Compositions comprising isolated endothelial progenitor cells for treating von willebrand disease

Abstract

The present technology comprises isolated endothelial progenitor cell (EPC) populations and mesenchymal stem cell (MSC) populations, and methods of making and using the same for treating bleeding disorders, including Von Willebrand Disease, and associated symptoms.

Description

COMPOSITIONS COMPRISING ISOLATED ENDOTHELIALPROGENITOR CELLS FOR TREATING VON WILLEBRANDDISEASECROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 681 ,376, filed August 9, 2024, the contents of which are incorporated herein by reference in their entirety.BACKGROUND

[0002] The most common inherited bleeding disorder, Von Willebrand Disease (VWD), may be caused by quantitative or qualitative defects of Von Willebrand Factor (VWF). As a monogenic disorder, VWD may be an ideal target for cell-based gene therapy. However, successful treatment has been hampered by the large size of VWF transgenes, complex post-translational processing of VWF, and insufficient engraftment of engineered cells. Recent therapeutic efforts include liver-directed viral approaches, where viral vectors expressing VWF are hepatically transduced into VWD subjects. However, the thereapeutic effects of such approaches are temporary, attributed to the limiting size of VWF complementary deoxyribonucleic acid (cDNA), failure of normal VWF post-translational multimerization, and the inability to redose subjects due to immune responses to the viral vectors. As such, additional VWD therapeutic treatments which overcome these pitfalls are needed.SUMMARY

[0003] In some embodiments, the present technology comprises a method of treating Von Willebrand Disease (VWD) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated endothelial progenitor cell (EPC) population comprising Protein C Receptor (PROCR)+ Platelet-Derived Growth Factor Receptor Alpha (PDGFRA)+ EPCs.

[0004] In some embodiments, the present technology comprises a method of treating VWD in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+Z- PDGFRA+Z- EPCs.

[0005] In some embodiments, the present technology comprises a method of treating VWD in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising mesenchymal stem cells (MSCs).

[0006] In some embodiments, the present technology comprises a method of treating VWD in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0007] In some embodiments, the present technology comprises a method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

[0008] In some embodiments, the present technology comprises a method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0009] In some embodiments, the present technology comprises a method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0010] In some embodiments, the present technology comprises a method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0011] In some embodiments, the present technology comprises a method of increasing a blood clotting level in a subject in need thereof relative to a control,comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

[0012] In some embodiments, the present technology comprises a method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0013] In some embodiments, the present technology comprises a method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0014] In some embodiments, the present technology comprises a method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0015] In some embodiments, the present technology comprises a method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

[0016] In some embodiments, the present technology comprises a method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0017] In some embodiments, the present technology comprises a method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs

[0018] In some embodiments, the present technology comprises a method of increasing a VWF protein level in a subject in need thereof relative to the control,comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0019] In some embodiments, the VWF protein level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0020] In some embodiments, the VWF protein level is increased in a plasma sample from the subject, relative to the control.

[0021] In some embodiments, the present technology comprises a method of increasing a VWF gene expression level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0022] In some embodiments, the present technology comprises a method of increasing a VWF gene expression level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0023] In some embodiments, the present technology comprises a method of increasing a VWF gene expression level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0024] In some embodiments, the VWF gene expression level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0025] In some embodiments, the VWF gene expression level is increased in a plasma sample from the subject, relative to the control.

[0026] In some embodiments, the present technology comprises a method of increasing a VWF protein activity level in a subject in need thereof relative to the control,comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0027] In some embodiments, the present technology comprises a method of increasing a VWF protein activity level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0028] In some embodiments, the present technology comprises a method of increasing a VWF protein activity level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0029] In some embodiments, the VWF protein activity level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0030] In some embodiments, the VWF protein activity level is increased in a plasma sample from the subject, relative to the control.

[0031] In some embodiments, the present technology comprises a method of increasing a VWF protein multimer level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0032] In some embodiments, the present technology comprises a method of increasing a VWF protein multimer level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0033] In some embodiments, the present technology comprises a method of increasing a VWF protein multimer level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs;and (b) a second isolated cell population comprising MSCs.ln some embodiments, the VWF protein multimer level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0034] In some embodiments, the VWF protein multimer level is increased in a plasma sample from the subject, relative to the control.

[0035] In some embodiments, the present technology comprises a method of reducing one or more symptoms of VWD in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs, wherein the one or more symptoms are selected from the group consisting of blood loss, platelet clumping, and bruising.

[0036] In some embodiments, the reduction in the blood loss level comprises a reduction in a duration or a frequency of epistaxis.

[0037] In some embodiments, the reduction in the blood loss level comprises an increase in a blood clotting level, relative to the control.

[0038] In some embodiments, the increase in the blood clotting level or the reduction in the blood loss level comprises an increase in blood clotting speed during or after an injury, relative to the control.

[0039] In some embodiments, the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a bleeding level during or after an injury, relative to the control.

[0040] In some embodiments, the injury comprises a surgical injury.

[0041] In some embodiments, the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a bleeding level during or after childbirth, relative to the control.

[0042] In some embodiments, the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a menstrual bleeding level, relative to the control.

[0043] In some embodiments, the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a stool blood level or a rectal bleeding level, relative to the control.

[0044] In some embodiments, the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a bruising level, relative to the control.

[0045] In some embodiments, the reduction in the bruising level comprises a reduction in a raised bruising level, relative to the control.

[0046] In some embodiments, the present technology comprises a method of increasing a Weibel-Palade body (WPB) level in a subject in need thereof, relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

[0047] In some embodiments, the present technology comprises a method of increasing a WPB level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0048] In some embodiments, the present technology comprises a method of increasing a WPB level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0049] In some embodiments, the present technology comprises a method of increasing a WPB level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0050] In some embodiments, the increase in the WPB level comprises an increase in WPB number or a WPB area.

[0051] In some embodiments, the method does not increase a VWF alloantibody level in the subject, relative to the control.

[0052] In some embodiments, the subject has been diagnosed with VWD.

[0053] In some embodiments, the subject has a VWD selected from the group consisting of Type 1 VWD, Type 2A VWD, Type 2B VWD, Type 2M VWD, Type 2N VWD, and Type 3 VWD.

[0054] In some embodiments, the method increases a Factor VIII gene expression level or a Factor VIII protein expression level, relative to a control.

[0055] In some embodiments, the isolated EPC population or the first isolated cell population comprises a CD45- / CD34+ phenotype.

[0056] In some embodiments, the isolated EPC population or the first isolated cell population comprises endothelial colony-forming cells (ECFCs).

[0057] In some embodiments, the isolated EPC population or the first isolated cell population is cocultured with an isolated MSC population.

[0058] In some embodiments, the isolated EPC population or the first isolated cell population comprises an increase in a vasculogensis level, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0059] In some embodiments, the vasculogenesis level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0060] In some embodiments, the increase in the vasculogenesis level occurs during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing.

[0061] In some embodiments, the isolated EPC population or the first isolated cell population comprises an increase in a CD90 gene expression level, a VWF gene expression level, a DLL4 gene expression level, a SLUG gene expression level, a CD34 gene expression level, a CD31 gene expression level, or a NOTCH gene expression level, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0062] In some embodiments, the increase in the CD90 gene expression level, the VWF gene expression level, the DLL4 gene expression level, the SLUG gene expression level, the CD34 gene expression level, the CD31 gene expression level, or the NOTCH gene expression level comprises an increase by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0063] In some embodiments, the increase in the CD90 gene expression level, the VWF gene expression level, the DLL4 gene expression level, the SLUG gene expression level, the CD34 gene expression level, the CD31 gene expression level, or the NOTCH gene expression level occurs during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing.

[0064] In some embodiments, the isolated EPC population or the first isolated cell population comprises an increase in a CD90 protein level, a VWF protein level, a DLL4 protein level, a SLUG protein level, a CD34 protein level, a CD31 protein level, or a NOTCH protein level, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0065] In some embodiments, the increase in the CD90 protein level, the VWF protein level, the DLL4 protein level, the SLUG protein level, the CD34 protein level, the CD31 protein level, or the NOTCH protein level comprises an increase by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0066] In some embodiments, the increase in the CD90 protein level, the VWF protein level, the DLL4 protein level, the SLUG protein level, the CD34 protein level, the CD31 protein level, or the NOTCH protein level occurs during coculturing or at leastabout 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing.

[0067] In some embodiments, a VWF gene expression level in the isolated EPC population or the first isolated cell population increases during or after coculturing.

[0068] In some embodiments, the VWF gene expression level increases by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a VWF gene expression level in an isolated EPC population that is not cocultured with an isolated MSC population.

[0069] In some embodiments, a VWF protein level in the isolated EPC population or the first isolated cell population increases during or after coculturing.

[0070] In some embodiments, the VWF protein level increases by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a VWF protein level in an isolated EPC population that is not cocultured with an isolated MSC population.

[0071] In some embodiments, the pharmaceutical composition is a first composition formulated for administration before, during, or after administration of a second composition comprising an isolated MSC population to a subject in need thereof.

[0072] In some embodiments, the first isolated cell population and the second isolated cell population are present in the composition at a ratio of about 1 :1.

[0073] In some embodiments, the first isolated cell population and the second isolated cell population are present in the composition at a ratio of at least about 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 , 12:1 , 15:1 , 20:1 , 30:1 , 40:1 , or 50:1.

[0074] In some embodiments, the MSCs are CD45- / CD34+ cells.

[0075] In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the second isolated cell population is a CD34+ / CD45- isolated cell population.

[0076] In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the isolated cell population or the second isolated cell population express PROCR and PDGFRA.

[0077] In some embodiments, the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from a donor.

[0078] In some embodiments, the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from two or more donors.

[0079] In some embodiments, the isolated cell population, the first isolated cell population, or the second isolated cell population comprise allogenic cells.

[0080] In some embodiments, the isolated cell population, the first isolated cell population, or the second isolated cell population are present in a medium.

[0081] In some embodiments, the medium comprises a liquid or frozen medium.

[0082] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle.

[0083] In some embodiments, the pharmaceutically acceptable vehicle is phosphate-buffered saline.

[0084] In some embodiments, the subject is administered one or more doses of the pharmaceutical composition.

[0085] In some embodiments, the subject is administered two or more doses of the pharmaceutical composition.

[0086] In some embodiments, the pharmaceutical composition is administered to the subject intravenously.BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIGS. 1A-1 D show single cell RNA-sequencing analyses of murine aortic endothelial compartment. FIG. 1A shows clustering across 3 aortic samples. FIG. 1 B shows SingleR unbiased labelling analysis. FIG. 1 C shows top differentially expressedgenes in an endothelial progenitor cell (EPC) cluster. FIG. 1 D shows top differentially expressed genes in mesemchymal stem cell (MSC) clusters.

[0088] FIGS. 2A-2F show single-cell RNA-sequencing and flow cytometry that demonstrate PROCR and PDGFRA are markers in endothelial populations. FIG. 2A shows a flow cytometric gating strategy showing the isolation of the endothelial hierarchy. Flow cytometry plots of cells further gated on Procr, CD157, Abcg2-YFP, and Sox18-YFP are be shown in FIG. 2B endothelial progenitor cells (EPC) and FIG. 2C mature differentiated endothelial cell populations. FIG. 2D shows quantification of EPC and mature differentiated endothelial cells positive for cell surface markers (i) PROCR (***p= 0.0008; n = 5), (ii) PDGFRA (*** p = 0.0005; n=5), (iii) CD157 (ns, p = 0.0805; n=2), (iv) Abcg2 (* p = 0.0490; n=3), and (v) Sox18 (*, p = 0.0452; n=3). FIG. 2E shows an alternative gating strategy showing live cells first gated as PROCR+PDGFRA+, followed by Lin’VE’cadherin+and finally gated as EPC and mature differentiated endothelial cells based on CD31 and CD34 expression. FIG. 2F shows quantification of percent of EPCs gating using gating strategy in FIG. 2B (****, p<0.0001 , n=3).

[0089] FIGS. 3A-3C show that PROCR+EPCs show increased endothelial colony formation capacity in vitro and engraftment potential in vivo compared to other cell populations. FIG. 3A shows representative IF images of collagen gels containing (i) PROCR+EPCs or (ii) PROCR’ EPCs FACS-sorted from CAG-EGFP mice aortae collected following 7 days of implantation in NOD-scid H2rynullB2mnull (NSG) mice (scale bars = 500 pm). FIG. 3B show the percentage of GFP+ area of each gel upon collection measured via IF (* p<0.05; n=3). FIG. 3C shows representative IF images of sections from (i) PROCR+EPC and (ii) PROCR’ EPC collagen gels collected after 7 days stained with DAPI, GFP, CD34 and Isolectin (scale bars = 250 pm).

[0090] FIGS. 4A-4C show that PROCR+EPCs form a niche in the thoracic aorta displaying increased clonogenic capacity. Shown are aortae collected from Cdh5- CreERT2 / ROSA-EYFP mice, opened and rolled lengthwise. FIG. 4A shows a 60x magnification of the thoracic aorta section; white arrows indicate regions of overlap between DAPI, PROCR, and YFP. FIG. 4B shows the percentage of PROCR+length in abdominal and thoracic aorta (** p=0.005; n=5). FIG. 4C shows quantification of colonies grown from thoracic and abdominal aorta (**, p=0.0052, n=13).

[0091] FIGS. 5A-5C show PROCFT PDGFRA+EPCs from Pdgfra- MerCreMer / Rosa-YFP differentiate into mature endothelial cells in homeostatic aorta and in an injury model of full-skin excisional wounds, respectively. FIG. 5A shows the percentage of PROCR+PDGFRA+EPCs and matured differentiate endothelial cells in the Lin_YFP+fraction of the aorta between D1 and D84 (** p<0.01 , *** p<0.001 ; n=4). FIG. 5B shows the percent of PROCR+PDGFRA+EPCs and mature differentiated endothelial cells in the Lin’YFP+ fraction of the wounds between D1 and D5 (* p<0.05, ** p<0.01 ; n=7). FIG. 5C shows quantification of (i) colony counts and (ii) colony types formed from cultured PROCR’ or PROCR+EPCs.

[0092] FIGS. 6A-6C show additional single cell RNA-sequencing analyses and flow cytometric FMOs for human datasets (aorta, n=3). FIGS. 6A and 6B show dot plots of top differentially expressed genes in EPC (FIG. 6A) and MSC (FIG. 6D) clusters. FIG. 6E shows FMO controls for human term placenta FACS-sorting.

[0093] FIG. 7 shows a schematic of a fetal EPC and MSC cell population isolation method involving the additional selection of PROCR+markers to identify fetal EPCs and the selection of PROCR’ to identify fetal MSCs.

[0094] FIG. 8 shows a schematic of a PROCR+PDGFRA+EPC and MSC cell population isolation method requiring the selection of PDGFRA and PROCR markers to identify PROCR+PDGFRA+EPCs and PROCR’ PDGFRA+MSCs.

[0095] FIG. 9 shows flow cytometry results of human placental PROCR+ cells sorted for various expression markers.

[0096] FIG. 10 shows gene expression levels for CD31 , CD34, VE-Cadherin, and PDGFRA in control endothelial colony forming cells (ECFCs) (ECFC-KK and ECFC- MG), MSCs, and PROCR+ ECFCs.

[0097] FIG. 11 shows representative images of PROCR+ ECFCs alone, fetal placental mesenchymal stem / stromal cell (fPL-MSC) alone, or co-cultured in EGM2 for 2 and 5 days. Arrows indicate the ECFCs. Scale bars = 100 pm

[0098] FIG. 12 shows flow cytometry results of CD31 + cells sorted from PROCR+ECFCs cocultured with fPL-MSCs.

[0099] FIGS. 13A-13D show changes in gene expression levels between PROCR+ ECFCs that are not cocultured with MSCs and PROCR+ ECFCs that arecocultured with MSCs. FIG. 13A shows CD31 and CD34 gene expression levels. FIG. 13B shows SLUG and SNAIL gene expression levels. FIG. 13C shows DLL4 and NOTCH1 gene expression levels. FIG. 13D shows VWF gene expression levels using two different primer sets (VWF-1 and VWF -2).DETAILED DESCRIPTION

[0100] The present technology comprises pharmaceutical compositions comprising or consisting of isolated endothelial progenitor cell (EPC) populations, and methods of making and using the same to treat or otherwise ameliorate bleeding disorders (e.g., Von Willebrand Disease (VWD)). In some embodiments, the pharmaceutical compositions further comprise an isolated cell population comprising mesenchymal stem cells (MSCs).Definitions

[0101] While the present technology is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present technology is to be considered as an exemplification of the present technology and is not intended to limit the present technology to the specific embodiments illustrated. Headings may be provided for convenience only and may be not to be construed to limit the present technology in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

[0102] The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, may be stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about.” The term “about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by acceptable levels in the art. Typically, such variation may be as much 10% above and below a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length and such variation may be influenced by standard applicable measurement practices. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.

[0103] Also, the ranges of the present technology may be intended as a continuous range, including every value between the minimum and maximum values recited, as well as any ranges that may be formed by such values. The present technology comprises any and all (and ranges of any such ratios) that may be formed by dividing a numeric value of the present technology into any other numeric value of the present technology. Accordingly, the skilled person will appreciate that many such ratios, ranges, and ranges of ratios may be unambiguously derived from the numerical values presented herein and in all instances, such ratios, ranges, and ranges of ratios represent various embodiments of the present technology.

[0104] Any reference to “endothelial progenitor cell,” as used herein should be understood as a reference to any cell that exhibits the potentiality to develop to a cell exhibiting one or more of the functional or structural characteristics that may be exhibited by an endothelial cell. Still, without limiting the present technology in any way, a reference to “endothelial cell” should be understood as a reference to the squamous epithelial cells that line the blood vessels, lymphatics or other serous cavities such as fluid-filled cavities. The phrase “endothelial cells” should also be understood as a reference to cells that exhibit one or more of the morphology, phenotype and / or functional activity of endothelial cells and is also a reference to mutants or variants thereof. Said endothelial cells may be at any differentiative stage of development subsequent to the endothelial progenitor cell stage. “Variants” include, but may be not limited to, cells exhibiting some but not all of the morphological or phenotypic features or functional activities of endothelial cells. “Mutants” include, but may be not limited to, endothelial cells which may be genetically modified, such as endothelial cells derived from endothelial progenitor cells which may be genetically modified subsequently to isolation by the method of the present technology but prior to undergoing directed differentiation along the endothelial cell lineage. In some embodiments, the subject endothelial cells may be blood vessel endothelial cells (i.e. , endothelial cells which form blood vessels) or may be an immature form of endothelial cells which would proliferate and differentiate to form a blood vessel, but which may be nevertheless more mature than an endothelial progenitor cell.

[0105] Reference to a “mesenchymal stem cell” as used herein refers to to any cell which exhibits the potentiality to develop to a cell exhibiting one or more of the functional or structural characteristics which may be exhibited by a mesenchymal ormesenchymal-derived cell but not a non-mesenchymal-derived cell such as an endodermal or mesodermal derived cell type. Mesenchymal stem cells may be also alternatively known as “stromal stem cells,” “fetal stem cells,” “adult stem cells,” “adipose derived stem cells,” “lipoaspirate derived stem cells,” and “post natal stem cells.” To this end, reference to “mesenchymal-derived cell” should be understood as a reference to cell types that may be more differentiated than a pluripotent mesenchymal cell and which have arisen from a mesenchymal stem cell. These cells will correspond to cells of the tissues to which mesenchymal cells may be known to give rise and which have been detailed hereinbefore. For example, the subject mesenchymal-derived cell may be a cell which is irreversibly committed to differentiating along a particular cell lineage, such as a myocytic precursor cell or adipocytic precursor cell, or it may correspond to a partially or terminally differentiated form of a specific cellular subtype of one of these lineages. Accordingly, mesenchymal stem cells exhibit the ability to differentiate to a cell type of one or more of the mesenchymal lineages under appropriate conditions. Standards to define Mesenchymal Stem Cells (MSCs) have been proposed by The International Society for Cellular Therapy (ISCT).

