulting in the outgrowth of EPC-derived discrete, adherent colonies by day 5 to day 14 termed herein as "colony- forming unit-ECs" (CFU-ECs). Conditions for such cultures can be found in the Example section and Current Protocols in Stem Cell Biology, 2008, 2C 1.1 - 2C1.27. CFU-ECs display phenotypic and functional characteristics of adult endothelial cells, including expression of cell surface markers, CD31, CD 105, CD 144, CD 146, vWF, and KDR, uptake of AcLDL (acetylated low-density lipoprotein), upregulate VCAM-1, and form capillary- like tubes when plated on Matrigel (Lin et al., 2000; Gulati et al., 2003; Hur et al., 2004; Ingram et al., 2004; Yoder et al., 2007). In some embodiments, the individual endothelial cell colonies or CFU-ECs thus formed can be clonally isolated and serially subcultured or replated, using techniques known to the skilled artisan.

[0061] As described herein, the use of a combination of agents specific for the cell- surface markers CD31, CD 105, CD 117, CD44, and Sca-1 for the isolation or enrichment of adult-derived EPCs results in an increased yield of CFU-ECs when compared to a method of isolation or enrichment of EPCs using a combination of agents specific only for the cell- surface markers CD31 and CD 105. In some embodiments, the increased yield is at least 5 fold greater. Also described herein, CFU-ECs obtained using a combination of agents specific for the cell- surface markers CD31, CD 105, CD 117, CD44, and Sca-1 for the isolation or enrichment of adult-derived EPCs have an increased ability to form new colonies when serially subcultured, when compared to CFU-EPCs obtained using a method of isolation or enrichment of adult-derived EPCs using a combination of agents specific only for the cell-surface markers CD31 and CD 105. In some embodiments, the increased ability to form new colonies is at least 6 fold greater. [0062] The terms "increased," "increase," or "enhance" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased," "increase," or "enhance" mean an increase, as compared to a reference level, of at least about 10%, of at least about 15%, of at least about 20%, of at least about 25%, of at least about 30%, of at least about 35%, of at least about 40%, of at least about 45%, of at least about 50%, of at least about 55%, of at least about 6o%, of at least about 65%>, of at least about 70%>, of at least about 75%>, of at least about 80%>, of at least about 85%>, of at least about 90%>, of at least about 95%>, or up to and including a 100%, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, at least about a 6-fold, or at least about a 7-fold, or at least about a 8- fold, at least about a 9-fold, or at least about a 10-fold increase, or any increase of 10-fold or greater ,as compared to a control sample or level. A control sample or level is used herein to describe a population of cells obtained from the same biological source that has been treated with identical media, culture condition, temperature, confluency, flask size, pH, etc., with the exception of the methods of isolation using a combination of agents specific for CD31, CD 105, CD117, CD44, and Sca-1 described herein. For example, a control sample can be isolated using only CD31 and CD105, without CD117, CD44, or Sca-1. [0063] In some embodiments, the CFU-ECs generated using the EPCs isolated by the methods described herein, can be tested for the expression of specific cell-surface markers including CD31, CD105, CD144, CD146, vWF, and KDR, and the lack of expression of the hematopoietic cell specific surface antigen CD45 or

monocyte/macrophage marker CD14. Such testing can be done using a flow cytometer or fluorescence microscope.

[0064] In some embodiments, the CFU-ECs generated using the EPCs isolated by the methods described herein, are tested for the ability to uptake AcLDL (acetylated low- density lipoprotein). In such embodiments, fluorescently labeled AcLDL is contacted with a CFU-EC population and the ability of the CFU-EC population to uptake the fluorescently labeled AcLDL assayed using a flow cytometer.

[0065] In some embodiments, the CFU-ECs generated using the EPCs isolated by the methods described herein, can be tested for the ability to mediate de novo blood vessel formation in an in vivo model, using for example, tube formation assays. In such embodiments, the CFU-EPCs are cultured, and then used to prepare cellularized gel implants, using a gel such as a matrigel. The cellularized gel implants can be implanted into a recipient subject, such as an animal model, using methods known to one of skill in the art. In some embodiments, immunocompromised mouse strains are used as a recipient for the cellularized gel implant, including severe combined immunodeficient (SCID)/beige (Schechner et al, 2000; Enis et al, 2005; Shepherd et al, 2006) and nonobese diabetic (NOD)/SCID mice (Yoder et al, 2007). Immunocompromised animals can be used as recipients, to ensure that immune cells are not participating in the formation of the new blood vessels in the recipient subject. Uses of Isolated Adult-derived Endothelial Progenitor Cells in Regenerative Medicine

[0066] Adult-derived EPCs have the ability to contribute to new blood vessel formation and re-endothelialization of injured vessels during adult life. These features make adult-derived EPCs a useful cell source for numerous research activities in tissue engineering and regenerative medicine. Accordingly, adult-derived EPCs isolated using the methods described herein can be used for in vitro or in vivo tissue engineering of blood vessels, revascularization of ischaemic tissues and endothelialization of biomaterials to improve biocompatibility of blood contacting implants, e.g. vascular grafts, heart valves, stents and other medical devices such as artificial lungs, hearts, ventricular assist devices, etc.

[0067] Accordingly, provided herein are isolated adult-derived vascular endothelial progenitor cells that are positive for the cell-surface markers CD31, CD 105, CD117, CD44, and Sca-1, and negative for one or more lineage cell-surface markers, such as CD2, CD3, CD14, CD16, CD19, CD56, and CD235a. Such isolated adult-derived vascular endothelial progenitor cells can be genetically modified to express one or more therapeutic agents, for example, an anti-inflammatory agents. In some aspects, described herein are pharmaceutical compositions comprising isolated adult-derived vascular endothelial progenitor cells that are positive for the cell-surface markers CD31, CD 105, CD117, CD44, and Sca-1, and negative for one or more lineage cell-surface markers. Such isolated adult-derived vascular endothelial progenitor cells and pharmaceutical compositions comprising such cells can be used, for example, in subjects in need of vascularization or vascular regeneration. Accordingly, described herein are uses of isolated adult-derived vascular endothelial progenitor cells that are positive for the cell-surface markers CD31 , CD 105, CD117, CD44, and Sca-1, and negative for one or more lineage cell-surface markers, such as CD2, CD3, CD14, CD16, CD19, CD56, and CD235a in promoting vascular regeneration or vascularization in a subject or tissue in need thereof. In some embodiments, the isolated adult-derived vascular endothelial progenitor cells are autologous to the subject in need, while in other embodiments, the cells are allogeneic to the subject in need. [0068] In other aspects, methods for treating a subject in need of vascular regeneration are provided herein. In one aspect, a method of promoting vascular regeneration in a subject in need thereof is provided, comprising administering to a subject an effective amount of adult-derived endothelial progenitor cells isolated from a biological sample that are positive for the group of cell- surface markers comprising CD31, CD 105, and CDl 17, to promote vascular regeneration in the subject. In another aspect, a method of promoting vascularization in a tissue is provided, comprising administering to a tissue in need of vascularization an effective amount of adult-derived endothelial progenitor cells isolated from a biological sample that are positive for the group of cell-surface markers comprising CD31, CD105, and CDl 17, to promote vascular regeneration in the subject. In some embodiments of these aspects, the group of cell-surface molecules consists essentially of CD31, CD 105, and CDl 17. In some embodiments of these aspects, the group of cell-surface molecules consists of CD31, CD105, and CDl 17. In some embodiments of these aspects, the biological sample is autologous to the subject or tissue in need of vascularization. In other embodiments of these aspects, the biological sample is allogenic to the subject or tissue in need of vascularization.

[0069] In other embodiments of these aspects, the method further comprises the use of additional cell-surface markers, such as CD44, Seal (Ly6A/E), and lineage markers, to isolate the adult-derived EPCs from the biological sample for use in the subject or tissue in need.

[0070] Accordingly, in some embodiments of these aspects, the group of cell- surface markers comprises CD31, CD105, CDl 17, and CD44. In some embodiments of these aspects, the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, and CD44. In some embodiments of these aspects, the group of cell-surface markers consists of CD31, CD105, CDl 17, and CD44. In some embodiments of these aspects and all such aspects described herein, the EPCs are positive for the expression of CD31, CD 105, CDl 17, and CD44. [0071] In other embodiments of these aspects, the group of cell-surface markers comprises CD31, CD105, CDl 17, CD44, and one or more lineage markers. In some embodiments of these aspects, the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, CD44, and one or more lineage markers. In some embodiments of these aspects, the group of cell- surface markers consists of CD31, CD 105, CDl 17, CD44, and one or more lineage markers. In some embodiments of these aspects and all such aspects described herein, the EPCs are positive for the expression of CD31, CD105, CDl 17, and CD44, and negative for the expression of the one or more lineage markers, such as CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

[0072] In some embodiments of these aspects, the group of cell-surface markers comprises CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of these aspects, the group of cell-surface markers consists essentially of CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of these aspects, the group of cell- surface markers consists of CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of these aspects and all such aspects described herein, the EPCs are positive for the expression of CD31, CD 105, CDl 17, CD44, and Sca-1, and negative for the expression of the one or more lineage markers, such as CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

[0073] In some embodiments of these aspects described herein, the method further comprises expanding the isolated adult-derived endothelial progenitor cells in vitro in order to increase or expand the number of endothelial cell progenitors for use in the methods described herein. The isolated endothelial progenitor cells can be expanded to proliferate to form CFU-ECs, using the methods described herein or known to one of skill in the art. Such expansion can comprise, for example, the use of a low-cell density adherent semi- so lid matrix colony assay, as described in the Examples section herein. In such embodiments, the method comprises administering an effective amount of the expanded population of endothelial progenitor cells to the subject in need thereof. In such embodiments, the expression of any of the cell-surface markers described herein on the CFU-ECs can be determined prior to the administration to the subject in need.

[0074] The populations of substantially pure and enriched endothelial progenitor cells that can be obtained using the methods described herein can be used to promote vascular regeneration for the treatment of a variety of conditions.

[0075] As defined herein, "vascular regeneration" refers to the formation of new blood vessels or the replacement of damaged blood vessels (e.g., capillaries) after injuries or traumas, as described herein, including but not limited to, cardiac disease, or ischemia. "Angiogenesis" is a term that can be used interchangeably to describe such phenomena. [0076] In some embodiments of this aspect, the subject in need of vascular regeneration has had or is having ischemia or ischemic damage. As defined herein, "ischemia" is a restriction in blood supply, with resultant damage or dysfunction of tissue. Ischemia is also described herein as an inadequate flow of blood to a part of the body, caused by constriction or blockage of the blood vessels supplying it. Ischemia is a feature of heart diseases, transient ischemic attacks, cerebrovascular accidents, ruptured arteriovenous malformations, and peripheral artery occlusive disease. The heart, the kidneys, and the brain are among the organs that are the most sensitive to ischemic damage. Ischemia in brain tissue, for example due to stroke or head injury, causes a process called the ischemic cascade to be unleashed, in which proteolytic enzymes, reactive oxygen species, and other harmful chemicals damage and may ultimately kill brain tissue.

