Compositions comprising isolated endothelial progenitor cells and uses thereof

Abstract

The present technology comprises isolated endothelial progenitor cell (EPC) populations comprising PROCR+ / - PDGFRA+ / - EPCs and mesenchymal stem cell (MSC) populations, and methods of making and use thereof in treating hypoxic-ischemic encephalopathy (HIE) or brain injury in a subject.

Description

COMPOSITIONS COMPRISING ISOLATED ENDOTHELIALPROGENITOR CELLS AND USES THEREOFBACKGROUND

[0001] Vascularization is an essential process for both the growth of developing organs and the circulation of blood, whether in tissue maintenance or repair. Vasculogenesis and neovascularization both rely on a population of progenitor cells which self-renew, differentiate into mature endothelial cells, merging to form blood vessels de novo. As the endothelial layer is at continuous risk of defects, repair mechanisms may be permanently active via endothelial progenitor cells (EPCs). Numerous clinical trials using different cell types to promote vascular repair have been assesed. The most frequent sources utilized were early outgrowth EPCs from the peripheral circulation and bone marrow mononuclear cells (MNC). A major limitation in these early clinical studies relate to how EPCs were defined. 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 vascular endothelial (VE)-cadherin or CD31 . However, the combination of CD34, VEGFR2, CD133, VE-cadherin and / or CD31 markers has not produced a reliable or discriminatory marker set. As such, there remains a need to provide more effective therapeutic treatments based on the use of EPCs, either alone, or in combination with other cell types.SUMMARY

[0002] The present technology comprises pharmaceutical compositions comprising or consisting of isolated endothelial progenitor cell (EPC) populations, and methods of making and using the same.

[0003] In some embodiments, the present technology comprises a pharmaceutical composition comprising or consisting of an isolated endothelial progenitor cell (EPC) population comprising PROCR+ PDGFRA+ EPCs.

[0004] In some embodiments, the present technology comprises a pharmaceutical composition comprising or consisting of an isolated EPC population comprisingPROCR+ / - PDGFRA+ / - EPCs for use in treating neonatal hypoxic-ischemic encephalopathy (HIE) in a subject in need thereof.

[0005] In some embodiments, the present technology comprises a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0006] In some embodiments, the present technology comprises a pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs for use in treating a brain injury in a subject in need thereof.

[0007] In some embodiments, the isolated EPC population comprises an increase in a PROCR, a PDGFRA, or a VE-Cadherin protein expression level relative to an expression level in a non-isolated EPC population.

[0008] In some embodiments, the increase in the PDGFRA protein expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PDGFRA protein expression level in the nonisolated EPC.

[0009] In some embodiments, the increase in the PROCR protein expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PROCR protein expression level in the non-isolated EPC.

[0010] In some embodiments, the isolated EPC population comprises an increase in a PROCR, a PDGFRA, or a VE-Cadherin gene expression level relative to an expression level in a non-isolated EPC population.

[0011] In some embodiments, the increase in the PROCR gene expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PROCR gene expression level in the non-isolated EPC.

[0012] In some embodiments, the increase in the PDGFRA gene expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PDGFRA gene expression level in the non-isolated EPC.

[0013] In some embodiments, the increase in the VE-Cadherin gene expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a VE-Cadherin gene expression level in the nonisolated EPC.

[0014] In some embodiments, the isolated EPC population comprises an increased proliferative capacity relative to a non-isolated EPC population.

[0015] In some embodiments, the isolated EPC population comprises an increased angiogenic capacity relative to a non-isolated EPC population.

[0016] In some embodiments, the increase in the angiogenic capacity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to an angiogenic capacity level in the non-isolated EPC.

[0017] In some embodiments, the isolated EPC population comprises an increased in a colony forming capacity or a tube formation capacity relative to a nonisolated EPC population.

[0018] In some embodiments, the increase in the colony forming capacity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a colony forming capacity level in the non-isolated EPC.

[0019] In some embodiments, the increase in the tube forming capacity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a tube forming capacity level in the non-isolated EPC.

[0020] In some embodiments, the isolated EPC population comprises an increased engraftment potential relative to a non-isolated EPC population.

[0021] In some embodiments, the increase in the engraftment potential is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to an engraftment potential level in the non-isolated EPC.

[0022] In some embodiments, the isolated EPC population comprises one or more EPCs having a more elongated cell shape, relative to one or more EPCs in a nonisolated EPC population.

[0023] In some embodiments, the one or more EPCs are at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% more elongated compared to the non-isolated EPC population.

[0024] In some embodiments, the isolated EPC population comprises a CD45- / CD34+ phenotype.

[0025] 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 mesenchymal stem cell (MSC) population to a subject in need thereof.

[0026] In some embodiments, the present technology comprises a pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and (b) a second isolated cell population comprising MSCs.

[0027] In some embodiments, the present technology comprises 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 pharmaceutical composition for use in treating HIE in a subject in need thereof, the 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 HIE is neonatal HIE.

[0030] In some embodiments, the present technology comprises a pharmaceutical composition for use in treating a brain injury in a subject in need thereof, the pharmaceutical composition comprising (a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and (b) a second isolated cell population comprising MSCs.

[0031] In some embodiments, the brain injury comprises neurodegeneration.

[0032] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle.

[0033] In some embodiments, the pharmaceutically acceptable vehicle is phosphate-buffered saline.

[0034] In some embodiments, the pharmaceutical composition is formulated for intranasal delivery, intrathecal, intraarterial, intralesional, or intravenous delivery to a subject in need thereof.

[0035] In some embodiments, the brain injury comprises ischemic brain injury.

[0036] In some embodiments, the subject has received or is receiving a therapeutic hypothermia treatment.

[0037] 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.

[0038] 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.

[0039] In some embodiments, the MSCs are CD45- / CD34+ cells.

[0040] 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.

[0041] 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.

[0042] In some embodiments, the present technology comprises a method of isolating an EPC population comprising PROCR+ PDGFRA+ EPCs, the method comprising 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 aPROCR+ and PDGFRA+ phenotype, thereby obtaining the isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

[0043] In some embodiments, the present technology comprises a method of isolating an EPC population comprising PROCR+ / - PDGFRA+ / - EPCs, the method comprising 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 the isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

[0044] In some embodiments, step (ii) comprises selecting cell 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.

[0045] 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).

[0046] 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 population of cells which are CD45- / CD34+.

[0047] 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.

[0048] In some embodiments, the present technology comprises a method of isolating an EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs, the method comprising 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 ofPDGFRA+ cells, cells which express a PROCR+ phenotype, thereby obtaining the isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

[0049] In some embodiments, the present technology comprises a method of isolating an EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs, the method comprising 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.

[0050] In some emnodiments, 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.

[0051] 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.

[0052] In some embodiments, the present technology comprises a method of isolating an EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs, the method comprising 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.

[0053] In some embodiments, the present technology comprises a method of isolating an EPC population comprising or consisting of PROCR+ / -PDGFRA+ / - EPCs, the method comprising 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.

[0054] In some embodiments, step (ii) 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.

[0055] In some embodiments, step (iii) 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.

[0056] In some embodiments, the binding molecule comprises a protein.

[0057] In some embodiments, the protein comprises an antibody.

[0058] In some embodiments, removing the complex comprises microfluidic sorting.

[0059] In some embodiments, the microfluidic sorting comprises microbead sorting or flow cytometry.

[0060] In some embodiments, the flow cytometry comprises fluorescence- activated cell sorting.

[0061] In some embodiments, the steps 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.

[0062] In some embodiments, the steps further comprise step (v), separating the isolated EPC population from the cell population comprising ECFCs or the cell population comprising MSCs.

[0063] In some embodiments, step (v) occurs 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).

[0064] In some embodiments, the biological sample is a mammalian biological sample.

[0065] In some embodiments, the 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.

[0066] In some embodiments, the mammalian placenta is a whole mammalian placenta.

[0067] In some embodiments, the mammalian tissue-resident vascular endothelium is selected from the group consisting of a mammalian umbilical cord, a mammalian pulmonary artery endothelium, a mammalian aorta, and a mammalian lung tissue.

[0068] In some embodiments, the PROCR+ PDGFRA+ EPCs or the PROCR+ / - PDGFRA+ / - EPCs express one or more proteins selected from the group consisting of CD32, CDH5, CD34, CD31 , VEGFR2, VE-Cadherin and CD157.

[0069] In some embodiments, the PROCR+ PDGFRA+ EPCs or the PROCR+ PDGFRA+ EPCs do not express one or more hematopoietic proteins.

[0070] In some embodiments, the one or more hematopoietic proteins are selected from the group consisting of CD3e, CD11 b, CD45, and B220.

[0071] In some embodiments, the PROCR+ PDGFRA+ EPCs or the PROCR / -+ PDGFRA+ / - EPCs decrease a gene expression level of one of more of CD157, ABCG2, or SOX18 when differentiated, relative to an undifferentiated PROCR+ PDGFRA+ EPC or an undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0072] In some embodiments, the decrease in the gene expression level of CD157 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a gene expression level of CD157 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0073] In some embodiments, the decrease in the gene expression level of ABCG2 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a gene expression level of ABCG2 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0074] In some embodiments, the decrease in the gene expression level of SOX18 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a gene expression level of SOX18 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0075] In some embodiments, the PROCR+ PDGFRA+ EPCs or the PROCR / -+ PDGFRA+ / - EPCs decrease a protein expression level of one of more of CD157, ABCG2, or SOX18 when differentiated, relative to an undifferentiated PROCR+ PDGFRA+ EPC or an undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0076] In some embodiments, the isolated cell population, the first isolated cell population, or the second isolated cell population comprises a mammalian cell.

[0077] In some embodiments, the mammalian cell is a human cell.

[0078] In some embodiments, the mammalian cell is a placental cell.

[0079] In some embodiments, the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from a donor.

[0080] 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.

[0081] In some embodiments, the isolated cell population, the first isolated cell population, or the second isolated cell population comprise autologous or allogenic cells.

[0082] In some embodiments, the isolated cell population, the first isolated cell population, or the second isolated cell population are present in a medium.

[0083] In some embodiments, the medium comprises a liquid medium or a frozen medium.BRIEF DESCRIPTION OF THE DRAWINGS

[0084] FIGS. 1 A-1 D shows 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 expressed genes in an endothelial progenitor cell (EPC) cluster. FIG. 1 D shows top differentially expressed genes in mesemchymal stem cell (MSC) clusters.

[0085] FIGS. 2A-2I shows single-cell RNA-sequencing and flow cytometry that demonstrate PROCR and PDGFRA may be markers in endothelial populations. FIG. 2A shows single-cell RNA sequencing data of endothelial from murine aorta showing aEPCs, mature differentiated endothelial cells and MSC clusters of interest (n=3). FIG. 2B shows individual plots of expression of markers of interest in each cluster. FIG. 2C shows relative expression of genes of interest across clusters. FIG. 2D shows 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 may be shown for FIG. 2E endothelial progenitor cells (EPC) and FIG. 2F maturedifferentiated endothelial cell populations. FIG. 2G 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. 2H 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. 2I shows quantification of percent of EPCs gating using gating strategy in FIG. 2E (****, p<0.0001 , n=3).

[0086] FIGS. 3A-3G show that PROCR+EPCs show increased endothelial colony formation capacity in vitro and engraftment potential in vivo compared to other populations. FIG. 3A shows endothelial cells from C57BI / 6 aorta were FACS sorted based on cell surface expression of PROCR as depicted in graphical representation. FIG. 3B shows representative brightfield images (i-ii) depicting endothelial (i) and elongated (ii) cell morphology types at day 12 (4x magnification; scale bar = 1 mm) and IF (iii-iv) of endothelial (iii) and elongated (iv) colonies on day 12 with Isolectin BSL-I (10x magnification; scale bar = 150 pm). FIG. 3C shows the percentage of colonies formed in each condition based on number of wells plated with all experiments normalized to 10 cells per well (n=14; * p<0.05). FIG. 3D shows a graphical representation depicting experimental procedure for in vivo collagen gel engraftment assay. FIG. 3E 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. 3F show the percentage of GFP+ area of each gel upon collection measured via IF (* p<0.05; n=3). FIG. 3G 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).