[0106] An “isolated population” typically refers to cells suspended in a culture medium and free of other components such as carriers and excipients. An “isolated population” may be suspended in a liquid culture medium and may exist at any appropriate temperature and conditions suitable for the viability of the cells. An “isolated population” may be suspended in a frozen medium and may exist in a cryopreserved state at any appropriate temperature and conditions suitable for the viability of the cells. A population may be present in a growth matrix or immobilized on a surface as discussed further herein. Any number of the relevant cells may be present in a population. For example, a population may comprise at least about 5 x 105of the relevant cells. The population may comprise at least about 1 x 106, at least about 2 x 106, at least about 5 x 106, at least about 1 x 107, at least about 2 x 107, at least about 5 x 107, at least about 1 x 108or at least about 2 x 108of the relevant cells. In some instances, the population may comprise at least about 1.0 x 107, at least about 1.0 x 108, at least about 1.0 x 109, at least about 1.0 x 101°, at least about 1.0 x 1011or at about least 1 .0 x 1012of the relevant cells or even more.

[0107] Cells of the present technology, including EPCs, mammalian mature differentiated endothelial cells (D), and MSCs may be defined according to “markerprofile.” This is a standard way of defining cells, such as progenitor and stem cells, which will be immediately apparent, recognizable and understandable to a skilled person in this technical fields. A “marker,” or “biomarker” is typically a cell surface molecule, such as a receptor or ligand or other molecule. A “marker,” or “biomarker” may alternatively be a molecule that is not a cell surface molecule, e.g., an intracellular molecule. In the context of the cells of the present technology, a “marker,” or “biomarker” may be a cell surface molecule.

[0108] In the context of the cells of the present technology, it should be understood that reference to any specific marker, such as “CD45,” “CD34,” “CD31 ,” “PROCR,” and “PDGFRA” is a reference to all forms of these molecules and to functional fragments, mutants or variants thereof. It should also be understood to include reference to any isoform that may arise from alternative splicing of e.g., CD45, CD34, and CD31 mRNA or isomeric or polymorphic forms of these molecules. A reference to a biomarker may be a reference to the wild-type form of the biomarker.

[0109] Reference to “phenotypic profile,” “expressing a phenotype,” or “cells which expresses the phenotype,” etc., should be understood as a reference to the presence or absence of the transcription of the genes encoding the subject markers and / or the cell surface expression of the expression product translated therefrom. A skilled person will appreciate that although most cells falling within the scope of the cells of the present technology will be characterized by the presence or absence of the subject marker as a cell surface anchored expression product, some cells falling within the defined populations may initially exhibit changes only at the transcriptome level, such as when the transcription of a given marker has been upregulated but may not yet have resulted in a cell surface anchored expression product. In general, cells which progress to a new differentiative stage will transiently exhibit gene expression changes which may be not yet evident in the context of changes to levels of an expression product. However, these cells nevertheless may be defined in accordance with a marker profile, although they may not be isolatable by a cell surface marker expression occurs. For any marker identified in connection with any of the cells of the present technology, the marker may be “a cell surface anchored expression product” or “a cell surface anchored protein” or a cell surface anchored polypeptide.”

[0110] Unless the context clearly dictates otherwise, reference to the expression of a marker is taken to mean the detectable expression of the marker or the presence of the marker at detectible levels of expression.

[0111] The terms “+” and may be well known in the art and refer to the expression level of the cell marker of interest, in that the expression level of the cell marker corresponding to “+” is high or intermediate and the expression level of the cell marker correspondingis null. Cells in the top 2, 3, 4, or 5% of staining intensity may be often designated “hi,” with those falling in the top half of the population categorized as being “+.” Those cells falling below 50% of fluorescence intensity may be designated as “Io” cells and below 1 % as cells.

[0112] The termdenotes a population of cells having a proportion of cells which express the “+” phenotype and a proportion of cells which express the phenotype. For example, a population of PROCR+ / - EPCs comprises a mixed population comprising a proportion of cells which express the PROCR+ phenotype and a proportion of cells which express the PROCR- phenotype.

[0113] The term “high” or “hi” or “bright” is well known in the art and refers to the expression level of the cell marker of interest, in that the expression level of the cell marker is high by comparison with the expression level of that cell marker in the population of cells being analyzed as a whole.

[0114] Although most markers, such as the CD45 and CD34 cell surface markers, may be defined by reference to the presence or absence of the marker on the cell surface, the expression of CD31 is defined by reference to the level of expression, specifically a low level of expression (herein referred to as “CD31 Io / -”). In the cells of the present technology, the “CD31 Io / -” subpopulation is based on defining a FACS gate based on an isotype control. In this exemplified embodiment, only the isotype control for CD31 is used and all other antibodies may be kept equal. Three populations may be seen based on CD31 level of expression. The first is negative for CD31 that gives rise to the fetal mesenchymal stem cells. The second population that gives rise to the endothelial progenitor cells is where the positive gate starts. Finally, there is a CD31 + population that has limited proliferative capacity. It would be appreciated by the skilled person that the specific manner in which the analysis is set up and the logs that may be used may vary according to the voltage of the FACS. However, these parameters maybe established as a matter of routine procedure by the skilled person. The term “Io / -” as used in relation to “CD31 Io / -” is well known in the art and refers to the expression level of CD31 , in that the expression level of this cell surface marker is low by comparison with the expression level of that marker in the population of cells being analyzed as a whole. The term “Io” in relation to CD31 lo refers to a distinct cell or population of cells that expresses CD31 at a lower level than one or more other distinct cells or populations of cells. Thus, the terms “CD31 Io / -” and “CD31 Io” may be used interchangeably herein to refer to the endothelial progenitor cells resulting from the subject isolation methods. The level of CD31 expressed by a CD31 Io cell or population of CD31 Io cells is less than 50% (and less than 49% to no less than 1 % and all integer percentages in between, suitably less than 40% to no less than 1 % and all integer percentages in between, suitably less than 30% to no less than 1 % and all integer percentages in between, suitably less than 20% to no less than 1 % and all integer percentages in between, even more suitably less than 10% to no less than 1 % and all integer percentages in between of the level of CD31 expressed by a HUVEC or HUVEC population.

[0115] A skilled person will also appreciate that although the cells of the present technology may be characterized by the defined phenotypic profiles, these cells will express a range of other intracellular and / or cell surface markers which may be not relevant in terms of phenotypically characterizing and isolating the cellular population of interest. Still further, to the extent that a given cell population may comprise a range of subpopulations, these subpopulations may exhibit variations in the expression of intracellular or cell surface markers other than those of the profiles defined herein.

[0116] Standard methods known in the art may be used to determine the detectable expression, low expression or lack thereof of the various markers discussed herein. Suitable methods include, but may be not limited to, immunocytochemistry, immunoassays, flow cytometry, such as fluorescence activated cells sorting (FACS), and polymerase chain reaction (PCR), such as reverse transcription PCR (RT-PCR). Suitable immunoassays include, but may be not limited to, Western blotting, enzyme- linked immunoassays (ELISA), enzyme-linked immunosorbent spot assays (ELISPOT assays), enzyme multiplied immunoassay techniques, radioallergosorbent (RAST) tests, radioimmunoassays, radiobinding assays and immunofluorescence. Western blotting, ELISAs and RT-PCR may be all quantitative and so may be used to measure the level of expression of the various markers if present. The use of FACS is disclosedin the Examples. Antibodies and fluorescently-labelled antibodies for all of the various markers discussed herein may be commercially-available.

[0117] Reference to “enriching” should be understood as a reference to increasing the ratio of cells expressing the desired phenotype relative to the cells not expressing the desired phenotype in the starting sample. This is achieved by removing or otherwise reducing the number of cells that do not express the desired phenotype. It should be understood that reference to “enrichment” is not limited to an enrichment step that removes all the cells not expressing the desired phenotype from the sample / cell population. Rather, it is a reference to decreasing the concentration of these suitably undesired cells in the sample / cell population. The decrease in concentration may therefore be of varying degrees. The methods of the present technology should be understood to extend to conducting one or more repeated sequential enrichment steps in order to improve the purity of the desired subpopulation (such as by performing two or more sequential enrichment steps). The decision as to whether one or more enrichment steps may be required to be performed at any given stage may be made by a person skilled in the art on a case-by-case basis. When target endothelial progenitor cell numbers may be relatively high (such as in a placenta sample), a single enrichment step may be sufficient to enrich for the desired subpopulation. However, where a sample such as blood is used (with very low numbers of endothelial progenitor cells), it may be desirable to perform two or more of each enrichment steps in order to maximize the purity of the desired cell population.

[0118] In any method of the present technology, the term “enriching for cells which expresses the phenotype” may also be referred to as “selecting for cells which expresses the phenotype” or “isolating cells which expresses the phenotype.” These terms may be used interchangeably. A skilled person in this particular field will readily appreciate the meaning of these terms and how to implement them.

[0119] Reference to these terms should be understood as achieving a highly enriched population of cells. Although it is desirable that the isolated cell population is pure, this may not be 100% achievable since in any biological system cellular contamination may occur. Accordingly, there may still be a small proportion of contaminating cells. However, it has been determined by the present inventors that the level of contamination that may exist is so low that it is not considered significant.

[0120] “Enriching,” “selecting,” or “isolating” may be achieved by any suitable method known to the skilled person, such as by FACS sorting.

[0121] As used herein, the term “gene” refers to any and all discrete coding regions of a genome, as well as associated non-coding and regulatory regions. The gene is also intended to mean an open reading frame encoding one or more specific polypeptides, and optionally comprising one or more introns, and adjacent 5' and 3' noncoding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control signals such as promoters, enhancers, termination and / or polyadenylation signals that may be naturally associated with a given gene, or heterologous control signals. Accordingly, the term “gene” includes and encompasses a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, siRNA, shRNA, miRNA, and the like. Genes may or may not be capable of being used to produce a functional protein. Genes may include both coding and noncoding regions.

[0122] The term “heterologous gene” is used herein to refer to genetic material that has been or is about to be artificially introduced into a genome of a host cell (e.g., an EPC or MSC in a population of the present technology) and that is transmitted to the progeny of that host cell. The heterologous gene will typically comprise a polynucleotide that is capable of being transcribed into RNA and optionally, translated and / or expressed under appropriate conditions. In some embodiments, it confers a desired property to the recombinant host cell into which it is introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In some embodiments, it is transcribed into a molecule that interferes with transcription or translation (e.g., antisense molecule) or mediates RNA interference (e.g., siRNA or shRNA).

[0123] As used herein, the term “effective amount,” which may also be used interchangeably with “therapeutically effective amount,” refers to an amount or concentration of any agent referred to herein, such as a cell, a cell composition, an isolated cell population, a pharmaceutical composition, which is effective in reducing, eliminating, treating, preventing or controlling the symptoms of a condition, disorder, or disease affecting a mammal. The term controlling is intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the condition, disorder or disease affecting the mammal. However, controlling doesnot necessarily indicate a total elimination of all condition, disorder, or disease symptoms, and is intended to include prophylactic treatment.

[0124] As used herein the term “endothelial cell mitogen” means any protein, polypeptide, mutein, or portion that is capable of, directly or indirectly, inducing endothelial cell growth. Such proteins include, for example, acidic and basic fibroblast growth factors (aFGF and bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factor a and [3 (TGF-a and TGF-[3), platelet- derived endothelial growth factor (PD-EGF), platelet-derived growth factor (PDGF), tumor necrosis factor a (TNF-a), hepatocyte growth factor (HGF), insulin like growth factor (IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF (M- CSF), granulocyte / macrophage CSF (GM-CSF), and nitric oxide synthase (NOS). See, (1 ) Klagsbrun M and D'Amore PA. Regulators of angiogenesis. Annu Rev Physiol. 1991 ;53:217-39.; (2) Folkman J and Shing Y. Angiogenesis. J Biol Chem. 1992 Jun 5;267(16):10931 -4; and (3) Symes JF and Sniderman AD. Angiogenesis: potential therapy for ischaemic disease. Curr Opin Lipidol. 1994 Aug;5(4):305-12, each incorporated herein by reference in their entireties. Muteins or fragments of a mitogen may be used as long as they induce or promote EC cell growth.Endothelial Progenitor Cells (EPCs)

[0125] The present technology comprises compositions, including pharmaceutical compositions and cell compositions, having isolated EPC populations. EPCs may comprise a population of cells that circulate in the blood and may have ability to differentiate into endothelial cells. Non-isolated EPCs may be rare or occurring at low quantities in blood.

[0126] The isolated EPC populations of the present technology may comprise one or more isolated EPC cells. In some embodiments, the isolated EPC population comprises 2 or more isolated EPC cells.Isolated EPC Population Purity

[0127] In some embodiments, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the cells in the isolated EPC population are isolated EPCs.

[0128] In some embodiments, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the cells in the isolated EPC population are isolated EPCs.

[0129] In some embodiments, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated EPC population are isolated EPCs.Isolated EPC Population Markers

[0130] The isolated EPC populations may be a mixed population of isolated EPCs which may express a PROCR+ phenotype or a PROCR- phenotype, and either of these cell types may separately express a PDGFRA+ phenotype or a PDGFRA- phenotype. In other words, the mixed population of PROCR+ / - PDGFRA+ / - EPCs express any of the following four phenotypes having regard to PROCR and PDGFRA: PROCR+ PDGFRA+, PROCR+ PDGFRA-, PROCR- PDGFRA+, or PROCR- PDGFRA-.

[0131] The isolated EPC populations may comprise or consist of PROCR+PDGFRA+endothelial progenitor cells (PROCR+PDGFRA+EPCs). In some embodiments, the EPC populations comprise or consist of PROCR+ / _PDGFRA+ / _endothelial progenitor cells (PROCR+ / _PDGFRA+ / _EPCs). In some embodiments, the isolated EPC populations comprise a CD45- / CD34+ phenotype.

[0132] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated EPC population are PROCR+.

[0133] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated EPC population are PROCR+.

[0134] In some embodiments, 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 95%, at least about 98%,at least about 99%, or at least about 100% of the cells in the isolated EPC population are PROCR+.

[0135] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated EPC population are PDGFRA+.

[0136] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated EPC population are PDGFRA+.

[0137] In some embodiments, 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 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated EPC population are PDGFRA+.

[0138] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated EPC population are PROCR+ PDGFRA+.

[0139] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated EPC population are PROCR+ PDGFRA+.

[0140] In some embodiments, 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 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated EPC population are PROCR+ PDGFRA+.

[0141] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated EPC population are PROCR-.

[0142] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated EPC population are PROCR-.

[0143] In some embodiments, 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 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated EPC population are PROCR-.

[0144] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated EPC population are PDGFRA-.

[0145] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated EPC population are PDGFRA-.

[0146] In some embodiments, 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 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated EPC population are PDGFRA-.

[0147] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated EPC population are PROCR- PDGFRA-.

[0148] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated EPC population are PROCR- PDGFRA-.

[0149] In some embodiments, 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 about70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated EPC population are PROCR- PDGFRA-.

[0150] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated EPC population are PROCR+ PDGFRA-.

[0151] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated EPC population are PROCR+ PDGFRA-.

[0152] In some embodiments, 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 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated EPC population are PROCR+ PDGFRA-.

[0153] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated EPC population are PROCR- PDGFRA+.

[0154] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated EPC population are PROCR- PDGFRA+.

[0155] In some embodiments, 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 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated EPC population are PROCR- PDGFRA+.

[0156] The isolated EPC populations may express the phenotype VE-cadherin (CDH5)+, CD34+, PROCR+, CD31 lo, VEGFR2lo, lineage (lin)-, CD45- and PDGFRA-.In some embodiments, the isolated EPC popualtions express the phenotype VE- cadherin (CDH5)+, CD34+, PROCR+ / -, CD31 lo, VEGFR2lo, lineage (lin)-, CD45- and PDGFRA+ / -.Differences Relative to Non-lsolated EPC Populations

[0157] The isolated EPC population may comprise protein expression changes, relative to a non-isolated EPC population. For example, the isolated PROCR+ PDGFRA+ EPC population may comprise an increase in a PROCR (National Center for Biotechnoloy Information (NCBI) Accessions: XP_047295786.1 ; XP_011526798.2; XP_047295787.1 ), a PDGFRA (NCBI Accessions: AAH63414.1 , AAH15186.1 ), a VE- Cadherin (NCBI Accession: CAA56306), a VWF (NCBI Accession: P04275.4), or a Factor VIII (NCBI Accession: AAA52484.1 ) protein expression level relative to an expression level in a non-isolated EPC population.

[0158] The isolated EPC population may comprise gene expression changes, relative to a non-isolated EPC population. For example, the isolated PROCR+ PDGFRA+ EPC population may comprise an increase in a PROCR (NCBI Gene ID: 10544), a PDGFRA (NCBI Gene ID: 5156), a VE-Cadherin (NCBI Gene ID: 1003), a VWF (NCBI Gene ID: 7450), or a Factor VIII (NCBI Gene ID: 2157) gene expression level relative to an expression level in a non-isolated EPC population.

[0159] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a PROCR protein expression level or a gene expression level by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-isolated EPC population.

[0160] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a PROCR protein expression level or a gene expression level by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0161] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a PROCR protein expression level or a gene expression level by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0162] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a PDGFRA protein expression level or a gene expression level by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-isolated EPC population.

[0163] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a PDGFRA protein expression level or a gene expression level by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0164] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a PDGFRA protein expression level or a gene expression level by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0165] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a VE-Cadherin protein expression level or a gene expression level by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-isolated EPC population.

[0166] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a VE-Cadherin protein expression level or a gene expression level by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0167] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a VE-Cadherin protein expression level or a gene expression level by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0168] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a VWF protein expression level or a gene expression level by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0169] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a Factor VIII protein expression level or a gene expression level by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0170] The isolated EPC populations of the present technology may further comprise cellular traits that are different from non-isolated EPC populations. Nonlimiting examples include an increased prolierative capacity, an increased angioenic capacity, an increased colony forming capacity, an increased tube forming capacity, an increased engraftment potential and / or capacity, an increased cell elongation, an increase in collagen production, or an increase in a WPB level relative to the non-isolated EPC populations.

[0171] In some embodiments, the isolated EPC population comprises an increase in a prolierative capacity by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a nonisolated EPC population.

[0172] In some embodiments, the isolated EPC population comprises an increase in a prolierative capacity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0173] In some embodiments, the isolated EPC population comprises an increase in a prolierative capacity by at least about 5%, 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 about150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0174] In some embodiments, the isolated EPC population comprises an increase in a angiogenic capacity by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a nonisolated EPC population.

[0175] In some embodiments, the isolated EPC population comprises an increase in a angiogenic capacity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0176] In some embodiments, the isolated EPC population comprises an increase in a angiogenic capacity by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0177] In some embodiments, the isolated EPC population comprises an increase in a colony forming capacity by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a nonisolated EPC population.

[0178] In some embodiments, the isolated EPC population comprises an increase in a colony forming capacity by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0179] In some embodiments, the isolated EPC population comprises an increase in a colony forming capacity by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0180] In some embodiments, the isolated EPC population comprises an increase in a Weibel-Palade body (WPB) level. The increase in the WPB level may comprise an increase in WPB number or a WPB area, relative to the WPB level in a non-isolated EPC population.

[0181] In some embodiments, the increase in the WPB level is an increase by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-isolated EPC population.

[0182] In some embodiments, the increase in the WPB level is an increase by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0183] In some embodiments, the increase in the WPB level is an increase by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0184] The colony forming capacity may be measured using an in vitro colony formation assay. In some embodiments, the isolated EPC population is capable of producing colonies which stain positive for isolectin (Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I)) in an in vitro colony formation assay.

[0185] In some embodiments, the isolated EPC population is capable of producing an increased number of colonies which stain positive for isolectin (Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I)) in an in vitro colony formation assay compared to the number of colonies produced by a non-isolated EPC population when tested in the same assay.

[0186] In some embodiments, the isolated EPC population is capable of producing an increase of about 39x in the mean number of colonies which stain positive for isolectin (Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I)) in an in vitro colony formation assay compared to the number of colonies produced by a non-isolated EPC population tested in the same assay. In some embodiments, the increase is an increase of about 39.27x.

