Compositions comprising isolated endothelial progenitor cells and uses thereof

Abstract

The present technology includes an isolated population of endothelial progenitor cells (EPCs) comprising PROCR + / -PDGFRA + / -EPC and a population of mesenchymal stem cells (MSCs), as well as methods of preparation and use thereof in the treatment of hypoxic ischemic encephalopathy (HIE) or brain injury in a subject.

Description

Background Technology

[0001] Vascularization is essential for the growth and blood circulation of developing organs, both in terms of tissue maintenance and repair. Angiogenesis and neovascularization depend on a population of progenitor cells that self-renew, differentiate into mature endothelial cells, and fuse to form blood vessels de novo. Because the endothelial layer is constantly at risk of defects, repair mechanisms can be permanently activated via endothelial progenitor cells (EPCs). Numerous clinical trials using different cell types to promote vascular repair have been evaluated. The most commonly used sources are early-growing EPCs from the peripheral circulation and bone marrow mononuclear cells (MNCs). A major limitation of these early clinical studies relates to how EPCs are defined. In addition to more classic endothelial markers such as vascular endothelial (VE)-cadherin or CD31, flow cytometry using CD34, VEGFR2 (KDR / FLK-1), and / or CD133 is routinely used to identify the number of circulating endothelial progenitor cells. However, combinations of CD34, VEGFR2, CD133, VE-cadherin, and / or CD31 biomarkers have not yet yielded reliable or differentially differentiated biomarker sets. Therefore, more effective therapeutic approaches remain based on the use of EPC alone or in combination with other cell types. Summary of the Invention

[0002] This technology includes pharmaceutical compositions containing isolated endothelial progenitor cell (EPC) populations or composed of isolated endothelial progenitor cell (EPC) populations, as well as methods for preparing and using these pharmaceutical compositions.

[0003] In some embodiments, the technology includes a pharmaceutical composition containing or composed of isolated endothelial progenitor cell (EPC) populations, said isolated endothelial progenitor cell (EPC) populations comprising PROCR+PDGFRA+EPC.

[0004] In some embodiments, the technology includes a pharmaceutical composition comprising or consisting of: a group of isolated EPCs containing PROCR+ / - PDGFRA+ / - EPCs, the pharmaceutical composition being used in the treatment of neonatal hypoxic-ischemic encephalopathy (HIE) in a subject of need.

[0005] In some embodiments, the technology includes a pharmaceutical composition containing isolated endothelial progenitor cell (EPC) populations or composed of isolated endothelial progenitor cell (EPC) populations, said isolated EPC populations comprising PROCR+ / - PDGFRA+ / - EPC.

[0006] In some embodiments, the technology includes a pharmaceutical composition comprising or consisting of: isolated EPC clusters, said isolated EPC clusters comprising PROCR+ / - PDGFRA+ / - EPC, said pharmaceutical composition for use in treating brain injury in a subject of need.

[0007] In some embodiments, the isolated EPC population includes increased expression levels of PROCR protein, PDGFRA protein, or VE-cadherin protein, relative to the expression levels in the unisolated EPC population.

[0008] In some embodiments, the increase in PDGFRA protein expression level relative to the PDGFRA protein expression level in the unseparated EPC 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%.

[0009] In some embodiments, the increase in the PROCR protein expression level relative to the PROCR protein expression level in the unseparated EPC 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%.

[0010] In some implementations, the isolated EPC population includes increased expression levels of the PROCR gene, PDGFRA gene, or VE-cadherin gene, relative to the expression levels in the unisolated EPC population.

[0011] In some embodiments, the increase in the PROCR gene expression level relative to the PROCR gene expression level in the unseparated EPC 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%.

[0012] In some embodiments, the increase in PDGFRA gene expression level relative to the PDGFRA gene expression level in the unseparated EPC 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%.

[0013] In some embodiments, the increase in the VE-cadherin gene expression level relative to the VE-cadherin gene expression level in the unseparated EPC 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%.

[0014] In some implementations, isolated EPC populations contain increased proliferation capacity compared to unisolated EPC populations.