[0077] In other embodiments of this aspect, the subject in need of vascular regeneration has heart disease. As used herein, heart disease includes cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, viral infection, wounds, hypertensive heart disease, valvular disease, congenital heart disease, myocardial infarction, congestive heart failure, arrhythmias, etc. The methods described herein are particularly useful in treating diseases of the heart involving damage to cardiac tissue due to restricted blood flow, i.e., cardiac ischemia.

[0078] In some embodiments of this aspect, the subject in need of vascular regeneration has had or is having cardiac ischemia. As defined herein, "cardiac ischemia" or "myocardial ischemia" refers to a lack or reduction of oxygen flow to the heart which results in cardiac ischemic damage. As used herein, the phrase myocardial ischemic damage includes damage caused by reduced blood flow to the myocardium. Non-limiting examples of causes of myocardial ischemia and myocardial ischemic damage include: decreased aortic diastolic pressure, increased intraventricular pressure and myocardial contraction, coronary artery stenosis (e.g., coronary ligation, fixed coronary stenosis, acute plaque change (e.g., rupture, hemorrhage), coronary artery thrombosis, vasoconstriction), aortic valve stenosis and regurgitation, and increased right atrial pressure. Non-limiting examples of adverse effects of myocardial ischemia and myocardial ischemic damage include: myocyte damage (e.g., myocyte cell loss, myocyte hypertrophy, myocyte cellular hyperplasia), angina (e.g., stable angina, variant angina, unstable angina, sudden cardiac death), myocardial infarction, and congestive heart failure. Damage due to myocardial ischemia may be acute or chronic, and consequences may include scar formation, cardiac remodeling, cardiac hypertrophy, wall thinning, dilatation, and associated functional changes. The existence and etiology of acute or chronic myocardial damage and/or myocardial ischemia may be diagnosed using any of a variety of methods and techniques well known in the art including, e.g., non-invasive imaging (e.g., MRI, echocardiography), angiography, stress testing, assays for cardiac-specific proteins such as cardiac troponin, and clinical symptoms. These methods and techniques as well as other appropriate techniques may be used to determine which subjects are suitable candidates for the treatment methods described herein.

[0079] In other embodiments of this aspect, a subject in need of vascular regeneration has a wound. Endothelial progenitor cells are useful in wound healing as they can increase vascularization, increase cell migration to the site of injury, decrease the amount of scarring, and increase tissue regeneration. As defined herein, a "wound" refers to a lesion caused by an injury or damage, usually restricted to those with disruption of the normal continuity of structures, such as the epidermal or dermal layers of the skin, continuity of the vasculature, or any damage to a layer of a tissue, such as a corneal epithelial layer. A wound can be an acute wound or a chronic wound. As defined herein, "acute wounds" are those wounds that heal promptly, within 30 days in a normal subject. Non-limiting examples of acute wounds that can be treated with the endothelial progenitor cell isolated using the methods described herein include abrasions, avulsions, contusions, crush wounds, cuts, lacerations, projectile wounds and puncture wounds. "Chronic wounds," as defined herein, are those wounds that take longer than 30 days to heal, and include, but are not limited to, diabetic skin sores, pressure sores, surgical wounds, spinal injury wounds, burns, chemical-induced wounds and wounds due to blood vessel disorders.

[0080] A "skin wound" is defined herein as a break in the continuity of skin tissue that is caused by direct injury to the skin. Several classes including punctures, incisions, excisions, lacerations, abrasions, atrophic skin, or necrotic wounds and burns generally characterize skin wounds. The methods described herein are useful for enhancing the healing of all wounds of the skin, including wounds in diabetics, normal patients and surgical patients. A "tissue wound" as used herein is a wound to an internal organ, such as a blood vessel, intestine, colon, etc. [0081] The endothelial progenitor cell isolated using the methods described herein are useful for enhancing the wound healing process in tissue wounds, whether they arise naturally or as the result of surgery. For instance, during the repair of arteries, the vessels need to be sealed and wound healing must be promoted as quickly as possible. The substantially pure and enriched endothelial progenitor cells isolated using the methods described herein can accelerate such processes.

[0082] The endothelial progenitor cells used in the methods described herein can be obtained from any biological source or sample. However, the cells are typically isolated from a sample from the subject in need of treatment so that there are no rejection issues (i.e., autologous transplantation). The cells, for example, may be isolated from the peripheral blood of the subject, from the bone marrow, from a tissue sample, etc. Besides autologous transplantation, the cells may be isolated from a biological source or sample from a relative, an MHC-matched donor, a donor of the same blood type, or any donor of the same species. In certain embodiments, cross-species cells are used (i.e., xenogeneic transplantation). As would be appreciated by one of skill in this art, immunosuppression may be required if the isolated cells are not from the subject or a related donor. The isolated endothelial progenitor cells may also be treated or modified to reduce their immunogenicity. For example, the MHC class I molecules on the endothelial progenitor cells may be masked or modified to limit their immunogenicity. Accordingly, in some embodiments of the aspects described herein, the biological sample is an autologous sample obtained from the subject in need. In other embodiments of these aspects, the biological sample is an allogenic sample. In some embodiments of these aspects, the biological sample is a peripheral blood sample obtained from a subject.

[0083] In other embodiments of these aspects, the methods for promoting vascular regeneration through the administration of endothelial progenitor cells can further comprise the administration of one or more therapeutic agents. The therapeutic agent may be a protein, a peptide, a polynucleotide, an aptamer, a virus, a small molecule, a chemical compound, a cell, etc.

[0084] In certain embodiments of these aspects, the therapeutic agent is a "pro- angiogenic factors," which refers to factors that directly or indirectly promote new blood vessel formation. These factors can be expressed and secreted by, for example, normal and tumor cells. In one embodiment, the pro-angiogenic factors include, but are not limited to epidermal growth factor (EGF), E-cadherin, VEGF, angiogenin, angiopoietin-1, fibroblast growth factors: acidic (aFGF) and basic (bFGF), fibrinogen, fibronectin, heparanase, hepatocyte growth factor (HGF), angiopoietin, hypoxia- inducible factor- 1 (HIF-1), insulinlike growth factor- 1 (IGF-1), IGF, BP-3, platelet-derived growth factor (PDGF), VEGF-A VEGF-C, pigment epithelium-derived factor (PEDF), vascular permeability factor (VPF), vitronection, leptin, trefoil peptides (TFFs), CYR61 (CCN1) and NOV (CCN3), leptin, midkine, placental growth factor platelet-derived endothelial cell growth factor (PD- ECGF), platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN), progranulin, proliferin, transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), c-Myc, granulocyte colony- stimulating factor (G-CSF), stromal derived factor 1 (SDF-1), scatter factor (SF), osteopontin, stem cell factor (SCF), matrix metalloproteinases (MMPs), thrombospondin-1 (TSP-1), pleitrophin, proliferin, follistatin, placental growth factor (PIGF), midkine, platelet-derived growth factor-BB (PDGF), and fractalkine, and inflammatory cytokines and chemokines that are inducers of angiogenesis and increased vascularity, e.g.

interleukin-3 (IL-3), interleukin-8 (IL-8), CCL2 (MCP-1), interleukin-8 (IL-8) and CCL5 (RANTES).

[0085] In certain embodiments, combinations of the above pro-angiogenic factors are used. Derivatives or modified versions of these pro-angiogenic factors are also useful in the invention. These modified versions are typically 75%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the wild protein or peptide. In certain embodiments, these modified versions show at least 50%>, 75%>, 80%>, or 90%> overall identity and share recognized or conserved sequence elements. Modified versions, fusions, or derivatives also include forms in which at least conserved or characteristic sequence elements have been placed in non- natural environments. In certain embodiments, the modified versions or derivatives have enough of the sequence of a pro-angiogenic factor to have the substantially the same activity as the naturally occurring factor. Any other pro-angiogenic factors known or discovered in the future may be used in the methods described herein.

[0086] The endothelial progenitor cells used in the methods described herein may also be genetically engineered, using any techniques known in the art. For example, the genomes of the cells may be altered permanently, or the cells may be altered to express a gene only transiently. In certain embodiments, the endothelial progenitor cells are genetically engineered to produce a therapeutic agent, such as a pro-angiogenic peptide or protein. In certain embodiments, the administration of cells engineered to express at least one therapeutic agent constitutes both the administration of a therapeutic agent and administration of endothelial progenitor cells. Such a therapeutic agent may be expressed constitutively, or it may be expressed upon a certain stimulus. [0087] As used herein, the terms "treat" or "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow the development of the disease. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a disease, such as, but not limited to, cardiac dysfunction of myocardial infarction. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced as that term is defined herein. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0088] "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. For example, those in need of treatment include those already diagnosed with cardiac dysfunction, as well as those likely to develop cardiac dysfunction, such as those at risk of myocardial infarction. Used in the context of cardiac dysfunction, the term treating as used herein refers to a reduction of a symptom of cardiac dysfunction of myocardial infarction and/or a reduction of at least one biochemical marker of cardiac dysfunction of myocardial infarction by at least 10%. For example but are not limited to, a reduction in a biochemical marker of cardiac dysfunction of myocardial infarction, for example a reduction in, as an illustrative example only, at least one of the following biomarkers as disclosed in U.S Patent Application 2005/0250156, which is incorporated herein by reference, include for example, protein biomarkers in the blood such as; troponin I and T (Tnl/TnT), creatine kinase-MB isoform (CK0MB), myoglobin (MYO), hsCRP, H-FABP, MPO, BNP, p-selectin, sCD40L, GPIIb/IIIa, PTF 1.2, DD, TAT, BTG, PF4, PECAM-1, TPP, IL-6, IL-18, PIGF, PaPP-A, glutathione peroxidase, plasma thioredoxin, cyctatin C, and serum deoxyribonuclease I and ATP/ADP, i.e. a reduction in the bio markers by at least 10%. As alternative examples, a reduction in a symptom of or a reduction in the size of infarct for example for myocardial infarction by 10% or reduction in myocardial infarct, would be considered effective treatments by the methods as disclosed herein, or a reduction in a symptom of cardiac dysfunction, for example a reduction in a symptom of acute coronary symptom (ACS) or a reduction of a symptom of Congestive Heart Failure (CHF), such as for example change in symptoms include but are not limited to, a reduction in high blood pressure by at least about 10%>, a reduction in chest pain by at least about 10%, an increase in heart contraction by at least about 10%, an increase in efficiency of heart pumping by about 10%, a increase in exercise tolerance by at least 10%, and decrease in shortness of breath by at least about 10% would also be considered as affective treatments by the methods as disclosed herein.