[0187] In some embodiments, the isolated EPC population is capable of producing an increase of at least 39x in the mean number of colonies which stain positive for isolectin (Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I)) in an in vitro colony formation assay compared to the number of colonies produced by a non-isolated EPC population tested in the same assay. In some embodiments, the increase is an increase of at least 39.27x.

[0188] In some embodiments, the isolated EPC population is capable of producing an increase of at least about 39x in the mean number of colonies which stain positive for isolectin (Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I)) in an in vitro colony formation assay compared to the number of colonies produced by a non-isolated EPC population tested in the same assay. In some embodiments, the increase is an increase of at least about 39.27x.

[0189] In some embodiments, the isolated EPC population comprises an increase in a tube forming capacity by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a nonisolated EPC population.

[0190] In some embodiments, the isolated EPC population comprises an increase in a tube forming capacity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0191] In some embodiments, the isolated EPC population comprises an increase in a tube forming capacity by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0192] In some embodiments, the isolated EPC population comprises an increase in an engraftment potential by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a nonisolated EPC population.

[0193] In some embodiments, the isolated EPC population comprises an increase in an engraftment potential by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 60a0%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0194] In some embodiments, the isolated EPC population comprises an increase in an engraftment potential by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0195] In some embodiments, the isolated EPC population comprises one or more EPCs that are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 60%, about 65%, about 70%, or about 75% more elongated, relative to one or more EPCs in a non-isolated EPC population.

[0196] In some embodiments, the isolated EPC population comprises one or more EPCs that are at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 60%, at least 65%, at least 70%, or at least 75% more elongated, relative to one or more EPCs in a non-isolated EPC population.

[0197] In some embodiments, the isolated EPC population comprises one or more EPCs that are at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% more elongated, relative to one or more EPCs in a non-isolated EPC population.

[0198] In some embodiments, the isolated EPC population comprises an increase in an engraftment capacity by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a nonisolated EPC population.

[0199] In some embodiments, the isolated EPC population comprises an increase in an engraftment capacity by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0200] In some embodiments, the isolated EPC population comprises an increase in an engraftment capacity by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0201] In some embodiments, the isolated EPC population comprises an increase in a collagen production level by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a nonisolated EPC population.

[0202] In some embodiments, the isolated EPC population comprises an increase in a collagen production level by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0203] In some embodiments, the isolated EPC population comprises an increase in a collagen production level by at least about 5%, 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 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0204] In some embodiments, the collagen production level and / or the engraftment capacity of the isolated EPC population may be assessed using an in vivo assay, including an an in vivo collagen plug engraftment assay. In some embodiments, the isolated EPC populations are capable of producing an increase of least 7x or at least 7.8x in the average GFP positive area per collagen plug in an in vivo collagen plug engraftment assay, relative to the non-isolated EPC population.

[0205] In some embodiments, the isolated EPC population is capable of coexpressing CD34 and isolectin in an engrafted cell. The engrafted cell may be a cell in an in vivo engraftment colocalization assay. The expression of CD34 and / or isolectin may be increased relative to the non-isolated EPC population.

[0206] In some embodiments, the isolated EPC population do not express one or more hematopoietic proteins. Nonlimiting examples of hematopoietic proteins include CD3 epsilon chain (CD3e) (NCBI Accession: NP_000724), Integrin Alpha M (CD11 b) (NCBi Accessions: AAB24821.1 ; XP_054236247.1 ; XP_054236246.1 ;XP_054236245.1 ; XP_054236244.1 ; XP_016878705.1 ; 1 BHO_1 ; 1 BHO_2), Protein tyrosine phosphatase, receptor type C (CD45) (NCBI Accessions: AAS46922.1 ; AAS46930.1 ; AAS46938.1 ; AAS46946.1 ; AAS46954.1 ; AAS46962.1 ; P08575.3), and B220 (NCBI Accesions: XP_054193920.1 ; XP_054193921 .1 ; XP_054193922.1 ; XP_054193924.1 ; XP_054193926.1 ; XP_054193928.1 ).Differentiation

[0207] The isolated EPC populations of the present technology may be differentiated. During or after differentiation, the isolated EPC populations may comprise a decrease in a gene expression level or a protein expression level, relative to undifferentiated EPC populations. The undifferentiated EPC population may comprise an undifferentiated non-isolated EPC population or an undifferentiated isolated EPC population. Nonlimiting examples of such proteins or genes include Bone marrow stromal antigen 1 (CD157) (NCBI Accessions: 1 ISM_A; 1 ISM_B; NP_004325.2; XP_054206734.1 ; XP_054206733.1 ; XP_054206732.1 ;XP_054206728.1 ; XP_054206729.1 ; XP_054206730.1 ; XP_054206731 .1 ; NCBI Gene ID: 683), ATP-binding cassette sub-family G member 2 (ABCG2) (NCBI Accesions: NP_004818.2; NP_001244315.1 ; NCBI Gene ID: 9429), and SRY-BOX Transcription Factor 18 (SOX18) (NCBI Accession: NP_060889; NCBI Gene ID: 54345).

[0208] In some embodiments, the reduction in the CD157 protein expression level or the CD157 gene expression level is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to a CD157 protein expression level or a CD157 gene expression level in the undifferentiated EPC population.

[0209] In some embodiments, the reduction in the CD157 protein expression level or the CD157 gene expression level is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, relative to a CD157 protein expression level or a CD157 gene expression level in the undifferentiated EPC population.

[0210] In some embodiments, the reduction in the CD157 protein expression level or the CD157 gene expression level is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, relative to a CD157 protein expression level or a CD157 gene expression level in the undifferentiated EPC population.

[0211] In some embodiments, the reduction in the ABCG2 protein expression level or the ABCG2 gene expression level is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to a ABCG2 protein expression level or a ABCG2 gene expression level in the undifferentiated EPC population.

[0212] In some embodiments, the reduction in the ABCG2 protein expression level or the ABCG2 gene expression level is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, relative to a ABCG2 proteinexpression level or a ABCG2 gene expression level in the undifferentiated EPC population.

[0213] In some embodiments, the reduction in the ABCG2 protein expression level or the ABCG2 gene expression level is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, relative to a ABCG2 protein expression level or a ABCG2 gene expression level in the undifferentiated EPC population.

[0214] In some embodiments, the reduction in the SOX18 protein expression level or the SOX18 gene expression level is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to a SOX18 protein expression level or a SOX18 gene expression level in the undifferentiated EPC population.

[0215] In some embodiments, the reduction in the SOX18 protein expression level or the SOX18 gene expression level is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, relative to a SOX18 protein expression level or a SOX18 gene expression level in the undifferentiated EPC population.

[0216] In some embodiments, the reduction in the SOX18 protein expression level or the SOX18 gene expression level is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, relative to a SOX18 protein expression level or a SOX18 gene expression level in the undifferentiated EPC population.Engineering Isolated EPC populations

[0217] The isolated EPC populations of the present technology may be engineered to increase a Von Willebrand Factor (VWF) gene expression level (National Center for Biotechnology Information (NCBI) Gene ID: 7450) or a VWF protein level (NCBI Accessions: AAB59458.1 ; CCQ25771.1 ; KAI4064161 .1 ; 1403304A; 1 FE8_A; 1 FE8_B; 1 FE8_C), relative to a non-isolated EPC population. Nonlimiting examples of methods which may be employed to increase the VWF gene expression level or the VWF protein epression include plasmid trasfections, transposon systems, electroporation, microinjection, nanoparticle mediated delivery, and gene editing techniques. In some embodiments, the isolated EPC populations are engineered to comprise a VWF overexpression construct. In some embodiments, the isolated EPC populations are engineered to comprise a genetically edited VWF locus, relative to a control (e.g., a non-isolated EPC population or the isolated EPC population at baseline).Methods of Generating Isolated EPC populations

[0218] The isolated EPC populations of the present technology may be generated by various methods. In some embodiments, the isolated EPC populations are generated by the steps of:(i) obtaining a biological sample from a subject;(ii) enriching for cells in the biological sample which comprise a CD45- phenotype, thereby obtaining a population CD45- cells;(iii) selecting from the population CD45- cells, cells which comprise a CD34+ phenotype, thereby obtaining a population of CD45- / CD34+ cells; and(iv) selecting from the population of CD45- / CD34+ cells, cells which express a PROCR+ and PDGFRA+ phenotype, thereby obtaining an isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

[0219] In some embodiments, the isolated EPC populations are generated by the steps of:(i) obtaining a biological sample from a subject;(ii) enriching for cells in the biological sample which comprise a CD45- phenotype, thereby obtaining a population CD45- cells;(iii) selecting from the population CD45- cells, cells which comprise a CD34+ phenotype, thereby obtaining a population of CD45- / CD34+ cells; and(iv) selecting from the population of CD45- / CD34+ cells, cells which express a PROCR+ / - and PDGFRA+ / - phenotype, thereby obtaining an isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

[0220] In some embodiments, step (ii) comprises selecting cells that express a CD45+ phenotype, removing the cells that express the CD45+ phenotype from the biological sample of step (i) and discarding the cells that expresses the CD45+ phenotype, thereby obtaining a population of CD45- cells.

[0221] In some embodiments, the step of selecting the cells that express the CD45+ phenotype comprises contacting a cell or cells that express a CD45 surface protein with a CD45-binding molecule to form a complex, and removing the complex from the biological sample in step (i).

[0222] In some embodiments, step (iii) comprises contacting a cell or cells that express CD34 surface protein with a CD34-binding molecule to form a complex, removing the complex from the population of CD45- cells of step (ii), and retaining the complex, thereby obtaining the second population of cells which are CD45- / CD34+.

[0223] In some embodiments, step (iv) comprises contacting a cell or cells that express a PROCR surface protein with a PROCR-binding molecule to form a complex, removing the complex from the population of cells and retaining the complex.

[0224] In some embodiments, the isolated EPC population is generated by the steps of:(i) obtaining a biological sample from a subject;(ii) enriching for cells in the biological sample which comprise a PDGFRA+ phenotype, thereby obtaining a population of PDGFRA+ cells;(iii) selecting from the population of PDGFRA+ cells, cells which express a PROCR+ phenotype, thereby obtaining an isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

[0225] In some embodiments, the isolated EPC population is generated by the steps of:(i) obtaining a biological sample from a subject;(ii) enriching for cells in the biological sample which comprise a PDGFRA+ / - phenotype, thereby obtaining a population of PDGFRA+ / - cells;(iii) selecting from the population of PDGFRA+ / - cells, cells which express a PROCR+ / - phenotype, thereby obtaining the isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

[0226] In some embodiments, step (iii) comprises contacting a cell or cells that express a PROCR surface protein with a PROCR-binding molecule to form a complex, removing the complex from the population of cells and retaining the complex.

[0227] In some embodiments, step (ii) comprises contacting a cell or cells that express a PDGFRA surface protein with a PDGFRA -binding molecule to form a complex, removing the complex from the population of cells and retaining the complex.

[0228] In some embodiments, step (iii) comprises contacting a cell or cells that express a PROCR surface protein with a PROCR-binding molecule to form a complex, removing the complex from the population of cells and retaining the complex.

[0229] In some embodiments, the isolated EPC population is generated by the steps of:(i) obtaining a biological sample from a subject;(ii) enriching for cells in the biological sample which comprise a PROCR+ phenotype, thereby obtaining a population of PROCR+ cells;(iii) selecting from the population of PROCR+ cells, cells which express a PDGFRA+ phenotype, thereby obtaining the isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

[0230] In some embodiments, the isolated EPC population is generated by the steps of:(i) obtaining a biological sample from a subject;(ii) enriching for cells in the biological sample which comprise a PROCR+ / - phenotype, thereby obtaining a population of PROCR+ / - cells;(iii) selecting from the population of PROCR+ / - cells, cells which express a PDGFRA+ / - phenotype, thereby obtaining the isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

[0231] In some embodiments, step (ii) comprises contacting one or more cells that express a PROCR surface protein with a PROCR-binding molecule to form a complex, removing the complex from the population of cells and retaining the complex.

[0232] In some embodiments, step (iii) comprises contacting one or more cells that express a PDGFRA surface protein with a PDGFRA -binding molecule to form a complex, removing the complex from the population of cells and retaining the complex.

[0233] The binding molecule in any of the methods of generating isolated EPC populations comprises a protein. The protein may be an antibody.

[0234] Removing the complex in any of the methods of generating isolated EPC populations may comprise microfluidic sorting. The microfluidic sorting may comprise microbead sorting or flow cytometry. In some embodiments, the flow cytometry comprises fluorescence-activated cell sorting.

[0235] In some embodiments, the method of generating isolated EPC populations further comprise step (iv), culturing or contacting the isolated EPC population with a cell population comprising endothelial colony forming cells (ECFCs) or a cell population comprising MSCs and / or step (v), separating the isolated EPC population from the cell population comprising ECFCs or the cell population comprising MSCs. Step (v) may occur at least about 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after step (iv).

[0236] In some embodiments, the isolated EPC populations and / or the isolated MSC populations are generated by one or more steps disclosed by PCT publication no. WO 2014 / 138793, the contents of which are incorporated herein by reference in its entirety.Additional Methods for isolating EPC populations and MSC populations

[0237] Flow cytometry with CD34, VEGFR2 (KDR / FLK-1 ) and / or CD133 is conventionally used to identify the number of circulating endothelial progenitor cells, in addition to more classical endothelial markers such as VE-cadherin or CD31. Analternate approach to isolate endothelial progenitor cells involves partially differentiated endothelial progenitor cells after short term culture on fibronectin, resulting in spindle shaped cells able to digest acetylated low-density lipoprotein and stain for several specific lectins appearing within 3 days. Both methods however result in considerable contamination by hematopoietic cells.

[0238] To isolate cell populations in accordance with the methods of the present technology, various well-known techniques may be performed. Antibodies and other CD45, CD34, PROCR, and / or PDGFRA specific cell surface binding molecules may be particularly useful. For example, antibodies may be attached to a solid support to allow for separation.

[0239] Flow cytometry may be used in combination with any of the methods of the present technology. For example, through flow cytometry only CD34+ cells may be gated, to remove any contaminating CD45+ cells from the initial CD45- population.

[0240] Other techniques providing particularly accurate separation include fluorescence activated cell sorting. Fluorescence activated cell sorting (FACS) is a specialized form of flow cytometry based upon the specific light scattering and fluorescent characteristics of each cell. FACS is also applicable to the separation of cells based on morphological characteristics which may be discernible by forward vs side light scatter.

[0241] In another example, but specifically in the context of the CD45 negative selection step, rather than physically separating the CD45- cellular subpopulation from the CD45+ cellular population, one may utilize a method which labels the CD45+ cells and then delivers a targeted lysis signal which lyses the labelled CD45+ cells, such as a cytolytic, apoptotic or toxic signal. In another example, opsonization with an antibody followed by complement administration may achieve the same outcome.

[0242] Additional negative selection techniques include, but may be not limited to, the site-directed administration of a cytolytic, apoptotic or otherwise toxic agent. This may be most conveniently achieved via the coupling of such an agent to a monoclonal antibody in order to facilitate its directed delivery. In another example, opsonization with an antibody followed by complement administration may achieve the same outcome.

[0243] Procedures for separation may include magnetic separation, using antibody magnetic beads, affinity chromatography, “panning” with antibody attached to a solid matrix or any other convenient technique such as Laser Capture Microdissection. For example, CD45 antibodies labelled with magnetic beads may be used in combination with a magnetic column to provide a CD45- enriched population.Mesenchymal Stem Cells (MSCs)

[0244] The present technology comprises compositions, including pharmaceutical compositions and cell compositions, comprising an isolated mesenchymal stem cell (MSC) population. The isolated MSC populations of the present technology may comprise one or more isolated MSC cells. In some embodiments, the isolated MSC population comprises 2 or more isolated MSC cells.Isolated MSC Population Purity

[0245] In some embodiments, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the cells in the isolated MSC population are isolated MSCs.

[0246] In some embodiments, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the cells in the isolated MSC population are isolated MSCs.

[0247] In some embodiments, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated MSC population are isolated MSCs.Isolated MSC Population Markers

[0248] In some embodiments, the isolated MSC population expresses the phenotype CD105+, CD73+, CD90+, CD45-, CD34-, CD14- or CD11 b-, CD79a- or CD19-, and HLA-DR-.

[0249] The isolated MSC populations may be a mixed population of isolated MSCs which may express a PROCR+ phenotype or a PROCR- phenotype, and either of these cell types may separately express a PDGFRA+ phenotype or a PDGFRA- phenotype. In other words, the mixed population of PROCR+ / - PDGFRA+ / - EPCs express any ofthe following four phenotypes having regard to PROCR and PDGFRA: PROCR+ PDGFRA+, PROCR+ PDGFRA-, PROCR- PDGFRA+, or PROCR- PDGFRA-.

[0250] The isolated MSC populations may comprise or consist of PROCR+PDGFRA+mesenchymal stem cells (PROCR+PDGFRA+MSCs). In some embodiments, the MSC populations comprise or consist of PROCR+ / _PDGFRA+ / _mesenchymal stem cells (PROCR+ / _PDGFRA+ / _MSCs). In some embodiments, the isolated EPC populations comprise a CD45- / CD34+ phenotype.

[0251] In some embodiments, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated MSC population are CD45- / CD34+ cells.

[0252] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated MSC population are CD45- / CD34+ cells.

[0253] In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated MSC population are CD45- / CD34+ cells.

[0254] The isolated MSC population may be coformulated in a composition with the isolated EPC populations of the present technology or may be present in a composition that does not comprise an isolated MSC population.

[0255] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated MSC population are PROCR+.

[0256] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated MSC population are PROCR+.

[0257] In some embodiments, 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 95%, at least about 98%,at least about 99%, or at least about 100% of the cells in the isolated MSC population are PROCR+.

[0258] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated MSC population are PDGFRA+.

[0259] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated MSC population are PDGFRA+.

[0260] In some embodiments, 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 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated MSC population are PDGFRA+.

[0261] In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% of the cells in the isolated MSC population are PROCR+ PDGFRA+.

[0262] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the cells in the isolated MSC population are PROCR+ PDGFRA+.

[0263] In some embodiments, 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 95%, at least about 98%, at least about 99%, or at least about 100% of the cells in the isolated MSC population are PROCR+ PDGFRA+.Additional Isolated MSC Population Features

[0264] The isolated MSC populations of thre present technology may be capable of differentiating into osteoblasts, adipocytes, and chondroblasts in vitro or in vivo.-M-Sources of Isolated EPC Populations and Isolated MSC Populations

[0265] The isolated MSC populations may be isolated from a biological sample. In some embodiments, the biological is a mammalian biological sample. The mammalian biological sample may be a human biological sample.Mammalian Biological Samples

[0266] In some embodiments, mammalian biological sample is selected from the group consisting of a mammalian placenta, mammalian cord blood, mammalian peripheral blood, and mammalian tissue-resident vascular endothelium. The mammalian placenta may comprise a whole mammalian placenta. The mammalian tissue-resident vascular endothelium may be selected from the group consisting of a mammalian umbilical cord, a mammalian pulmonary artery endothelium, a mammalian aorta, and a mammalian lung tissue.

[0267] The mammalian biological sample may be provided directly or may require some form of prior treatment. For example, a biopsy or surgical sample may require homogenization or other form of cellular dispersion. Further, to the extent that the biological sample is not in liquid form, it may require the addition of a reagent, such as a buffer, to mobilize the sample and create a cell suspension. Alternatively, it may require some other form of pretreatment such a heparinization, where the sample is a whole blood sample, in order to prevent clotting. A skilled person will readily appreciate the steps required to provide a suitable and appropriate mammalian biological sample according to the required circumstances.

[0268] The mammalian biological sample may be in the form of a single cell suspension or a cell aggregate which has been freshly isolated from an individual (such as an individual who may be the subject of treatment) or it may have been sourced from a non-fresh source, such as from a culture (for example, where cell numbers were expanded) or a frozen stock of cells which had been isolated at some earlier time point either from an individual or from another source. It should also be understood that the initial mammalian biological sample provided may have undergone some other form of treatment or manipulation, such as but not limited to enrichment or purification.