[0087] FIGS. 4A-4F show that PROCR+EPCs form a niche in the thoracic aorta displaying increased clonogenic capacity. FIGS. 4A-4C show aortae collected from Cdh5-CreERT2 / ROSA-EYFP mice, opened and rolled lengthwise. FIGS. 4A and 4B show representative images of sections of thoracic and abdominal aorta respectively. FIG. 4C shows a 60x magnification of the thoracic aorta section shown in FIG. 4A; white arrows indicate regions of overlap between DAPI, PROCR, and YFP. FIG. 4D shows the percentage of PROCR+length in abdominal and thoracic aorta (** p=0.005; n=5).FIG. 4E shows representative brightfield image of colony grown from thoracic aorta of Zs-Green / ROSA-EYFP mice in Matrigel following 12 days; scale bar = 500 pm. FIG. 4F shows quantification of colonies grown from thoracic and abdominal aorta (**, p=0.0052, n=13).

[0088] FIGS. 5A-5E show PROCR+PDGFRA+EPCs from Pdgfra- MerCreMer / Rosa-YFP differentiate into mature endothelial cells in homeostatic aorta. FIG. 5A (i-ii) shows the gating strategy for isolating the YFP+PROCR+PDGFRA+EPCs and mature differentiated endothelial cells from the aorta of PDGFRa-MerCreMer / Rosa- YFP mice; (iii) Percent of PROCR+PDGFRA+EPCs and mature differentiated endothelial cells in the Lin_YFP+fraction of the adult homeostatic aorta (****, p<0.0001 , n=4). FIG. 5B shows representative IF image showing YFP+PROCR+PDGFRA+EPCs cultured for 12 days from PDGFRa-MerCreMer / Rosa-YFP mice. FIG. 5C shows representative IF image showing aorta section from D1 (i) and D84 (ii; scale bars: large image = 200 pm, zoomed images = 10 pm). FIG. 5D shows Lin_YFP+compartment of homeostatic aorta from mice injected with tamoxifen at 4 weeks of age showing changes in proportions of PROCR+PDGFRA+EPCs; (ii-iv) shown in red oval, and mature differentiated endothelial cells; (ii-iv) shown in black) between D1 and D84. FIG. 5E 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).

[0089] FIGS 6A-6E show PROCR+EPCs from Pdgfra-MerCreMer / Rosa-YFP differentiate into mature endothelial cells in an injury model of full-skin excisional wounds. FIGS. 6A and 6B show representative IF images showing aorta section from D1 and D84 with endothelial markers. FIG. 6B shows results with scale bars: large image = 200 pm, zoomed images = 10 pm). FIG. 6C and 6D show Lin_YFP+compartment of full-skin excisional wounds from adult mice showing changes in proportions of PROCR+PDGFRA+EPCs ((ii-iv) shown in red oval in FIG. 6C); (ii-iv) shown in black in FIG. 6D) between D1 and D5. FIG. 6E 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).

[0090] FIGS. 7A-7F show PROCR is expressed in human aorta scRNA-seq data and leads to increased clonogenic capacity in a human term placental model ofendothelial colony forming cells (ECFCs). FIG. 7A shows single-cell RNA-sequencing data from human control aorta, clustered and filtered to remove hematopoietic clusters and label primary populations (n=3). FIG. 7B shows markers of interest shown across clusters. FIG. 7C shows flow cytometry plots showing the gating strategy of PROCR+ / ’ EPCs from human term placenta. FIG. 7D shows brightfield images of colonies growing from cultured PROCR’ (i) or PROCR+(ii) EPCs (scale bar = 200 pm). FIG. 7E shows immunofluorescent staining of PROCR+colony at P6. FIG. 7F shows quantification of (i) colony counts and (ii) colony types formed from cultured PROCR’ or PROCR+EPCs.

[0091] FIGS. 8A-8E show additional single cell RNA-sequencing analyses and flow cytometric FMOs for human datasets. FIG. 8A shows clustering across three samples of normal human aorta. FIG. 8B shows SingleR unbiased labelling analysis. FIGS. 8C and 8D show dot plots of top differentially expressed genes in EPC (FIG. 8C) and MSC (FIG. 8D) clusters. FIG. 8E shows FMO controls for human term placenta FACS-sorting.

[0092] FIG. 9 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.

[0093] FIG. 10 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.

[0094] FIG 11 . shows a photograph of the experimental set up and sample data. Oxygen saturation, arterial blood pressure, end-tidal CO2 and rectal temperature were acquired via the Marquette data acquisition system.

[0095] FIG. 12 shows a schematic of the experimental set up and sample data from FIG. 11

[0096] FIG. 13 shows increased survival in the piglets treated with combined hypothermia and PROCR+ / ’ PDGFRA+ / ’ EPC and MSC cell therapy (HHS group). Statistical comparisons: Mantel-Cox test of equality of survival distribution between groups. HHS: hypothermia and stem cell treated groups; HHV: hypothermia and stem vehicle treated groups.

[0097] FIG. 14 shows mean aEEG score and background amplitude score in HI injured piglets. Statistical comparisons: Welch's t-test, adjusted using Holm-Sidak method. HHS: hypothermia and stem cell treated groups; HHV: hypothermia and stem vehicle treated groups.

[0098] FIGS. 15A-15J show MRI, MRS and neurobehavioral outcome measures at P8. FIG. 15A shows representative T2 maps and ADC maps for hypothermia and stem cell (PROCR+ / _PDGFRA+ / _EPC and MSC) treated (HHS) and hypothermia and stem vehicle treated (HHV) groups at P8. White circles indicate the voxel region used for quantification. FIGS. 15B and 15C show quantitative MRI measures T2 relaxation time and ADC value between hypothermia and stem cell (PROCR+ / _PDGFRA+ / _EPC and MSC) treated (HHS) and hypothermia and stem vehicle treated (HHV) groups respectively. FIG. 15D shows representative magnetic resonance spectroscopy of the brain at P8. T2-weighted image shows the voxel's location for proton magnetic resonance spectroscopy from the coronal and sagittal views. FIGS. 15E-15I show ratios of NAA / Cho, NAA / Cr, Cho / Cr, Lac / NAA and Lac / Cr respectively. FIG. 15J shows mean neurobehavioral scores over time with the mean neurobehavioural scores significantly reduced at P3 following the insult. The dashed line indicates a perfect score.

[0099] FIGS. 16A-16C show representative hematoxylin and eosin staining. FIGS. 16A-16C show reduction in regional neuropathology within the parasagittal frontal cortex. High magnification images (FIGS. 16A’-16C’) show the presence of eosinophilic neurons (tailed arrow) indicative of acute neuronal injury that may be not present in the hypothermia and stem cell (PROCR+ / _PDGFRA+ / _EPC and MSC) treated (HHS) group or the control group. Immune cells may be also shown (untailed arrow). Scale bar: 50pm on high magnification images.

[0100] FIGS. 17A-17R show altered expression of neuropathological markers in the frontal cortex of HI injured piglets at P8. FIGS. 17A-17C show immunofluorescent labelling of mature neurons using the neuron-specific nuclear marker NeuN in piglet brains. Scale bar: 250 pm. FIGS. 17D, 171, and 17N show NeuN+cells per mm2, FJC+cells per mm2and C-caspase 3+cells per mm2quantification respectively in the frontal cortex. FIGS. 17E, 17J, and 170 show NeuN+cells per mm2, FJC+cells per mm2and C-caspase 3+cells per mm2quantification respectively in the putamen. FIGS. 17F to 17H show representative staining of degenerating neurons with Fluoro-Jade Cobserved in both hypothermia and stem cell (PROCR+ / _PDGFRA+ / _EPC and MSC) treated (HHS) and hypothermia and stem vehicle treated (HHV) brains (scale bar: 100 pm). Quantification demonstrated regional differences in numbers of degenerating neurons may be shown in FIGS. 171 and 17J. FIGS. 17P-17R show imaging overlays for each group. C: Control; HHS: hypothermia and stem cell treated groups; HHV: hypothermia and stem vehicle treated groups; FC; Frontal Cortex: Put: putamen.

[0101] FIGS. 18A-18O show representative images of lba-1 labelling in the frontal cortex showing examples of resting-state microglia (thin tailed arrows) in the control (FIGS. 18 A and FIGS. 18A’) and the hypothermia and stem cell (PROCR+ / _PDGFRA+ / _EPC and MSC) treated (HHS) (FIGS. 18C, FIGS. 18C’) groups and activated microglia (thick tailed arrows) in the hypothermia and stem vehicle treated (HHV) (FIGS. 18B, FIGS. 18B’) group. Classification of microglial morphology is shown in both the frontal cortex (FIGS. 18D and 18E) and the putamen (FIGS. 18H and 181) in the hypothermia and stem cell (PROCR+ / _PDGFRA+ / _EPC and MSC) treated (HHS) treated group. FIGS. 18F and 18J show an analysis of the total number of microglia and stem vehicle treated (HHV) groups in the frontal cortex (FIG. 18F) or the putamen (FIG. 18J). FIGS. 18K to 18M show representative images of astrocytes (GFAP) in the intragyral white matter. FIGS. 18K to 18M show astrocytes in HHS group animals displayed long extended processes and large cell bodies, like those observed in the healthy controls (indicated by the thin arrows). FIG. 18N show no significant difference in the IGWM. FIG. 180 shows that HHV differed significantly from HHS in the PVWM. Column heights denote the median and error bars denote 95%CI [lower, upper], C: Control; HHS: hypothermia and stem cell treated groups; HHV: hypothermia and stem vehicle treated groups; FC: frontal cortex; Put: putamen.

[0102] FIG. 19 shows gene expression analysis of various inflammatory markers at P8. C: Control; HHS: hypothermia and stem cell treated groups; HHV: hypothermia and stem vehicle treated groups.

[0103] FIG. 20 shows colocalization of TNFa and IL-1 [3 with lba-1 in HHV and HHS. Thin tailed arrows indicate TNFa colocalised with lba-1. Thick tailed arrows indicate IL-1 [3 colocalised with lba-1. Scale bar: 100 pm. C: Control; HHS: hypothermia and stem cell treated groups; HHV: hypothermia and stem vehicle treated groups.

[0104] FIG. 21 shows differential colocalization of NF-KB p65 between hypothermia and stem cell (PROCR+ / _PDGFRA+ / _EPC and MSC) treated (HHS) and hypothermia and stem vehicle treated (HHV) groups. Low power image scale bar: 200pm. C: Control; HHS: hypothermia and stem cell treated groups; HHV: hypothermia and stem vehicle treated groups.

[0105] FIG. 22 shows representative images of CD34 labelling in the parasagittal region of the frontal cortex. C: Control; HHS: hypothermia and stem cell treated groups; HHV: hypothermia and stem vehicle treated groups.

[0106] FIG. 23 shows flow cytometry results of human placental PROCR+ cells sorted for various expression markers.

[0107] FIG. 24 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.

[0108] FIG. 25 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

[0109] FIG. 26 shows flow cytometry results of CD31 + cells sorted from PROCR+ECFCs cocultured with fPL-MSCs.DETAILED DESCRIPTION

[0110] The present technology comprises pharmaceutical compositions comprising or consisting of isolated endothelial progenitor cell (EPC) populations, and methods of making and using the same. In some embodiments, the pharmaceutical compositions further comprise an isolated cell population comprising mesenchymal stem cells (MSCs). These, and additional features of the technology, are described throughout the application.Definitions

[0111] 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 presenttechnology 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.

[0112] 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.

[0113] 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.

[0114] 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 invention 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 areference 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 invention 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.

[0115] 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 or mesenchymal-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).

[0116] 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. A population may comprise at least about 5 x 105 of 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 108 or at least about 2 x 108 of 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 1010, at least about 1.0 x 1011 or at about least 1 .0 x 1012 of the relevant cells or even more.

[0117] Cells of the present technology, including EPCs, mammalian mature differentiated endothelial cells (D), and MSCs may be defined according to “marker profile.” This is a standard way of defining cells, such as progenitor and stem cells, which will be immediately apparent, recognisable 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.

[0118] 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.

[0119] 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.”

[0120] 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.

[0121] 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 corresponding to is 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.

[0122] 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+Z- 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.

[0123] 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.

[0124] 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 may be 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 lo / -” and “CD31 lo” 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.

[0125] 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.

[0126] 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 disclosed in the Examples. Antibodies and fluorescently-labelled antibodies for all of the various markers discussed herein may be commercially-available.

[0127] 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 bya 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.

[0128] In any method defined or described herein, 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.

[0129] 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.

[0130] “Enriching,” “selecting,” or “isolating” may be achieved by any suitable method known to the skilled person as described in more detail herein, such as by FACS sorting.

[0131] 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 non-coding regions.

[0132] The term “heterologous gene” is used herein to describe 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).

[0133] 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 does not necessarily indicate a total elimination of all condition, disorder, or disease symptoms, and is intended to include prophylactic treatment.