[0089] The term "effective amount" as used herein refers to the amount of adult endothelial progenitor cells needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect, i.e., vascular regeneration. The term "therapeutically effective amount" therefore refers to an amount of the adult endothelial progenitor cells isolated using the methods as disclosed herein that is sufficient to effect vascular regeneration when administered to a typical subject, such as one who has a cardiac dysfunction. An effective amount as used herein would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not possible to specify the exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. In embodiments where the adult endothelial progenitor cells are used for the treatment of cardiac dysfunction, such as cardiac ischemia, the efficacy of the methods described herein can be judged using an experimental animal model of cardiac dysfunction, e.g., mice or rats, or for example, induction of myocardial infarction in animal models, or an animal model which has been genetically modified to develop cardiac abnormalities. An effective amount can be assessed in an animal models of ischemia/reperfusion injury when adult endothelial progenitor cells are administered just before reperfusion, such as disclosed in Smits et al, J Pharmacol Exp Ther 1998;286:611-618 ; McVey et al., ,J Cardiovasc Pharmacol

1999;33:703-710; Budde et al, Cardiovasc Res 2000;47:294-305 and Xu et al, J Mol Cell Cardiol 2000;32:2339-2347, which are incorporated herein in their entirety by reference. Further, in some embodiments an experimental model could be an in vitro model, such as organ culture, cells or cell lines.

[0090] A therapeutically or prophylatically significant reduction in a symptom is, e.g. at least about 10%, at least about 20%, at least about 30%>, at least about 40%>, at least about 50%), at least about 60%>, at least about 70%>, at least about 80%>, at least about 90%>, at least about 100%, at least about 125%, at least about 150% or more in a measured parameter as compared to a control or non-treated subject. Measured or measurable parameters include clinically detectable markers of disease, for example, elevated or depressed levels of a clinical or biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for a disease or disorder. It will be understood, however, that the total daily usage of the compositions and formulations as disclosed herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated. [0091] The adult-derived endothelial progenitor cells described herein can be administered by any appropriate route which results in an effective treatment in the subject. As used herein, the terms "administering," and "introducing" are used interchangeably and refer to the placement of the adult-derived endothelial progenitor cells, isolated using the methods as disclosed herein, into a subject by a method or route which results in at least partial localization of the cells at a desired site, such as a site of ischemic damage. For example, in some embodiments of the aspects described herein, the endothelial progenitor cells are administered to the site of cardiac tissue damaged following a heart attack. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. The phrases

"systemic administration," "administered systemically", "peripheral administration" and "administered peripherally" as used herein mean the administration of pharmaceutical compositions other material other than directly into the diseased tissue, such as cardiac tissue, such that it enters the subjects system and, thus, is subject to metabolism and other like processes. [0092] For the clinical use of the methods described herein, administration of the adult endothelial progenitor cells of the invention can include formulation into

pharmaceutical compositions or pharmaceutical formulations for parenteral administration, e.g., intravenous; mucosal, e.g., intranasal; enteral, e.g., oral; topical, e.g., transdermal; ocular, or other mode of administration. The adult endothelial progenitor cells of the present invention can be administered along with any pharmaceutically acceptable compound, material, or composition which results in an effective treatment in the subject. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in maintaining the activity of or carrying or transporting the subject adult human endothelial progenitor cells from one organ, or portion of the body, to another organ, or portion of the body. In addition to being "pharmaceutically acceptable" as that term is defined herein, each carrier must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The

pharmaceutical formulation contains a compound of the invention in combination with one or more pharmaceutically acceptable ingredients. The carrier can be in the form of a solid, semi-solid or liquid diluent, cream or a capsule. These pharmaceutical preparations are a further object of the invention. Usually the amount of active compounds is between 0.1- 95% by weight of the preparation, preferably between 0.2-20% by weight in preparations for parenteral use and preferably between 1 and 50% by weight in preparations for oral administration.

Targeting Adult-derived Endothelial Progenitor Cells for Blocking Angiogenesis

[0093] Described herein are methods for targeting adult-derived endothelial progenitor cells for the inhibition or amelioration of angiogenesis-dependent diseases or disorders. We have found that in angiogenic vasculature, such as those found in tumors, the numbers and percentages of adult-derived endothelial progenitor cells expressing the cell-surface markers CD31, CD 105, CD 117, CD44, and Sca-1 are greatly increased, relative to quiescent vasculature. [0094] Accordingly, provided herein are methods for blocking angiogenesis comprising targeting one or more angiogenesis inhibitors to a cell that is positive for the cell-surface markers comprising CD31, CD 105, CDl 17, CD44, and Sca-1 and negative for one or more lineage markers, such as CD2, CD3, CD14, CD16, CD19, CD56, and CD23a. In some embodiments of these aspects the cell is positive for the cell-surface markers comprising CDl 17 and CD44.

[0095] In some embodiments, the methods described herein comprise targeting an adult-derived endothelial progenitor cell using one or more antibodies, or a mixture of antibodies, specific for the cell-surface markers CD31, CD 105, CDl 17, CD44, and Sca-1, to block angiogenesis in a subject or tissue in need. In those embodiments where one or more antibodies are used to target the adult-derived endothelial progenitor cell, the antibody or antibodies can further comprise a tag, such as a toxin, that can be used to lyse or destroy the cell expressing the combination of cell-surface markers. For example, the one or more antibodies will bind the cell-surface antigen(s) present on the adult-derived endothelial progenitor cell, and the toxin delivered by the antibody enters the cytosolic part of the adult-derived endothelial progenitor cell to kill the cell. The toxins can be any toxin, such as the glycosidases diphtheria toxin, pseudomonas exotoxin (PE), and ricin. In other embodiments, the method further comprises targeting one or more angiogenesis inhibitors to the adult-derived endothelial progenitor cell. [0096] The term "angiogenesis" refers to the sprouting of new blood vessels from pre-existing blood vessels or through the proliferation of adult-derived endothelial progenitor cells, as described herein, characterized by endothelial cell proliferation and migration triggered by pro-angiogenic factors. Angiogenesis can be a good and necessary process, for example, in wound healing, or it can be an aberrant and undesired process with detrimental consequences, such as the growth of solid tumors and metastasis, and hemangiomas. Aberrant angiogenesis can lead to certain pathological conditions such as death, blindness, and disfigurement.

[0097] Accordingly, "angiogenesis-dependent disease or disorder" refers to diseases or disorders that are dependent on a rich blood supply and blood vessel proliferation for the diseases' pathological progression (e.g. metastatic tumors) or diseases or disorders that are the direct result of aberrant blood vessel proliferation (e.g. diabetic retinopathy and hemangiomas). Examples include abnormal vascular proliferation, ascites formation, psoriasis, age-related macular degeneration, thyroid hyperplasia, preeclampsia, rheumatoid arthritis and osteoarthritis, Alzheimer's disease, obesity, pleural effusion, atherosclerosis, endometriosis, diabetic/other retinopathies, ocular neovascularizations such as neovascular glaucoma and corneal neovascularization. [0098] A variety of angiogenesis inhibitors, drugs, or treatments are available for use in the methods described herein, and are known to one of skill in the art. There are three main types of anti-angiogenic drugs that are currently approved by the United State Food and Drug Administration (FDA) for the treatment of cancer and tumors: (1) Drugs that stop new blood vessels from sprouting (angiogenesis inhibitors); (2) Drugs that attack a tumor's established blood supply (vascular targeting agents); and (3) Drugs that attack both the cancer cells as well as the blood vessel cells (the double-barreled approach).

[0099] In some embodiments, the angiogenesis inhibitors for use in the methods described herein include but are not limited to monoclonal antibody therapies directed against specific pro-angiogenic growth factors and/or their receptors. Examples of these are: bevacizumab (Avastin®), cetuximab (Erbitux®), panitumumab (Vectibix™), and trastuzumab (Herceptin®).

[00100] In some embodiments, the angiogenesis inhibitors for use in the methods described herein include but are not limited to small molecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor receptors. The three TKIs that are currently approved as anti-cancer therapies are erlotinib (Tarceva®), sorafenib

(Nexavar®), and sunitinib (Sutent®).

[00101] In some embodiments, the angiogenesis inhibitors for use in the methods described herein include but are not limited to inhibitors of mTOR (mammalian target of rapamycin) such as temsirolimus (Toricel™), bortezomib (Velcade®), thalidomide (Thalomid®), and Doxycyclin,

[00102] In other embodiments, the angiogenesis inhibitors for use in the methods described herein include one or more drugs that target the VEGF pathway. Bevacizumab (Avastin®) was the first drug that targeted new blood vessels to be approved for use against cancer. It is a monoclonal antibody that binds to VEGF, thereby blocking VEGF from reaching the VEGF receptor (VEGFR). Other drugs, such as sunitinib (Sutent®) and sorafenib (Nexavar®), are small molecules that attach to the VEGF receptor itself, preventing it from being turned on. Such drugs are collectively termed VEGF inhibitors. As the VEGF/VPF protein interacts with the VEGFRs, inhibition of either the ligand VEGF, e.g. by reducing the amount that is available to interact with the receptor; or inhibition of the receptor's intrinsic tyrosine kinase activity, blocks the function of this pathway. This pathway controls endothelial cell growth, as well as permeability, and these functions are mediated through the VEGFRs.

[00103] Accordingly, as described herein, "VEGF inhibitors" for use as

angiogenesis inhibitors include any compound or agent that produces a direct or indirect effect on the signaling pathways that promote growth, proliferation and survival of a cell by inhibiting the function of the VEGF protein, including inhibiting the function of VEGF receptor proteins. These include any organic or inorganic molecule, including, but not limited to modified and unmodified nucleic acids such as antisense nucleic acids, RNAi agents such as siRNA or shRNA, peptides, peptidomimetics, receptors, ligands, and antibodies that inhibit the VEGF signaling pathway. The siRNAs are targeted at components of the VEGF pathways and can inhibit the VEGF pathway. Preferred VEGF inhibitors, include for example, AVASTIN® (bevacizumab), an anti-VEGF monoclonal antibody of Genentech, Inc. of South San Francisco, CA, VEGF Trap (Regeneron / Aventis). Additional VEGF inhibitors include CP-547,632 (3-(4-Bromo-2,6-difiuoro- benzyloxy)-5-[3-(4-pyrrolidin 1-yl- butyl)-ureido]-isothiazole-4- carboxylic acid amide hydrochloride; Pfizer Inc. , NY), AG13736, AG28262 (Pfizer Inc.), SU5416, SU11248, & SU6668 (formerly Sugen Inc., now Pfizer, New York, New York), ZD-6474

(AstraZeneca), ZD4190 which inhibits VEGF-R2 and -Rl (AstraZeneca), CEP-7055 (Cephalon Inc., Frazer, PA), PKC 412 (Novartis), AEE788 (Novartis), AZD-2171), NEXAVAR® (BAY 43-9006, sorafenib; Bayer Pharmaceuticals and Onyx

Pharmaceuticals), vatalanib (also known as PTK-787, ZK-222584: Novartis & Schering: AG), MACUGEN® (pegaptanib octasodium, NX-1838, EYE-001, Pfizer

Inc./Gilead/Eyetech), IM862 (glufanide disodium, Cytran Inc. of Kirkland, Washington, USA), VEGFR2-selective monoclonal antibody DC 101 (ImClone Systems, Inc.), angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron

(Emeryville, California), Sirna-027 (an siRNA-based VEGFR1 inhibitor, Sirna

Therapeutics, San Francisco, CA) Caplostatin, soluble ectodomains of the VEGF receptors, Neovastat (Sterna Zentaris Inc; Quebec City, CA), ZM323881 (CalBiochem. CA, USA), pegaptanib (Macugen) (Eyetech Pharmaceuticals), an anti-VEGF aptamer and

combinations thereof.