[0269] The initial mammalian biological sample may be obtained from mammalian whole placenta. Whole placenta should be understood as a reference to some or all of the heterogeneous population of cells that make up the placenta. In humans, the placenta averages 22 cm in length and 2-2.5 cm in thickness, with the center being the thickest and the edges being the thinnest. It typically weighs approximately 500 grams. It exhibits a dark reddish-blue or crimson color and connects to the fetus by an umbilical cord of approximately 55-60 cm in length. The umbilical cord contains two umbilical arteries and one umbilical vein. The umbilical cord inserts into the chorionic plate. Vessels branch out over the surface of the placenta and further divide to form a network covered by a thin layer of cells. This results in the formation of villous tree structures. On the maternal side, these villous tree structures may be grouped into lobules called cotyledons. In humans, the placenta usually has a disc shape, but size varies vastly between different mammalian species. The placenta begins to develop upon implantation of the blastocyst into the maternal endometrium. The outer layer of the blastocyst becomes the trophoblast, which forms the outer layer of the placenta. This outer layer is divided into two further layers: the underlying cytotrophoblast layer and the overlying syncytiotfophoblast layer. The syncytiotrophoblast is a multinucleated continuous cell layer that covers the surface of the placenta. It forms as a result of differentiation and fusion of the underlying cytotrophoblast cells, a process that continues throughout placental development. The syncytiotrophoblast (otherwise known as syncytium) thereby contributes to the barrier function of the placenta. The placenta grows throughout pregnancy. Development of the maternal blood supply to the placenta is complete by the end of the first trimester of pregnancy (approximately 12-13 weeks).

[0270] The initial mammalian biological sample may be obtained from the cellular population of the cotyledons. A post-parturition placenta may used, such as an intact placenta, e.g., following a caesarean section. The decidual component may be dissected away in order to isolate the placental cotyledons. These cotyledons may then be digested in a cocktail of enzymes, such as collagenase, dispase and DNAse, and thereafter filtered in order to obtain the initial mammalian biological sample.

[0271] In order to obtain the initial mammalian biological sample, one may use placenta at any stage of development. Although post-parturition placenta is most conveniently obtained, placentas from earlier stages of pregnancy may also be used,such as where a miscarriage or other termination of pregnancy occurs. Placenta in particular and umbilical cord blood provide a good source of endothelial progenitor cells and mesenchymal stem cells. This provides the possibility of women routinely isolating and storing either placental / umbilical cord tissue or blood (for example) for future endothelial progenitor cell harvesting or else freshly harvesting and then freezing endothelial progenitor cells for future use. This therefore provides the possibility of either autologous endothelial progenitor cell treatment or, for individuals related to the donor, more closely MHC-matched endothelial progenitor cells than might otherwise be accessible. In both of these cases the donor endothelial progenitor cells may be defined as being histocompatible with respect to the recipient of those cells.Donors

[0272] The isolated EPC populations and / or the isolated MSC populations may be derived from a donor. In some embodiments, the isolated EPC populations and / or the isolated MSC populations are derived from one or more donors. In some embodiments, the isolated EPC populations and / or the isolated MSC populations are derived from two or more donors.

[0273] In some embodiments, the isolated EPC populations and / or the isolated MSC populations comprise allogenic cells. The allogenic cells may be derived from a subject that is immunologically compatible with the subject into which the cells will be administered.

[0274] The isolated EPC populations and / or the isolated MSC populations may be present in a medium, including but not limited to, a liquid medium or a frozen medium.Culture and Maintenance of Isolated EPC Populations and Isolated MSC Populations

[0275] The isolated EPC populations and / or isolated MSC populations of the present technology may be cultured or maintained according to any means routine in the art. Means may be provided for routinely and reliably producing isolated populations of the relevant cells in vitro on either a small scale or a larger scale. Methods for culturing EPCs and MSCs have been previously established (see Chand, K.K et al. (2021 ) npj Regen Med 6(75), pp1 -15), inorportated herein by reference in its entirety.

[0276] The methods of the present technology may be particularly suitable for producing populations of cells for a given individual and in the context of a specific condition. In terms of large-scale production, one means of achieving such production is via the use of a bioreactor.

[0277] Bioreactors may be designed to provide a culture process that may deliver medium and oxygenation at controlled concentrations and rates that mimic nutrient concentrations and rates in vivo. Bioreactors have been available commercially for many years and employ a variety of types of culture technologies. Of the different bioreactors used for mammalian cell culture, most have been designed to allow for the production of high-density cultures of a single cell type. Typical application of these high-density systems is to produce as the end product, a conditioned medium produced by the cells. This is the case, for example, with hybridoma production of monoclonal antibodies and with packaging cell lines for viral vector production. However, these applications differ from applications where the therapeutic end product is the harvested cells themselves, as in the present case.

[0278] Once operational, bioreactors provide automatically regulated medium flow, oxygen delivery, and temperature and pH controls, and they generally allow for production of large numbers of cells. Bioreactors thus provide economies of labour and minimization of the potential for mid-process contamination, and the most sophisticated bioreactors allow for set-up, growth, selection, and harvest procedures that involve minimal manual labour requirements and open processing steps. Such bioreactors optimally may be designed for use with a homogeneous cell mixture or aggregated cell populations. Suitable bioreactors for use include but may be not limited to those described in U.S. Pat. No. 5,763,194, U.S. Pat. Nos. 5,985,653 and 6,238,908, U.S. Pat. No. 5,512,480, U.S. Pat. Nos. 5,459,069; 5,763,266; 5,888,807 and 5,688,687, each of which are incorporated herein by reference in their entireties.

[0279] With any large volume cell culture, several fundamental parameters require tight control. Cultures should be provided with the medium that allows for, where appropriate, stem cell maintenance, endothelial progenitor cell proliferation, endothelial progenitor cell differentiation (perhaps in the context of several separate differentiation cultures and conditions) as well as final cell culture / preservation. Typically, the various media may be delivered to the cells by a pumping mechanism in the bioreactor, feedingand exchanging the medium on a regular basis. The exchange process allows for byproducts to be removed from the culture. Growing cells or tissue also requires a source of oxygen. Different cell types may have different oxygen requirements. Accordingly, a flexible and adjustable means for providing the relevant requirements to the cells is a desired component.

[0280] Depending on the particular culture, even distribution of the cell population and medium supply in the culture chamber may be an important process control. Such control is often achieved by use of a suspension culture design, which may be effective where cell-to-cell interactions may be not important. Examples of suspension culture systems include various tank reactor designs and gas-permeable plastic bags. For cells that do not require assembly into a three-dimensional structure or require proximity to a stromal or feeder layer such suspension designs may be used.

[0281] Efficient collection of the cells at the completion of the culture process is an important feature of an effective cell culture system. One approach for production of cells as a product is to culture the cells in a defined space, without physical barriers to recovery, such that simple elution of the cell product results in a manageable, concentrated volume of cells amenable to final washing in a commercial, closed system cell washer designed for the purpose. Optimally, the system would allow for addition of a pharmaceutically acceptable carrier, with or without preservative, or a cell storage compound, as well as provide efficient harvesting into appropriate sterile packaging. Optimally the harvest and packaging process may be completed without breaking the sterile barrier of the fluid path of the culture chamber.

[0282] When the product cells may be to be transplanted into subjects (often at a time when the subject is ill or immunocompromised), absence of microorganisms is mandated. Once the culture is initiated, the culture chamber and the fluid pathway in the bioreactor systems should be maintained in a sterile, closed environment, to maintain sterility.Coculturing of Isolated EPC Populations and Isolated MSC Populations

[0283] The isolated EPC populations and the isolated MSC populations of the present technology may be cocultured. For example, an isolated EPC population may be added to a culture comprising an isolated MSC population, an isolated MSC population may be added to a culture comprising an isolated EPC population, anisolated EPC population and an isolated MSC population may be added to a culture or otherwise combined at about the same time or simultaneously, or an isolated EPC population and an isolated MSC population may be combined into a coculture. Coculturing these populations may enhance one or more features of the isolated EPC populations and / or the isolated MSC populations of the present technology, relative to an isolated EPC population cultured without an isolated MSC population or to an isolated MSC population cultured without an isolated EPC population. Nonlimiting examples include enhancement of a gene expression level, protein change, and / or physical property that is modified in an isolated EPC and / or MSC population of the present technology, relative to a non-isolated EPC and / or MSC population.

[0284] The isolated EPC population cocultured with the isolated MSC population may comprise or consist of endothelial colony-forming cells (ECFCs). The ECFCs may comprise PROCR+ ECFCs. The percentage of PROCR+ ECFCs in the isolated EPC population may increase during or after coculturing with an isolated MSC population, relative to a percentage of PROCR+ ECFCs in an isolated EPC population that is not cocultured with an isolated MSC population. The PROCR+ ECFCs derived from the coculture may be PDGFRA+ or PDGFRA-. The increase in the percentage of PROCR+ ECFCs may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing. In some embodiments, the vasculogenesis level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a percentage of PROCR+ ECFCs in a non-cocultured isolated EPC population.

[0285] The isolated EPC population cocultured with the isolated MSC population may comprise CD31 + CD90+ ECFCs (CD31 NCBI Accession: AAA60057.1 ; CD90 NCBI Accession: AAH65559.1 . The percentage of CD31 + CD90+ ECFCs in the isolated EPC population may increase during or after coculturing with an isolated MSC population, relative to a percentage of PROCR+ ECFCs in an isolated EPC population that is not cocultured with an isolated MSC population. The CD31 + CD90+ ECFCs derived from the coculture may be PDGFRA+ or PDGFRA-. The increase in thepercentage of CD31 + CD90+ ECFCs may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing. In some embodiments, the vasculogenesis level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a percentage of CD31 + CD90+ ECFCs in a non-cocultured isolated EPC population.

[0286] In some embodiments, the isolated EPC populations and / or the isolated MSC population from the cocultured isolated EPC population and isolated MSC population comprise an increased CD90 gene expression level (NCBI Gene ID: 7070) or a CD90 protein level (NCBI Accession: AAH65559.1 ), relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. The increase in CD90 gene expression or protein levels may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing. In some embodiments, the CD90 gene expression level or the CD90 protein level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. In some embodiments, the cocultured isolated EPC population is an isolated ECFC population.

[0287] In some embodiments, the isolated EPC populations and / or the isolated MSC population from the cocultured isolated EPC population and isolated MSC population comprise an increased DLL4 gene expression level (NCBI Gene ID: 54567) or a DLL4 protein level (NCBI Accession: AAQ89253.1 ), relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. The increase in DLL4 gene expression or protein levels may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks,or 4 weeks after coculturing. In some embodiments, the DLL4 gene expression level or the DLL4 protein level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. In some embodiments, the cocultured isolated EPC population is an isolated ECFC population.

[0288] In some embodiments, the isolated EPC populations and / or the isolated MSC population from the cocultured isolated EPC population and isolated MSC population comprise an increased VWF gene expression level or a VWF protein level relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. The increase in VWF gene expression or protein levels may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing. In some embodiments, the VWF gene expression level or the VWF protein level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. In some embodiments, the cocultured isolated EPC population is an isolated ECFC population.

[0289] In some embodiments, the isolated EPC populations and / or the isolated MSC population from the cocultured isolated EPC population and isolated MSC population comprise an increased SLUG gene expression level (NCBI Gene ID: 6591 ) or a SLUG protein level (NCBI Accession: NP_003059.1 ), relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. The increase in SLUG gene expression or protein levels may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing. In some embodiments, the SLUG gene expression level or the SLUG protein level is increased by at least about 5%, about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. In some embodiments, the cocultured isolated EPC population is an isolated ECFC population.

[0290] In some embodiments, the isolated EPC populations and / or the isolated MSC population from the cocultured isolated EPC population and isolated MSC population comprise an increased CD34 gene expression level (NCBI Gene ID: 947) or a CD34 protein level (NCBI Accession: XP_054195678.1 ), relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. The increase in CD34 gene expression or protein levels may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing. In some embodiments, the CD34 gene expression level or the CD34 protein level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. In some embodiments, the cocultured isolated EPC population is an isolated ECFC population.

[0291] In some embodiments, the isolated EPC populations and / or the isolated MSC population from the cocultured isolated EPC population and isolated MSC population comprise an increased CD31 gene expression level (NCBI Gene ID: 5175) or a CD31 protein level (NCBI Accession(s): XP_054172404.1 ; XP_054172405.1 ; XP_054172407.1 ; XP_054172412.1 ; XP_054172411 .1 ; XP_054172415.1 ;XP_054172416.1 ; XP_054172398.1 ), relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. The increase in CD31 gene expression or protein levels may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing. In some embodiments, the CD31 gene expression level or the CD31protein level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. In some embodiments, the cocultured isolated EPC population is an isolated ECFC population.

[0292] In some embodiments, the isolated EPC populations and / or the isolated MSC population from the cocultured isolated EPC population and isolated MSC population comprise an increased NOTCH1 gene expression level (NCBI Gene ID: 4851 ) or a NOTCH1 protein level (NCBI Accession: CAG33502.1 ), relative to a non- cocultured isolated EPC population and / or non-cocultured isolated MSC population. The increase in NOCTH1 gene expression or protein levels may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing. In some embodiments, the NOTCH1 gene expression level or the NOTCH1 protein level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. In some embodiments, the cocultured isolated EPC population is an isolated ECFC population.

[0293] In some embodiments, the isolated EPC populations and / or the isolated MSC population from the cocultured isolated EPC population and isolated MSC population comprise one or more physical properties, including but not limited to an increased vasculogenesis level, relative to a non-cocultured isolated EPC population and / or non-cocultured isolated MSC population. The increase in the vasculogenesis level may occur during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing. In some embodiments, the vasculogenesis level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a non-cocultured isolated EPC population and / or noncocultured isolated MSC population. In some embodiments, the cocultured isolated EPC population is an isolated ECFC population.Modification of Cells

[0294] The isolated EPC populations and / or isolated MSC populations of the present technology may be genetically engineered or molecularly modified to express a heterologous gene, illustrative examples of which include factors or proteins that, for example, directly or indirectly inhibit thrombogenesis, restenosis or platelet adhesion, or that enhance cell viability or that have anti-inflammatory properties. Heterologous genes may be introduced into cells by means well known in the art. For example, a vector (e.g., a viral vector, such as an adenoviral vector, an adeno-associated viral vector, an AAV chimeric vector or a retroviral vector or pseudotyped viral vector) may be constructed comprising an expression cassette containing a gene, pseudogene, mutant gene, such as dominant negative gene, or a gene-silencing construct, e.g., short hairpin RNA (shRNA) or microRNA (miRNA). Suitable expression cassettes may be constructed using an array of conventional cloning methods. While the use of gene delivery via viral vectors is preferred, non-viral methodologies may also be used, e.g., plasmid or cosmid DNA delivery via liposomal reagents, lipoplexes or polyplexes, electroporation, sonoporation, hydrodynamic gene delivery, use of a ‘gene gun’, and nucleofector techniques and nanoparticle delivery.

[0295] The genetic engineering of the subject cells is not restricted to overexpressing or addition of advantageous genes, but also includes the inhibition, downregulation and ‘knockout’ of disadvantageous genes. This may be achieved by standard means known in the art, such as by use of CRISPR and / or related gene editing technologies.

[0296] In some embodiments, the isolated EPC populations are genetically engineered or molecularly modified after co-culturing with an isolated MSC population. In some embodiments, the isolated MSC populations are genetically engineered or molecularly modified after co-culturing with an isolated EPC population.Pharmaceutical Compositions and Formulations

[0297] The present technology comprises pharmaceutical compositions and cell compositions comprising the isolated EPC populations and / or the isolated MSC populations of the present technology. In some embodiments, an isolated EPC population is present in a composition, such as a pharmaceutical composition or a cell composition, with an isolated MSC population.

[0298] In some embodiments, the isolated EPC population is present at a ratio of about 1 :1 , about 2:1 , about 3: 1 , about 4: 1 , about 5: 1 , about 6: 1 , about 7:1 , about 8: 1 , about 9: 1 , about 10:1 , about 12:1 , about 15:1 , about 20: 1 , about 30: 1 , about 40: 1 , or about 50:1 with the isolated MSC population.

[0299] In some embodiments, the isolated EPC population is present at a ratio of at least about 1 : 1 , at least about 2: 1 , at least about 3: 1 , at least about 4: 1 , at least about 5:1 , at least about 6: 1 , at least about 7:1 , at least about 8:1 , at least about 9:1 , at least about 10:1 , at least about 12:1 , at least about 15:1 , at least about 20: 1 , at least about 30: 1 , at least about 40: 1 , or at least about 50: 1 with the isolated MSC population.

[0300] In some embodiments, the isolated EPC population is present at a ratio of at least 1 :1 , at least 2:1 , at least 3:1 , at least 4:1 , at least 5:1 , at least 6:1 , at least 7:1 , at least 8:1 , at least 9:1 , at least 10:1 , at least 12:1 , at least 15:1 , at least 20: 1 , at least 30: 1 , at least 40: 1 , or at least 50: 1 with the isolated MSC population.

[0301] In some embodiments an isolated EPC population is present in a first composition, such as a pharmaceutical composition or a cell composition, and an isolated MSC population is present in a second composition. The second composition may be formulated for delivery before, during, or after administration of the first composition.

[0302] The pharmaceutical compositions may further comprise a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutically acceptable vehicle is phosphate-buffered saline.Medical Devices and Tissue Engineering Applications

[0303] The isolated EPC populations and / or isolated MSC populations of the present technology may used in combination with an appropriate medical device.

[0304] The isolated EPC populations and / or isolated MSC populations of the present technology may be combined with an implantable cell support substrate, device and / or pharmaceutically acceptable carrier.

[0305] The cell support substrate may be a polymer matrix. Illustrative examples include gels such as a solubilized basement membrane matrix (e.g., a solubilized basement membrane matrix extracted from mouse tumor). In other embodiments, the gel may be a collagen I gel. Such a gel may also include other extracellular matrix (ECM) components, such as glycosaminoglycans, fibrin, fibronectin, proteoglycans, and glycoproteins. The gel may also include basement membrane components such as collagen TV and laminin. Enzymes such as proteinases and collagenases may be added to the gel, as may cell response modifiers such as growth factors and chemotactic agents.

[0306] Any of the isolated cell populations and compositions as of the present technology may be combined with a stent.

[0307] The stent may be seeded with the isolated EPC poulations and / or isolated MSC populations. Blood vessels treated with such stents may exhibit accelerated re- endothelialization, preventing restenosis in the injured vessel.

[0308] In some embodiments, any of the isolated EPC poulations and / or isolated MSC populations may be seeded into a polymeric sheet and wrapped around the outside of a blood vessel that has undergone angioplasty or stent insertion. The cells may also be mixed with a gel and infused into the polymer sheet instead of directly seeded onto the matrix.

[0309] In some embodiments, any of the isolated EPC poulations and / or isolated MSC populations may be seeded onto a polymer matrix, for example, a sponge or mesh, which is then implanted into the desired tissue site. Alternatively, the cells may be mixed with a gel which is then absorbed onto the interior and exterior surfaces of the matrix and which may fill some of the pores of a spongy or other porous matrix. Capillary forces will retain the gel on the matrix before hardening, or the gel may be allowed to harden on the matrix to become more self-supporting. Illustrative biocompatible polymer matrices include any biocompatible synthetic, semi-synthetic material, including plastics and other polymers. In some embodiments, the biocompatible polymer matrix may be made from absorbable or non-absorbable materials. Materials useful for makingbiocompatible polymer matrices include, for example, poly(ethylene), polyesters, poly(propylene), poly(propylene) polyesters such as poly(propylene) fumarate, polystyrene, polytetrafluoroethylene (PTFE), nylon, polypropylene / PTFE, polypropylene / cellulose, polypropylene / monochryal, polyester / collagen, poly(acrylate), poly(methyl methacrylate), poly(hydroxyethyl methacrylate), poly(vinyl alcohol), poly(carbonate), poly(trimethylene carbonate), poly(ethylene-co-vinyl acetate), polypther urethane), poly(ester urethane), poly(arylate), poly(imide), poly(anhydride-co- imide), poly(amino acid), polydepsipeptide, poly(phospbazene), poly(glycolic acid), poly(lactic acid), poly(lactide-co-glycolide), poly(£-caprolactone), poly(p-dioxanone), poly(lactide-co- glycolide), poly(£-caprolactone-co-glycolide), poly(glycolide-co- trimethylene carbonate), lactide / tetramethylglycolide copolymer, lactide / trimethylene carbonate copolymer, lactide-5-valerolactone copolymer, lactide (£-captrolactone copolymer), poly(lactide) / polyethylene oxide copolymer, unsymmetrically 3,6- substituted poly(1 ,4-dioxane-2, 5-dione), poly([3-alkanoic acids) such as poly(|3- hydroxybutyrate), poly([3-hydroxybutyrate) / ([3-hydroxyvalerate) copolymer, poly(|3- maleic acid) and poly([3-hydroxypropionate), poly(b-valerolatone), methylmethacrylate- N-vinyl pyrrolidone copolymer, polyesteramide, polyesters of oxalic acid, polydihydropyran, polyalkyl-2-cyanoacrylate, composites thereof, cellulosic materials, and combinations thereof.