[0015] In some implementations, isolated EPC clusters contain increased angiogenic capacity compared to unisolated EPC clusters.

[0016] In some embodiments, the increase in angiogenesis capacity relative to the level of angiogenesis capacity in the unseparated EPC 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%.

[0017] In some implementations, a separated EPC cluster includes an increase in cluster-forming or pipe-forming capabilities compared to an unseparated EPC cluster.

[0018] In some implementations, the increase in colony-forming capacity relative to the colony-forming capacity level in the unseparated EPC 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%.

[0019] In some embodiments, the increase in tube forming capability relative to the tube forming capability level in the unseparated EPC 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%.

[0020] In some implementations, a separated EPC cluster contains increased implantation potential compared to an unseparated EPC cluster.

[0021] In some embodiments, the increase in implantation potential relative to the implantation potential level in the unseparated EPC 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%.

[0022] In some implementations, a separated EPC group comprises one or more EPCs having a more elongated cell shape, relative to one or more EPCs in an unseparated EPC group.

[0023] In some implementations, the one or more EPCs are elongated by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% compared to the unseparated EPC group.

[0024] In some implementations, the isolated EPC groups have the CD45- / CD34+ phenotype.

[0025] In some embodiments, the pharmaceutical composition is a first composition formulated for administration before, during, or after administration to a subject in need of a second composition comprising isolated mesenchymal stem cell (MSC) populations.

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

[0027] In some embodiments, the technology includes a pharmaceutical composition comprising (a) a first isolated cell population containing PROCR+ / - PDGFRA+ / - EPC; and (b) a second isolated cell population containing MSCs.

[0028] In some embodiments, the technology includes a pharmaceutical composition for use in treating HIE in a subject of need, the pharmaceutical composition comprising (a) a first isolated cell population containing PROCR+ / - PDGFRA+ / - EPC; and (b) a second isolated cell population containing MSCs.

[0029] In some embodiments, HIE is neonatal HIE.

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

[0031] In some implementations, brain injury includes neurodegeneration.

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

[0033] In some implementations, the pharmaceutically acceptable medium is phosphate-buffered saline.

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

[0035] In some implementations, brain injury includes ischemic brain injury.

[0036] In some implementations, the subjects have received or are receiving 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 approximately 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 implementations, MSCs are CD45- / CD34+ cells.

[0040] In some implementations, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the second isolated cell population are CD34+ / CD45- isolated cell populations.

[0041] In some implementations, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated cell population or the second isolated cell population express PROCR and PDGFRA.

[0042] In some embodiments, the technology includes a method for isolating an EPC population containing PROCR+ PDGFRA+ EPC, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) enriching cells containing the CD45- phenotype in the biological sample to obtain a CD45- cell population; (iii) selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) selecting cells expressing both the PROCR+ and PDGFRA+ phenotypes from the CD45- / CD34+ cell population to obtain an isolated EPC population containing or consisting of PROCR+ PDGFRA+ EPC.

[0043] In some embodiments, the technology includes a method for isolating an EPC population comprising PROCR+ / - PDGFRA+ / - EPCs, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) enriching cells containing the CD45- phenotype in the biological sample to obtain a CD45- cell population; (iii) selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) selecting cells expressing both PROCR+ / - and PDGFRA+ / - phenotypes from the CD45- / CD34+ cell population to obtain an isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

[0044] In some implementations, step (ii) includes selecting cells expressing the CD45+ phenotype, removing cells expressing the CD45+ phenotype from the biological sample of step (i), and discarding cells expressing the CD45+ phenotype to obtain a CD45- cell population.

[0045] In some embodiments, the step of selecting cells expressing the CD45+ phenotype includes contacting one or more cells expressing the CD45 surface protein with CD45-binding molecules to form a complex, and removing the complex from the biological sample in step (i).

[0046] In some embodiments, step (iii) includes contacting one or more cells expressing the CD34 surface protein with a CD34 binding molecule to form a complex, removing the complex from the CD45- cell population of step (ii), and retaining the complex to obtain a second cell population of CD45- / CD34+.