[00104] VEGF inhibitors are also disclosed in US Patent No. 6,534,524 and

6,235,764, both of which are incorporated in their entirety. Additional VEGF inhibitors are described in, for example in WO 99/24440 (published May 20, 1999), International Application PCT/IB99/00797 (filed May 3, 1999), in WO 95/21613 (published August 17, 1995), WO 99/61422 (published December 2, 1999), U.S. Pat. Publ. No. 20060094032 "siRNA agents targeting VEGF", U.S. Patent 6, 534,524 (discloses AG13736), U.S. Patent 5,834,504 (issued November 10, 1998), WO 98/50356 (published November 12, 1998), U.S. Patent 5, 883,113 (issued March 16, 1999), U.S. Patent 5, 886,020 (issued March 23, 1999), U.S. Patent 5,792,783 (issued August 11, 1998), U.S. Patent No. US 6,653,308 (issued November 25, 2003), WO 99/10349 (published March 4, 1999), WO 97/32856 (published September 12, 1997), WO 97/22596 (published June 26, 1997), WO 98/54093 (published December 3, 1998), WO 98/02438 (published January 22, 1998), WO 99/16755 (published April 8, 1999), and WO 98/02437 (published January 22, 1998), WO 01/02369 (published January 11, 2001); U.S. Provisional Application No. 60/491,771 piled July 31, 2003); U.S. Provisional Application No. 60/460,695 (filed April 3, 2003); and WO

03/106462A1 (published December 24, 2003). Other examples of VEGF inhibitors are disclosed in International Patent Publications WO 99/62890 published December 9, 1999, WO 01/95353 published December 13, 2001 and WO 02/44158 published June 6, 2002.

[0100] In other embodiments, the angiogenesis inhibitors for use in the methods described herein include anti-angiogenic factors such as alpha-2 antiplasmin (fragment), angiostatin (plasminogen fragment), antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59 complement fragment, endostatin (collagen XVIII fragment), fibronectin fragment, gro-beta ( a C-X-C chemokine), heparinases heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, interferon inducible protein (IP- 10), interleukin-12, kringle 5 (plasminogen fragment), beta- thrombo globulin, EGF (fragment), VEGF inhibitor, endostatin, fibronection (45 kD fragment), high molecular weight kininogen (domain 5), NK1, NK2, NK3 fragments of HGF, PF-4, serpin proteinase inhibitor 8, TGF-beta-1, thrombospondin-1, prosaposin, p53, angioarrestin, metalloproteinase inhibitors (TIMPs), 2-Methoxyestradiol, placental ribonuclease inhibitor, plasminogen activator inhibitor, prolactin 16kD fragment, proliferin-related protein (PRP), retinoids, tetrahydrocortisol-S transforming growth factor- beta (TGF-b), vasculostatin, and vasostatin (calreticulin fragment) .pamidronate

thalidomide, TNP470, the bisphosphonate family such as amino-bisphosphonate zoledronic acid, bombesin/gastrin-releasing peptide (GRP) antagonists such as RC-3095 and RC-3940-II (Bajol AM, et. al, British Journal of Cancer (2004) 90, 245-252), anti- VEGF peptide RRKRRR (dRK6) (Seung-Ah Yoo, J.Immuno, 2005, 174: 5846-5855).

[0101] In some embodiments, the methods described herein include the use of more than one anti-angiogenic factor or angiogenesis inhibitor. The therapy can also be administered in conjunction with other anti-cancer treatment such as biological, chemotherapy and radiotherapy. Biological therapies use the body's immune system, either directly or indirectly, to fight cancer or to lessen the side effects that may be caused by some cancer treatments. Immune response modifying therapies such as the administration of interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents are also envisioned as anti-cancer therapies. [0102] In some embodiments, the methods described herein are directed at angiogenic diseases and disorders. When angiogenesis occurs at inappropriate locations, is aberrant, and/or uncontrolled and results in undesirable effects, then that angiogenesis is considered pathological. The pathological angiogenic diseases and disorders include but are not limited to cancer, ascites formation, psoriasis, age-related macular degeneration, thyroid hyperplasia, preeclampsia, rheumatoid arthritis and osteoarthritis, Alzheimer's disease, obesity, pleura effusion, atherosclerosis, endometriosis, diabetic/other

retinopathies, neovascular glauocoma, age-related macular degeneration, hemangiomas, and corneal neovascularization.

[0103] Accordingly, in some embodiments, the pathological angiogenic disease or disorder is cancer, where the rapidly dividing neoplastic cancer cells require an efficient blood supply to sustain their continual growth of the tumor. As used herein, cancer refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths. The blood vessels provide conduits to metastasize and spread elsewhere in the body. Upon arrival at the metastatic site, the cancer cells then work on establishing a new blood supply network. Inhibition of angiogenesis at the primary tumor site and secondary tumor site serve to prevent and limit the progression of the disease.

[0104] Encompassed in the methods disclosed herein are subjects that are treated for cancer, including but not limited to all types of carcinomas and sarcomas, such as those found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum,

endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus. The types of carcinomas include papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sino nasal undifferentiated carcinoma. The types of sarcomas include soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermato fibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma. Abnormal build up and growth of blood vessels in the skin or internal organs in the form of hemangiomas can also be treated and/or monitored according to the methods described herein.

[0105] In other embodiments, the methods described herein are used in blocking pathological angiogenesis that occurs in age-related macular degeneration. It is known that VEGF contributes to abnormal blood vessel growth from the choroid layer of the eye into the retina, similar to what occurs during the wet or neo vascular form of age-related macular degeneration. Macular degeneration, often called AMD or ARMD (age-related macular degeneration), is the leading cause of vision loss and blindness in Americans aged 65 and older. New blood vessels grow (neovascularization) beneath the retina and leak blood and fluid. This leakage causes permanent damage to light-sensitive retinal cells, which die off and create blind spots in central vision or the macula. Accordingly, encompassed in the methods disclosed herein are subjects treated for age-related macular degeneration with anti-angiogenic therapy.

[0106] In other embodiments, the methods described herein are used in blocking pathological angiogenesis that occurs in diabetic retinopathy, where abnormal blood vessel growth is associated with diabetic eye diseases and diabetic macular edema. VEGF inhibitors can block and/or reduce the activity of VEGF and pathologic angiogenesis. Released by the retina (light-sensitive tissue in back of the eye) when normal blood vessels are damaged by tiny blood clots due to diabetes, VEGF turns on its receptor, igniting a chain reaction that culminates in new blood vessel growth. However, the backup blood vessels are faulty; they leak (causing edema), bleed and encourage scar tissue that detaches the retina, resulting in severe loss of vision. Such growth is the hallmark of diabetic retinopathy, the leading cause of blindness among young people in developed countries. Therefore, encompassed in the methods disclosed herein are subjects treated for diabetic retinopathy and/or diabetic macular edema. [0107] In some embodiments, the methods described herein are used in blocking pathological angiogenesis that occurs in rheumatoid arthritis. Rheumatoid arthritis (RA) is characterized by synovial tissue swelling, leukocyte ingress and angiogenesis, or new blood vessel growth. The expansion of the synovial lining of joints in rheumatoid arthritis (RA) and the subsequent invasion by the pannus of underlying cartilage and bone necessitate an increase in the vascular supply to the synovium, to cope with the increased requirement for oxygen and nutrients. Angiogenesis is now recognized as a key event in the formation and maintenance of the pannus in RA (Paleolog, E. M., Arthritis Res. 2002;4 Suppl 3:S81-90; Afuwape AO, Histol Histopathol. 2002;17(3):961-72). Even in early RA, some of the earliest histological observations are blood vessels. A mononuclear infiltrate characterizes the synovial tissue along with a luxuriant vasculature. Angiogenesis is integral to formation of the inflammatory pannus and without angiogenesis, leukocyte ingress could not occur (Koch, A. E., Ann. Rheum. Dis. 2000, 59 Suppl l :i65-71).

Disruption of the formation of new blood vessels would not only prevent delivery of nutrients to the inflammatory site, it could also reduce joint swelling due to the additional activity of VEGF, a potent proangiogenic factor in RA, as a vascular permeability factor. Anti-VEGF hexapeptide RRKRRR (dRK6) can suppress and mitigate the arthritis severity (Seung-Ah Yoo, et. al.,2005, supra). Accordingly, encompassed in the methods disclosed herein are subjects treated for rheumatoid arthritis. [0108] In some embodiments, the methods described herein are used in blocking pathological angiogenesis that occurs in Alzheimer's disease. Alzheimer's disease (AD) is the most common cause of dementia worldwide. AD is characterized by an excessive cerebral amyloid deposition leading to degeneration of neurons and eventually to dementia. The exact cause of AD is still unknown. It has been shown by epidemiological studies that long-term use of non-steroidal anti-inflammatory drugs, statins, histamine H2 -receptor blockers, or calcium- channel blockers, all of which are cardiovascular drugs with an anti- angiogenic effects, seem to prevent Alzheimer's disease and/or influence the outcome of AD patients. Therefore, AD angiogenesis in the brain vasculature can play an important role in AD. In Alzheimer's disease, the brain endothelium secretes the precursor substrate for the beta-amyloid plaque and a neurotoxic peptide that selectively kills cortical neurons. Moreover, amyloid deposition in the vasculature leads to endothelial cell apoptosis and endothelial cell activation which leads to neovascularization. Vessel formation could be blocked by the VEGF antagonist SU 4312 as well as by statins, indicating that anti- angiogenesis strategies can interfere with endothelial cell activation in AD (Schultheiss C, el. al, 2006; Grammas P., et. al., 1999) and can be used for preventing and/or treating AD. Accordingly, encompassed in the methods disclosed herein are subjects treated for

Alzheimer's disease.

[0109] In other embodiments, the methods described herein are used in blocking pathological angiogenesis that occurs in obesity. Adipogenesis in obesity involves interplay between differentiating adipocytes, stromal cells, and blood vessels. Close spatial and temporal interrelationships between blood vessel formation and adipogenesis, and the sprouting of new blood vessels from preexisting vasculature was coupled to adipocyte differentiation. Adipogenic/angiogenic cell clusters can morphologically and

immunohistochemically be distinguished from crown-like structures frequently seen in the late stages of adipose tissue obesity. Administration of anti-vascular endothelial growth factor (VEGF) antibodies inhibited not only angiogenesis but also the formation of adipogenic/angiogenic cell clusters, indicating that the coupling of adipogenesis and angiogenesis is essential for differentiation of adipocytes in obesity and that VEGF is a key mediator of that process. (Satoshi Nishimura et. al., 2007, Diabetes 56:1517-1526). It has been shown that the angiogenesis inhibitor, TNP-470 was able to prevent diet-induced and genetic obesity in mice (Ebba Brakenhielm et. al, Circulation Research, 2004;94: 1579). TNP-470 reduced vascularity in the adipose tissue, thereby inhibiting the rate of growth of the adipose tissue and obesity development. Accordingly, encompassed in the methods disclosed herein are subjects treated for obesity.