[0310] In some embodiments, the polymer matrix is biodegradable. Suitable biodegradable matrices may be well known in the art and include collagenglycosaminoglycan (GAG), collagen, fibrin, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and PLA-PGA co-polymers. Additional biodegradable materials include poly(anhydrides), poly(hydroxy acids), poly(ortho esters), poly(propylfumerates), poly(caprolactones), polyamides, polyamino acids, polyacetals, biodegradable polycyanoacrylates, biodegradable polyurethanes and polysaccharides. Non- biodegradable polymers may also be used as well. Other non-biodegradable, yet biocompatible polymers include polypyrrole, polyanilines, polythiophene, polystyrene, polyesters, non-biodegradable polyurethanes, polyureas, poly(ethylene vinyl acetate), polypropylene, polymethacrylate, polyethylene, polycarbonates, and poly(ethylene oxide). Those skilled in the art will recognize that this is an exemplary, not a comprehensive, list of polymers appropriate for tissue engineering applications.

[0311] In some embodiments, the matrix may be formed with a microstructure similar to that of the ECM that is being replaced. Mechanical forces imposed on the matrix by the surrounding tissue will influence the cells on the artificial matrix and promote the regeneration of ECM with the proper microstructure. The cross-link density of the matrix may also be regulated to control both the mechanical properties of the matrix and the degradation rate (for degradable scaffolds). The shape and size of the final implant should be adapted for the implant site and tissue type. The matrix may serve simply as a delivery vehicle for the cells or may provide a structural or mechanical function. The matrix may be formed in any shape, for example, as particles, a sponge, a tube, a sphere, a strand, a coiled strand, a capillary network, a film, a fiber, a mesh, or a sheet.

[0312] The isolated EPC populations and / or isolated MSC popualtions of the present technology may be combined with a tubular substrate. The tubular substrate may be seeded with the relevant cells. For example, the polymer matrix may be formed into a tube or network. Such tubes may be formed of natural or synthetic ECM materials such as PLA or collagen or may come from natural sources, for example, decellularized tubular grafts. The cells may coat the inside of the tube, forming an artificial channel.

[0313] The cells may be allowed to proliferate on the polymer matrix or tubular substrate before being implanted in an animal. During proliferation, mechanical forces may be imposed on the implant to stimulate particular cell responses or to simulate the mechanical forces the implant will experience in the animal. For example, a medium may be circulated through a tubular substrate in a pulsatile manner (i.e. , a hoop stress) or with sufficient speed to exert a sheer stress on cells coating the inside of the tube (see, Kaushall S et al., Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med. 2001 Sep;7(9): 1035-40), incorporated herein by reference in its entirety. Alternatively, a hydrostatic force or compressive force may be imparted on an implant that will be deposited within an organ such as the liver, or a tensile stress may be imparted on an implant that will be used in a tissue that experiences tensile forces.

[0314] Medical devices that may be coated with the relevant cells include “implants” or “implantable medical devices.” Implants may be introduced, temporarily or permanently, into a mammal for the prophylaxis or therapy or diagnosis of a medicalcondition, as well as wireless monitoring of physiologic parameters. Such implants include, but may be not limited to, vascular prostheses, vascular grafts, fixtures for connecting prosthetic organs to vascular circulation, stents including vascular and nonvascular stents (e.g., gastrointestinal, pulmonary or biliary stents), covered stents, artificial heart valves, artificial hearts, cardiac prosthesis (e.g., an artificial heart valve), a biological heart valve prosthesis (e.g., derived from animals such as pigs - xenografts may be coated with the relevant cells to render them more biocompatible and less thrombic), venous valves, abdominal aortic aneurysm grafts, vascular filters (e.g., vena cava filter), catheters, guide wires, balloons, devices to protect against pulmonary embolism (e.g., embolic coils, embolic materials for vascular embolization, etc.), orthopedic implants (e.g., bone or joint prostheses), vascular sutures, scaffolds, smooth or porous implants, intraluminal devices, vascular prosthetic filters, pacemakers, pacemaker lead, electrodes, defibrillators, subcutaneous and / or intramuscular implants, vascular occlude, ventricular shunt, vascular sheath, drug delivery devices and ports, septal closure devices, sutures, neurological stimulators, implantable wireless sensors (e.g., blood glucose and blood pressure monitors), artificial filtration systems or other artificial organs, insulin pumps, artificial oxygenators and the like. Other illustrative examples of suitable medical device includemechanical circulatory assist device (MCAD) (e.g., a left ventricular assist device (LVAD), including its inflow and outflow cannula and adapters), hemodialysis grafts, dental implants, orthopedic implants, reconstructive prostheses, implantable wireless biosensors that measure parameters including, but not limited to, pH and blood oxygenation, blood pressure, blood glucose level (for application of blood sugar control in diabetics), implantable insulin pumps, implantable artificial oxygenators, implantable artificial kidneys or filtration systems, artificial or tissue engineered urinary bladders and / or ureters, other implantable artificial organs, implantable electric devices (e.g., pacemakers), or wireless Micro-Electro- Mechanical System (MEMS). The medical device may be made, for example, of titanium or a titanium alloy, which includes shape memory alloys (e.g., Nitinol (NiTi), aluminum and vanadium alloys (Ti6A14V) and (Ti6A14V ELI), as well as niobium alloys (Ti6A17Nb), iron alloys (Ti5A12.5Fe), including, but not limited to, titanium alloys containing Nb, Ta, Zr, Mo, Fe, Si). The device may also be made of other metals, e.g., stainless steel.MethodsBlood-related Disorders

[0315] The present technology comprises methods of treating or otherwise ameliorating a bleeding disorder and / or anemia in a subject in need thereof. The bleeding disorder may comprise an inhereited bleeding disorder. Nonlimiting examples of inherited bleeding disorders include Von Willebrand Disease (VWD).

[0316] In some embodiments, the methods comprise treating, reducing, preventing, or otherwise ameliorating one or more symptoms of VWD. VWD may comprise Type 1 VWD, Type 2A VWD, Type 2B VWD, Type 2M VWD, Type 2N VWD, or Type 3 VWD. Nonlimiting examples of symptoms of VWD include blood loss, platelet clumping, and bruising.

[0317] In some embodiments, the present technology comprises methods of increasing a blood clotting level and / or reducing a blood loss level in a subject in need thereof.

[0318] In some embodiments, the blood clotting level is increased in the subject by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0319] In some embodiments, the blood clotting level is increased in the subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0320] In some embodiments, the blood clotting level is increased in the subject by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0321] In some embodiments, the blood loss level in the subject is reduced by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0322] In some embodiments, the blood loss level in the subject is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0323] In some embodiments, the blood loss level in the subject is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0324] The increase in the blood clotting level or the reduction in the blood loss level may comprise an increase in blood clotting speed and / or a reduction in a bleeding level during or after an injury (e.g., surgical injury), menstruation, or childbirth relative to the control.

[0325] In some embodiments, the blood clotting speed is increased in the subject by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0326] In some embodiments, the blood clotting speed is increased in the subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0327] In some embodiments, the blood clotting speed is increased in the subject by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0328] In some embodiments, the bleeding level during or after an injury, menstruation, or childbirth in the subject is reduced by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0329] In some embodiments, the bleeding level during or after an injury, menstruation, or childbirth in the subject is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0330] In some embodiments, the bleeding level during or after an injury, menstruation, or childbirth in the subject is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0331] In some embodiments, the increase in the blood clotting speed or the reduction in the blood loss level comprises a reduction in a stool blood level or a rectal bleeding level, relative to the control. In some embodiments, the stool blood level or the rectal bleeding level in the subject is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0332] In some embodiments, the increase in the blood clotting speed or the reduction in the blood loss level comprises a reduction in a bruising level (e.g., a raised bruising level), relative to the control.

[0333] In some embodiments, a bruising level in the subject is reduced by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0334] In some embodiments, a bruising level in the subject is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0335] In some embodiments, a bruising level in the subject is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0336] The increase in the blood clotting level or the reduction in the blood loss level may comprise a reduction in a duration or a frequency of epistaxis.

[0337] In some embodiments, the duration or the frequency of epistaxis in the subject is reduced by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0338] In some embodiments, the duration or the frequency of epistaxis in the subject is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.

[0339] In some embodiments, the duration or the frequency of epistaxis in the subject is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to the control.Gene Expression and Proteins

[0340] The present technology comprises methods of increasing gene expression or protein levels in a subject in need thereof, relative to a control. In some embodiments, the present technology comprises methods of increasing a VWF gene expression level or a VWF protein level in the subject, relative to the control (e.g. a sample from the subject at baseline or a sample from a subject subjected to a method lacking one or more steps relative to the methods of the present technology).

[0341] In some embodiments, the increase in the VWF gene expression level or VWF protein level is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0342] In some embodiments, the increase in the VWF gene expression level or VWF protein level is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0343] In some embodiments, the increase in the VWF gene expression level or VWF protein level is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0344] In some embodiments, the increase in the VWF gene expression level or protein level is increased in a plasma sample from the subject.

[0345] In some embodiments, the methods of the present technology comprise increasing a VWF protein activity level in a subject in need thereof, relative to a control.

[0346] In some embodiments, the increase in the VWF protein activity level is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0347] In some embodiments, the increase in the VWF protein activity level is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0348] In some embodiments, the increase in the VWF protein activity level is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0349] In some embodiments, the increase in the VWF protein activity level is increased in a plasma sample from the subject.

[0350] In some embodiments, the methods of the present technology comprise increasing a VWF protein multimer level in a subject in need thereof, relative to a control. In some embodiments, the increase in the VWF protein multimer level comprises an increase in normal multimeric structure of the VWF protein mulitmers in the subject, relative to the multimeric structure of the VWF protein multimers in the control.

[0351] In some embodiments, the VWF protein multimer level is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0352] In some embodiments, the VWF protein multimer level is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0353] In some embodiments, the VWF protein multimer level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0354] The VWF protein multimer level may be increased in a plasma sample from the subject, relative to the control.

[0355] In some embodiments, the methods of the present technology comprise increasing a Weibel-Palade body (WPB) level in a subject in need thereof, relative to a control. The increase in the WPB level may comprise an increase in WPB number or a WPB area, relative to the control.

[0356] The WPB level may be increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0357] The WPB level may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0358] The WPB level may be increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0359] The methods of the present techology may increase a Factor VIII gene expression level (NCBI Gene ID: 2157) or a Factor VIII protein expression level (NCBI Accession: AAA52484.1 ) in the subject in need thereof, relative to the control.

[0360] In some embodiments, the Factor VIII gene expression level or protein level is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0361] In some embodiments, the Factor VIII gene expression level or protein level is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0362] In some embodiments, the Factor VIII gene expression level or protein level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0363] The Factor VIII gene expression level or protein level may be increased in a plasma sample from the subject, relative to the control.

[0364] In some embodiments, the methods of the present technology do not increase a VWF alloantibody level in the subject, relative to the control.Administration

[0365] In some embodiments, the methods comprise administering to the subject a pharmaceutical composition or a cell composition of the present technology. In some embodiments, the methods comprise a step of measuring a VWF protein level or a VWF gene expression level in the subject before, during, and / or after administration of the pharmaceutical composition or the cell composition. The VWF protein level or the VWF gene expression level in the subject may be measured in a plasma sample from the subject.

[0366] In some embodiments, the subject at baseline (i.e., prior to commencement or completion of the methods of the present technology) may comprise a VWF protein level or a VWF gene expression level that is at least about 5%, 10%, 15%, 20%, 25%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% less than a VWF protein level or a VWF gene expression level in a control that does not have a bleeding disorder (e.g., VWD or hemophilia) or a symptom thereof.

[0367] In some embodiments, the subject at baseline may comprise a VWF protein level or a VWF gene expression level that is about 5%, 10%, 15%, 20%, 25%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% less than a VWF protein level or a VWF gene expression level in a control that does not have a bleeding disorder or a symptom thereof.

[0368] In some embodiments, the subject at baseline may comprise a VWF protein level or a VWF gene expression level that is at least 5%, 10%, 15%, 20%, 25%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% less than a VWF protein level or a VWF gene expression level in a control that does not have a bleeding disorder or a symptom thereof.

[0369] In some embodiments, one or more cells of the isolated EPC population are assessed for a VWF protein level or a VWF gene expression level prior to formulation in a pharmaceutical composition or a cell composition. In some embodiments, one or more cells of the isolated EPC population formulated in a pharmaceutical omposition or a cell compoisiton are assessed for a VWF protein level or a VWF gene expression level prior to administration of the pharmaceutical composition or the cell composition to the subject.

[0370] In some embodiments, one or more cells of the isolated EPC population comprise a VWF protein level or a VWF gene expression level that is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control (e.g., one or more cells of a non-isolated EPC population).

[0371] In some embodiments, one or more cells of the isolated EPC population comprise a VWF protein level or a VWF gene expression level that is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.

[0372] In some embodiments, one or more cells of the isolated EPC population comprise a VWF protein level or a VWF gene expression level that is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.

[0373] In some embodiments, the one or more cells of the isolated EPC population comprise an average VWF protein level or an aneverage VWF gene expression level for the total cells in the isolated EPC population.

[0374] The methods may comprise administering to the subject, a pharmaceutical composition or a cell composition comprising an isolated EPC population of the present technology, wherein the isolated EPC population comprises or consists of PROCR+ / _PDGFRA+ / _EPCs. The methods may further comprise administering to the subject, a pharmaceutical composition or a cell composition comprising the isolated MSCs of the present technology. The MSCs may be coformulated with or in a separate composition than the composition comprising the PROCR+ / - PDGFRA+ / - EPCs.

[0375] The pharamaceutical compositions of the present technology may be administered to a subject in need thereof as single or multiple doses. In some embodiments, the methods comrprise administering one or more doses of the pharmaceutical composition or the cell composition to the subject in need thereof.

[0376] In some embodiments, the methods comrprise administering two or more doses of the pharmaceutical composition or the cell composition to the subject in need thereof.

[0377] In some embodiments, the pharmaceutical composition or the cell composition is formulated for administration to the subject intravenously, intraarterially, or intramuscularly.Subjects

[0378] In some embodiments, the subject of the present technology has been diagnosed with VWD. In some embodiments, the subject has a VWD selected from the group consisting of Type 1 VWD, Type 2A VWD, Type 2B VWD, Type 2M VWD, Type 2N VWD, and Type 3 VWD.Pre-treatment Prior to Therapeutic Use

[0379] The isolated EPC populations and / or isolated MSC populations of the present technology may be cultured under endothelial cell-inducing conditions prior to administering the cells to the subject. Illustrative examples include inducing endothelial cell production prior to transplantation. For instance, the isolated cell populations and compositions may be induced to form vascular endothelial cells, e.g., on a medical or surgical device, scaffold or matrix or other structure (e.g., a tube), and then may be transplanted into a subject at a site in need of endothelial cells. Any convenient endothelial cell producing condition may be employed in such embodiments.Medical Devices

[0380] The present technology comprises methods of coating blood-contacting surfaces of implantable medical devices with the isolated EPC populations and / or the isolated MSC populations of the present technology.

[0381] In order to increase the rate of cell spreading, blood-contacting surfaces of implantable devices may be pre-coated with an extracellular matrix protein, such as fibronectin, collagen, vitronectin, laminin, fibrin, or any of the following components containing molecules, proteins or constructs, including proteoglycans, such as heparan sulfate, chondroitin sulfate, keratin sulfate, or non-proteoglycan polysaccharide containing molecules, such as hyaluronic acid, or any combination thereof. Bloodcontacting surfaces may also be pre-coated with gelatin or a gelatin matrix or gelatin foam, cellulose, microfibrillar collagen, thrombin, e.g., recombinant human thrombin (Recothrom, ZymoGenetics), a fibrin sealant, e.g. , Tisseel (Baxter), or fibrin gel, fibrin glue, fibrinolytically inhibited fibrin glue, adhesive glue or sealant, hydrogel. Bloodcontacting surfaces may also be pre-coated with a serum protein or other blood component, or growth factor or hormone, e.g., platelet-derived growth factor BB, basic fibroblast growth factor, acidic fibroblast growth factor, or transforming growth factor betal . Pre-coating may also be effected using a synthetic polymer, e.g., polymer of lysine, ornithine or arginine, or polymethylmethacrylate, polyacrylic acid, or L-glutamic acid-treated construct, or glutaraldehyde-preserved cellular matrix, or a biodegradable binder or coating, such as poly(DL-lactide-co-glycolide), or biodegradable polyester, e.g., polyhydroxyalkanoate, polysorbate, or poly amino acids, e.g., poly-L-lysine, or chitosan, fetuin, or cationic silica microbeads, other types of microbeads or carbon- deposition surface coating, polyethyleneterephthalate with or without alteration by plasma discharge surface modification, or covalently-attached avidin, biotinylation, or RGD peptide sequence containing molecules, structures or constructs or peptides, which may be cross-linked to RGD peptides, or molecules specific to one or more EPC specific integrin binding site or synergistic binding site, e.g., the amino acid sequence DRVPHSRN or antibodies, peptides or aptamers specific to EPC, or any combination of the above. The aforementioned molecules may be physiosorbed or covalently bound to the underlying surface, e.g., titanium / titanium alloy surface, for the latter a variety of methods may be available, including silanization (e.g., linkage of steel to an aminosilane crosslinker), biotinylation, covalent linkage to dopamine, etc. In addition to the proteins, molecules, polymers, structures and artificial constructs mentioned above, other celltypes may be used to pre-coat the blood-contacting device surfaces to provide for a suitable matrix for EPCs, including, but not limited to, fibroblasts, smooth muscle cells, stem cells, mesothelial cells, mesenchymal cells, progenitor cells, myocytes, or other cell type. It is generally desirable to pre-coat the implantable device with autologous cells to avoid rejection. Fibroblasts, for example, may be easily harvested for this purpose from a sample of the subject’s skin.

[0382] The biocompatible implants of the present technology may comprise at least one bioactive agent, representative examples of which include growth factors, analgesics / antipyretics, antiasthamatics, antibiotics, antidepressants, antidiabetics, antifungal agents, antihypertensive agents, anti-inflammatories, antineoplastics, antianxiety agents, immunosuppressive agents, antimigraine agents, sedatives / hypnotics, antipsychotic agents, anti manic agents, antiarrhythmics, antiarthritic agents, antigout agents, anticoagulants, thrombolytic agents, antifibrinolytic agents, antiplatelet agents and antibacterial agents, antiviral agents, antimicrobials, anti-infectives, and combinations thereof. The bioactive agent may be a cell response modifier such as a growth factor or a chemotactic agent. Exemplary growth factors include epidermal growth factor, bone morphogenetic protein, TGF-[3, hepatocyte growth factor, platelet-derived growth factor, TGF-a, IGF-I and II, hematopoietic growth factors, heparin binding growth factor, peptide growth factors, basic and acidic fibroblast growth factors, nerve growth factor (NGF), muscle morphogenic factor (MMP) and vascular endothelial growth factor (VEGF). The particular growth factor employed should be appropriate to the desired cell activity. For example, VEGF may be used to promote differentiation of the EPCs. Alternatively, the growth factor may be selected to recruit cells to the implant or to promote or inhibit specific metabolic activities of cells recruited to the implant. The regulatory effects of a large family of growth factors may be well known to those skilled in the art.