[0134] 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, Klagsbrun et al (1991 ) Annu. Rev. Physiol., 53, 217-239; Folkmnan et al. (1992) J. Biol. Chem., 267,10931 -10934 and Symes et al. (1994) Current Opinion in Lipidology, 5, 305-312, 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)

[0135] 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.

[0136] 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

[0137] 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.

[0138] 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.

[0139] 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

[0140] 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-.

[0141] 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.

[0142] 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+.

[0143] 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+.

[0144] 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+.

[0145] 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+.

[0146] 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+.

[0147] 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+.

[0148] 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+.

[0149] 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+.

[0150] 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+.

[0151] 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-.

[0152] 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-.

[0153] 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-.

[0154] 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-.

[0155] 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-.

[0156] 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-.

[0157] 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-.

[0158] 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-.

[0159] 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-.

[0160] 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-.

[0161] 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-.

[0162] 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-.

[0163] 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+.

[0164] 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+.

[0165] 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+.

[0166] 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

[0167] 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 ), or a VE-Cadherin (NCBI Accession: CAA56306) protein expression level relative to an expression level in a non-isolated EPC population.

[0168] 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), or a VE-Cadherin (NCBI Gene ID: 1003) gene expression level relative to an expression level in a non-isolated EPC population.

[0169] In some embodiments, the isolated PROCR+ PDGFRA+ EPC population comprises an increase in a PROCR protein expression level or a gene expression levelby 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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%, atleast 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%, relative to a non-isolated EPC population.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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 about800%, at least about 900%, or at least about 1000%, relative to a non-isolated EPC population.

[0178] 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, relative to the non-isolated EPC populations.

[0179] 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.

[0180] 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.

[0181] 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 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.

[0182] 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%, about600%, about 700%, about 800%, about 900%, or about 1000%, relative to a nonisolated EPC population.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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%, 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.

[0187] 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%, atleast 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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%, orabout 75% more elongated, relative to one or more EPCs in a non-isolated EPC population.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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%, 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.

[0202] 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 about700%, at least about 800%, at least about 900%, or at least about 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 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] In some embodiments, the isolated EPC population do not express one or more hematopoietic proteins. Nonlimiting examples of hematopoietic proteins include CD3e (NCBI Accession: NP_000724), 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), 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

[0209] 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 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), ABCG2 (NCBI Accesions: NP_004818.2; NP_001244315.1 ; NCBI Gene ID: 9429), and SOX18 (NCBI Accession: NP_060889; NCBI Gene ID: 54345).

[0210] 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.

[0211] 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 proteinexpression level or a CD157 gene expression level in the undifferentiated EPC population.

[0212] 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.

[0213] 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.

[0214] 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 protein expression level or a ABCG2 gene expression level in the undifferentiated EPC population.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.Methods of Generating Isolated EPC populations

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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).

[0223] 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+.

[0224] 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.

[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 an isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[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, 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.

[0232] 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.

[0233] 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.

[0234] The binding molecule in any of the methods of generating isolated EPC populations comprises a protein. The protein may be an antibody.

[0235] Removing the complex in any of the methods of generating isolated EPC populations can comprise microfluidic sorting. The microfluidic sorting may comprise microbead sorting or flow cytometry. In some embodiments, the flow cytometry comprises fluorescence-activated cell sorting.

[0236] 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).

[0237] In some embodiments, the isolated EPC populations and / or the isolated MSC populations are generated by one or more steps disclsoed by WO2014 / 138793, incorporated herein by reference in its entirety.Additional Methods for isolating EPC populations and MSC populations

[0238] 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. An alternate 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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 asa cytolytic, apoptotic or toxic signal. In another example, opsonization with an antibody followed by complement administration may achieve the same outcome.

[0243] 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.

[0244] 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)

[0245] 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

[0246] 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.

[0247] 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.

[0248] 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

[0249] 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-.

[0250] 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 of the following four phenotypes having regard to PROCR and PDGFRA: PROCR+ PDGFRA+, PROCR+ PDGFRA-, PROCR- PDGFRA+, or PROCR- PDGFRA-.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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+.

[0257] 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+.

[0258] 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+.

[0259] 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+.

[0260] 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+.

[0261] 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+.

[0262] 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+.

[0263] 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+.

[0264] 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 MSC population are PROCR+ PDGFRA+.Additional Isolated MSC Population Features

[0265] The isolated MSC populations of thre present technology may be capable of differentiating into osteoblasts, adipocytes, and chondroblasts in vitro or in vivo.Sources of Isolated EPC Populations and Isolated MSC Populations

[0266] 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

[0267] 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.

[0268] 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.

[0269] 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 wereexpanded) 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.

[0270] 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).

[0271] 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 thenbe digested in a cocktail of enzymes, such as collagenase, dispase and DNAse, and thereafter filtered in order to obtain the initial mammalian biological sample.

[0272] 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

[0273] 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.

[0274] In some embodiments, the isolated EPC populations and / or the isolated MSC populations comprise autologous cells. The autologous cells may be derived from a subject into which the cells will be administered for therapeutic treatment.

[0275] 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.

[0276] 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 Populaitons

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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 US Pat. No. 5,763,194, US Pat. Nos. 5,985,653 and 6,238,908, US Pat.No. 5,512,480, US Pat. Nos. 5,459,069; 5,763,266; 5,888,807 and 5,688,687, each incorporated herein by reference in their entireties.

[0281] 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, feeding and 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.

[0282] 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.

[0283] 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.

[0284] 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 ismandated. 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.Modification of Cells

[0285] The isolated EPC populations and / or isolated MSC populations of the present technology may be genetically engineered or molecularly modified to express an 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.

[0286] 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.Pharmaceutical Compositions and Formulations

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] The pharmaceutical compositions may further comprise a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutically acceptable vehicle is phosphate-buffered saline.

[0293] The pharmaceutical compositions or the cell compositions may be formulated for administration to a subject in need thereof. Nonlmiting examples include formualtion for intranasal delivery, intrathecal, intraarterial, intralesional, or intravenous delivery.Medical Devices and Tissue Engineering Applications

[0294] The isolated EPC populations and / or isolated MSC populations of the present technology may used in combination with an appropriate medical device.

[0295] 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.

[0296] 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.

[0297] Any of the isolated cell populations and compositions as of the present technology may be combined with a stent.

[0298] 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.

[0299] 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.

[0300] 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 making biocompatible 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.

[0301] In some embodiments, the polymer matrix is biodegradable. Suitable biodegradable matrices may be well known in the art and include collagen-GAG, collagen, fibrin, PLA, 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.

[0302] 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 thematrix 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.

[0303] 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.

[0304] 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 et al. (2001 ) Nat Med. 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.

[0305] 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 medical condition, 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 - xenograftsmay 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 include 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.Methods

[0306] The present technology comprises methods of treating a subject in need thereof. In some embodiments, the methods comprise treating hypoxic-ischemic encephalopathy (HIE), including neonatal HIE, or a brain injury in the subject. In some embodiments, the brain injury comprises ischemic brain injury, a perinatal brain injury, and / or neurodegeneration. The methods 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 comprisesor 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.

[0307] The subject in need thereof may have or be at risk of developing a disease selected from the group consisting of myocardial infarction, congestive heart failure, peripheral vascular obstructive disease, peripheral artery disease (PAD), ischemia, cardiac ischemia, limb ischemia, ischaemic retinopathy, diabetic retinopathy, stroke, transient ischemia, reperfusion injury, inherited bleeding disorders, von Willebrand disease (vWD), Haemophilia A, intrauterine growth restriction (IUGR), bronchopulmonary dysplasia (BPD), retinopathy of prematurity, acute kidney injury (AKI), pulmonary arterial hypertension (PAH), bone repair and wounds, inclusive of skin wounds, diabetic foot or ulcers, gangrene, diabetic wounds, neuronal injury, neuronal injury to the brain, neuronal injury to the spinal cord, neuronal injury to peripheral nerve cells, neuronal injury to central nerve cells, brain injury, a neurodegenerative disease, Alzheimer's disease, Ataxia, Huntington’s disease, Parkinson’s disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, inflammatory bowel disease Crohn’s disease, ulcerative colitis, and microscopic colitis.

[0308] In some embodiments, the subject has received or is receiving a therapeutic hypothermia treatment.Pre-treatment Prior to Therapeutic Use

[0309] 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

[0310] 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.

[0311] 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.

[0312] 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.

[0313] 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.

[0314] 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 in by U.S. Pat. No. 5,652,225, incorporated herein by reference in its entirety.

[0315] 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, incorporated herein by reference in its entirety.

[0316] 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.).

[0317] 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 , incorporated herein by reference in its entirety.

[0318] 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.

[0319] Cell-seeded implants of the present invention 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.

[0320] 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.

[0321] 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 as described above. 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.

[0322] 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

[0323] 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 onthe phenotypic or functional state of any of the isolated EPC population and / or MSC populations 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.

[0324] Placental tissues were processed, and single-cell suspension was prepared as described in Patel et al., Stem Cells Trans Med. 2013 Nov;2(11 ):839-47) and Patel et al., Placenta. 201 Nov;35(11 ):969-71 ), incorporated herein by reference in its 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 (see Patel et al., Placenta. 2014 Nov;35(11 ):969-71 , incorporated herein by reference in its entirety). 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

[0325] Single-cell RNA sequencing was analyzed using RStudio (RStudio, MA, USA) with the package Seurat (Version 4.2.0) according to Hao et al., Cell, 2021. 184(13): p. 3573-3587. e29, incorporated herein by reference in its entirety. Data frompreviously conducted scraps of the murine aorta and publicly available scRNA-seq of the human aorta was re-analyzed (see Shafiee et al., Stem Cell Reports, 2018. 10(3): p. 890-904 and Lukowski et al. Cell Reports, 2019. 27(9): p. 2748-2758. e3, each 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 (Aran et al., Nature Immunology, 2019. 20(2): p. 163-172, Mabbott et al., BMC Genomics, 2013. 14: p. 632 and Monaco etal., Cell Rep, 2019. 26(6): p. 1627-1640. e7, each incorporated herein by reference in their entireties).Animals

[0326] 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 / ROSA-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

[0327] Aortae were dissected and prepared for en face IF as previously described by Zhao et al., Nature Communications, 2021. 12(1 ): p. 2564, incorporated herein by reference in its entirety. Samples were sectioned perpendicularly to the cut face to analyze the length of the aorta.Immunofluorescence

[0328] Tissues were prepared as described according to Zhao et al., Nature Communications, 2021. 12(1 ): p. 2564. Primary antibodies used included: rat antimouse CD31 ; rabbit anti-mouse CD34, rabbit anti-mouse 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.

[0329] 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

[0330] 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.

[0331] 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.

[0332] 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 negativepopulations and set appropriate gates. All analysis was conducted using FlowJo® software (FlowJo LLC, USA).In Vitro Colony Formation Assay

[0333] 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).

[0334] 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 DAP I solution before imaging.In Vivo Vessel Generation and Collagen Plugs

[0335] 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 in 96-well plates at 37°C for 90-120 minutes before topping with EGM2 and incubating overnight.

[0336] The following day, gels were rinsed in PBS and implanted into NOD-scid H2rynullB2mnull (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

[0337] 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 as described. 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 as previously described, with the addition of a PROCR+ / _gate on each of the 4 populations of varying CD31 expression.

[0338] 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

[0339] Data were analyzed using GraphPad Prism8 (GraphPad, United States) software. Paired t-tests, unpaired t-tests, and Mann-Whitney tests were used for single comparison 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

[0340] 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 subpopulationsemerged, allowing the analysis of gene expression within previously delegated clusters (FIG. 2A). 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, FIG. 2B (i-ii)). 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 (FIG. 2B (iii-v)) 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; FIG. 2B (vi-viii)). Pathway analysis and dot plots of genes of interest were conducted on cluster 4 for further characterization of differentially expressed (DE) genes (FIG. 2C, FIGS. 1 B-1 D).Endothelial progenitor cells highly express PROCR and PDGFRA

[0341] Flow cytometry was performed on adult C57BL / 6 mouse aortae using the markers highlighted in single-cell RNA seguencing analysis above in conjunction with the 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 etal., 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. 2D). EPCs and mature differentiated endothelial cell populations were then further evaluated for expression levels of PROCR, PDGFRA, and CD157 (FIGS. 2E and 2F). PROCR and PDGFRA were 1.4-fold (p<0.001 ) and 3.3-fold (p<0.001 ) more freguently expressed in EPCs as compared to differentiated endothelial cells, respectively, whereas CD157 was 5.9-fold (n.s.) more highly expressed in differentiated endothelialcells than in EPCs (FIG. 2G). In addition to these markers, two additional mouse strains were used based on the studies described above 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. 2E-2I).