[0110] In some embodiments, the methods described herein are used in blocking pathological angiogenesis that occurs in endometriosis. Excessive endometrial

angiogenesis is proposed as an important mechanism in the pathogenesis of endometriosis (Healy, DL., et. al, Hum Reprod Update. 1998 Sep-Oct;4(5):736-40). The endometrium of patients with endometriosis shows enhanced endothelial cell proliferation. Moreover there is an elevated expression of the cell adhesion molecule integrin vB3 in more blood vessels in the endometrium of women with endometriosis when compared with normal women. The U.S. Patent No. 6,121,230 described the use of anti-VEGF agents in the treatment of endometriosis and is Patent is incorporated hereby reference. Accordingly, encompassed in the methods disclosed herein are subjects treated for endometriosis.

Definitions

[0111] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0112] As used herein, the term "biological sample" refers to a cell or population of cells or a quantity of tissue or fluid obtained from a subject. Most often, the sample has been removed from a subject, but the term "biological sample" can also refer to cells or tissue analyzed in vivo, i.e. without removal from the subject. A biological sample or tissue sample includes, but is not limited to, blood, plasma, serum, lymph fluid, bone marrow, tumor biopsy, urine, stool, sputum, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, lung tissue, adipose tissue, connective tissue, sub-epithelial tissue, epithelial tissue, liver tissue, kidney tissue, uterine tissue, respiratory tissues, gastrointestinal tissue, and genitourinary tract tissue, tears, saliva, milk, nipple aspirates, cells (including, but not limited to, blood cells), biopsies, scrapes (e.g. buccal scrapes), tumors, organs, and also samples of an in vitro cell culture constituent. Often, a "biological sample" will contain cells from the subject, but the term can also refer to non- cellular biological material, such as non-cellular fractions of blood, saliva, or urine. In some embodiments, the sample is from a resection, bronchoscopic biopsy, or core needle biopsy of a primary or metastatic tumor, or a cell block from pleural fluid. In addition, fine needle aspirate samples are used. Samples may be either paraffin-embedded or frozen tissue. [0113] As used herein, the term "stem cells" is used in a broad sense and includes traditional stem cells, progenitor cells, preprogenitor cells, reserve cells, and the like. The term "stem cell" or "progenitor cell" are used interchangeably herein, and refer to an undifferentiated cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiate daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term "stem cell" refers then, to a cell with the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating. In one embodiment, the term progenitor or stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. In many biological instances, stem cells are also "multipotent" because they can produce progeny of more than one distinct cell type, but this is not required for "stem-ness." Self-renewal is the other classical part of the stem cell definition, and it is essential as used in this document. In theory, self-renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Formally, it is possible that cells that begin as stem cells might proceed toward a differentiated phenotype, but then "reverse" and re-express the stem cell phenotype, a term often referred to as "dedifferentiation".

[0114] Exemplary stem cells include, but are not limited to, embryonic stem cells, adult stem cells, pluripotent stem cells, neural stem cells, liver stem cells, muscle stem cells, muscle precursor stem cells, endothelial progenitor cells, bone marrow stem cells, chondrogenic stem cells, lymphoid stem cells, mesenchymal stem cells, hematopoietic stem cells, central nervous system stem cells, peripheral nervous system stem cells, and the like. Descriptions of stem cells, including method for isolating and culturing them, may be found in, among other places, Embryonic Stem Cells, Methods and Protocols, Turksen, ed., Humana Press, 2002; Weisman et al, Annu. Rev. Cell. Dev. Biol. 17:387 403;

Pittinger et al, Science, 284: 143 47, 1999; Animal Cell Culture, Masters, ed., Oxford University Press, 2000; Jackson et al, PNAS 96(25): 14482 86, 1999; Zuk et al, Tissue Engineering, 7:211 228, 2001 ("Zuk et al."); Atala et al., particularly Chapters 33 41; and U.S. Pat. Nos. 5,559,022, 5,672,346 and 5,827,735. Descriptions of stromal cells, including methods for isolating them, may be found in, among other places, Prockop, Science, 276:71 74, 1997; Theise et al, Hepatology, 31 :235 40, 2000; Current Protocols in Cell Biology, Bonifacino et al, eds., John Wiley & Sons, 2000 (including updates through March, 2002); and U.S. Pat. No. 4,963,489.

[0115] The term "progenitor cell" is used herein to refer to cells that have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. Often, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate. [0116] As indicated above, there are different levels or classes of cells falling under the general definition of a "stem cell." These are "totipotent," "pluripotent" and

"multipotent" stem cells.

[0117] The term "totipotency" or "totipotent" refers to a cell with the degree of differentiation describing a capacity to make all of the cells in the adult body as well as the extra-embryonic tissues including the placenta. The fertilized egg (zygote) is totipotent as are the early cleaved cells (blastomeres)

[0118] The term "pluripotent" or a "pluripotent state" as used herein refers to a cell with the capacity, under different conditions, to differentiate to cell types characteristic of all three germ cell layers: endoderm (gut tissue), mesoderm (including blood, muscle, and vessels), and ectoderm (such as skin and nerve). Pluripotent cells are characterized primarily by their ability to differentiate to all three germ layers, using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers. In some embodiments, a pluripotent cell is an undifferentiated cell.

[0119] The term "multipotent" when used in reference to a "multipotent cell" refers to a cell that is able to differentiate into some but not all of the cells derived from all three germ layers. Thus, a multipotent cell is a partially differentiated cell. Multipotent cells are well known in the art, and examples of muiltipotent cells include adult stem cells, such as for example, hematopoietic stem cells and neural stem cells. Multipotent means a stem cell may form many types of cells in a given lineage, but not cells of other lineages. For example, a multipotent blood stem cell such as a "hematopoietic stem cells" refers to all stem cells or progenitor cells found inter alia in bone marrow and peripheral blood that are capable of differentiating into any of the specific types of hematopoietic or blood cells, such as erythrocytes, lymphocytes, macrophages and megakaryocytes. The term

"hematopoietic cells" also referred to as "HSCs" refers to all types of hematopoietic cells throughout their differentiation from self-renewing hematopoietic stem cells through immature precursor cells of the various blood lineages to and including the mature functioning blood cells as would be understood by persons skilled in the art. The term

"multipotency" refers to a cell with the degree of developmental versatility that is less than totipotent and pluripotent. [0120] In the context of cell ontogeny, the adjectives "differentiated", or

"differentiating" are relative terms. The term "differentiation" in the present context means the formation of cells expressing markers known to be associated with cells that are more specialized and closer to becoming terminally differentiated cells incapable of further differentiation. The pathway along which cells progress from a less committed cell, to a cell that is increasingly committed to a particular cell type, and eventually to a terminally differentiated cell is referred to as progressive differentiation or progressive commitment. Cell which are more specialized (e.g., have begun to progress along a path of progressive differentiation) but not yet terminally differentiated are referred to as partially

differentiated. Differentiation is a developmental process whereby cells assume a specialized phenotype, e.g., acquire one or more characteristics or functions distinct from other cell types. In some cases, the differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway (a so called terminally differentiated cell). In many, but not all tissues, the process of differentiation is coupled with exit from the cell cycle. In these cases, the terminally differentiated cells lose or greatly restrict their capacity to proliferate. However, we note that in the context of this specification, the terms "differentiation" or "differentiated" refer to cells that are more specialized in their fate or function than at a previous point in their development, and includes both cells that are terminally differentiated and cells that, although not terminally differentiated, are more specialized than at a previous point in their development. The development of a cell from an uncommitted cell (for example, a stem cell), to a cell with an increasing degree of commitment to a particular differentiated cell type, and finally to a terminally differentiated cell is known as progressive differentiation or progressive commitment. A cell that is "differentiated" relative to a progenitor cell has one or more phenotypic differences relative to that progenitor cell. Phenotypic differences include, but are not limited to morphologic differences and differences in gene expression and biological activity, including not only the presence or absence of an expressed marker, but also differences in the amount of a marker and differences in the co-expression patterns of a set of markers. [0121] The term "lineages" as used herein refers to a term to describe cells with a common ancestry or cells with a common developmental fate, for example cells that are derived from the same CFU-ECs or progeny thereof. [0122] As used herein, the terms "proliferating", "proliferation", "expanding", and

"expansion" refer to an increase in the number of cells in a population (growth) by means of cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to the environment, including growth factors and other mitogens. Cell proliferation may also be promoted by release from the actions of intra- or extracellular signals and mechanisms that block or negatively affect cell proliferation.

[0123] The term "regeneration" means regrowth of a cell population, organ or tissue after disease or trauma. For example, vascular regeneration refers to the regrowth of vascular components following disease or trauma to a tissue.

[0124] The term "media" as referred to herein is a medium for maintaining a tissue or cell population, or culturing a cell population (e.g. "culture media") containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art.

[0125] As used herein, the term "donor" refers to a subject to which a organ, tissue or cell to be transplanted is harvested from. As used herein, the term "recipient" refers to a subject which will receive a transplanted organ, tissue or cell. The term "graft" as used herein refers to the process whereby a free (unattached) cell, tissue, or organ integrates into a tissue following transplantation into a subject. The term "allograft" refers to a transplanted cell, tissue, or organ derived from a different animal of the same species. The term "xenograft" or "xenotransplant" as used herein refers to a transplanted cell, tissue, or organ derived from an animal of a different species. In some embodiments, a xenograft is a surgical graft of tissue from one species to an unlike species, genus or family. By way of an example, a graft from a baboon to a human is a xenograft.

[0126] The terms "subject" and "individual" are used interchangeably herein, and refer to an animal, for example a human, from whom the adult endothelial progenitor cells isolated using the methods described herein can be isolated and collected from. A subject can also be the recipient of the isolated adult endothelial progenitor cells. For treatment of disease states which are specific for a specific animal such as a human subject, the term "subject" refers to that specific animal. The terms "non-human animals" and "non-human mammals" are used interchangeably herein, and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g. dog, cat, horse, and the like, or production mammal, e.g. cow, sheep, pig, and the like are also encompassed in the term subject.

[0127] The term "tissue" refers to a group or layer of similarly specialized cells which together perform certain special functions. The term "tissue-specific" refers to a source or defining characteristic of cells from a specific tissue.

[0128] The term "agent" as used herein means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An "agent" can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc. In certain embodiments, agents are small molecule having a chemical moiety. For example, chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Compounds can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds. [0129] As used herein, the term "small molecule" refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (e.g., including heterorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0130] The term "disease" or "disorder" is used interchangeably herein, and refers to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort,

dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder can also related to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, indisposition or affection.

[0131] The term "pathology" as used herein, refers to symptoms, for example, structural and functional changes in a cell, tissue, or organs, which contribute to a disease or disorder. For example, the pathology may be associated with a particular nucleic acid sequence, or "pathological nucleic acid" which refers to a nucleic acid sequence that contributes, wholly or in part to the pathology, as an example, the pathological nucleic acid may be a nucleic acid sequence encoding a gene with a particular pathology causing or pathology-associated mutation or polymorphism. The pathology may be associated with the expression of a pathological protein or pathological polypeptide that contributes, wholly or in part to the pathology associated with a particular disease or disorder. In another embodiment, the pathology is for example, is associated with other factors, for example ischemia and the like. [0132] The term "drug" or "compound" as used herein refers to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject to treat or prevent or control a disease or condition. The chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, for example, an oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siR As, lipoproteins, aptamers, and modifications and combinations thereof.