[0047] In some embodiments, step (iv) includes contacting one or more cells expressing the PROCR surface protein with the PROCR binding molecule to form a complex, removing the complex from the cell population, and retaining the complex.

[0048] In some embodiments, the technology includes a method for isolating an EPC population comprising PROCR+ PDGFRA+ EPC, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) enriching cells containing the PDGFRA+ phenotype in the biological sample to obtain a PDGFRA+ cell population; and (iii) selecting cells expressing the PROCR+ phenotype from the PDGFRA+ cell population to obtain an isolated EPC population comprising PROCR+ PDGFRA+ EPC.

[0049] In some embodiments, the technology includes a method for 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 cells containing the PDGFRA+ / - phenotype in the biological sample to obtain a PDGFRA+ / - cell population; and (iii) selecting cells expressing the PROCR+ / - phenotype from the PDGFRA+ / - cell population to obtain an isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

[0050] In some embodiments, step (ii) includes contacting one or more cells expressing the PDGFRA surface protein with PDGFRA binding molecules to form a complex, removing the complex from the cell population and retaining the complex.

[0051] In some embodiments, step (iii) includes contacting one or more cells expressing the PROCR surface protein with the PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0052] In some embodiments, the technology includes a method for isolating an EPC population comprising or consisting of PROCR+ PDGFRA+ EPC, the method comprising the steps of: (i) obtaining a biological sample from a subject; (ii) enriching cells containing the PROCR+ phenotype in the biological sample to obtain a PROCR+ cell population; and (iii) selecting cells expressing the PDGFRA+ phenotype from the PROCR+ cell population to obtain an isolated EPC population comprising or consisting of PROCR+ PDGFRA+ EPC.

[0053] In some embodiments, the technology includes a method for 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 cells containing the PROCR+ / - phenotype in the biological sample to obtain a PROCR+ / - cell population; and (iii) selecting cells expressing the PDGFRA / -+ phenotype from the PROCR+ / - cell population to obtain an isolated EPC population comprising or consisting of PROCR+ / - PDGFRA+ / - EPCs.

[0054] In some implementations, step (ii) includes contacting one or more cells expressing the PROCR surface protein with the PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0055] In some embodiments, step (iii) includes contacting one or more cells expressing the PDGFRA surface protein with PDGFRA binding molecules to form a complex, removing the complex from the cell population and retaining the complex.

[0056] In some implementations, the binding molecule contains a protein.

[0057] In some implementations, the protein contains an antibody.

[0058] In some implementations, removing the complex includes performing microfluidic sorting.

[0059] In some implementations, microfluidic sorting includes bead sorting or flow cytometry.

[0060] In some implementations, flow cytometry includes fluorescence-activated cell sorting.

[0061] In some embodiments, the steps further include step (iv), culturing or contacting the isolated EPC population with a cell population containing endothelial colony-forming cells (ECFCs) or a cell population containing MSCs.

[0062] In some implementations, the steps further include step (v), separating the isolated EPC population from a cell population containing ECFCs or a cell population containing MSCs.

[0063] In some implementations, step (v) occurs at least approximately 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 implementations, the biological sample is a mammalian biological sample.

[0065] In some implementations, mammalian biological samples are selected from the group consisting of mammalian placenta, mammalian umbilical cord blood, mammalian peripheral blood, and mammalian tissue-resident vascular endothelium.

[0066] In some implementations, the mammalian placenta is a complete mammalian placenta.

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

[0068] In some implementations, PROCR+ PDGFRA+ EPC or PROCR+ / - PDGFRA+ / - EPC expresses one or more proteins selected from the group consisting of CD32, CDH5, CD34, CD31, VEGFR2, VE-cadherin, and CD157.

[0069] In some implementations, PROCR+PDGFRA+EPC or PROCR+PDGFRA+EPC does not express one or more hematopoietic proteins.