[0342] 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. 2H; ** 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.

[0343] 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 of both 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

[0344] 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’ differentiatedendothelial cells (FIG. 3A). Colonies were characterized based on their morphology at day 12, with two major morphologies seen: a classic endothelial morphology (FIG. 3B (i)) with positive expression of endothelial marker Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I) - Rhodamine (Isolectin) in immunofluorescence (IF; FIG. 3B (iii)), and an elongated morphology (FIG. 3B (ii)) that was not labelled by Isolectin (FIG. 3B (iv)). 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 (FIG. 3C i-ii; *, p < 0.05). Differentiated endothelial cell wells (PROCR+ / _) never formed endothelial colonies based on positive staining for Isolectin.

[0345] 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 (FIG. 3D). 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. 3E (ii), FIG. 3F; 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 was conducted 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. 3G). 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 conqenic capacity

[0346] 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%; FIGS. 4A and 4C) as compared to the abdominal aorta (21.10%; FIGS. 4B and 4D; **, 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 (FIGS. 4E and 4F; **, p = 0.0052, n=3).PROCR+ EPCs form differentiated endothelial cells in vivo in homeostasis and injury

[0347] 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 than differentiated endothelial cells (FIG. 5A; 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 (FIG. 5B).

[0348] 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 (FIG. 5C (i)). 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 (FIG. 5C (ii)). 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 (FIGS. 5D and 5E; n = 5, ** p<0.01 , *** p<0.001 ).

[0349] 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 (FIG. 6A), while IF at D5 demonstrated co-expression of endogenous YFP with differentiated endothelial markers CD31 and ERG (FIG. 6B). 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 D1 to 1.19% at D5 (FIGS. 6C-6E, 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

[0350] 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 et al., Circulation, 2020. 142(14): p. 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 (FIG. 7A, FIGS. 8A and 8B). 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 and FIG. 8B).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

[0351] 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 (FIG. 7C, FIGS. 8C and 8D). Major endothelial genes PECAM1 and CDH5 were most highly expressed in differentiated endothelial cluster 8 along with ABCG2 and S0X18 (FIG. 7C (i-iv)). CD34, PROCR, and PDGFRA were expressed in EPC clusters 3 and 12 as well as differentiated endothelial cell cluster 8 (FIG. 7B (v-vii)).

[0352] 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 et al., STAR Protoc, 2022. 3(2): p. 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) (FIGS. 7C, FIG. 8E). 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. 7D (i), FIG. 7F (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. 7D (ii), FIG. 7F (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 (FIG. 7E). The remaining 2 / 5 colonies formed ECs (FIG. 7F (i-ii)).

[0353] 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 on positive staining for endothelial marker Isolectin. PROCR+ mature differentiatedendothelial 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.

[0354] 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.

[0355] 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

[0356] 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

[0357] Placental tissues were processed, and single-cell suspension was prepared to isolate a PDGFRA+single cell population using a PDGFRA antibody.

[0358] 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. 9 and 10).Example 5: Method for isolating a PROCR* and PDGFRA* cells

[0359] Placental tissues were processed, and single-cell suspension was prepared to isolate a PROCR+single cell population using human PROCR PE antibody.

[0360] 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. 9 and 10)..Example 6: Placental stem cell augmentation of hypothermia therapy for hypoxic- ischemic encephalopathyAnimals and Human Tissue

[0361] Human placenta 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.

[0362] Large white piglets (n = 23) aged < 24h of age (postnatal day (P)1 ), and body weight between 1.32kg and 2.18kg were used in this project. An additional five P8 control animals between 2.25kg and 2.90kg were used in this project.Experimental setup

[0363] Piglets were anesthetized, intubated, and ventilated as detailed elsewhere (Miller et al., Journal of Neurochemistry. 2016;139(3):471-84, incorporated herein by reference in its entirety). All piglets received an intramuscular (i.m) dose of vitamin K (Konakion, 2.0 mg, i.m) and prophylactic intravenous (i.v) cephalothin (DBL TM Cephalothin Sodium, 0.2 mg / kg i.v), gentamicin (Gentamicin Sulphate, 0.25 mg / kg i.v) and penicillin (Penicillin G, 12 mg / kg i.v) (FIG. 11 and FIG. 12). The rectal temperature was maintained at 38.5 ± 0.2°C using an overhead radiant heater until the commencement of HTH. Arterial pressure was recorded via an umbilical artery catheter and the temperature was monitored via a rectal thermometer (Marquette). Two-channel EEG (Unique CFM 6.0, Inspiration Healthcare, United Kingdom) and bipolar ECG were continuously recorded at 256 samples per second. Throughout intensive care monitoring, arterial blood gas analysis was performed to inform the active titration of pH, HCO3 and glucose (glu). Ventilator settings were adjusted for the period of anesthesia to maintain arterial oxygen saturation > 96% and end-expiratory pressure of carbon dioxide at 35-45 mmHg. An intravenous infusion of glucose (10% at 3 mL / kg / h) was titrated to maintain blood glucose levels within the normal range (2.6 - 8.3 mmol / L). Piglets were randomized to either the hypoxic-ischemic, hypothermia and stem cell (PROCR+ / _PDGFRA+EPCs and MSCs) treated group (HHS; n=7) or the hypoxic- ischemic, hypothermia and stem vehicle treated group (HHV; n=4) (control n=5) (FIG. 11 and FIG. 12).

[0364] The cerebral function monitoring system acquiree video, 2-channel electroencephalogram (EEG) and 1 -channel electrocardiogram (ECG). Glucose and dopamine infusions were delivered via the mammary vein, and propofol / alfentanil infusion was delivered via the great auricular vein. Arterial blood was sampled via the umbilical artery for blood gas analysis. The rectal temperature was controlled via an overhead heater until the initiation of the hypothermia protocol, where the temperature was controlled using the Tecotherm Neo (pictured top right). The Tecotherm Neo temperature control system was used to induce and maintain hypothermia and return the animal to normothermia (FIG. 11 and 12).

[0365] Two hours after the induction of anaesthesia, Hypoxic-ischaemic (HI) insult was induced by decreasing the fraction of inspired oxygen (FiO2) to 4%. FiO2 was manipulated (2-10%) as necessary to maintain low amplitude EEG (< 5 pV) and elicit hypotension (<30 mmHg) (Bjorkman et al., Brain Res. 2006 Jul 19; 1100(1 ):110-7). After the HI period, FiO2 was returned to 21 %, and piglets were maintained under light sedation.Cell Sorting

[0366] Cells were freshly isolated from human term placenta (S.L.S) according to Example 1 (Patel et al., Stem Cells Transl Med. 2013 Nov;2(11 ):839-47).Stem Cell administration

[0367] Two hours post-insult, animals received a single dose of freshly isolated human placental stem cells (250,000-750,000 cells, 1 :1 ratio MSC: PROCR+ / _EPC) suspended in 1 mL of phosphate-buffered saline (PBS, pH 7.4). Animals in the vehicle- treated group received 1 mL of PBS administered i.v. Animal treatment was randomized by coin flip, and researchers were blinded until the conclusion of data analysis.Hypothermia therapy (HTH) administration

[0368] Immediately following stem cell administration, whole-body HTH was initiated at 2h post-insult using a servo-controlled cooling device (Tecotherm, Inspiration Healthcare, Leicester, UK). Animals were wrapped in a pre-cooled mattress, and their body temperature was reduced from average piglet body temperature (~38.5°C) to33.5°C. HTH was maintained for 24 hours (2-26h post-HI). Animals were then rewarmed by 0.5°C per hour for 10h (26-36h post-insult).Post-insult recovery & monitoring

[0369] Anesthesia was ceased / terminated after rewarm, weaned from ventilation, and extubated. Animals were housed in pairs following recovery from anesthesia and fed artificial pig milk (Woombaroo Pig milk replacer, ProviCo, Australia) every 3-4 hours via an orogastric tube or bottle-fed.Neurobehavioral scoring

[0370] Following recovery from anesthesia, animals were assessed on neurobehavioral criteria daily until the end of the experiment (FIG. 15A-15J). The neurobehavioral score criteria include assessment for respiration, consciousness, walking and limb control, overall activity, and presence of clinical seizures (Bjorkman et al., Neuroscience. 2010; 166(1 ): 157-67).Magnetic resonance (MR) methods

[0371] At P8 piglets were anesthetized with isoflurane (1 -3 %) mixed with oxygen for the duration of the scanning protocol. Following induction, the animals were positioned inside the magnet bore in the prone position. A 150mm volumetric head coil was used to acquire sagittal, coronal, and axial slices. Images were obtained at TR / TE 8500 / 60ms, 1.6mm slices thickness, 64x64 acquisition matrix. Bilateral regions of interest over the frontal cortex were defined on the T2 map and applied to the apparent diffusion coefficient (ADC) map and raw values extracted (see Bjorkman et al., Neuroscience. 2010 2010 / 03 / 10 / ; 166(1 ): 157-67, incorporated herein by reference in its entirety).

[0372] 1 H-MR spectra were obtained on a 7T Bruker / Siemens whole-body scanner. A single spectrum was acquired P8 in the frontoparietal region of the brain from a 10 mm3 voxel using single-voxel spectroscopy with the following parameters: TR=6000 ms, TE = 60 ms and 128 averages. Metabolite spectra were exported, processed, and analyzed 149 using the AMARES tool within jMURI (see Vanhamme et al., Journal of Magnetic Resonance.1997 1997 / 11 / 01 / ;129(1 ):35-43, incorporated herein by reference in its entirety). Spectra were manually phased, apodization (10 Hz)was applied to the spectrum, and Lorentzian peaks were fitted to regions corresponding to N-acetyl aspartate (NAA), creatine (Cr), choline (Cho) peaks, and lactate (lac) peaks. Peak area ratios were calculated for NAA / Lac, NAA / Cho, NAA / Cr, Lac / Cr, Lac / Cho, Lac / NAA and Cho / Cr.Euthanasia and tissue processing

[0373] Following MRI / S animals were administered an overdose of 120 mg / kg sodium pentobarbitone 325 mg / ml via intraperitoneal injection. The brain was transcardially flushed with 0.9 % saline, removed, sliced coronally into 3 mm sections and hemisected. The right hemisphere and brainstem were fixed in 4% paraformaldehyde (PFA) / 0.1 M PBS (pH 7.4) overnight. Brain regions from the left hemisphere were isolated and snap-frozen in liquid nitrogen and stored at -80°C. The lungs, heart, liver, spleen, and kidneys were removed for weighing and gross pathological inspection before fixation in 2% PFA / 0.1 % PBS (pH 7.4) (Bjdrkman et al., Neuroscience. 2010 2010 / 03 / 10 / ; 166(1 ): 157-67).Hematoxylin and eosin

[0374] Tissue sections were dewaxed in xylene and rehydrated through graded alcohols. Sections were stained with haematoxylin and eosin with an automated system (Leica ST5010 AutostainerXL, Leica Biosystems North Ryde, NSW, AUS). Histological injury from three slides 48pm apart for each brain section was examined and qualitatively described.Immunolabelling

[0375] Paraffin-embedded coronal slices were sectioned at 6 pm. Sections were dewaxed in xylene and rehydrated through graded alcohols using an automated system (Leica ST5010 AutostainerXL, Leica Biosystems North Ryde, NSW, Australia). Antigen retrieval was performed in a decloaking chamber (Biocare Medical) with 10mM citrate buffer (pH 6) or TRIS-EDTA buffer (pH 9) at 90°C for 20 minutes before cooling to room temperature (RT). A hydrophobic barrier was drawn around the tissue, followed by nonspecific blocking with 5% donkey serum in PBS with 0.5% Triton-X 100 and 0.05% tween-20 for 1 h at RT. Primary antibodies (C-cas3, GFAP, lba-1 , IL-1 [3, NeuN, NFKB- p65, TNFa) were incubated according to the conditions in Table 2. Slides were washed in PBS followed by incubation with species-specific secondary fluorophores at RT for1 h. Sections were washed with PBS, counterstained with 4',6-diamidino-2-phenylindole (DAPI), and mounted with Prolong Gold antifade (Molecular Probes, Invitrogen Australia, Victoria, Australia). A Zeiss Axio Microscope (Axioscope 5; Zeiss Microscopy, Australia) with the Plan-Apochromat 10x / 0.45 M27 objective lens (878.94 pm x 662.84 pm), the EC Plan-Neofluar 20x / 0.50 M27 objective lens (439.47 pm x 331.42 pm), or EC Plan-Neofluar 40x / 0.75 M27 objective lens (219.74 pm x 165.71 pm) were used to visualize stained and labelled sides. Photomicrographs were captured with an Axiocam503 camera (Zeiss Microscopy, Australia). Secondary only (negative) controls were also run to rule out non-specific binding (data not shown).Table 2: Primary AntibodyFluoro-Jade C staining (FJC)

[0376] Sections were dewaxed in xylene and rehydrated through graded alcohols using an automated system (Leica ST5010 Autostainer XL, Leica Biosystems NorthRyde, NSW, Australia). Degenerating neurons were observed using Fluoro-Jade C (FJC). Slides were transferred to a solution of FJC (Merck Millipore, Germany) dissolved in acetic acid vehicle, containing DAP I to counterstain nuclei FJC positive cells were counted and were averaged for each piglet prior to visualization.Gene expression analysis

[0377] Snap frozen brain tissue (frontal cortex and basal ganglia) was disrupted using a needle and syringe. Total RNA was extracted and cDNA was synthesized, using random hexamers.