[0133] The terms "decrease" , "reduced", "reduction" , "decrease" or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount.

However, for avoidance of doubt, ""reduced", "reduction" or "decrease" or "inhibit" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%>, or at least about 40%>, or at least about 50%>, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level. [0134] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0135] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0136] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. [0137] As used in this specification and the appended claims, the singular forms

"a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. [0138] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

EXAMPLES [0139] The origin of new endothelial cells (ECs) during lifelong cellular turnover to compensate for cell loss as well as in angiogenic situations such as cancer growth is not well defined or understood. Creation of new ECs in adults could occur by yet undiscovered vascular endothelial stem cells (VESCs), as has been documented for the stem cells for skin or epithelia (1-3), or by the duplication of existing differentiated cells, as has been described for pancreatic beta-cells (4).

[0140] Here, we describe the use multi-parameter cell sorting and limiting dilution analyses to discover and identify within the adult vascular wall endothelium a rare population of stem cells for ECs. We have surprisingly discovered that CD 117^" expressing ECs from the blood vessel wall endothelium comprise a subpopulation of EC progenitors with a very high proliferative potential that are able to produce discrete EC colonies in low-cell density adherent cultures. We demonstrate that a single vessel wall endothelial stem cell by the phenotype Lin— CD31 CD105 Scal⁺CDl 17⁺ can produce several hundreds of thousands of endothelial daughter cells and generate a functional blood vessel after transplantation in vivo. Transplanted adult VESCs contributed to endothelium in D6 embryos, and in functional neovasular blood vessels in matrigel plugs or melanoma tumors inoculated into adult mice. Moreover, we have discovered that VESCs have long-term self- renewal capacity, as shown by repeated isolation and serial transplantation in vivo. We have also surprisingly found that VESCs can be expanded in suitable conditions ex vivo for several weeks and subsequently be used for in vivo transplantations forming functional blood vessels.

[0141] Our data prove the existence of self-renewing adult vascular endothelial stem cells defined by a CD117⁺ phenotype that can be expanded ex vitro and form functional blood vessels in vivo both in physiological settings, and in pathological neoangiogenesis such as occurring during cancer growth. While these cells are very rare in quiescent endothelium, we have found that in neoangiogenic vessels and tumor vasculature CD117⁺ ECs are abundant and form complete blood vessels segments. Our results provide new approaches for personalized medicine, i.e., individual cell-based therapy, using VESCs to restore tissue vascularization, as well as for novel approaches to block tumor angiogenesis by targeted inhibition of VESC functions.

Results [0142] The early blood vessels of the embryo and yolk sac in mammals develop by aggregation of de-novo-formmg angioblasts into a primitive vascular plexus

(vasculogenesis). Blood vessels arise from endothelial precursors, which share an origin with haematopoietic progenitors (5, 6). In adults, the growth of blood vessels (a process known as angiogenesis) is essential for organ growth and repair. In many disorders, angiogenesis becomes excessive. The best-known conditions in which angiogenesis is switched on are malignant, ocular, and inflammatory disorders (7).

[0143] To first better understand the need for new ECs to maintain adult blood vessel homeostasis, we estimated the average EC turnover rate in healthy non-angiogenic tissues. To detect the newly created ECs on blood vessel endothelium in the lungs, thoracic aortas, and subcutaneous tissues in wild-type male C57BL/6 mice, BrdU was given with the drinking water for 30, 60, or 90 days, and specific staining for BrdU and vWF was employed as described in Methods (Figure 1A). Our results indicated that to compensate for cell loss during normal vascular maintenance, after one month an average of 5.8% (SD ±0.29) of ECs in the lungs (Figure IB), 5.5% (SD ±1.48) in subcutaneous tissues, and 8.6%) in aortas (SD ±1.94) are replaced by a newly formed EC. These cells new cells could be produced by rare vascular endothelial stem cells (VESCs) with a very high capacity to produce endothelial daughter cells and residing within the endothelium or possibly elsewhere. Alternatively, novel ECs could be produced by duplication of large numbers of the existing ECs. In passaged human aortic ECs not all cells in the monolayers proliferate at an equal rate (8). Previous work has also indicated that very low numbers of cells with endothelial characteristics and high proliferative potential may be found in umbilical cord blood or in peripheral blood (9-12).

[0144] We wanted to assess if all ECs in adult tissues have an equal potential to produce progeny. We found that rare endothelial colony- forming cells (CFCs) were routinely detected when CD31⁺CD105⁺ ECs are isolated from single cell suspensions prepared by enzymatic digestion of adult mouse tissues, and cultured in vitro in low-cell density adherent semi- so lid matrix colony assays supplemented with the endothelial growth factor VEGF.

[0145] To further investigate whether the endothelial CFCs might reside in the vascular wall endothelium, we perfused adult C57BL/6 mice with PBS to wash out the circulating cells, and subsequently isolated CD31⁺CD105⁺ ECs from the lung vasculature and tested them in in vitro colony assays. Lung vasculature was selected as the source for ECs for technical reasons: the lung tissues are richly vascularized and easy to process to obtain large numbers of viable single cells. After the remaining contaminating

hematopoietic (CD5, CD45R (B220), CDl lb, Gr-1 (Ly-6G/C), 7-4, and Ter-119) cells, described herein as being "Lin ", were removed using immunomagnetic lineage depletion, endothelial CFCs could be recovered from the tissues at a sevenfold higher frequency. In ISO lated Lin^~CD31 CD105 mouse lung ECs, we found that CFCs were detected at a mean frequency of 0.19% (SD ±0.032) corresponding to approximately two colony forming units (CFUs) per thousand isolated ECs (Fig. lc) The colonies were expressing EC markers CD31, CD 105, and vWF while negative for the pan- hematopoietic marker CD45. To further characterize the cells in the EC colonies, we picked up individual colonies, resuspended them in EC growth medium, and cultured them as monolayers in standard 2D EC cultures.

[0146] To study in vitro within EC monolayers the growth patterns of individual CFCs, we isolated CD31⁺CD105⁺ ECs from transgenic C57BL/6-Tg(ACTB-EGFP)10sb/J mice where all the tissues, including the blood vessels, are GFP⁺. Subsequently, we seeded a mixture of one CFU of GFP⁺ CD31⁺CD105⁺ ECs and 20 CFUs of wt ECs on a 2D EC culture and let the cell grow until the formed monolayer was confluent, typically 12 days. The resulting confluent monolayers of wt ECs contained on average one clonal, circular GFP⁺ batch per culture (Figure ID).

[0147] To further characterize the CFC subpopulation within ECs, we analyzed their distribution in various subpopulations of Lin^"CD31 CD105⁺ ECs isolated from enzymatically digested mouse lung vasculature using fluorescence activated cell sorting (FACS). Phenotypic markers examined included surface molecules previously described to identify various different adult stem cell populations (13-16). We first prepared CD117- enriched and CD117-depleted fractions of isolated Lin^"CD31⁺CD105⁺ ECs, and assayed them in vitro for CFCs. The CD117 enriched fraction contained CFCs with an almost tenfold frequency (0.42%, SD ±0.054 vs 0.045%, SD ±0.037; PO.0001; Figure 2A) compared to CDl 17 depleted ECs that contained only few or no CFCs.

[0148] We next picked up the colonies, prepared single cell suspensions, and re- plated the cells again on a second colony forming assay. In the second assay, 58% of the colonies from the CDl 17-enriched EC population formed one or more new colonies. The number of these second assay colonies formed from one CDl 17⁺ primary colony ranged from 0 to 10, median 1. This indicates that the majority of the daughter cells created in the primary colonies have very limited proliferative potential, and that the high proliferative potential is retained only by one or a few CFCs within a colony. In contrast to CDl 17⁺ ECs, only 6% of the colonies from the CDl 17-depleted EC population, and 14% from ECs not selected for CDl 17 formed new colonies when re-plated on the second assay (a total of 28 colonies not selected for CDl 17, 118 CDl 17⁺ colonies, and 84 CDl 17^" colonies were picked up and re-assayed in 5 independent experiments).

[0149] Based on our screens where we sought for CFU-containing EC

subpopulations, we selected three surface markers (Sca-1, CDl 17, CD44) and used them in simultaneous evaluation for CFU distribution in distinct EC subpopulations.

Multiparameter FACS sorting of lung Lin^"CD31 CD105⁺ ECs into fractions enriched or depleted for Sca-1, CDl 17, or CD44 in a single sort produced the following EC

subpopulations: Sca-1⁺ CD117 CD44^", Sca-1⁺CD117^"CD44⁺, Sca-1⁺CD117⁺ CD44^", and Sca-1⁺CD117⁺ CD44⁺. The gating strategy for the FACS sorting of the lineage depleted cells is shown in Figure 5). Because almost all isolated Lin^" cells were Sca-1⁺,

subpopulations depleted for Sca-1 were impossible to obtain in large enough cell numbers to enable further analysis. The relative distributions and overlap of these 3 phenotypic markers within the iso lated Lin^"CD31 CD105 ECs shows that while Sca-1 and CDl 17 were found in a large fraction of the ECs, the CD44⁺ subpopulation is very small, constituting only 5% of the total Lin^"CD31⁺CD105⁺ ECs (Figure 2B). The highest proportion of CFUs were found in the Lin^"CD31⁺CD105⁺Scal⁺CDl 17⁺CD44⁺

subpopulation as these cells contained as many as 0.56% (SD ±0.068) of colony forming ECs (Figure 2C). This EC subpopulation of is very small, containing only 3% (SD ±1) of all Lin^"CD31⁺CD105⁺ ECs (Figure 2B). When lung tissues of C57BL/6 mice were studied using multichannel high resolution laser confocal scanning, occasional

Scal⁺CD117⁺CD44⁺ ECs were observed in the lung vascular endothelium (Figure 2D), corresponding to the low frequency of these cells based on the FACS analyses. Similarly, infrequent Scal⁺CD117 CD44⁺ ECs were observed also in the blood vessel endothelium in subcutaneous tissues (Figure 2E).

[0150] Based on these results, we continued our experiments using Lin^"

CD31⁺CD105⁺Scal⁺ CDl 17⁺ ECs. To learn if blood vessels could originate from one single Lin^" CD31⁺CD105⁺Scal⁺ CDl 17⁺ cell, we performed in vivo transplantations of GFP-tagged ECs originating from a single Lin^"CD31⁺CD105 Scal⁺ CDl 17⁺ colony forming cell (Figure 3A). GFP-tagged Lin^"CD31⁺CD105⁺Scal⁺ CDl 17⁺ cells were first isolated from transgenic C57BL/6-Tg(ACTB-EGFP)10sb/J mice, and the single cell suspension was plated in adherent colony forming assays at one CFC per plate. The plates that later contained a single colony were utilized in cell transplantations. To expand the cell number prior to transplantation, the distinct colonies were cultured for twelve days in adherent matrix, and subsequently picked up, resuspended, and transplanted into wild-type C57BL/6 mice.