[0070] In some implementations, one or more hematopoietic proteins are selected from the group consisting of CD3e, CD11b, CD45, and B220.

[0071] In some implementations, compared to undifferentiated PROCR+ PDGFRA+ EPC or undifferentiated PROCR+ / - PDGFRA+ / - EPC, PROCR+ PDGFRA+ EPC or PROCR / -+ PDGFRA+ / - EPC reduces the expression level of one or more of CD157, ABCG2, or SOX18 during differentiation.

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

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

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

[0075] In some implementations, compared to undifferentiated PROCR+ PDGFRA+ EPC or undifferentiated PROCR+ / - PDGFRA+ / - EPC, PROCR+ PDGFRA+ EPC or PROCR / -+ PDGFRA+ / - EPC reduces the expression level of one or more of the proteins CD157, ABCG2, or SOX18 upon differentiation.

[0076] In some implementations, the isolated cell population, the first isolated cell population, or the second isolated cell population contains mammalian cells.

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

[0078] In some implementations, the mammalian cells are placental cells.

[0079] In some implementations, the isolated cell population, the first isolated cell population, or the second isolated cell population is derived from the donor.

[0080] In some implementations, the isolated cell population, the first isolated cell population, or the second isolated cell population is derived from two or more donors.

[0081] In some implementations, the isolated cell population, the first isolated cell population, or the second isolated cell population contains autologous cells or allogeneic cells.

[0082] In some implementations, the isolated cell population, the first isolated cell population, or the second isolated cell population is present in the culture medium.

[0083] In some implementations, the culture medium includes liquid culture medium or frozen culture medium. Attached Figure Description

[0084] Figure 1A-1D Single-cell RNA sequencing analysis of the rat aortic endothelial compartment is shown. Figure 1A Clustering was shown across three aortic samples. Figure 1B The SingleR unbiased labeling analysis is shown. Figure 1C The differentially expressed genes in the endothelial progenitor cell (EPC) cluster were shown. Figure 1D The top differentially expressed genes in mesenchymal stem cell (MSC) clusters were shown.

[0085] Figure 2A-2I Single-cell RNA sequencing and flow cytometry demonstrated that PROCR and PDGFRA can be markers in the endothelial population. Figure 2A Single-cell RNA sequencing data from the endothelium of a mouse aorta displaying aEPC, mature differentiated endothelial cells, and target MSC clusters (n=3) are shown. Figure 2B A separate graph showing the representation of the target markers in each cluster is displayed. Figure 2C The relative expression of the target gene across clusters is shown. Figure 2D A flow cytometry-gated strategy demonstrating the separation of endothelial layers is shown. (Targeting...) Figure 2E Endothelial progenitor cells (EPCs) and Figure 2F The mature and differentiated endothelial cell population can be visualized by flow cytometry, showing cells further gated on Procr, CD157, Abcg2-YFP, and Sox18-YFP. Figure 2G This demonstrates the quantification of endothelial progenitor cells (EPCs) and mature differentiated endothelial cells that are positive for the following 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). Figure 2H An alternative gating strategy was demonstrated: first, live cells were gated to PROCR. + PDGFRA + Secondly, the gating is Lin - VE - cadherin + Ultimately, it is gated to EPCs and mature differentiated endothelial cells based on CD31 and CD34 expression. Figure 2I Showing the use Figure 2E Quantitative analysis of the EPC gating percentage in the gating strategy (in the context of gating strategies) (p < 0.0001, n = 3).