[0378] Quantitative polymerase chain reaction (qPCR) was performed. Results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data were analyzed using the AACT method and presented as log fold changes (log(2)2A(AACT)) and 95%CI [upper, lower] Livak ef al., Methods. 2001 Dec;25(4):402-8).Statistical methods

[0379] Results from pilot studies using the same insult model resulted in an effect size of f = 0.62 for the number of NeuN positive cells, this indicated 10 animals per group were required (power 80%; p = 0.05). Power analyses were conducted in G*Power Version 3.1.9.4. All analyses were completed using the R software version 5.3.2. Mantel-Cox log-rank test was used to examine the difference in survival between the two groups. Animals that did not make the P8 experimental endpoint were excluded from further analyses. Initial data explorations were performed using the Shapiro-Wilks 223 tests for normality and Levene’s test for the assumption of equal variance. No deviations from normality were observed, but occasional deviations from the assumption of equal variance were observed between physiological EEG measures and neurobehavioural measures. Welches’ T-test was used to compare group means between physiological measures, EEG measures and neurobehavioral scores; multiple comparisons were controlled for using the Holm-Sidak method. These data may be presented as mean ± standard deviation (SD), as these may be parametric comparisons. Significant departures from the normality and equal variance assumptions were observed in MRI, MRS, immunofluorescence, and qPCR data. As this reduces the validity of parametric tests, these measures were analyzed with either the Mann-Whitney U test or Kruskal-Wallis H tests with a two-stage linear step-upprocedure of Benjamini, Krieger and Yekutieli. As these results may be non-parametric, they may be presented median [Cl 95% lower, upper]. Statistical significance was accepted at q < 0.05, or p < 0.05 where appropriate. Figures were generated Graph Pad Prism 9, or IBM SPSS Statistics for Windows, version 27.0.Animal Study Group Characteristics

[0380] Twenty-three piglets were used in this study. Two male piglets were excluded before the administration of stem cells because they succumbed to the HI injury. A further three males in the HHV group were excluded because they died before the experimental endpoint. No animals in the HHS group died. Statistical analysis of the survival outcomes did not reach statistical significance (FIG. 13). Thus, for final analyses there were four animals in the HHV (4 males) group, and seven in the HHS group (3 males, 4 females) with all exclusions applied. To address the need for a healthy biological comparison, brains from five healthy piglets (2 males, 3 females) were obtained and culled at P8 (C). No significant difference in P1 bodyweights were observed between the HHV and HHS. Significant differences were observed between groups across bodyweight, brain weight, heart weight or liver weight (Table 3). Post- hoc analyses showed the control group was significantly different to HHV groups across all measures. The control group was also significantly different to the HHS group in P8 body weight, heart weight and liver weight. Liver weight was significantly higher in the HHS group than the HHV group. No other significant differences were observed between the HHV and HHS groups. No significant differences at any time point were observed across the measured heart rate and blood pressure metrics (Table 4), as well as arterial blood pH, lactate concentration (lac), arterial base excess (ABE) and HCO3-, and glucose (Glu) concentration (FIG. 14). aEEG score was reduced to 0 following HI in all animals. Group means were not significantly different from baseline after two hours following HI. There were no differences in aEEG score between groups at any time point. EEG background was significantly reduced in both hypothermia and stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treated (HHS) animals and hypothermia and stem vehicle treated (HHV) animals at 0 (adjusted p = 0.00045) and 1 hour (adjusted p = 0.012) post HI, but not at any other time point.Table 3: Tissue WeightsTable 4: Heart rate and blood pressure metricsElectroencephalographic outcomes following HI insult

[0381] HI insult resulted in a reduction of background amplitude from a baseline of 9.00pV ± 0.70pV in the HHV group and 10.76 pV ± 1 .81 pV in the HHS group to 1 ,37pV ± 0.20pV and 1.42pV ± 0.36pV respectively. No significant difference in background amplitude between was observed at any timepoint. One electrographic seizure was observed in the HHS group that lasted 12 seconds. Three animals in the HHV group had electrographic seizures ranging from 2 seizures to 22 seizures, with an average seizure burden of 00:07:14 ranging from 11 to 22 minutes. Burst suppression patterns following injury were observed in two out of three piglets that experienced seizures in the HHV group and none in the HHS group.MRI and MRS outcomes following HI insult

[0382] MRI scans were collected from eleven piglets. One piglet from each group was excluded due to motion artefact, leaving three piglets in the HHV group and six piglets in the HHS group (FIG. 15A). Due to the low group numbers and a true outlier 269 in the HHV group, the Mann-Whitney U test was used. There were no significant differences in the mean T2 relaxation time (Mann-Whitney U = 8, n1 = 4, n2 = 6, p = 0.47; FIG. 15B) or ADC value (Mann-Whitney U = 4, n1 = 4, n2 = 6, p = 0.11 ; see FIG. 15C) between HHV (T2, 75.76 272 [54.66, 180.8]; ADC, 317.10 [301 , 734.3]) and HHS (T2 71.65 [66.62, 76.10]; ADC, 290.5[267.7, 273 274 315.4]). MR spectroscopy was measured and analyzed from eight piglets, three from the HHV group and five from the HHS group (FIG. 15D). Due to the low group numbers and a true large outlier in the HHV group, it was inappropriate to use Welch’s T-test and the Mann-Whitney U test was more appropriate. A significant difference was observed in NAA / Cho ratio (Mann- Whitney U = 10.5, n1 = 3, n2 = 7, P = 0.036 two-tailed; HHV, 1.450 [-0.6374, 1.947]; HHS, 1.947 [1.715, 2.392]; FIG. 15E). No significant differences were found for any other measured ratio (FIG. 15F-15I). A significant difference was only observed between hypothermia and stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treated (HHS) and hypothermia and stem vehicle treated (HHV) groups in NAA / Cho ratio. The hypothermia and stem cell (PROCR+Z- PDGFRA+Z- EPC and MSC) treated (HHS)group had significantly higher neurobehavioral scores at P4 and P5, but not at any time after that. For a full description of the results, see Table 5.Table 5: Experimental ResultsImproved functional recovery following HI

[0383] Neurobehavioral scores were decreased from pre-HI assessment scores of 22 ± 1 in the HHV group and HHS group, to 6 ± 8 and 17 ± 4 respectively at P3 (see FIG. 15J). Neurobehavioral scores were significantly higher in the HHS group on P4 (HHV: 9 ± 8 vs. HHS: 21 ± 1 ; adjusted p = 0.044)) and P5 (HHV: 9 ± 8 vs. HHS: 21 ± 1 ; adjusted p = 0.0311 ). There were no significant differences at any other time point.Stem cell administration reduces histological neuropathology following HI

[0384] Trends and differences in the EEG, MRI and neurobehavioral scoring were reflected in the brain histopathology. For a healthy biological comparison age-matched controls (C) were used. No instances of Hl-associated neuropathology were observed in the control group (FIGS. 16A and 16A’). Animals in the HHV group had multiple instances of major tissue injury in all brain areas examined. Diffuse coagulated ischaemic neurons were observed frontal cortex that were not present in any animal in the HHS group. All HHV piglet brains showed infiltrating macrophages in the white matter and laminar cortical neuronal layers of the frontal cortex (FIGS. 16B and 16B’). Neuropathology in this area became more severe in deeper regions of the gyri. This region did not exhibit neuropathology in the HHS group (FIGS. 16C and 16C’).Stem cell administration reduces measures of neuropathology following HI

[0385] Trends and differences in the MRI / S and neurobehavioral scoring were reflected in the brain immunofluorescence analyses examining the numbers of mature neurons with NeuN (FIGS. 17A-17C), degenerating neurons with FJC (FIGS. 17F-17H), and apoptotic cells with cleaved caspase3 labelling (FIGS. 17K-17JM). Frontal cortext (FC) (FIGS. 17D, 171, 17N) and putamen (OUT) (FIGS.17E, 17J, 170) were assessed. Exploratory analyses of these immunofluorescent data showed significant deviations from normality and sphericity that could not be accommodated by log transform or Box- Cox transform. Therefore, the data were analyzed using the Kruskal-Wallis test. A significant difference in numbers of NeuN positive cells were observed in frontal cortex between groups H(2) = 11.25, p = 0.0002. Post-hoc comparisons indicate the median NeuN number in the controls (979 [893, 1021 ]), were significantly higher 299 than the HHV group (703 [-53, 1163]; C vs HHV, g = 0.010) and the HHS group (849 [788, 874]; C vs HHV g = 0.0152). Similarly, a significant difference between HHV and HHS group (HHV vs HHS, g = 0.0471 ) was found. No significant differences were observed in the putamen H(2) = 2.757, p = 0.26. Through FJC staining analyses significant differences were found in the number of degenerating neurons in the frontal cortex H(2) = 6.285, p = 0.041 . Post-hoc analyses showed no significant difference between the control group (0 [0,0], mean rank = 4.5) and the HHV group (26 [-21 , 78], mean rank = 10.75) and the HHS group (20 [2, 46], mean rank = 10.07) (C vs HHV, g = 0.057; C 307 vs HHS, g = 0.574). Furthermore, no significant differences were observed between the HHV and the HHS group (HHV vs HHS, g = 0.84). No significant difference was observed in the putamen H(2) = 309 3.359, p = 0.1753. No significant difference in the number of apoptotic cells (cleaved casp-3) were observed in the frontal cortex H(2) = 0.9274, p = 0.656 or the putamen H(2) = 2.70, p = 0.2774. Colocalization studies demonstrated that the cleaved caspase-3 positive cells predominantly colocalized with astrocytes in all experimental groups (FIG. 17P-17R).

[0386] Hypothermia and stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treated (HHS) brains displayed similar levels of mature neuron throughout the frontal cortex, while HHV consistently showed fewer neurons. Significantly fewer NeuN-positive cells were observed in the HHV group. This reduction was in the hypothermia and stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treated (HHS) group in the frontal cortex but not the putamen. No FJC staining was observed in the frontal cortex of controls.Stem cell treatment is associated with alterations to glial cell activation

[0387] Increased neuropathology may be associated with glial cell activation caused by local neuroinflammation, so the morphology of microglia and astrocytes was examined, lba-1 -positive microglia in control brains displayed stellate morphology with light cell bodies and fine extended processes indicative of a resting state (FIGS. 18A and 18A’). In comparison, many of the lba-1 -positive microglia in HHV brains resembled the morphology of activated microglia with darker cell bodies and thickened retracted processes (FIGS. 18B and 18B’). Conversely, microglia in the HHS brains were like those of the controls (FIGS. 18C, 18C’).