[0383] To further enhance angiogenesis, endothelial cell mitogens may also be administered to the subject in conjunction with, or subsequent to, the administration of any of the isolated cell populations and compositions as of the present technology. Endothelial cell mitogens may be administered directly, e.g., intra-arterially, intramuscularly, or intravenously, or nucleic acid encoding the mitogen may be used.

[0384] The nucleic acid encoding the EC mitogen may be administered to a blood vessel perfusing the ischemic tissue or to a site of vascular injury via a catheter, for example, a hydrogel catheter, as described, for example, by U.S. Pat. No. 5,652,225, the contents of which are incorporated herein by reference in their entirety.

[0385] The nucleic acid also may be delivered by injection directly into the ischemic tissue using the method described in U.S. Pat. No. 6,121 ,246, the contents of which are incorporated herein by reference in their entirety.

[0386] The endothelial cell mitogen may contain a secretory signal sequence that facilitates secretion of the protein. Proteins having native signal sequences, e.g., VEGF, may be desirable. Proteins that do not have native signal sequences, e.g., bFGF, may be modified to contain such sequences using routine genetic manipulation techniques (e.g. Nabel et al. (1993) Nature, 362, 844.).

[0387] A DNA segment encoding the desired endothelial cell mitogen may be chemically synthesized or, alternatively, such a DNA segment may be obtained using routine procedures in the art, e.g., PCR amplification. A DNA encoding VEGF is disclosed in U.S. Pat. No. 5,332,671 , the contents of which are incorporated herein by reference in their entirety.

[0388] In some embodiments, it may be desirable to use nucleic acids encoding two or more different proteins in order optimize the therapeutic outcome. For example, DNA encoding two proteins, e.g., VEGF and bFGF, may be used to provide improvement over the use of bFGF alone. In some embodiments, an angiogenic factor may be combined with other genes or their encoded gene products to enhance the activity of targeted cells, while simultaneously inducing angiogenesis, including, for example, nitric oxide synthase, L-arginine, fibronectin, urokinase, plasminogen activator, and heparin.

[0389] Cell-seeded implants of the present technology may be implanted into any tissue including connective, muscle, nerve, and organ tissues. For example, an implant placed into a bony defect will attract cells from the surrounding bone, which will synthesize ECM, while the EPCs form blood vessels. The blood supply for the new bone will be provided as the new ECM is formed and mineralized. An implant placed into a skin defect will promote dermis formation and provide a vascular network to supply nutrients to the newly formed skin.

[0390] Cells that may be recruited to the implant may also differentiate into other cell types. Bone cell precursors migrating into a bone implant may differentiate into osteoblasts. Mesenchymal stem cells migrating into a blood vessel may differentiate into muscle cells. Endothelial cells forming tubular networks in liver may induce the formation of liver tissue.

[0391] Any of the isolated cell populations and compositions as of the present technology may be mixed with another cell type before implantation. The cell mixture may be suspended in a carrier such as a culture medium or in a gel. Alternatively, the cells may be co-seeded onto a polymer matrix or combined with a gel that is absorbed into the matrix. For some applications, it may be desirable to seed one cell type directly onto the matrix and add the second cell type via a gel. Any ratio of EPC to the other cell type or types may be used. A skilled person will recognize that this ratio may be easily optimized for a particular application. Exemplary ratios of EPC to other cells may be at least 10% (e.g., 1 :9), at least 25%, at least 50% (e.g., 1 :1 ), at least 75%, and at least 90%. Smaller ratios, for example, less than 10%, may also be employed.

[0392] Any cell type, including connective tissue cells, nerve cells, muscle cells, organ cells, or other stem cells, may be combined with any of the isolated cell populations and compositions as of the present technology. For example, osteoblasts may be combined with the fetal endothelial cells to promote the co-production of bone and its vasculature in a large defect. Fibroblasts combined with fetal endothelial cells and inserted into skin will produce fully vascularized dermis. Other exemplary cells that may be combined with the fetal endothelial cells of the present technology include hematopoietic cells (including hematopoietic stem cells), ligament cells, lung cells, epithelial cells, smooth muscle cells, cardiac muscle cells, skeletal muscle cells, islet cells, nerve cells, hepatocytes, kidney cells, bladder cells, and bone-forming cells.In Vitro Screening

[0393] The present technology comprises isolated EPC populations and isolated MSC populations. Provision of these populations and compositions comprising the same may facilitate in vitro based screening systems for testing the effectiveness and toxicity of existing or potential treatment or culture regimes. As such, the present technology comprises methods of assessing effects of a treatment or culture regime on the phenotypic or functional state of any of the isolated EPC population and / or MSCpopulations of the present technology and the pharmaceutical compositions and cell compoisitons comprising the same. In some embodiments, the method comprisessubjecting the the isolated EPC population and / or MSC populations of the present technology and / or the pharmaceutical compositions or cell compoisiton, to a treatment regime and screening for an altered functional or phenotypic state, relative to non-isolated cell populations or compositions comprising non-isolated cell populations.EXAMPLESExample 1: Methods for isolating a CD34+CD45' cell population from human placenta.

[0394] Placental tissues were processed, and single-cell suspension was prepared as described in Patel J et al., Prospective surface marker-based isolation and expansion of fetal endothelial colony-forming cells from human term placenta. Stem Cells Transl Med. 2013 Nov;2(11 ):839-47 and Patel J at al., Novel isolation strategy to deliver pure fetal-origin and maternal-origin mesenchymal stem cell (MSC) populations from human term placenta. Placenta. 2014 Nov;35(11 ):969-71 , the contents of which are incorporated herein by reference in their entirety. The isolated placental cluster of differentiation (CD)34+ single-cell suspension was incubated with human CD34- phycoerythrin (PE), human CD45-FITC, and human CD31 -V450 for 20min at 4 °C. Cells were flow-sorted using Fluorescence-activated cell sorting (FACS). Cell doublets were removed, and 7-amino-actinomycin D (7AAD) was used to exclude dead cells. Fluorescence minus one (FMO) control was used in gating the population of interest. To remove any remaining contaminating CD45+ cells from the hematopoietic lineage, only the CD45’ CD34+population was gated. All cells that were CD45’ CD34+were then FACS sorted directly into 100% fetal bovine serum. The fraction of cells sorted was considered to contain fetal EPCs and MSCs.Example 2: Identification of PROCR+and PDGFRA+EPC subgroup.Materials and MethodsSingle-Cell RNA Sequencing Analysis

[0395] Single-cell RNA sequencing was analyzed using RStudio (RStudio, MA, USA) with the package Seurat (Version 4.2.0) according to Hao Y et al., Integratedanalysis of multimodal single-cell data. Cell. 2021 Jun 24;184(13):3573-3587.e29, incorporated herein by reference in its entirety. Data from previously conducted scraps of the murine aorta and publicly available scRNA-seq of the human aorta was reanalyzed (see Shafiee A et al., Meso-Endothelial Bi potent Progenitors from Human Placenta Display Distinct Molecular and Cellular Identity. Stem Cell Reports. 2018 Mar 13;10(3):890-904 and Lukowski SW et al., Single-Cell Transcriptional Profiling of Aortic Endothelium Identifies a Hierarchy from Endovascular Progenitors to Differentiated Cells. Cell Rep. 2019 May 28;27(9):2748-2758.e3, each of which are incorporated herein by reference in their entireties). Data was filtered using the following criteria: >200 and <3000 gene counts per cell, <20% mitochondrial genes, and >3 cells present. The data was then normalized and integrated correcting for batch effects using the standard Seurat pipeline, with a scaling factor of 10,000. Principal component analysis was conducted using RunPCA on the integrated datasets. The first 30 principal components were used to compute nearest neighbours and clusters using FindNeighbors and FindClusters, respectively, with a resolution of 0.4 (mouse) or 0.5 (human) for optimal distinction between clusters. Clustering plots were made using the two-dimensional Uniform Manifold Approximation and Projection (UMAP) algorithm in Seurat. Differentially expressed (DE) genes were identified in each cluster. Clusters were annotated using the Bioconductor package SingleR with reference to the Mouse RNA Sequencing Data and Human Primary Cell Atlas Data databases, respectively ((1 ) Aran D et al., Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat Immunol. 2019 Feb;20(2): 163-172; (2) Mabbott NA et al., An expression atlas of human primary cells: inference of gene function from coexpression networks. BMC Genomics. 2013 Sep 20;14:632; and (3) Monaco G et al., RNA-Seq Signatures Normalized by mRNA Abundance Allow Absolute Deconvolution of Human Immune Cell Types. Cell Rep. 2019 Feb 5;26(6):1627-164O.e7, each of which are incorporated herein by reference in their entireties).Animals

[0396] Mixed sex adult C57BI / 6 mice and NOD scid H2rynullB2mnull (NSG) mice were used for experimentation. Mixed sex adult CAG-EGFP, Cdh5-CreERT2 / ROSA- EYFP, Pdgfra-MerCreMer / ROSA-YFP, Abcg2-lres-CreERT2 / ROSA-YFP, and Sox18- Cre / ROSA-YFP mice were supplied from in-house breeding colonies. Cdh5-CreERT2 / R0SA-EYFP, Cdh5-CreERT2 / ROSA-ZsGreen, Pdgfra-MerCreMer / ROSA- YFP, Abcg2-lres-CreERT2 / ROSA-YFP and Sox18-Cre / ROSA-YFP mice were injected (intraperitoneal) with 100 pL of 20 mg / mL Tamoxifen (Sigma-Aldrich, Ml, USA) in 90% corn oil and 10% ethanol for 5 consecutive days to induce recombination of the yellow fluorescent protein (YFP) in target cells prior to tissue collection (only 3 days of injections were used for Cdh5-CreERT2 / ROSA-ZsGreen mice as per standard protocol).En Face / Aorta Length Preparation

[0397] Aortae were dissected and prepared for en face IF as previously described by Zhao J et al., Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors. Nat Commun. 2021 May 7; 12(1 ):2564, the contents of which are incorporated herein by reference in their entirety. Samples were sectioned perpendicularly to the cut face to analyze the length of the aorta.Immunofluorescence

[0398] Tissues were prepared as described according to Zhao J et al., Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors. Nat Commun. 2021 May 7; 12(1 ):2564. Primary antibodies used included: rat anti-mouse CD31 ; rabbit anti-mouse CD34, rabbit antimouse ERG, rat anti-mouse PROCR, chicken anti-GFP, and Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I) - Rhodamine. Secondary antibodies conjugated to Alexa-fluor 488, 568, or 647 were used for fluorescence detection.

[0399] Fluorescence imaging was performed using an Olympus FV3000 confocal microscope (Olympus, Shinjuku, Japan) and a Nikon / Spectral Spinning Disc confocal microscope (Nikon, New York, USA). Brightfield imaging was conducted using a Nikon Eclipse 50i Brightfield Microscope (Nikon, New York, USA). Image analysis was conducted using the Olympus Fluoview FV31 S-SW software (Olympus, Shinjuku, Japan) as well as ImageJ (National Institute of Health).Flow Cytometry and Fluorescence-Activated Cell Sorting

[0400] Aortae and full-skin excisional wounds were digested according to Zhao et al., Nature Communications, 2021. 12(1 ): p. 2564. For comparison of colony formation between thoracic and abdominal aorta, aortae were divided at the diaphragm.

[0401] Antibodies used to assess the endothelial hierarchy and subpopulations included, in murine aorta and full-skin excisional wounds respectively: Hematopoietic Lineage Cocktail PerCP-Cy5.5, VE-Cadherin BV421 , CD34 Alexa Fluor 647, CD31 PE- Cy7, PROCR PE, PDGFRA BV605, CD157 PE, and 7-AAD PE-Cy5.

[0402] Flow cytometry analysis and FACS were conducted. Single stain controls were used to acquire cytometer voltage settings and to compensate data. Fluorescence-minus-one (FMO) controls were used to distinguish positive and negative populations and set appropriate gates. All analysis was conducted using FlowJo® software (FlowJo LLC, USA).In Vitro Colony Formation Assay

[0403] Cells from fluorescence-activated cell sorting and Endothelial Growth Medium-2 (EGM2; Lonza, Basel, Switzerland) were deposited on top of cross-linked Matrigel® (Coming® Matrigel® Basement Membrane Matrix, Phenol Red-free, LDEV- free; Corning, New York, USA). Cells were divided to allow the deposition of 10 or 100 cells into each well, depending on experimental conditions. Plates were then incubated at 37°C and media was replaced twice weekly. Cells were imaged intermittently using a Nikon Eclipse 50i Brightfield Microscope (Nikon, New York, USA).

[0404] On day 12, wells were fixed and permeabilized before blocking in PBST. Cells were then stained with the aforementioned primary antibody solutions overnight at 4°C. The following day, cells were incubated in secondary antibody solutions and stained with a DAPI solution before imaging.In Vivo Vessel Generation and Collagen Plugs

[0405] Following FACS sorting, 100 cells of desired populations isolated from CAG-EGFP mice were mixed with gel solutions prepared on ice by mixing 80% collagen (3% PurCol; Advanced Biomatrix, Carlsbad, CA, USA) with 10% DMEM, 5-7% sodium bicarbonate to reach a pH of 7.2-7.4, and water. Gel + cell solutions were incubated in96-well plates at 37°C for 90-120 minutes before topping with EGM2 and incubating overnight.

[0406] The following day, gels were rinsed in PBS and implanted into NOD-scid I I2rynu II B2m nul I (NSG) mice. Lateral incisions were made on each dorsal flank to create a pocket below the skin and above the muscle, with a total of two plugs being implanted into each mouse. Incisions were sutured and mice were monitored daily. Plugs were collected after 7 days and analyzed using wholemount microscopy as well as cryosections for IF. For wholemount microscopy, gels were rinsed in PBS before clearing in RapiClear® (SunJin Lab Co, Taiwan) for 30-60 minutes and imaging immediate. Gels were then subject to a sucrose gradient as described above to prepare for cryo-sectioning.Human Term Placental Cells

[0407] Frozen single cells suspensions of human term placenta samples previously processed as per the protocol outlined in Nano et al., STAR Protoc, 2022. 3(2): p. 101354 were thawed and prepared for flow cytometry / FACS-sorting. In addition to the markers outlined in Nano et al., STAR Protoc, 2022. 3(2): p. 101354, PROCR PE (1 :25) was added to panel. Cells were then gated and sorted, with the addition of a PROCR+ / _gate on each of the 4 populations of varying CD31 expression.

[0408] Cells that were FACS-sorted were then plated onto collagen-coated plates as previously described at a 1000 cells per-well density in 48-well plates with EGM2. Cells were cultured and allowed to expand for up to 30 days to evaluate colony formation capacity (no colony, endothelial colony (EC, <50 cells), low-proliferative potential ECFC (LPP-ECFC, <1000 cells) or high-proliferative potential ECFC (HPP- ECFC, >1000 cells, ability to form secondary colonies)). HPPs were then further passaged to limiting dilution assay conditions (1000 cells per well in a 6-well plate) for evaluation of further colony formation, serially passaged for expansion, or stained to evaluate immunofluorescent expression using rabbit-anti-human VECAD (1 :100) and mouse-anti-human CD31 (1 :100).Statistical Analysis

[0409] Data were analyzed using GraphPad Prism8 (GraphPad, United States) software. Paired t-tests, unpaired t-tests, and Mann-Whitney tests were used for singlecomparison results, depending on experimental conditions. For multiple comparisons, Friedman one-way ANOVA, and Kruskal-Wallis tests were conducted in accordance with data. Results were shown as an average with error bars representing the standard deviation (SD), with a significance threshold set at p < 0.05. A minimum of three biological replicates were used for all significance testing.Single-cell RNA-sequencing reveals key markers in endothelial populations

[0410] Single-cell RNA-sequencing done on the Lin- CD34+compartment of the aortae from 3 C57BI / 6 mice was re-analyzed in order to illuminate highly expressed genes in endothelial and mesenchymal clusters of interest (FIG. 1A). By increasing the resolution when re-clustering this sequencing data, more distinctive subpopulations emerged, allowing the analysis of gene expression within previously delegated clusters. Clusters 0, 10, and 12 were characterized as mature differentiated endothelial cells based on expression of known major endothelial markers classically used to gate the endothelial compartment including Pecaml and Cdh5 and confirmed using SingleR labeling analysis (FIG. 1 B). Clusters 1 , 2, and 3 showed upregulation of mesenchymal markers leading to their designation of mesenchymal (M) clusters, whereas cluster 4 showed expression of both mesenchymal and endothelial markers, leading to the designation of this cluster as the putative endothelial progenitor cell (EPC) population. Neither group possessed expression of hematopoietic cell markers (confirmed with SingleR). Among candidate progenitor genes studied, Cd157 (adj. p = 5.16 x 10-9 in Cluster 10), Sox18 (adj. p = 4.77 x 10-85 in Cluster 0, adj. p = 1.1 x 10-14 in Cluster 10), and Abcg2 (adj. p = 2.21 x 10-76 in Cluster 0, adj. p = 1.47 x 10-50 in Cluster 12) showed significant upregulation in the differentiated endothelial cell clusters while Procr (adj. p = 2.07 x 10-121 in Cluster 4) and Pdgfra showed expression in EPC cluster 4, with mesenchymal marker Pdgfra being most upregulated in the MSC clusters (adj. p = 3.18 x 10-103 in Cluster 1 , adj. p = 6.38 x 10-178 in Cluster 2). Pathway analysis and dot plots of genes of interest were conducted on cluster 4 for further characterization of differentially expressed (DE) genes (FIGS. 1 B-1 D).Endothelial progenitor cells highly express PROCR and PDGFRA

[0411] Flow cytometry was performed on adult C57BL / 6 mouse aortae using the markers highlighted in single-cell RNA sequencing analysis above in conjunction withthe markers used to characterize the EPC population mentioned previously. From total aorta cells, the endothelial hierarchy was segregated based on cell surface marker profiles and the original gating strategy outlined in Patel et al., Circulation, 2017. 135(8): p. 786-805. EPCs were identified as Lin / E-cadherin+CD34+CD31l0 / ’, and differentiated endothelial cells were identified as Lin’VE-cadherin+CD34+CD31+cells (FIG. 2A). EPCs and mature differentiated endothelial cell populations were then further evaluated for expression levels of PROCR, PDGFRA, and CD157 (FIGS. 2B and 2C). PROCR and PDGFRA were 1 .4-fold (p<0.001 ) and 3.3-fold (p<0.001 ) more frequently expressed in EPCs as compared to differentiated endothelial cells, respectively, whereas CD157 was 5.9-fold (n.s.) more highly expressed in differentiated endothelial cells than in EPCs (FIG. 2D). In addition to these markers, two additional mouse strains were used based on the studies described to test further gene expressional differences between EPCs and differentiated endothelial cells. Aortae from adult Abcg2-lres-CreERT2 / ROSA- EYFP and Sox18-Cre ERT2 / ROSA-EYFP mice were collected for flow cytometry following 5 days of tamoxifen injections. Further characterization of these populations based on YFP expression showed that ABCG2 and SOX18 were 2.3-fold (p<0.05) and 4.3-fold (p<0.05) more frequently expressed in differentiated endothelial cells compared to EPCs, respectively (FIGS. 2B-2F).

[0412] These findings suggested that in both flow cytometry and single-cell RNA- sequencing, PDGFRA and PROCR were additional markers that could enrich progenitors within the already described EPC population. Among EPC cells, an average of 78.04% were PROCR+and 82.28% were PDGFRA+, suggesting that these markers may allow refining of the progenitor definition. An alternative gating strategy on live aortic cells co-expressing both PROCR and PDGFRA showed that an average of 91.43% (**) were Lin’VE-cadherin+, and from here a further 94.97% (****) were EPCs (FIG. 2E; ** p =0.0012, **** p <0.0001 , n=3). This demonstrates the powerful ability of PROCR and PDGFRA expression alone to mark the same population as the classic EPC gating strategy to a high degree of confidence.