[0151] Self-renewal is one defining functional property of adult stem cells. To determine whether the endothelial CFCs would retain their capacity to generate functional blood vessels, we performed repeated isolations and serial transplantations in vivo. We inoculated C57BL/6 mice with B16 melanoma tumors mixed with 15 CFUs of GFP-tagged CD31 CD105⁺ ECs, and performed repeated isolations and serial transplantations of lineage depleted single cell suspensions from the tumors every time after two weeks of tumor growth. GFP⁺ blood vessels were observed in secondary, tertiary, and quaternary transplants (Fig. 3c). These findings provide direct evidence that the GFP- tagged ECs contained endothelial stem cells with self-renewal capacity. Taken together, our results provide novel and surprising evidence for adult endothelial stem cell hierarchy and the existence of a slowly cycling self-renewing Sca-1⁺CD44⁺CD117⁺ adult vascular endothelial stem cell (VESC) that resides at the blood vessel endothelium (Figure 3D). CDl 17 identify the ECs with stem cell potential since they are less abundantly expressed in ECs with limited or no proliferative potential. According to our, practically all VESCs are comprised in the CDl 17⁺ ECs subpopulation ECs. However, especially CD44 is very rarely found on quiescent endothelium, thus providing a very discriminative phenotypic marker for ECs with stem or progenitor cell potential.

[0152] Finally, we studied CDl 17⁺CD44⁺ VESCs in angiogenic blood vessels.

Nonangiogenic vasculature in intact subcutaneous tissues was studied first. We found that CD117⁺ or CD44⁺ ECs were very rare in quiescent vasculature (Figure 4A). Only 38 ± 10% and 2 ± 1% of all ECs were estimated to be CC117⁺ or CD44⁺, respectively (three independent experiments). We found that CD117 CD117⁺ ECs were more infrequent. However, in angiogenic vasculature both CD117⁺ and CD44⁺ ECs were very numerous (Figures 4B and 4C). Both in the neoangiogenic vessels in matrigel plugs and in intratumoural vasculature of B16 melanomas, CD117⁺ and CD44⁺ ECs constituted the majority of all ECs, with long segments of vasculature being composed of CD117⁺CD44⁺ ECs.

[0153] We have also found that a single endothelial progenitor cell can be expanded and cultured ex vivo for over 20 passages and over 3 months. The culture and expansion of the single endothelial progenitor cell involves first culturing in matrigel colony assays, followed by 2D monolayer cultures. Such methods allow a single endothelial progenitor cell to give rise to tens of millions of endothelial cells that can be used in a variety of downstream applications, such as stem cell therapy and personalized medicine.

[0154] Since our results show that CD117⁺ ECs are highly enriched for cells with stem or progenitor cell potential, these results suggest for an activation of normally slowly cycling VESCs and/or expansion of a transient amplifying EC progenitor pool during angiogenic situations and cancer. The results also indicate that stem and progenitor cells for vascular ECs are a novel cellular target for therapeutic approaches to inhibit excessive angiogenesis and to block tumor growth.

In vivo labeling of proliferating mouse cells with BrdU and determination of the proportion of newly formed ECs.

[0155] Male C56BL/6 mice of 8 weeks of age were given BrdU (lmg/ml) mixed in the drinking water for one, two, or three months (n=4 in each group). The BrdU mixture was made up freshly, protected from light, and changed every 3 days. Mice were sacrificed and perfused with PBS. The tissues (lung, ear, thoracic aorta) were fixed with 2% PFA and cryoprotected in 20% sucrose for 24h. After cryopreservation the tissues were embedded in OCT compound (Tissue-Tek; Sakura Finetek Europe) and frozen at -70°C. Sections (5-10 μιη) were cut and antigen retrieval was performed using 2M HC1 for 15min at room temperature followed by neutralization in 0.1 M sodium borate for lOmin. Rinsing 3 times with PBS for 5min was followed by blocking with 5% goat serum for 30 min at room temperature. Subsequently, the sections were incubated with FITC-labeled rat anti-BrdU mAb to BrdU (Abeam) at 1 :50) and rabbit polyclonal anti-vWF at 1 :700 overnight at 4°C. Negative controls were performed by omitting the primary antibodies or using irrelevant controls of the same isotype. Subsequently the vWF signal detected with fluorochrome- conjugated secondary antibodies for 30 min at RT. Finally, the sections were fixed with 4% PFA for 2 min, stained with DAPI for 20 min, and mounted with antifading medium (Vectashield). The samples were analyzed and photographed with a Zeiss Axioplan 2 immunofluorescence microscope using 20x (NA = 0.5) and 40x (NA = 0.75) Plan- Neofluar objectives, AxioCam Hrc camera, and Axiovision 4.3 software (Carl Zeiss,

Gottingen, Germany). Total ECs were manually counted and the number of BrdU- labeled ECs recorded on five different randomly selected sections of each tissue sample from each mouse. A mean number of 321 (range, 42 -1261) of total ECs was counted on each section. The scoring was performed blinded to avoid bias. Isolation of mouse lung endothelial cells

[0156] Mouse lung endothelial cells were isolated from lungs dissected from adult wild type C57BL/6 or GFP-tagged transgenic C57BL/6-Tg(ACTB-EGFP)10sb/J mice. Mice were anesthetized with Rompun vet (Bayer) and Ketaminol vet (intervet). The chest was opened through a midline sternotomy. The left ventricle was identified and the ventricular cavity was entered through the apex with a 27-gauge needle. The right ventricle was identified and an incision was made in the free wall to exsanguinate the animal and to allow the excess perfusate to exit the vascular space. The animal was perfused with approximately 2-6 ml of PBS at approximately 10 ml/min. After sacrifice, the lungs were collected, fat tissue was removed and lung tissue was minced. The tissue fragments were then digested with DMEM medium containing Dispase II (0.8U/ml, Roche), Collagenase H (1 mg/ml, Roche), Pen/Strep (lOOU/lOOg/ml), 2% FCS, 2mM glutamine at 37° C for 1 h after which the suspension was homogenized by pipetting. The homogenate was filtered through a lOOum nylon mesh filcon (BD Biosciences) and subsequently through a 30 μιη nylon mesh falcon (BD Biosciences) and pelleted by centrifugation (300g for 6

minutes). The erythrocytes were then lysed by lysis buffer containing lOmM KHCO3,

155mM NH₄CI, 0.1 mM EDTA, pH 7.5 at room temperature for 2 minutes. The cell pellets were resuspended in DMEM medium containing Pen/Strep (lOOU/lOOg/ml), 2% FCS, and 2mM glutamine. If lineage depletion was used, Mouse Lineage Cell Depletion Kit (Miltenyi Biotec) was used according to manufacturer's instructions. The lineage-depleted fraction was then incubated with anti-mouse CD16/CD32 blocker (BD PharMingen) according to the manufacturer's instructions to reduce Fcyll/III receptor-mediated antibody binding stained for FACS. The antibodies used included APC anti-mouse CD31 (BD Pharmingen) and PE anti-mouse CD 105 (eBioscience), V450 Sca-1, PerCP-Cy5.5 CD44 and PE-Cy7 CD117. Compensation adjustments were performed with single color positive controls. Dead cells and cell debris were excluded by gating the population according to the forward and side light scatters. The number of positive cells were compared with the number of cells positive in the staining with the IgG isotype controls (BD PharMingen) and determined with FACSAria flow cytometer (Becton Dickinson) and sorted. Before each sorting, laser compensations were adjusted automatically with FACSDiva software version 4.1.2 (Becton Dickinson). For some of the in vitro assays, CD31+CD105+ ECs were also isolated using anti-fluorochrome multisort kit (Miltenyi Biotec) according to the instructions of the manufacturer. In vitro colony assays

[0157] Mouse lung endothelial cells obtained from wild type C57BL/6 mice were plated in triplicate in 1ml of 0.8% methylcellulose containing 15% FCS, 1% L-glutamine, 1% BSA, 10-4mM 2-Mercaptoethanol, 0.2mg/ml human transferrin, O.Olmg/ml recombinant human insulin and 100 ng/ml recombinant murine VEGF (Invitrogen).

Scoring of colonies was performed with an inverted microscope. Colonies containing 15 or more cells on day 7 were counted. In preliminary experiments the isolated cell populations were plated at various plating densities, and an ideal plating density for each subpopulation was determined.

Induction of angiogenesis by syngeneic B16 melanoma tumors or matrigel plugs [0158] The B 16-F 1 melanoma cell line (ATCC) was maintained in DMEM supplemented with 2 mM L-glutamine, Pen/Strep (lOOU/lOOg/ml), and 10%> fetal bovine serum (PromoCell). The mice were injected in the ear with B16 cells (2>< 10⁶ cells in 30 μΐ). In some experiments, GFP+ isolated mouse lung endothelial cells were mixed with B6 cells prior to injection. The tumors were allowed to grow for 10-20 days, the mice were killed, and the tissues were processed for analyses. Matrigel plugs (400μ1 per injection Basement Membrane Matrix; BD PharMingen) supplemented with recombinant murine VEGF164 (100 ng/ml; R&D Systems) were injected close to the dorsal midline of the ventral side of the mouse. The plugs were excised and processed for tissue analyses at 1-2 weeks after injection. To verify that detected blood vessels are actually functional vessels, Fluorescent carboxy late-modified microspheres, 0.2 um, red fluorescent (580/605) (FluoSpheres, Molecular Probes) were used diluted 1 :6 with PBS. Mice were anesthetized with Rompun vet (Bayer) and Ketaminol vet (Iintervet). Tridil (nitroglycerin, Orion Oyj) was included in the anesthesia mixture at 50 mg/ml to allow maximal vasodilation of the peripheral vasculature. The chest was opened through a midline sternotomy. The left ventricle was identified and the ventricular cavity was entered through the apex with a 27- gauge needle. The right ventricle was identified and an incision was made in the free wall to exsanguinate the animal and to allow the excess perfusate to exit the vascular space. The animal was perfused with approximately 2-6 ml of PBS at approximately 10 ml/min and then with the fluorescent microspheres.