[0388] Quantification of the numbers of resting and activated microglia found significant differences between groups in the frontal cortex Hresting (2) = 12.21 , p < 0.0001 ; Hactivated (2) = 10.27, p < 0.0013 (FIGS. 18D, 18E) and the putamen Hresting (2) = 10.33, p = 0.0013, Hactivated (2) = 10.64, p = 0.0009 (FIGS. 18F, 18H, 181). The number of resting microglia in the control group (237 [218, 249]) were significantly greater than the HHV (95 [6, 151 ]; C vs HHV, qresting = 0.0013) and the HHS group (140 [114, 156]; C vs HHS, qresting = 0.038). This was paired with observations of significantly fewer activated microglia in the control group (40 [32, 56]) compared with the HHV group (230, [77, 474]; C, HHV, q = 0.004) and HHS group (222 [162, 241 ]; C vs HHS, q = 0.179). Similarly, in the putamen, post-hoc analyses of the morphology of microglia showed the numbers of resting microglia was significantly higher in the control group (234 [188, 252]; C vs HHV, q = 0.004) compared with the HHV (114 [-5.9, 207]; C vsHHV, q = 0.004) and HHS groups (138 [115, 152]; C vs HHS, q = 332 0.0043). No significant difference in the numbers of resting microglia was observed between the HHV and HHS group (HHV vs HHS, q = 0.27). Post-hoc analyses showed significantly fewer activated microglia in the control group (47 [39, 55]) compared with HHV group (241 [84, 498]; C vs HHV, q = 0.0051 ) and the HHS group (218 [184, 256]; C vs HHV, q = 0.0051 ). Analysis of the total number of microglia showed significant differences in the frontal cortex HFC total (2) = 12.11 , p < 0.0001 and the putamen HPLIT total (2) = 8.153, p = 0.0083. Post-hoc analysis showed significantly more microglia in the HI injured groups (HHV, [330, 503]; HHS, 328 [314, 371 ]) compared with the control group (276 [263, 288]; C vs HHV, q = 0.0011 ; C vs HHS, q = 0.039), and no significant difference between the HHV and the HHS group (HHV vs HHS, q = 0.055) (FIG. 18F). A similar relationship was seen in the putamen with the control group exhibitingsignificantly fewer microglia (282 [238, 295]), than the HHV (400 [251 , 536], C vs HHS, q = 0.009) or the HHS group (349 [314, 384]; C vs HHS, q = 0.011 ). No significant difference was observed between the HHV and HHS (HHV vs HHS, q = 0.18; see FIG. 18J).

[0389] GFAP-positive astrocytes were observed throughout the white matter regions. In both the C and HHS brain, GFAP-positive cells demonstrated multiple long branching processes from the cell body typical of normal astrocyte morphology (FIG. 18K’, 18M’;). In the HHV brains, many of the GFAP-positive astrocytes displayed reactive morphology with retracted processes and large cell bodies (FIG. 20L’). Astrocytes in HHV white matter displayed morphology characteristic of astrocytes in an activated state, with enlarged cell bodies and short thickened processes. Differences between groups were observed between levels of astrocyte coverage These qualitative descriptions were captured quantitatively by measuring the average astrocyte coverage in each region. A significant difference in GFAP coverage was observed between the HHV and the HHS group (HHV vs. HHS, q= 0.045). No significant difference was observed in GFAP coverage was observed in the IGWM H(2) = 2.586, p = 0.116 (FIG. 18N). A significant difference in GFAP coverage was observed in the PVWM H(2) = 6.659, p = 0.01 (FIG. 180). Post-hoc analyses showed control group (17.25 [15.62, 19.53]) differed significantly with HHV group (13.84 [11.93, 16.33]) but not the HHS group (15.88 [14.90, 17.30]) (C vs HHV, q = 0.006; C vs HHS, q = 0.0683) (FIGS. I SA- 180).Analysis of pro-inflammatory cytokine mRNA expression

[0390] There were significant differences in frontal cortex mRNA expression of several inflammatory markers between experimental groups for all markers except CCR5 (FIG. 19). Post-hoc analyses found no significant differences between HHV or HHS for any inflammatory marker (see Table 6). To discern the cellular source of the pro-inflammatory cytokines, colocalization studies were performed with antibodies raised against TNFa, IL-1 [3 and NF-KB with lba-1 and GFAP. Both TNFa and IL-1 [3 colocalized with lba-1 in HHV and HHS groups (FIG. 20). NF-KB colocalized with Iba- 1 in the HHV group but not the HHS group (FIG. 21 ). As shown in FIG. 21 , (Upper) In the frontal cortex, NF-KB was preferentially expressed in microglia in the HHV group (triangle). HHS treated animals had predominant expressed activated NF-KB in neurons(small-tailed arrows). High power image scale bar = 50pm. (Lower) NF-KB expression was generally higher in the deep cortical regions of the HHV group (region white arrows) than in (right) the HHS group. The predominant source of NF-KB in the HHS were neurons.

[0391] Hypoxic-ischemic insult was associated with significant increases in the inflammatory marker transcription compared with the control group. No significant differences were observed between the HS and the HHV groups in any inflammatory marker.Table 6: Inflammatory MarkersStem cell treatment restore reductions in vascular coverage

[0392] Vascular coverage was measured to give an indication of vascular protection. Significant differences in vascular coverage were discovered in the FC [x2 = 8.250, p = 0.0073] and the IGWM [y2 = 10.89, p = 0.0002] (6.21 ). Post hoc analysis in the FC showed the CD34 coverage in HHV (2.356 [2.142, 2.582]) was significantly less than C (2.881 [2.551 , 9.974]) (C vs. HHV; q = 0.0065). CD34 coverage in HHS brains (2.688 [2.563, 2.947]) was significantly higher than CD34 coverage in HHV brains (q = 0.0065). No significant difference was observed between C and HHS (q = 0.35). Post hoc analysis in the IGWM showed the CD34 coverage in HHV (1.922 [1.149,2.731 ]) and HHS group (2.695 [2.031 , 3.169]) was significantly less than C (3.167 [3.039, 3.438])(C vs. HHV; q = 0.0023; C vs. HHS; q = 0.0457). No significant difference was observed between HHV and HHS (q = 0.093) (FIG. 22). As shown in FIG. 22, Quantification of CD34 coverage shows the HHS group has significantly higher coverage than the HHV group in the frontal cortex and is not significantly different from the C. Intragyral white matter comparisons show the control group is significantly higher than HHV and HHS. There was no significant difference between HHV and HHS. Values are presented as median [lower, upper].Example 7: Human Placental Cells

[0393] 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. 10(3): p. 890-904, incorporated herein by reference in its entirety.

[0394] 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. 23). Cells were selected based CD45-CD34+CD31 Int phenotypes.

[0395] 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. 24). 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. 25). Control ECFCs were isolated as a CD45-CD34+CD31 Int population.

[0396] 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, incorporated herein by reference in its entirety. fPL-MSC and PROCR+ ECFCs were cocultured together incollagen 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.

[0397] After 5 days of coculture (or single culture in control groups), the cells were detached using dissociation reagent, washed, and resuspend in FACS buffer and transferred 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.

[0398] 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. 26).Additional Embodiments

[0399] The present technology includes, but is not limited to, the following specific embodiments set forth herein below in paragraphs

[0400] -

[0514] :

[0400] 1. A pharmaceutical composition comprising or consisting of an isolated endothelial progenitor cell (EPC) population comprising PROCR+ PDGFRA+ EPCs.

[0401] 2. A pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs for use in treating neonatal hypoxic-ischemic encephalopathy (HIE) in a subject in need thereof.

[0402] 3. A pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

[0403] 4. A pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs for use in treating a brain injury in a subject in need thereof.

[0404] 5. The pharmaceutical composition of embodiment 1 , wherein the isolated EPC population comprises an increase in a PROCR, a PDGFRA, or a VE- Cadherin protein expression level relative to an expression level in a non-isolated EPC population.

[0405] 6. The pharmaceutical composition of embodiment 5, wherein the increase in the PROCR protein expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PROCR protein expression level in the non-isolated EPC.

[0406] 7. The pharmaceutical composition of embodiment 5, wherein the increase in the PDGFRA protein expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PDGFRA protein expression level in the non-isolated EPC.

[0407] 8. The pharmaceutical composition of any one of embodiments 1 -7, wherein the isolated EPC population comprises an increase in a PROCR, a PDGFRA, or a VE-Cadherin gene expression level relative to an expression level in a non-isolated EPC population.

[0408] 9. The pharmaceutical composition of embodiment 8, wherein the increase in the PROCR gene expression level is at least about 5%, 10%, 15%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PROCR gene expression level in the non-isolated EPC.

[0409] 10. The pharmaceutical composition of embodiment 8, wherein the increase in the PDGFRA gene expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PDGFRA gene expression level in the non-isolated EPC.

[0410] 11. The pharmaceutical composition of embodiment 8, wherein the increase in the VE-Cadherin gene expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a VE- Cadherin gene expression level in the non-isolated EPC.

[0411] 12. The pharmaceutical composition of any one of embodiments 1 -11 , wherein the isolated EPC population comprises an increased proliferative capacity relative to a non-isolated EPC population.

[0412] 13. The pharmaceutical composition of any one of embodiments 1 -12, wherein the isolated EPC population comprises an increased angiogenic capacity relative to a non-isolated EPC population.

[0413] 14. The pharmaceutical composition of embodiment 13, wherein the increase in the angiogenic capacity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to an angiogenic capacity level in the non-isolated EPC.

[0414] 15. The pharmaceutical composition of any one of embodiments 1 -14, wherein the isolated EPC population comprises an increased in a colony forming capacity or a tube formation capacity relative to a non-isolated EPC population.

[0415] 16. The pharmaceutical composition of embodiment 15, wherein the increase in the colony forming capacity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%,400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a colony forming capacity level in the non-isolated EPC.

[0416] 17. The pharmaceutical composition of embodiment 15, wherein the increase in the tube forming capacity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a tube forming capacity level in the non-isolated EPC.

[0417] 18. The pharmaceutical composition of any one of embodiments 1 -17, wherein the isolated EPC population comprises an increased engraftment potential relative to a non-isolated EPC population.

[0418] 19. The pharmaceutical composition of embodiment 18, wherein the increase in the engraftment potential is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to an engraftment potential level in the non-isolated EPC.

[0419] 20. The pharmaceutical composition of any one of embodiments 1 -19, wherein the isolated EPC population comprises one or more EPCs having a more elongated cell shape, relative to one or more EPCs in a non-isolated EPC population.

[0420] 21. The pharmaceutical composition of embodiment 20, wherein the one or more EPCs are at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% more elongated compared to the non-isolated EPC population.

[0421] 22. The pharmaceutical composition of any one of embodiments 1 -21 , wherein the isolated EPC population comprises a CD45- / CD34+ phenotype.

[0422] 23. The pharmaceutical composition of any one of embodiments 3-22, wherein the pharmaceutical composition is a first composition formulated for administration before, during, or after administration of a second composition comprising an isolated mesenchymal stem cell (MSC) population to a subject in need thereof.

[0423] 24. A pharmaceutical composition comprising(a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and(b) a second isolated cell population comprising MSCs.

[0424] 25. A pharmaceutical composition comprising(a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and(b) a second isolated cell population comprising MSCs.

[0425] 26. A pharmaceutical composition for use in treating HIE in a subject in need thereof, the pharmaceutical composition comprising(a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and(b) a second isolated cell population comprising MSCs.

[0426] 27. The pharmaceutical composition of embodiment 26, wherein theHIE is neonatal HIE.

[0427] 28. A pharmaceutical composition for use in treating a brain injury in a subject in need thereof, the pharmaceutical composition comprising(a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and(b) a second isolated cell population comprising MSCs.

[0428] 29. The pharmaceutical composition of embodiment 28, wherein the brain injury comprises neurodegeneration.

[0429] 30. The pharmaceutical composition of any one of embodiments 1-29, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle.

[0430] 31. The pharmaceutical composition of embodiment 30, wherein the pharmaceutically acceptable vehicle is phosphate-buffered saline.

[0431] 32. The pharmaceutical composition any one of embodiments 3-31 , wherein the pharmaceutical composition is formulated for intranasal delivery, intrathecal, intraarterial, intralesional, or intravenous delivery to a subject in need thereof.

[0432] 33. The pharmaceutical composition of embodiment 28, wherein the brain injury comprises ischemic brain injury.

[0433] 34. The pharmaceutical composition of any one of embodiments 26-33, wherein the subject has received or is receiving a therapeutic hypothermia treatment.

[0434] 35. A method of treating HIE 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 MSCs.

[0435] 36. The method of embodiment 35, wherein the HIE is neonatal HIE.

[0436] 37. A method of treating brain injury 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 MSCs.

[0437] 38. The method of embodiment 37, wherein the brain injury comprises neurodegeneration.

[0438] 39. The method of embodiment 37, wherein the brain injury comprises ischemic brain injury.

[0439] 40. A cell composition for use in a treatment of neonatal HIE, the composition comprising a mixture of two isolated cell populations, wherein the first isolated cell population consists PROCR+ / - PDGFRA+ / - EPCs, and the second isolated cell population consists of MSCs.

[0440] 41 . A method of treating neonatal HIE in a subject in need thereof, the method comprising administering to the subject a cell composition comprising a mixture of two cell isolated populations, wherein the first isolated cell population consists of PROCR+ / - PDGFRA+ / - EPCs, and the second isolated cell population consists of MSCs.

[0441] 42. The method of any one of embodiments 35-41 , wherein the subject has received or is receiving a therapeutic hypothermia treatment.