[0413] Given the significant overlap of three distinct strategies to identify progenitor cells in the endothelium, we next called this population the PROCR+endovascular progenitor cell population and proceeded to their functional analysis. The use of single-cell RNA-seq suggested that cluster 4 was representative of this cell population. Differential gene expression defining this cluster included the expression ofboth major endothelial and mesenchymal genes as seen in the analyses of top differentially expressed genes from this cluster (FIGS. 1 B-1 D).PROCR+EPCs show increased endothelial colony formation capacity in vitro and increased enqraftment potential in vivo

[0414] To begin analyzing the functional capacity of the PROCR+endovascular progenitor cells versus other EPCs and differentiated endothelial cell populations, aorta cells were collected and FACS-sorted based on cell surface expression of PROCR for colony formation assays. Four groups were sorted in each experiment: PROCR+EPCs, PROCR- EPCs, PROCR+differentiated endothelial cells, and PROCR’ differentiated endothelial cells. Colonies were characterized based on their morphology at day 12, with two major morphologies seen: a classic endothelial morphology with positive expression of endothelial marker Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I) - Rhodamine (Isolectin) in immunofluorescence (IF), and an elongated morphology that was not labelled by Isolectin. Colonies emanating from PROCR+EPCs showed an exclusively endothelial morphology, while all other conditions showed colonies with an elongated morphology which did not express Isolectin. PROCR+EPCs possessed the greatest colony formation capacity with a mean of 17.67% of wells plated per mouse forming colonies, followed by 8.21 % of PROCR’ EPC wells, 1.16% of PROCR+differentiated endothelial cell wells, and 0.45% PROCR’ differentiated endothelial cell wells (*, p < 0.05). Differentiated endothelial cell wells (PROCR+ / ’) never formed endothelial colonies based on positive staining for Isolectin.

[0415] To distinguish the potency of PROCR+EPCs and PROCR’ EPCs, these populations were then challenged with a more stringent in vivo assay where collagen gels containing 100 PROCR+EPCs, PROCR’ EPCs, or total differentiated endothelial cells, respectively, from CAG-EGFP mice were transplanted into the dorsal flanks of NOD-scid-ll2rynullB2mnull (NSG) recipient mice in addition to gels containing no cells as a control. Whole mount images of collagen gels after 7 days revealed that PROCR+EPCs had the highest engraftment potential with 9 / 15 gels engrafting and an average of 5.67% GFP+ area per plug (FIG. 1 E (i), FIG. 1 F), while only 2 / 10 PROCR- EPC gels engrafted with an average of 0.79% GFP+ area per plug (FIG. 3A (ii), FIG. 3B; p < 0.05). Differentiated endothelial cells were never able to engraft (0 / 6 gels), showing 0% GFP+ area, identical to the results of the gels containing no cells. Furthermore, IF staining wasconducted on sections of these gels in order to further characterize the cells that had engrafted. GFP+ PROCR+EPCS co-expressed endothelial markers CD34 and Isolectin while no overlap of these endothelial markers was seen with GFP+cells from collagen gels containing PROCR’ EPCs (FIG. 3C). These findings more robustly pointed to key functional differences between EPC and differentiated endothelial cells as reported before, but more remarkably between EPCs expressing PROCR and EPCs devoid of PROCR.PROCR+EPCs form a niche in the thoracic aorta and display increased congenic capacity

[0416] To confirm that aortic endothelial cells express PROCR and to find their anatomical distribution in situ, aortae from Cdh5’ CreERT2 / ROSA-EYFP mice, where endothelial cells may be labelled with YFP upon administration of tamoxifen, were harvested for ex vivo analysis. IF staining showed greater co-expression of PROCR and YFP in the thoracic aorta (68.18%; FIG. 4A) as compared to the abdominal aorta (21.10%; FIG. 4B; **, p = 0.005, n = 5). Upon uncovering the spatial difference of PROCR expression in the aorta, further investigation was needed to confirm if this correlated with a spatial difference in functionality in terms of clonogenic capacity as well. YFP+ EPCs from both the thoracic and abdominal aortae of Cdh5’ CreERT2 / ROSA-ZsGreen mice were FACS-sorted and plated in Matrigel® to compare the colony forming capacity between the two populations. Interestingly, 7 / 13 wells containing YFP+ EPCs from the thoracic aorta formed branching endothelial colonies while 0 / 13 wells from the abdominal aorta formed colonies (FIG. 4C; **, p = 0.0052, n=3).PROCR+ EPCs form differentiated endothelial cells in vivo in homeostasis and injury

[0417] As PROCR and mesenchymal marker PDGFRA showed a high degree of overlap in in PROCR+EPCs in flow cytometry and scRNA-sequencing results, Pdgfra- MerCreMer / Rosa-EYFP mice were used to trace the fate of CEPCs. Animals were administered tamoxifen to label PDGFRA-expressing cells permanently with YFP and trace this population in tissues of interest over time. In the aorta of adult homeostatic mice after a short pulse of tamoxifen, flow cytometry first confirmed that the Lin- PDGFRA(YFP)+ subpopulation of aorta largely consists of EPCs rather thandifferentiated endothelial cells (90.85% compared to 2.87%, n=4, p<0.0001 ). To confirm that the population being traced in this model was indeed the same endothelial population as previous studies, YFP+ PROCR+EPC colonies cultured from both Cdh5_CreERT2 / ROSA-EYFP and Pdgfra-MerCreMer / Rosa-EYFP aortae were compared and found to have no difference morphologically or phenotypically with both expressing endothelial markers Isolectin and ERG in immunofluorescent analysis.

[0418] Few studies in the past have been able to identify a single Cre reporter system distinguishing progenitors from differentiated cells. This provided a unique opportunity to demonstrate the lineage relationship between in PROCR+EPCs and differentiated endothelial cells. Lineage tracing was conducted on the homeostatic aorta following tamoxifen induction at 4 weeks of age to label PDGFRA-expressing cells. Aorta were then assessed from juvenile age to adulthood to trace the fate of YFP+ cells. IF staining of aorta collected from day 1 post-tarn oxifen course (D1 ) showed YFP+ cells in the intima without any co-expression of mature endothelial markers. This further showed that at least a fraction of PDGFRA-expressing cells as labelled by YFP may be endothelial as in intimal position and not simply in the mesenchymal layers of the aorta. When examining the fate of these PDGFRA-expressing cells labelled at D1 , IF at D84 demonstrated co-expression of endogenous YFP with CD31 and ERG in the intima, confirming endothelial fate of these cells. This result was confirmed quantitatively using flow cytometry where it was seen that the percentage of CEPCs between D1 and D84 ranged from an average of 69.58% to 88.24% of the Lin_YFP+ compartment, while the percentage of differentiated endothelial cells increased significantly from 0.74% at DO to 4.67% at D84 (FIG. 5A; n = 5, ** p<0.01 , *** p<0.001 ).

[0419] To analyze this mechanism in the context of injury, we similarly conducted a course of tamoxifen injections on adult Pdgfra-MerCreMer / Rosa-EYFP mice before performing full skin excisional wounding at DO and collecting the wounds across the wound healing timeframe. IF staining at D1 showed YFP expression was mostly focused on fibroblast-looking populations and did not overlap with mature endothelial markers, while IF at D5 demonstrated co-expression of endogenous YFP with differentiated endothelial markers CD31 and ERG. Flow cytometry at each time point confirmed that the percentage of PROCR+EPCs between D1 and D5 ranged from an average of 29.43% to 43.29% of the Lin_YFP+ compartment, while the percentage of differentiated endothelial cells increased significantly from an average of 0.04% at D1to 1.19% at D5 (FIG. 6B, n = 7, * p<0.05; ** p<0.01 ). Overall, making use of PDGFRA expression as a reporter of PROCR+EPCs allowed tracing the fate of this population to demonstrate its contribution to differentiated endothelial cells both in homeostatic aorta and skin wounds.PROCR is expressed in human aorta scRNA-seq data and leads to increased clonogenic capacity in a human term placental model of ECFCs

[0420] To begin investigating whether this characterization applies to human tissues as well, publicly available human normal aorta single-cell RNA-sequencing data was reanalyzed to investigate the markers of interest to this study (see Li Y et al., SingleCell Transcriptome Analysis Reveals Dynamic Cell Populations and Differential Gene Expression Patterns in Control and Aneurysmal Human Aortic Tissue. Circulation. 2020 Oct 6; 142(14): 1374-1388, incorporated herein by reference in its entirety). Data from across 3 normal aortae samples was re-clustered before removing hematopoietic clusters based on known marker expression and SingleR labeling analysis. Remaining clusters were identified based on SingleR labelling as primarily mesenchymal (M), however the human counterparts to the EPC cluster specifically were identified as clusters 3 and 12 based on a high degree of overlap between top differentially expressed (DE) genes in these clusters and EPC cluster 4 in the murine sc-RNA seq dataset (Table 1 ).Table 1 : overlapping genes from top 100 differentially expressed genes of clusters 3 and 12 from human normal aorta dataset and murine aorta sc-RNA seq PROCR+ EPC cluster

[0421] These overlapping genes were found to be implicated in endothelial, mesenchymal, extracellular matrix, and cell cycle pathways, indicative of genes maintaining a population between endothelial and mesenchymal states. Genes such as ACKR3 (or CXCR7) may be be downstream of PDGFRa signalling and contributing to vasculogenesis, potentially highlighting their importance in a presumed endothelial progenitor population. Other genes listed play essential roles in endothelial identify or regulation of mesenchymal transition and fibrosis. Markers of interest outlined above were then analyzed in remaining endothelial, MSC, and EPC-like clusters usingFeaturePlots, Dot Plots of top DE genes, and pathway analysis (FIGS. 6A and 6B). Major endothelial genes PECAM1 and CDH5 were most highly expressed in differentiated endothelial cluster 8 along with ABCG2 and SOX18.CD34, PROCR, and PDGFRA were expressed in EPC clusters 3 and 12 as well as differentiated endothelial cell cluster 8.

[0422] Upon confirmation that the expression of markers of interest in human control aorta using scRNA-seq resembled the expression seen in mouse models described previously, functional assays were conducted to investigate whether progenitor capacity was increased in human cells expressing these markers as seen in murine studies. A model of human full-term placental cells was used in line with previously described experiments of the EPC population termed ECFCs. Placental cells were isolated and FACS-sorted as described previously (Nano R, et al., High-yield isolation of pure fetal endothelial colony forming cells and mesenchymal stem cells from the human full-term placenta. STAR Protoc. 2022 Apr 23;3(2):101354, incorporated herein by reference in its entirety), with the additional gating of PROCR+ / _for each population of varying CD31 expression (negative, low, intermediate, and high) (FIG. 6C). These populations were then cultured on collagen coated plates until colonies formed. Across all donors, only 1 / 13 wells containing CD31intPROCR’ cells formed a colony, only growing to <50 cells before dying therefore classifying as an endothelial cluster (EC; FIG. 7A (i-ii), n=3). Conversely, 5 / 7 wells containing CD31 intPROCR+ cells grew colonies across all donors, with 3 / 5 of these colonies continually expanding and reaching the size of high proliferative potential (HPP; >1000 cells) colonies (FIG. 7A (i- ii)). These HPP colonies were then passaged and showed the ability to continually expand through P6, as well as to form additional LPPs and ECs in limiting dilution. Moreover, IF staining of these colonies confirmed the endothelial nature of this population with positive expression of CD31 and VE-cadherin. The remaining 2 / 5 colonies formed ECs (FIG. 7A (i-ii)).

[0423] Beyond expressional confirmation, consensus EPCs were tested functionally in a series of assays. Consensus EPCs showed increased functional progenitor capacity compared to PROCR- EPCs or endothelial differentiated cell populations regardless of their expression of PROCR. Consensus EPCs consistently formed more colonies in vitro than all other populations, as well as being the only population to form strictly endothelial colonies both morphologically and based onpositive staining for endothelial marker Isolectin. PROCR+ mature differentiated endothelial cells formed 15-fold fewer colonies, with none demonstrating an endothelial phenotype, recapitulating that the overlap of both PROCR and EPC marker expression (Lin’CD34+VE-cadherin+CD31l0) in the endothelium may be required to enrich for functional progenitor capacity. Moreover, EPCs from murine aortae showed increased engraftment potential in a 3D collagen matrix in vivo, forming vascular networks within gels embedded in hosts that stained positive for mature endothelial markers after 7 days of implantation, to a significantly higher degree than PROCR’ EPCs while mature differentiated endothelial cells failed to engraft regardless of their PROCR expression. These experiments clearly highlight the superior self-renewal capacity of the CEPC population beyond EPC definition or PROCR staining alone.

[0424] Fate tracing of PROCR+PDGFRA+EPCs from DO timepoints in homeostatic aorta and full-skin excisional wounds using Pdgfra-MerCreMer / ROSA- EYFP mice both showed the ability, via IF and flow cytometry, to differentiate into mature differentiated endothelial cells. This formally demonstrates for the first time that mesenchymal marker PDGFRA marks a population capable of endothelial fate in both homeostasis and injury. Particularly, during homeostasis, few reporters may distinguish progenitors from differentiated cells in the endothelium. The intimal position of staining, the flow cytometric gating and the final endothelial fate of the PDGFRA-expressing YFP- labelled cells in the homeostatic aorta clearly point to their endothelial capacity. Finally, translation to human models showed using scRNA-seq that PROCR and PDGFRA may be expressed in an EPC-like population in the human normal aorta. Moreover, when further gated on positive PROCR expression, previously defined human term placental ECFCs show increased clonogenic capacity, forming colonies of higher yield and secondary colonies, and self-renewing to at least P6.

[0425] These data suggest that combining the expressional requirements of EPCs with expression of PROCR and PDGFRA characterizes a more specific progenitor population with increased functional capacity within the endothelial compartment of various tissue beds, both in murine and human models. The addition of stringent functional requirements as well as a wider array of cell surface markers allows for EPCs to be targeted more specifically, which may create possibilities for advances in both clinical applications of vascular therapeutics such as pro- and anti-angiogenics, as well as in the fields of tissue- and bio-engineering.Example 3: Method for isolating a PROCR* and PDGFRA+cells

[0426] Placental tissues were processed, and single-cell suspension was prepared as described in Patel et al., 2013. Stem Cells Transl Med. 2013 Nov;2(11 ):839-47 and Patel et al., Placenta. 2014 Nov;35(11 ):969-71. The isolated placental cluster of differentiation (CD)34+ single-cell suspension was incubated with human CD34-phycoerythrin (PE) (Bio-Rad (MCA1578PE); dilution 1 :25), human CD45- FITC (BioLegend (304006); dilution 1 :25) and human CD31 -V450 BD Biosciences (561653): dilution 1 :30) for 20min at 4 °C. In addition to the markers outlined in Example 1 , PROCR PE (1 :25) and PDGFRA antibody were added to panel. Cells were flow- sorted using FACS. Cell doublets were removed, and 7-amino-actinomycin D (7AAD) was used to exclude dead cells. Fluorescence minus one (FMO) control was used in gating the population of interest. Cells were then gated and sorted as previously described in Example 1 , with the addition of PROCR+ / _and PDGFRA+ / _gates.Example 4: Method for isolating a PROCR* and PDGFRA* cells

[0427] Placental tissues were processed, and single-cell suspension was prepared to isolate a PDGFRA+single cell population using a PDGFRA antibody.

[0428] The isolated placental PDGFRA+single-cell suspension was incubated with human PROCR PE. Cells were flow-sorted using FACS. Cell doublets were removed, and 7AAD was used to exclude dead cells. FMO control was used in gating the population of interest. Cells were then gated and sorted with a PROCR+ / - gate (FIGS. 7 and 8).Example 5: Method for isolating a PROCR* and PDGFRA* cells

[0429] Placental tissues were processed, and single-cell suspension was prepared to isolate a PROCR+single cell population using human PROCR PE antibody.

[0430] The isolated placental PROCR+single-cell suspension was incubated with human PDGFRA antibody. Cells were flow-sorted using FACS Cell doublets were removed, and 7AAD was used to exclude dead cells. FMO control was used in gating the population of interest. Cells were then gated and sorted with a PDGFRA+ / - gate FIGS. 7 and 8).Example 6: Human Placental Cells

[0431] Human placental tissues were obtained from healthy women undergoing caesarean deliveries at term (38-39 weeks of gestation), allowing for the isolation and use of any stem cell populations obtained from the placental tissue. PROCR+ cells were isolated from each sample, generating a single-cell suspension. Isolation was conducted as described in Shafiee, A et al., Meso-Endothelial Bipotent Progenitors from Human Placenta Display Distinct Molecular and Cellular Identity. Stem Cell Reports. 2018 Mar 13;10(3):890-904, the contents of which are incorporated herein by reference in their entirety.

[0432] The isolated placental PROCR+single-cell suspension was incubated with human PDGFRA antibody. Cells were flow-sorted using FACS Cell doublets were removed, and 7AAD was used to exclude dead cells. FMO control was used in gating the population of interest. Cells were then gated and sorted with a PDGFRA+ / - gate (FIG. 9). Cells were selected based CD45-CD34+CD31 Int phenotypes.

[0433] Colony formation was assessed upon FACS for present of ECFCs. Gene expression of PROCR+ ECFCs measured CD31 , CD34, VE-Cadherin, and PDGFRA expression in control ECFCs (ECFC-KK and ECFC-MG) compared to MSCs and PROCR+ cells (FIG. 10). The PROCR+ ECFCs were co-cultured with an equal amount of fpl-MSCs in endothelial growth medium for 5 days and assessed for live and dead cells (FIG. 11 ). Control ECFCs were isolated as a CD45-CD34+CD31 Int population.

[0434] Co-cultures were conducted according to Shafiee A et al., Priming of endothelial colony -forming cells in a mesenchymal niche improves engraftment and vasculogenic potential by initiating mesenchymal transition orchestrated by NOTCH signaling, The FASEB Journal, 2017. 31 (2): p. 610-624, the contents of which are incorporated herein by reference in their entirety. fPL-MSC and PROCR+ ECFCs were cocultured together in collagen coated flasks with 1 :1 ratio (5 x 105 cells: 5 x 105 cells) at a final concentration of 10 x 105 cells in endothelial growth medium (EGM2). In control groups, ECFCs and fPL-MSC were cultured alone (at a final concentration of 5 x 105 cells) in collagen coated flasks in EGM2. The cultures were maintained in EGM2 for the duration of the experiment and media was changed after 3 days.

[0435] After 5 days of coculture (or single culture in control groups), the cells were detached using dissociation reagent, washed, and resuspend in FACS buffer andtransferred to 2 ml microtubes. The cells were stained with mouse PE / Cy5 conjugated anti-human CD90 antibody and 7-AAD and V450 conjugated anti-human CD31 antibody at 4 °C. After 20 min, 1 ml FACS buffer was added to each microtube, and cells were centrifuged at 400 x g for 4 min and resuspended in 100 pl of ice-cold FACS buffer. Cells were filtered through a 40 pm cell strainer to reduce cell aggregates before running samples through FACS machine (BD Biosciences, USA). The CD31 + cells from PROCR+ECFCs cocultured with fPL-MSC were FACS sorted according to the PDGFRA+ / - gate.

[0436] The results demonstrated a distinct variation in cell populations between the ECFC alone group and the primed ECFC group. In the ECFC alone group, there were 49,831 cells in the CD31 + CD90- population and 2,424 cells in the CD31 + CD90+ population. Conversely, the primed ECFC group showed 19,066 cells in the CD31 + CD90- population and 8,146 cells in the CD31 + CD90+ population. Notably, only 4.6% of CD31 + cells were CD90+ in the ECFC alone group, whereas a significant 30% of CD31 + cells were CD90+ in the primed ECFC group. These findings indicate that priming ECFCs significantly increases the proportion of CD31 + CD90+ cells. The expression of this mesenchymal markers is an indicator of cellular mobility and potential for better vasculogenic capacity. Priming ECFCs significantly increased the proportion of CD31 + CD90+ cells. MSCs, and ECFCs were cultured alone, or co-cultured (primed ECFCs) for 5 days (FIG. 12).