Immunohistochemistry and Whole Mounts [0159] The primary antibodies used were rat anti-mouse CD31/PECAM-1 (BD

PharMingen), rat anti-mouse CD105/endoglin (BD PharMingen), rat anti-mouse VEGFR-2 (BD Pharmingen), rabbit anti-mouse/human von Willebrand Factor (vWF; DAKO), rat anti-mouse Sca-1 (BD Pharmingen), rat anti-mouse CD44 (BD Pharmingen), rabbit anti- mouse CD44 (Abeam), rabbit anti-mouse ki-67 (Abeam), and goat anti-mouse CD117 (R&D systems), n some stainings, rat anti-mouse CD45 and rat anti-mouse CD1 lb (both from BD Pharmingen) were used as negative controls. The secondary antibodies used were Alexa594 anti-rat, Alexa594 anti-rabbit, Alexa633 anti-rat, Alexa633 anti-rabbit, Alexa 594 anti-goat, Alexa 647 anti-goat, Alexa 647 anti-rabbit, Alexa488 anti-rat, Alexa 488 anti-rabbit (all from Molecular Probes). The correct detection of the endogenous GFP signal was controlled by also staining part of the samples with an anti-GFP-Alexa488 antibody (Molecular Probes). For staining of whole mounts, all samples were fixed in 4% PFA; blocked with PBS buffer containing 5% serum (Vector Laboratories), 0.2% BSA, 0.09% Na-Azide, 0.2% BSA and 0.3% Triton-X (Sigma-Aldrich), and incubated with the primary antibodies for 2 days at room temperature. Autofluorescent cartilage was removed from the ears before fixing. The samples were washed and incubated with fluorochrome conjugated secondary antibodies overnight at room temperature. Finally, the plugs were sliced, the ears were flattened, and the samples were mounted with antifading medium (Vectashield; Vector Laboratories). For immunohistochemistry of cryosections, samples were fixed for 1 h with 2% PFA and incubated in 20% sucrose/PBS overnight. After the cryopreservation, tissues were embedded in OCT compound (Tissue-Tek; Sakura Finetek Europe) and frozen at -70°C. Sections (8-80 μιη) were stained with the primary antibodies overnight at 4°C and subsequently detected with fluorochrome-conjugated secondary antibodies for 30 min at room temperature. Finally, the sections were mounted with antifading medium (Vectashield). The samples were analyzed with a Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss) using multichannel (sequential) scanning in frame mode and a 40x (NA = 1.3) Plan-Neofluar oil immersion objective (LSM 5 Software version 3.2). Single XY-scans typically had an optical slice thickness of 0.9 μιη or less. Additionally, the samples were analyzed and photographed with a Zeiss Axioplan 2 immunofluorescence microscope using 20x (NA = 0.5) and 40x (NA = 0.75) Plan- Neofluar objectives, AxioCam Hrc camera, and Axiovision 4.3 software (Carl Zeiss, Gottingen, Germany).

[0160] For staining of colonies in Methocult, methylcellulose was washed with PBS and cells were fixed in 4% PFA; blocked with PBS buffer containing 5% serum (Vector Laboratories), 0.2% BSA, 0.09% Na-Azide, 0.2% BSA and 0.3% Triton-X (Sigma-Aldrich); and incubated with the primary antibodies for overnight at 4°C and subsequently detected with fluorochrome-conjugated secondary antibodies for lh at room temperature.

Endothelial cell culture and proliferation assays

[0161] Freshly isolated ECs were cultured in gelatin coated plates with IMDM medium containing 15% FCS, 1% L-glutamine, 1% BSA, 10-4mM 2-Mercaptoethanol, 0.2mg/ml human transferring, O.Olmg/ml recombinant human insulin, 100 ng/ml recombinant murine VEGF (Invitrogen, lOOng/ml recombinant murine bFGF and lOOng/ml recombinant murine EGF). In cell proliferation assays, the seeding density was 2x l0⁴ cells per well (24-well plates). Measurements were performed at days 6, 8, 9, 10 and 12. 20μ1Λνβ11 of combined MTS/PMS solution was added. After 1 hour at 37°C in a humidified 5% C0₂ atmosphere, the absorbance at 492nm was measured using an ELISA plate reader. For calibration curve, cultured primary ECs were washed once in IMDM medium containing 15% FCS, 1% L-glutamine, 1% BSA, 10-4mM 2-Mercaptoethanol, 0.2mg/ml human transferrin, O.Olmg/ml recombinant human insulin, 100 ng/ml recombinant mouse VEGF (Invitrogen, lOOng/ml rm bFGF and lOOng/ml rm EGF) by centrifugation at 300^xg for 5 minutes. Cell number and viability (by trypan blue) were determined. Seeding density was 2>< 10⁴ cells/ml. The medium was allowed to equilibrate for 1 hour, then 20μ1Λνε11 of combined MTS/PMS solution was added. After 1 hour at 37°C in a humidified 5% C02 atmosphere, the absorbance at 492nm was measured.

Genetically tagged mice

[0162] The following strains were used as donors in cell transplantations:

C57BL/6-Tg(ACTB-EGFP)10sb/J (from Jackson Laboratory; Bar Harbor, ME. E-cadherin Provides a Cell-surface Marker for Further Enrichment of CD117+ Vascular Endothelial Stem Cells

[0163] In order to discover more powerful cell-surface markers for adult mouse

VESCs, lung EC subsets expressing surface molecules previously in adult stem cell populations were assayed for endothelial CFCs. A significant enrichment of endothelial CFCs was achieved when lin-CD31+CD105+Sca-l+CDl 17+ ECs were further selected for E-cadherin (Fig. 4A). The E-cadherin -enriched EC fraction encompassed as much as 0.7% (mean, SD+0.2) of CFCs in contrast to the E-cadherin -depleted lin-CD31+CD105+Sca- 1+CDl 17+ ECs that were greatly depleted of CFCs and contained only 0.05%> (mean, SD+0.01; P=0.0022; Fig. 4A) of colony-forming ECs. Correspondingly, also the CD117- E-cadherin- fraction of lin-CD31+CD105+Sca-l+ ECs was greatly depleted of CFCs

(mean 0.1%, SD+0.1; P=0.0079) Thus further enrichment for E-cadherin almost doubled the isolation efficiency compared to selecting against CD117 only. In FACS analyses, an average of 67%> of E-cadherin+ ECs were also CD117+ and this double positive population constituted 16% (SD+6) of all lin-CD31+ lung ECs (Fig. 6). REFERENCES

Tumbar, T. et al. (2004) Defining the epithelial stem cell niche in skin. Science 303, 359- 63.

Stingl, J. et al. (2006) Purification and unique properties of mammary epithelial stem cells. Nature 439, 993-7.

Barker, N. et al. (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003-7.

Dor, Y., Brown, J., Martinez, O. I., Melton, D. A. (2004) Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429, 41-6. Coultas, L., Chawengsaksophak, K.,Rossant, J. (2005) Endothelial cells and VEGF in vascular development. Nature 438, 937-45.

Eichmann, A. et al. (2005) Vascular development: from precursor cells to branched arterial and venous networks. Int J Dev Biol 49, 259-67.

Carmeliet, P. (2005) Angiogenesis in life, disease and medicine. Nature 438, 932-6. Ingram, D. A. et al. (2005) Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells. Blood 105, 2783-6.

Bompais, H. et al. (2004) Human endothelial cells derived from circulating progenitors display specific functional properties compared with mature vessel wall endothelial cells. Blood 103, 2577-84. Timmermans, F. et al. (2007) Endothelial outgrowth cells are not derived from CD133+ cells or CD45+ hematopoietic precursors. Arterioscler Thromb Vase Biol 27, 1572-9.

Ingram, D. A. et al. (2004) Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 104, 2752-60. Yoder, M. C. et al. (2007) Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 109, 1801-9.

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hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc Natl Acad Sci U S A 86, 4634-8. Liu, A. Y. et al. (1997) Cell-cell interaction in prostate gene regulation and

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Claims

1. A method for isolating and enriching a cell population of adult-derived

endothelial progenitor cells (EPCs) from an adult-derived cell sample comprising selecting from the adult-derived cell sample cells that are positive for cell-surface markers comprising CD31 (PECAM-1), CD 105 (endoglin) and

CDl 17(c-kit), thereby isolating and enriching the adult-derived cell sample for adult-derived EPCs.

2. The method of claim 1, wherein the cell surface markers further comprise CD44.

The method of any of the preceding claims, wherein the cell surface markers further comprise Sca-1.

The method of any of the preceding claims, wherein the adult-derived cell sample is further selected to be negative for at least one lineage marker selected from the group consisting of CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

The method of claim 4, wherein the selection is performed

immunomagnetic lineage depletion.

6. The method of any of the preceding claims, wherein the selection is performed using at least one antibody.

7. The method of any of the preceding claims, wherein the selection is further performed using fluorescence activated cell sorting.

8. The method of any of the preceding claims, wherein the adult-derived cell sample is derived from a solid tissue.

9. The method of any of the preceding claims, wherein the adult-derived cell sample is derived from a lung tissue.

10. The method of any of the preceding claims, wherein the adult-derived cell sample is derived from vascular endothelium.

11. The method of claim 10, wherein the vascular endothelium is lung vascular endothelium.

12. The method of any of the preceding claims, wherein the adult is a human.

13. The method of any of the preceding claims, wherein the cells are further

cultured in vitro using a low-cell density adherent semi-solid matrix colony assay.

14. The method of any of the preceding claims, wherein the cells are further

cultured in 2D monolayer cultures.

15. The method of any of the preceding claims, wherein the adult-derived EPCs are genetically engineered.

16. An isolated endothelial progenitor cell clone prepared by the method of any one of the preceding claims.

17. An isolated cell population comprising adult-derived vascular endothelial

progenitor cells that are positive for cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44 and Sca-1, and that are negative for at least one of the cell-surface markers CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

18. An isolated cell population, wherein said adult-derived vascular endothelial progenitor cells express surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44 and Sca-1 but not at least one of the cell-surface markers CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

19. An isolated cell population comprising adult-derived vascular endothelial

progenitor cells that are positive for cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit) and Sca-1, and that are negative for at least one of the cell-surface markers CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

20. An isolated cell population, wherein said adult-derived vascular endothelial progenitor cells express surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit) and Sca-1 but not at least one of the cell-surface markers CD2,

CD3, CD14, CD16, CD19, CD56, and CD235a.

21. The isolated cell population of any one of claims 16 to 20, wherein the cells are genetically modified.

22. A pharmaceutical composition comprising the isolated cell population of any one of claims 16 to 21.

23. A method of inducing vascularization in a tissue comprising administering to a tissue in need of vascularization an isolated cell population of any one of claims 16 to 21, or a pharmaceutical composition of claim 22.

24. The method of claim 23, wherein the isolated cell population is autologous to the tissue.

25. The method of claim 23, wherein the isolated cell population is allogenic to the tissue.

26. A method for blocking angiogenesis comprising targeting an angiogenesis

inhibitor to a cell that is positive for CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44, and Sca-1, and that is negative for at least one of the cell- surface markers CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

27. A method for blocking angiogenesis comprising targeting an angiogenesis

inhibitor to a cell that is positive for CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit) and Sca-1, and that is negative for at least one of the cell-surface markers CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.

28. The method of claim 26, wherein the targeting is performed using an antibody or a mixture of antibodies against CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44 and Sca-1.

29. The method of claim 27, wherein the targeting is performed using an antibody or a mixture of antibodies against CD31 (PECAM-1), CD 105 (endoglin),

CD117(c-kit) and Sca-1

30. Use of the isolated cell population of any one of claims 16 to 21, or the

pharmaceutical composition of claim 22, for inducing vacularization in a tissue.

31. The isolated cell population of any one of claims 16 to 21 for use as a

medicament.

32. A method of ex vivo clonal expansion of a single endothelial progenitor cell comprising isolating a single endothelial progenitor cell in one or more matrigel colony assay and one or more 2D monolayer cultures.