[0442] 43. A pharmaceutical composition comprising a cell composition and a pharmaceutically acceptable vehicle for use in a treatment of HIE wherein the cellcomposition comprises a mixture of two isolated cell populations, wherein the first isolated cell population consists of mammalian PROCR+ / - PDGFRA+ / - PROCR+ / - PDGFRA+ / - EPCs, and the second isolated cell population consists of MSCs.

[0443] 44. A pharmaceutical composition for use according to embodiment43, wherein the pharmaceutically acceptable vehicle is phosphate-buffered saline.

[0444] 45. An isolated cell population, a cell composition, a pharmaceutical composition, or a cell composition for use according to any one of embodiments 26, 27, 35, 36, or 41 , wherein the use in the treatment of HIE or the treatment of neonatal HIE comprises providing a therapeutic hypothermia treatment to a subject in need thereof, and additionally administering the isolated cell population, the isolated cell composition, the cell composition or the pharmaceutical composition to the subject before, during and / or after the therapeutic hypothermia treatment.

[0445] 46. The pharmaceutical composition, the method, or the cell composition of any one of embodiments 24-45, wherein the first isolated cell population and the second isolated cell population are present in the composition at a ratio of about 1 :1.

[0446] 47. The pharmaceutical composition, the method, or the cell composition of any one of embodiments 24-45, 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.

[0447] 48. The pharmaceutical composition, the method, or the cell composition of any one of embodiments 24-47, wherein the MSCs are CD45- / CD34+ cells.

[0448] 49. The pharmaceutical composition, the method, or the cell composition of embodiment 48, 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.

[0449] 50. The pharmaceutical composition, the method, or the cell composition of any one of embodiments 24-49, 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.

[0450] 51. An isolated EPC population comprising PROCR+ PDGFRA+EPCs, the isolated EPC population 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 the isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

[0451] 52. An isolated EPC population comprising PROCR+ / - PDGFRA+ / -EPCs, the isolated EPC population 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 the isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

[0452] 53. The isolated EPC population of embodiment 51 or 52, wherein step(ii) comprises selecting cell 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.

[0453] 54. The isolated EPC population of embodiment 53, wherein the step of selecting the cells that express the CD45+phenotype comprises contacting a cell orcells 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).

[0454] 55. The isolated EPC population of any one of embodiments 51 -54, wherein 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 population of cells which are CD45- / CD34+.

[0455] 56. The isolated EPC population of any one of embodiments 51 -55, wherein 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.

[0456] 57. A method of isolating an EPC population comprising PROCR+PDGFRA+ EPCs, the method comprising 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 the isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

[0457] 58. A method of isolating an EPC population comprising PROCR+ / -PDGFRA+ / - EPCs, the method comprising 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 the isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

[0458] 59. The method of embodiment 57 or 58, wherein step (ii) comprises selecting cell 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.

[0459] 60. The method of embodiment 59, wherein 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).

[0460] 61. The method of any one of embodiments 57-60, wherein 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 population of cells which are CD45- / CD34+.

[0461] 62. The method of any one of embodiments 57-61 , wherein 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.

[0462] 63. An isolated EPC population comprising or consisting of PROCR+PDGFRA+ EPCs, the isolated cell population 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.

[0463] 64. An isolated EPC population comprising or consisting of PROCR+ / -PDGFRA+ / - EPCs, the isolated EPC population 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.

[0464] 65. The isolated EPC population of embodiment 63 or 64, wherein 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.

[0465] 66. A method of isolating an EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs, the method comprising 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.

[0466] 67. A method of isolating an EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs, the method comprising 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.

[0467] 68. The method of embodiment 66 or 67, wherein 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.

[0468] 69. The method of any one of embodiments 66-68, wherein 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.

[0469] 70. An isolated EPC population comprising or consisting of PROCR+PDGFRA+ EPCs, the isolated EPC population 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.

[0470] 71 . An isolated EPC population comprising or consisting of PROCR+ / -PDGFRA+ / - EPCs, the isolated EPC population 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.

[0471] 72. The isolated EPC population of embodiment 70 or 71 , wherein step (ii) 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.

[0472] 73. The isolated EPC population of any one of embodiments 70-72, wherein step (iii) 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.

[0473] 74. A method of isolating an EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs, the method comprising 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.

[0474] 75. A method of isolating an EPC population comprising or consisting of PROCR+ / -PDGFRA+ / - EPCs, the method comprising 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.

[0475] 76. The method of embodiment 74 or 75, wherein step (ii) 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.

[0476] 77. The method of any one of embodiments 74-76, wherein step (iii) 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.

[0477] 78. The isolated EPC population or the method of any one of embodiments 54-56, 60-62, 65, 68, 69, 72, 73, 76, or 77, wherein the binding molecule comprises a protein.

[0478] 79. The isolated EPC population or the method of embodiment 78, wherein the protein comprises an antibody.

[0479] 80. The isolated EPC population or the method of any one of embodiments 54-56, 60-62, 65, 68, 69, 72, 73, 76, or 77-79, wherein removing the complex comprises microfluidic sorting.

[0480] 81. The isolated EPC population or the method of embodiment 80, wherein the microfluidic sorting comprises microbead sorting or flow cytometry.

[0481] 82. The isolated EPC population or the method of embodiment 81 , wherein the flow cytometry comprises fluorescence-activated cell sorting.

[0482] 83. The isolated EPC population or the method of any one of embodiments 51 -82, wherein the steps 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.

[0483] 84. The isolated EPC population or the method of embodiment 83, wherein the steps further comprise step (v), separating the isolated EPC population from the cell population comprising ECFCs or the cell population comprising MSCs.

[0484] 85. The isolated EPC population or the method of embodiment 84, wherein step (v) occurs 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).

[0485] 86. The isolated EPC population or the method of any one of embodiments 51 -85, wherein the biological sample is a mammalian biological sample.

[0486] 87. The isolated EPC population or the method of embodiment 86, wherein the 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.

[0487] 88. The isolated EPC population or the method of embodiment 87, wherein the mammalian placenta is a whole mammalian placenta.

[0488] 89. The isolated EPC population or the method of embodiment 87, wherein the mammalian tissue-resident vascular endothelium is selected from the group consisting of a mammalian umbilical cord, a mammalian pulmonary artery endothelium, a mammalian aorta, and a mammalian lung tissue.

[0489] 90. The isolated EPC population or the method of any one of embodiments 51 -89, wherein the isolated EPC population comprises an increase in a PROCR, a PDGFRA, or a VE-Cadherin protein expression level relative to an expression level in a non-isolated EPC population.

[0490] 91. The isolated EPC population or the method of any one of embodiments 51 -90, wherein the isolated EPC population comprises an increased proliferative capacity relative to a non-isolated EPC population.

[0491] 92. The isolated EPC population or the method of any one of embodiments 51 -91 , wherein the isolated EPC population comprises an increased angiogenic capacity relative to a non-isolated EPC population.

[0492] 93. The isolated EPC population or the method of any one of embodiments 51 -92, wherein the isolated EPC population comprises an increased in a colony forming capacity or a tube formation capacity relative to a non-isolated EPC population.

[0493] 94. The isolated EPC population or the method of any one of embodiments51 -93, wherein the isolated EPC population comprises an increased engraftment potential relative to a non-isolated EPC population.

[0494] 95. The isolated EPC population or the method of any one of embodiments 51 -94, wherein the isolated EPC population comprises one or more EPCs having a more elongated cell shape, relative to one or more EPCs in a non-isolated EPC population.

[0495] 96. The isolated EPC population or the method of embodiment 95, wherein the one or more EPCs are at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% more elongated.

[0496] 97. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of embodiments 1 -96, wherein the PROCR+ PDGFRA+ EPCs or the PROCR+ / - PDGFRA+ / - EPCs express one or more proteins selected from the group consisting of CD32, CDH5, CD34, CD31 , VEGFR2, VE-Cadherin and CD157.

[0497] 98. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of embodiments 1 -97, wherein the PROCR+ PDGFRA+ EPCs or the PROCR+ PDGFRA+ EPCs do not express one or more hematopoietic proteins.

[0498] 99. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 98, Wherein the one or morehematopoietic proteins are selected from the group consisting of CD3e, CD11 b, CD45, and B220.

[0499] 100. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of embodiments 1 -99, PROCR+ PDGFRA+ EPCs or the PROCR / -+ PDGFRA+ / - EPCs decrease a gene expression level of one of more of CD157, ABCG2, or SOX18 when differentiated, relative to an undifferentiated PROCR+ PDGFRA+ EPC or an undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0500] 101. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 100, wherein the decrease in the gene expression level of CD157 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a gene expression level of CD157 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0501] 102. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 100, wherein the decrease in the gene expression level of ABCG2 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a gene expression level of ABCG2 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0502] 103. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 100, wherein the decrease in the gene expression level of SOX18 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a gene expression level of SOX18 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0503] 104. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of embodiments 1 -99, PROCR+ PDGFRA+ EPCs or the PROCR / -+ PDGFRA+ / - EPCs decrease a protein expression level of one of more of CD157, ABCG2, or SOX18 when differentiated, relative to an undifferentiated PROCR+ PDGFRA+ EPC or an undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0504] 105. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 104, wherein the decrease in the protein expression level of CD157 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a protein expression level of CD157 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0505] 106. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 104, wherein the decrease in the protein expression level of ABCG2 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a protein expression level of ABCG2 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0506] 107. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 100, wherein the decrease in the protein expression level of SOX18 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a protein expression level of SOX18 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

[0507] 108. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of embodiments 1-107, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population comprises a mammalian cell.

[0508] 109. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 108, wherein the mammalian cell is a human cell.

[0509] 110. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 108 or 109, wherein the mammalian cell is a placental cell.

[0510] 111. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of embodiments 1-110, wherein theisolated cell population, the first isolated cell population, or the second isolated cell population are derived from a donor.

[0511] 112. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of embodiments 1 -111 , wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from two or more donors.

[0512] 113. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 111 or 112, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population comprise autologous or allogenic cells.

[0513] 114. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of embodiments 1 -113, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are present in a medium.

[0514] 115. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of embodiment 114, wherein the medium comprises a liquid medium or a frozen medium.

Claims

CLAIMSI / We claim:

1. A pharmaceutical composition comprising or consisting of an isolated endothelial progenitor cell (EPC) population comprising PROCR+ PDGFRA+ EPCs.

2. A pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs for use in treating neonatal hypoxic-ischemic encephalopathy (HIE) in a subject in need thereof.

3. A pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs.

4. A pharmaceutical composition comprising or consisting of an isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs for use in treating a brain injury in a subject in need thereof.

5. The pharmaceutical composition of claim 1 , wherein the isolated EPC population comprises an increase in a PROCR, a PDGFRA, or a VE-Cadherin protein expression level relative to an expression level in a non-isolated EPC population.

6. The pharmaceutical composition of claim 5, wherein the increase in the PROCR protein expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PROCR protein expression level in the non-isolated EPC.

7. The pharmaceutical composition of claim 5, wherein the increase in the PDGFRA protein expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PDGFRA protein expression level in the non-isolated EPC.

8. The pharmaceutical composition of any one of claims 1 -7, wherein the isolated EPC population comprises an increase in a PROCR, a PDGFRA, or a VE- Cadherin gene expression level relative to an expression level in a non-isolated EPC population.

9. The pharmaceutical composition of claim 8, wherein the increase in the PROCR gene expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PROCR gene expression level in the non-isolated EPC.

10. The pharmaceutical composition of claim 8, wherein the increase in the PDGFRA gene expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a PDGFRA gene expression level in the non-isolated EPC.

11. The pharmaceutical composition of claim 8, wherein the increase in the VE-Cadherin gene expression level is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a VE-Cadherin gene expression level in the non-isolated EPC.

12. The pharmaceutical composition of any one of claims 1 -11 , wherein the isolated EPC population comprises an increased proliferative capacity relative to a nonisolated EPC population.

13. The pharmaceutical composition of any one of claims 1 -12, wherein the isolated EPC population comprises an increased angiogenic capacity relative to a nonisolated EPC population.

14. The pharmaceutical composition of claim 13, wherein the increase in the angiogenic capacity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to an angiogenic capacity level in the nonisolated EPC.

15. The pharmaceutical composition of any one of claims 1 -14, wherein the isolated EPC population comprises an increased in a colony forming capacity or a tube formation capacity relative to a non-isolated EPC population.

16. The pharmaceutical composition of claim 15, wherein the increase in the colony forming capacity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a colony forming capacity level in the non-isolated EPC.

17. The pharmaceutical composition of claim 15, wherein the increase in the tube forming capacity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a tube forming capacity level in the non-isolated EPC.

18. The pharmaceutical composition of any one of claims 1 -17, wherein the isolated EPC population comprises an increased engraftment potential relative to a nonisolated EPC population.