[0437] Gene expression for CD31 , CD34, SLUG, SNAIL (NCBI Gene ID: 6615), DLL4, NOTCH1 , and VWF Awas assessed between M SC -cocultured and noncocultured PROCR+ECFCs using quantitative PCR (FIGS. 13A-D). These results demonstrate that MSC-cocultured ECFCs have increased expression in genes that may be implicated in vasculogenic ability.Additional Embodiments

[0438] Various embodiments of the present technology are set forth below in paragraphs

[0439] to

[0523] :

[0439] 1. A method of treating Von Willebrand Disease (VWD) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated endothelial progenitor cell (EPC) populationcomprising Protein C Receptor (PROCR)+ Platelet-Derived Growth Factor Receptor Alpha (PDGFRA)+ EPCs.

[0440] 2. A method of treating VWD in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0441] 3. A method of treating VWD in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising mesenchymal stem cells (MSCs).

[0442] 4. A method of treating VWD in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0443] 5. A method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

[0444] 6. A method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0445] 7. A method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0446] 8. A method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0447] 9. A method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceuticalcomposition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

[0448] 10. A method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0449] 11. A method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0450] 12. A method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0451] 13. A method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

[0452] 14. A method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0453] 15. A method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0454] 16. A method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0455] 17. The method of any one of embodiments 13-16, wherein the VWF protein level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0456] 18. The method of any one of embodiments 13-17, wherein the VWF protein level is increased in a plasma sample from the subject, relative to the control.

[0457] 19. A method of increasing a VWF gene expression level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0458] 20. A method of increasing a VWF gene expression level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0459] 21 . A method of increasing a VWF gene expression level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0460] 22. The method of any one of embodiments 19-21 , wherein the VWF gene expression level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0461] 23. The method of any one of embodiments 19-22, wherein the VWF gene expression level is increased in a plasma sample from the subject, relative to the control.

[0462] 24. A method of increasing a VWF protein activity level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0463] 25. A method of increasing a VWF protein activity level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0464] 26. A method of increasing a VWF protein activity level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0465] 27. The method of any one of embodiments 24-26, wherein the VWF protein activity level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0466] 28. The method of any one of embodiments 200-104, wherein the VWF protein activity level is increased in a plasma sample from the subject, relative to the control.

[0467] 29. A method of increasing a VWF protein multimer level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0468] 30. A method of increasing a VWF protein multimer level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0469] 31. A method of increasing a VWF protein multimer level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0470] 32. The method of any one of embodiments 29-31 , wherein the VWF protein multimer level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

[0471] 33. The method of any one of embodiments 29-32, wherein the VWF protein multimer level is increased in a plasma sample from the subject, relative to the control.

[0472] 34. A method of reducing one or more symptoms of VWD in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs, wherein the one or more symptoms are selected from the group consisting of blood loss, platelet clumping, and bruising.

[0473] 35. The method of embodiment 34, wherein the reduction in the blood loss level comprises a reduction in a duration or a frequency of epistaxis.

[0474] 36. The method of embodiment 34, wherein the reduction in the blood loss level comprises an increase in a blood clotting level, relative to the control.

[0475] 37. The method of any one of embodiments 34-36, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises an increase in blood clotting speed during or after an injury, relative to the control.

[0476] 38. The method of any one of embodiments 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a bleeding level during or after an injury, relative to the control.

[0477] 39. The method of embodiment 38, wherein the injury comprises a surgical injury.

[0478] 40. The method of any one of embodiments 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a bleeding level during or after childbirth, relative to the control.

[0479] 41. The method of any one of embodiments 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a menstrual bleeding level, relative to the control.

[0480] 42. The method of any one of embodiments 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a stool blood level or a rectal bleeding level, relative to the control.

[0481] 43. The method of any one of embodiments 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a bruising level, relative to the control.

[0482] 44. The method of embodiment 43, wherein the reduction in the bruising level comprises a reduction in a raised bruising level, relative to the control.

[0483] 45. A method of increasing a Weibel-Palade body (WPB) level in a subject in need thereof, relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

[0484] 46. A method of increasing a WPB level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0485] 47. A method of increasing a WPB level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0486] 48. A method of increasing a WPB level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0487] 49. The method of any one of embodiments 45-30, wherein the increase in the WPB level comprises an increase in WPB number or a WPB area.

[0488] 50. The method of any one of embodiments 1 -49, wherein the method does not increase a VWF alloantibody level in the subject, relative to the control.

[0489] 51 . The method of any one of embodiments 1 -50, wherein the subject has been diagnosed with VWD.

[0490] 52. The method of any one of embodiments 1 -51 , wherein the subject has a VWD selected from the group consisting of Type 1 VWD, Type 2A VWD, Type 2B VWD, Type 2M VWD, Type 2N VWD, and Type 3 VWD.

[0491] 53. The method of any one of embodiments 1 -52, wherein the method increases a Factor VIII gene expression level or a Factor VIII protein expression level, relative to a control.

[0492] 54. The method of any one of embodiments 1 -53, wherein the isolatedEPC population or the first isolated cell population comprises a CD45- / CD34+ phenotype.

[0493] 55. The method of any one of embodiments 1 -54, wherein the isolatedEPC population or the first isolated cell population comprises endothelial colony-forming cells (ECFCs).

[0494] 56. The method of any one of embodiments 1 -54, wherein the isolatedEPC population or the first isolated cell population is cocultured with an isolated MSC population.

[0495] 57. The method of embodiment 56, wherein the isolated EPC population or the first isolated cell population comprises an increase in a vasculogensis level, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0496] 58. The method of embodiment 57, wherein the vasculogenesis level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0497] 59. The method of embodiment 57 or 58, wherein the increase in the vasculogenesis level occurs during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing.

[0498] 60. The method of any one of embodiments 56-59, wherein the isolated EPC population or the first isolated cell population comprises an increase in a Thy-1 Cell Surface Antigen (CD90) gene expression level, a Von Willebrand Factor (VWF) gene expression level, a Delta-Like Canonical Notch Ligandal 4 (DLL4) gene expression level, a Snail Family Transcriptional Repressor 2 (SLUG) gene expression level, a CD34 Molecule (CD34) gene expression level, a Platelet endothelial cell adhesion molecule 1 (CD31 ) gene expression level, or a Neurogenic locus notch homolog (NOTCH) gene expression level, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0499] 61 . The method of embodiment 60, wherein the increase in the CD90 gene expression level, the VWF gene expression level, the DLL4 gene expression level, the SLUG gene expression level, the CD34 gene expression level, the CD31 gene expression level, or the NOTCH gene expression level comprises an increase by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0500] 62. The method of embodiment 60 or 61 , wherein the increase in theCD90 gene expression level, the VWF gene expression level, the DLL4 gene expression level, the SLUG gene expression level, the CD34 gene expression level, the CD31 gene expression level, or the NOTCH gene expression level occurs during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing.

[0501] 63. The method of any one of embodiments 56-62, wherein the isolated EPC population or the first isolated cell population comprises an increase in a CD90 protein level, a VWF protein level, a DLL4 protein level, a SLUG protein level, a CD34 protein level, a CD31 protein level, or a NOTCH protein level, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0502] 64. The method of embodiment 63, wherein the increase in the CD90 protein level, the VWF protein level, the DLL4 protein level, the SLUG protein level, theCD34 protein level, the CD31 protein level, or the NOTCH protein level comprises an increase by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

[0503] 65. The method of embodiment 63 or 64, wherein the increase in theCD90 protein level, the VWF protein level, the DLL4 protein level, the SLUG protein level, the CD34 protein level, the CD31 protein level, or the NOTCH protein level occurs during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing.

[0504] 66. The method of any one of embodiments 56-65, wherein a coculturing VWF gene expression level in the isolated EPC population or the first isolated cell population increases during or after coculturing.

[0505] 67. The method of embodiment 66, wherein the VWF gene expression level increases by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a VWF gene expression level in an isolated EPC population that is not cocultured with an isolated MSC population.

[0506] 68. The method of any one of embodiments 56-66, wherein a VWF protein level in the isolated EPC population or the first isolated cell population increases during or after coculturing.

[0507] 69. The method of embodiment 68, wherein the VWF protein level increases by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a VWF protein level in an isolated EPC population that is not cocultured with an isolated MSC population.

[0508] 70. The method of any one of embodiments 1 -62, wherein the pharmaceutical composition is a first composition formulated for administration before, during, or after administration of a second composition comprising an isolated MSC population to a subject in need thereof.

[0509] 71. The method of any one of embodiments 3, 4, 7, 8, 11 , 12, 15, 16,20, 21 , 45, 46, 30, 31 , 47, 48, or 70, wherein the first isolated cell population and the second isolated cell population are present in the composition at a ratio of about 1 :1.

[0510] 72. The method of any one of embodiments 3, 4, 7, 8, 11 , 12, 15, 16,20, 21 , 45, 46, 30, 31 , 47, 48, or 70, wherein the first isolated cell population and the second isolated cell population are present in the composition at a ratio of at least about 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 , 12:1 , 15:1 , 20:1 , 30:1 , 40:1 , or 50:1.

[0511] 73. The method of any one of embodiments 3, 4, 7, 8, 11 , 12, 15, 16,20, 21 , 45, 46, 30, 31 , 47, 48, or 70-72, wherein the MSCs are CD45- / CD34+ cells.

[0512] 74. The method of any one of embodiments 3, 4, 7, 8, 11 , 12, 15, 16,20, 21 , 45, 46, 30, 31 , 47, 48, or 70-73, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the second isolated cell population is a CD34+ / CD45- isolated cell population.

[0513] 75. The method of any one of embodiments 1 -74, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the isolated cell population or the second isolated cell population express PROCR and PDGFRA.

[0514] 76. The method of any one of embodiments 1 -74, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from a donor.

[0515] 77. The method of embodiment 76, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from two or more donors.

[0516] 78. The method of embodiment 76 or 77, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population comprise allogenic cells.

[0517] 79. The method of any one of embodiments 1 -78, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are present in a medium.

[0518] 80. The method of embodiment 79, wherein the medium comprises a liquid or frozen medium.

[0519] 81. The method of any one of embodiments 1 -80, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle.

[0520] 82. The method of embodiment 81 , wherein the pharmaceutically acceptable vehicle is phosphate-buffered saline.

[0521] 83. The method of any one of embodiments 1 -82, wherein the subject is administered one or more doses of the pharmaceutical composition.

[0522] 84. The method of embodiment 83, wherein the subject is administered two or more doses of the pharmaceutical composition.

[0523] 85. The method of any one of embodiments 1 -84, wherein the pharmaceutical composition is administered to the subject intravenously.

[0524] From the foregoing, it will be appreciated that specific embodiments of the present technology have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the present technology. Accordingly, the present technology is not limited except as by the appended claims.

Claims

CLAIMSI / We claim:1 . A method of treating Von Willebrand Disease (VWD) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated endothelial progenitor cell (EPC) population comprising Protein C Receptor (PROCR)+ Platelet-Derived Growth Factor Receptor Alpha (PDGFRA)+ EPCs.

2. A method of treating VWD in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

3. A method of treating VWD in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising mesenchymal stem cells (MSCs).

4. A method of treating VWD in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

5. A method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

6. A method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

7. A method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

8. A method of preventing or treating anemia in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

9. A method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

10. A method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

11. A method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

12. A method of increasing a blood clotting level in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

13. A method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

14. A method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

15. A method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceuticalcomposition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

16. A method of increasing a VWF protein level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

17. The method of any one of claims 13-16, wherein the VWF protein level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

18. The method of any one of claims 13-17, wherein the VWF protein level is increased in a plasma sample from the subject, relative to the control.

19. A method of increasing a VWF gene expression level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

20. A method of increasing a VWF gene expression level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.21 . A method of increasing a VWF gene expression level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

22. The method of any one of claims 19-21 , wherein the VWF gene expression level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

23. The method of any one of claims 19-22, wherein the VWF gene expression level is increased in a plasma sample from the subject, relative to the control.

24. A method of increasing a VWF protein activity level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

25. A method of increasing a VWF protein activity level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

26. A method of increasing a VWF protein activity level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

27. The method of any one of claims 24-26, wherein the VWF protein activity level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

28. The method of any one of claims 200-104, wherein the VWF protein activity level is increased in a plasma sample from the subject, relative to the control.

29. A method of increasing a VWF protein multimer level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

30. A method of increasing a VWF protein multimer level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.31 . A method of increasing a VWF protein multimer level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.-I OS-32. The method of any one of claims 29-31 , wherein the VWF protein multimer level is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to the control.

33. The method of any one of claims 29-32, wherein the VWF protein multimer level is increased in a plasma sample from the subject, relative to the control.

34. A method of reducing one or more symptoms of VWD in a subject in need thereof relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs, wherein the one or more symptoms are selected from the group consisting of blood loss, platelet clumping, and bruising.

35. The method of claim 34, wherein the reduction in the blood loss level comprises a reduction in a duration or a frequency of epistaxis.

36. The method of claim 34, wherein the reduction in the blood loss level comprises an increase in a blood clotting level, relative to the control.

37. The method of any one of claims 34-36, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises an increase in blood clotting speed during or after an injury, relative to the control.

38. The method of any one of claims 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a bleeding level during or after an injury, relative to the control.

39. The method of claim 38, wherein the injury comprises a surgical injury.

40. The method of any one of claims 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a bleeding level during or after childbirth, relative to the control.41 . The method of any one of claims 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a menstrual bleeding level, relative to the control.

42. The method of any one of claims 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a stool blood level or a rectal bleeding level, relative to the control.

43. The method of any one of claims 34-37, wherein the increase in the blood clotting level or the reduction in the blood loss level comprises a reduction in a bruising level, relative to the control.

44. The method of claim 43, wherein the reduction in the bruising level comprises a reduction in a raised bruising level, relative to the control.

45. A method of increasing a Weibel-Palade body (WPB) level in a subject in need thereof, relative to a control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ PDGFRA+ EPCs.

46. A method of increasing a WPB level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

47. A method of increasing a WPB level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

48. A method of increasing a WPB level in a subject in need thereof relative to the control, comprising administering to the subject a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

49. The method of any one of claims 45-30, wherein the increase in the WPB level comprises an increase in WPB number or a WPB area.

50. The method of any one of claims 1 -49, wherein the method does not increase a VWF alloantibody level in the subject, relative to the control.

51. The method of any one of claims 1 -50, wherein the subject has been diagnosed with VWD.-no-52. The method of any one of claims 1 -51 , wherein the subject has a VWD selected from the group consisting of Type 1 VWD, Type 2A VWD, Type 2B VWD, Type 2M VWD, Type 2N VWD, and Type 3 VWD.

53. The method of any one of claims 1 -52, wherein the method increases a Factor VIII gene expression level or a Factor VIII protein expression level, relative to a control.

54. The method of any one of claims 1 -53, wherein the isolated EPC population or the first isolated cell population comprises a CD45- / CD34+ phenotype.

55. The method of any one of claims 1 -54, wherein the isolated EPC population or the first isolated cell population comprises endothelial colony-forming cells (ECFCs).

56. The method of any one of claims 1 -54, wherein the isolated EPC population or the first isolated cell population is cocultured with an isolated MSC population.

57. The method of claim 56, wherein the isolated EPC population or the first isolated cell population comprises an increase in a vasculogensis level, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

58. The method of claim 57, wherein the vasculogenesis level is increased by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

59. The method of claim 57 or 58, wherein the increase in the vasculogenesis level occurs during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing.

60. The method of any one of embodiments 56-59, wherein the isolated EPC population or the first isolated cell population comprises an increase in a Thy-1 Cell Surface Antigen (CD90) gene expression level, a Von Willebrand Factor (VWF) gene-in-expression level, a Delta-Like Canonical Notch Ligandal 4 (DLL4) gene expression level, a Snail Family Transcriptional Repressor 2 (SLUG) gene expression level, a CD34 Molecule (CD34) gene expression level, a Platelet endothelial cell adhesion molecule 1 (CD31 ) gene expression level, or a Neurogenic locus notch homolog (NOTCH) gene expression level, relative to an isolated EPC population that is not cocultured with an isolated MSC population61. The method of claim 60, wherein the increase in the CD90 gene expression level, the VWF gene expression level, the DLL4 gene expression level, the SLUG gene expression level, the CD34 gene expression level, the CD31 gene expression level, or the NOTCH gene expression level comprises an increase by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

62. The method of claim 60 or 61 , wherein the increase in the CD90 gene expression level, the VWF gene expression level, the DLL4 gene expression level, the SLUG gene expression level, the CD34 gene expression level, the CD31 gene expression level, or the NOTCH gene expression level occurs during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing.

63. The method of any one of claims 56-62, wherein the isolated EPC population or the first isolated cell population comprises an increase in a CD90 protein level, a VWF protein level, a DLL4 protein level, a SLUG protein level, a CD34 protein level, a CD31 protein level, or a NOTCH protein level, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

64. The method of claim 63, wherein the increase in the CD90 protein level, the VWF protein level, the DLL4 protein level, the SLUG protein level, the CD34 protein level, the CD31 protein level, or the NOTCH protein level comprises an increase by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to an isolated EPC population that is not cocultured with an isolated MSC population.

65. The method of claim 63 or 64, wherein the increase in the CD90 protein level, the VWF protein level, the DLL4 protein level, the SLUG protein level, the CD34 protein level, the CD31 protein level, or the NOTCH protein level occurs during coculturing or at least about 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after coculturing.

66. The method of any one of claims 56-65, wherein a coculturing VWF gene expression level in the isolated EPC population or the first isolated cell population increases during or after coculturing.

67. The method of claim 66, wherein the VWF gene expression level increases by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a VWF gene expression level in an isolated EPC population that is not cocultured with an isolated MSC population.

68. The method of any one of claims 56-66, wherein a VWF protein level in the isolated EPC population or the first isolated cell population increases during or after coculturing.

69. The method of claim 68, wherein the VWF protein level increases by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, relative to a VWF protein level in an isolated EPC population that is not cocultured with an isolated MSC population.

70. The method of any one of claims 1 -62, wherein the pharmaceutical composition is a first composition formulated for administration before, during, or afteradministration of a second composition comprising an isolated MSC population to a subject in need thereof.71 . The method of any one of claims 3, 4, 7, 8, 11 , 12, 15, 16, 20, 21 , 45, 46, 30, 31 , 47, 48, or 70, wherein the first isolated cell population and the second isolated cell population are present in the composition at a ratio of about 1 :1.

72. The method of any one of claims 3, 4, 7, 8, 11 , 12, 15, 16, 20, 21 , 45, 46, 30, 31 , 47, 48, or 70, wherein the first isolated cell population and the second isolated cell population are present in the composition at a ratio of at least about 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 , 12:1 , 15:1 , 20:1 , 30:1 , 40:1 , or 50:1 .

73. The method of any one of claims 3, 4, 7, 8, 11 , 12, 15, 16, 20, 21 , 45, 46, 30, 31 , 47, 48, or 70-72, wherein the MSCs are CD45- / CD34+ cells.

74. The method of any one of claims 3, 4, 7, 8, 11 , 12, 15, 16, 20, 21 , 45, 46, 30, 31 , 47, 48, or 70-73, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the second isolated cell population is a CD34+ / CD45- isolated cell population.

75. The method of any one of claims 1 -74, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the isolated cell population or the second isolated cell population express PROCR and PDGFRA.

76. The method of any one of claims 1 -74, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from a donor.

77. The method of claim 76, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from two or more donors.

78. The method of claim 76 or 77, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population comprise allogenic cells.

79. The method of any one of claims 1 -78, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are present in a medium.

80. The method of claim 79, wherein the medium comprises a liquid or frozen medium.

81. The method of any one of claims 1 -80, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle.

82. The method of claim 81 , wherein the pharmaceutically acceptable vehicle is phosphate-buffered saline.

83. The method of any one of claims 1 -82, wherein the subject is administered one or more doses of the pharmaceutical composition.

84. The method of claim 83, wherein the subject is administered two or more doses of the pharmaceutical composition.

85. The method of any one of claims 1 -84, wherein the pharmaceutical composition is administered to the subject intravenously.

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