19. The pharmaceutical composition of claim 18, wherein the increase in the engraftment potential is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to an engraftment potential level in the non-isolated EPC.

20. The pharmaceutical composition of any one of claims 1 -19, wherein the isolated EPC population comprises one or more EPCs having a more elongated cell shape, relative to one or more EPCs in a non-isolated EPC population.

21. The pharmaceutical composition of claim 20, wherein the one or more EPCs are at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% more elongated compared to the non-isolated EPC population.

22. The pharmaceutical composition of any one of claims 1 -21 , wherein the isolated EPC population comprises a CD45- / CD34+ phenotype.

23. The pharmaceutical composition of any one of claims 3-22, wherein the pharmaceutical composition is a first composition formulated for administration before, during, or after administration of a second composition comprising an isolated mesenchymal stem cell (MSC) population to a subject in need thereof.

24. A pharmaceutical composition comprising(a) a first isolated cell population comprising PROCR+ PDGFRA+ EPCs; and(b) a second isolated cell population comprising MSCs.

25. 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 pharmaceutical composition for use in treating HIE in a subject in need thereof, the pharmaceutical composition comprising(a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and(b) a second isolated cell population comprising MSCs.

27. The pharmaceutical composition of claim 26, wherein the HIE is neonatal HIE.

28. A pharmaceutical composition for use in treating a brain injury in a subject in need thereof, the pharmaceutical composition comprising(a) a first isolated cell population comprising PROCR+ / - PDGFRA+ / - EPCs; and(b) a second isolated cell population comprising MSCs.

29. The pharmaceutical composition of claim 28, wherein the brain injury comprises neurodegeneration.

30. The pharmaceutical composition of any one of claims 1 -29, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle.

31. The pharmaceutical composition of claim 30, wherein the pharmaceutically acceptable vehicle is phosphate-buffered saline.

32. The pharmaceutical composition any one of claims 3-31 , wherein the pharmaceutical composition is formulated for intranasal delivery, intrathecal, intraarterial, intralesional, or intravenous delivery to a subject in need thereof.

33. The pharmaceutical composition of claim 28, wherein the brain injury comprises ischemic brain injury.

34. The pharmaceutical composition of any one of claims 26-33, wherein the subject has received or is receiving a therapeutic hypothermia treatment.

35. A method of treating HIE 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 MSCs.

36. The method of claim 35, wherein the HIE is neonatal HIE.

37. A method of treating brain injury 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 MSCs.

38. The method of claim 37, wherein the brain injury comprises neurodegeneration.

39. The method of claim 37, wherein the brain injury comprises ischemic brain injury.

40. A cell composition for use in a treatment of neonatal HIE, the composition comprising a mixture of two isolated cell populations, wherein the first isolated cellpopulation consists PROCR+ / - PDGFRA+ / - EPCs, and the second isolated cell population consists of MSCs.41 . A method of treating neonatal HIE in a subject in need thereof, the method comprising administering to the subject a cell composition comprising a mixture of two cell isolated populations, wherein the first isolated cell population consists of PROCR+ / - PDGFRA+ / - EPCs, and the second isolated cell population consists of MSCs.

42. The method of any one of claims 35-41 , wherein the subject has received or is receiving a therapeutic hypothermia treatment.

43. A pharmaceutical composition comprising a cell composition and a pharmaceutically acceptable vehicle for use in a treatment of HIE wherein the cell composition comprises a mixture of two isolated cell populations, wherein the first isolated cell population consists of mammalian PROCR+ / - PDGFRA+ / - PROCR+ / - PDGFRA+ / - EPCs, and the second isolated cell population consists of MSCs.

44. A pharmaceutical composition for use according to claim 43, wherein the pharmaceutically acceptable vehicle is phosphate-buffered saline.

45. An isolated cell population, a cell composition, a pharmaceutical composition, or a cell composition for use according to any one of claims 26, 27, 35, 36, or 41 , wherein the use in the treatment of HIE or the treatment of neonatal HIE comprises providing a therapeutic hypothermia treatment to a subject in need thereof, and additionally administering the isolated cell population, the isolated cell composition, the cell composition or the pharmaceutical composition to the subject before, during and / or after the therapeutic hypothermia treatment.

46. The pharmaceutical composition, the method, or the cell composition of any one of claims 24-45, wherein the first isolated cell population and the second isolated cell population are present in the composition at a ratio of about 1 :1.

47. The pharmaceutical composition, the method, or the cell composition of any one of claims 24-45, 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.

48. The pharmaceutical composition, the method, or the cell composition of any one of claims 24-47, wherein the MSCs are CD45- / CD34+ cells.

49. The pharmaceutical composition, the method, or the cell composition of claim 48, 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.

50. The pharmaceutical composition, the method, or the cell composition of any one of claims 24-49, 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.

51. An isolated EPC population comprising PROCR+ PDGFRA+ EPCs, the isolated EPC population 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 the isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

52. An isolated EPC population comprising PROCR+ / - PDGFRA+ / - EPCs, the isolated EPC population 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 the isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

53. The isolated EPC population of claim 51 or 52, wherein step (ii) comprises selecting cell 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.

54. The isolated EPC population of claim 53, wherein 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).

55. The isolated EPC population of any one of claims 51 -54, wherein 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 population of cells which are CD45- / CD34+.

56. The isolated EPC population of any one of claims 51 -55, wherein 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.

57. A method of isolating an EPC population comprising PROCR+ PDGFRA+ EPCs, the method comprising 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 the isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs.

58. A method of isolating an EPC population comprising PROCR+ / - PDGFRA+ / - EPCs, the method comprising 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 the isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

59. The method of claim 57 or 58, wherein step (ii) comprises selecting cell 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.

60. The method of claim 59, wherein 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).

61. The method of any one of claims 57-60, wherein 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 population of cells which are CD45- / CD34+.

62. The method of any one of claims 57-61 , wherein step (iv) comprises contacting a cell or cells that express a PROCR surface protein with a PROCR-bindingmolecule to form a complex, removing the complex from the population of cells and retaining the complex.

63. An isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs, the isolated cell population 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.

64. An isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs, the isolated EPC population 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.

65. The isolated EPC population of claim 63 or 64, wherein 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.

66. A method of isolating an EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs, the method comprising 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.

67. A method of isolating an EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs, the method comprising 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.

68. The method of claim 66 or 67, wherein 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.

69. The method of any one of claims 66-68, wherein 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.

70. An isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs, the isolated EPC population 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.

71. An isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs, the isolated EPC population 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.

72. The isolated EPC population of claim 70 or 71 , wherein step (ii) 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.

73. The isolated EPC population of any one of claims 70-72, wherein step (iii) 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.

74. A method of isolating an EPC population comprising or consisting of PROCR+ PDGFRA+ EPCs, the method comprising 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.

75. A method of isolating an EPC population comprising or consisting of PROCR+ / -PDGFRA+ / - EPCs, the method comprising 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.

76. The method of claim 74 or 75, wherein step (ii) 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.

77. The method of any one of claims 74-76, wherein step (iii) 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.

78. The isolated EPC population or the method of any one of claims 54-56, 60-62, 65, 68, 69, 72, 73, 76, or 77, wherein the binding molecule comprises a protein.

79. The isolated EPC population or the method of claim 78, wherein the protein comprises an antibody.

80. The isolated EPC population or the method of any one of claims 54-56, 60-62, 65, 68, 69, 72, 73, 76, or 77-79, wherein removing the complex comprises microfluidic sorting.

81. The isolated EPC population or the method of claim 80, wherein the microfluidic sorting comprises microbead sorting or flow cytometry.

82. The isolated EPC population or the method of claim 81 , wherein the flow cytometry comprises fluorescence-activated cell sorting.

83. The isolated EPC population or the method of any one of claims 51-82, wherein the steps further comprise step (iv), culturing or contacting the isolated EPCpopulation with a cell population comprising endothelial colony forming cells (ECFCs) or a cell population comprising MSCs.

84. The isolated EPC population or the method of claim 83, wherein the steps further comprise step (v), separating the isolated EPC population from the cell population comprising ECFCs or the cell population comprising MSCs.

85. The isolated EPC population or the method of claim 84, wherein step (v) occurs 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).

86. The isolated EPC population or the method of any one of claims 51 -85, wherein the biological sample is a mammalian biological sample.

87. The isolated EPC population or the method of claim 86, wherein the mammalian biological sample is selected from the group consisting of a mammalian placenta, mammalian cord blood, mammalian peripheral blood, and mammalian tissueresident vascular endothelium.

88. The isolated EPC population or the method of claim 87, wherein the mammalian placenta is a whole mammalian placenta.

89. The isolated EPC population or the method of claim 87, wherein the mammalian tissue-resident vascular endothelium is selected from the group consisting of a mammalian umbilical cord, a mammalian pulmonary artery endothelium, a mammalian aorta, and a mammalian lung tissue.

90. The isolated EPC population or the method of any one of claims 51 -89, wherein the isolated EPC population comprises an increase in a PROCR, a PDGFRA, or a VE-Cadherin protein expression level relative to an expression level in a nonisolated EPC population.

91. The isolated EPC population or the method of any one of claims 51-90, wherein the isolated EPC population comprises an increased proliferative capacity relative to a non-isolated EPC population.

92. The isolated EPC population or the method of any one of claims 51-91 , wherein the isolated EPC population comprises an increased angiogenic capacity relative to a non-isolated EPC population.

93. The isolated EPC population or the method of any one of claims 51-92, wherein the isolated EPC population comprises an increased in a colony forming capacity or a tube formation capacity relative to a non-isolated EPC population.

94. The isolated EPC population or the method of any one of claims 51-93, wherein the isolated EPC population comprises an increased engraftment potential relative to a non-isolated EPC population.

95. The isolated EPC population or the method of any one of claims 51-94, wherein the isolated EPC population comprises one or more EPCs having a more elongated cell shape, relative to one or more EPCs in a non-isolated EPC population.

96. The isolated EPC population or the method of claim 95, wherein the one or more EPCs are at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% more elongated.

97. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of claims 1-96, wherein the PROCR+ PDGFRA+ EPCs or the PROCR+ / - PDGFRA+ / - EPCs express one or more proteins selected from the group consisting of CD32, CDH5, CD34, CD31 , VEGFR2, VE- Cadherin and CD157.

98. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of claims 1-97, wherein the PROCR+ PDGFRA+ EPCs or the PROCR+ PDGFRA+ EPCs do not express one or more hematopoietic proteins.

99. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 98, Wherein the one or more hematopoietic proteins are selected from the group consisting of CD3e, CD11 b, CD45, and B220.

100. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of claims 1 -99, PROCR+ PDGFRA+ EPCs or the PROCR / -+ PDGFRA+ / - EPCs decrease a gene expression level of one of more of CD157, ABCG2, or SOX18 when differentiated, relative to an undifferentiated PROCR+ PDGFRA+ EPC or an undifferentiated PROCR+ / - PDGFRA+ / - EPC.

101. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 100, wherein the decrease in the gene expression level of CD157 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a gene expression level of CD157 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

102. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 100, wherein the decrease in the gene expression level of ABCG2 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a gene expression level of ABCG2 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

103. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 100, wherein the decrease in the gene expression level of SOX18 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a gene expression level of SOX18 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

104. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of claims 1 -99, PROCR+ PDGFRA+EPCs or the PROCR / -+ PDGFRA+ / - EPCs decrease a protein expression level of one of more of CD157, ABCG2, or S0X18 when differentiated, relative to an undifferentiated PROCR+ PDGFRA+ EPC or an undifferentiated PROCR+ / - PDGFRA+ / - EPC.

105. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 104, wherein the decrease in the protein expression level of CD157 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a protein expression level of CD157 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

106. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 104, wherein the decrease in the protein expression level of ABCG2 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a protein expression level of ABCG2 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

107. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 100, wherein the decrease in the protein expression level of SOX18 is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a protein expression level of SOX18 in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC.

108. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of claims 1 -107, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population comprises a mammalian cell.

109. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 108, wherein the mammalian cell is a human cell.

110. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 108 or 109, wherein the mammalian cell is a placental cell.

111. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of claims 1 -110, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from a donor.

112. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of claims 1 -111 , wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are derived from two or more donors.

113. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 111 or 112, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population comprise autologous or allogenic cells.

114. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of any one of claims 1 -113, wherein the isolated cell population, the first isolated cell population, or the second isolated cell population are present in a medium.

115. The pharmaceutical composition, the method, the isolated EPC population, or the cell composition of claim 114, wherein the medium comprises a liquid medium or a frozen medium.

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