[0086] Figure 3A-3G PROCR was displayed + Compared with other cell populations, EPC has a stronger ability to form endothelial cell colonies in vitro and a higher potential for in vivo implantation. Figure 3A The image shows FACS sorting of endothelial cells from the C57Bl / 6 aorta based on cell surface expression of PROCR as depicted in the figure. Figure 3B Representative bright-field images (i-ii) depicting endothelial (i) and elongation (ii) cell morphology types on day 12 (4x magnification; scale bar = 1 mm) and IF (iii-iv) of endothelial (iii) and elongation (iv) colonies under BSL-I isohemagglutinin on day 12 (10x magnification; scale bar = 150 μm). Figure 3C The percentage of colonies formed under each condition is shown, based on the number of inoculation wells. All experiments were normalized to 10 cells per well (n=14). p<0.05). Figure 3D A graphical representation depicting the experimental steps used in an in vivo collagen gel implantation trial is shown. Figure 3E The image shows (i) PROCR samples obtained by flow cytometry sorting from the aorta of CAG-EGFP mice collected 7 days after implantation into NOD-scid Il2rynullB2mnull (NSG) mice. + EPC or (ii) PROCR - A representative IF image from EPC (scale bar = 500 µm). Figure 3F The percentage of GFP+ area in each gel was measured by IF after collection. p<0.05; n=3). Figure 3G(i)PROCR samples were collected 7 days after staining with DAPI, GFP, CD34, and isolectins. + EPC and (ii) PROCR - Representative IF images of sections of EPC collagen gel (scale bar = 250 µm).

[0087] Figures 4A-4F PROCR was displayed + EPC forms a niche in the thoracic aorta, demonstrating increased clonogenic capacity. Figures 4A-4C The aorta, dissected and rolled up along its length, is shown from Cdh5-CreERT2 / ROSA-EYFP mice. Figure 4A and 4B Representative images of slices from the thoracic aorta and abdominal aorta are shown, respectively. Figure 4C Showing Figure 4A The thoracic aortic slice shown is magnified 60x; the white arrows indicate the overlapping area between DAPI, PROCR, and YFP. Figure 4D PROCR is shown in the abdominal aorta and thoracic aorta. + Percentage of length ( p=0.005; n=5). Figure 4E Representative bright-field images of colonies formed from the thoracic aorta of Zs-Green / ROSA-EYFP mice after 12 days of culture in Matrigel are shown; scale bar = 500 µm. Figure 4F This demonstrates the quantitative analysis of colonies formed by cultures from the thoracic and abdominal aortas. (p=0.0052, n=13).

[0088] Figures 5A-5E PROCR+ PDGFRA+ EPCs derived from Pdgfra-MerCreMer / Rosa-YFP differentiate into mature endothelial cells in a steady-state aorta. Figure 5A (i-ii) show the gating strategies used to separate YFP+ PROCR+ PDGFRA+ EPCs and mature differentiated endothelial cells from the aorta of PDGFRa-MerCreMer / Rosa-YFP mice; (iii) the percentage of PROCR+ PDGFRA+ EPCs and mature differentiated endothelial cells in the Lin-YFP+ fraction of the adult homeostatic aorta. (p < 0.0001, n = 4). Figure 5BRepresentative IF images of YFP+ PROCR+ PDGFRA+ EPC cultured for 12 days from PDGFRa-MerCreMer / Rosa-YFP mice are shown. Figure 5C Representative IF images of aortic slices from D1 (i) and D84 (ii; scale bar: large image = 200 μm, scaled image = 10 μm) are shown. Figure 5D The changes in the ratio of PROCR+ PDGFRA+ EPC cells shown in red ovals to mature differentiated endothelial cells shown in black ovals are displayed in the Lin-YFP+ compartment of the steady-state aorta of mice injected with tamoxifen at 4 weeks of age. Figure 5E This shows the percentage of PROCR+ PDGFRA+ EPC versus mature differentiated endothelial cells in the Lin-YFP+ segment of the aorta between D1 and D84. p<0.01, p<0.001; n=4).

[0089] Figures 6A-6E PROCR from Pdgfra-MerCreMer / Rosa-YFP is shown in a lesion model of a total skin excision wound. + EPCs differentiate into mature endothelial cells. Figure 6A and 6B The representative IF images shown depict aortic slices from D1 and D84 with endothelial markers. Figure 6B Results are shown with scale bars: Large image = 200 μm, Scaled image = 10 μm. Figure 6C and 6D The Lin-YFP+ compartment of the total skin excision wound in adult mice is shown in the PROCR+ PDGFRA+ EPC ((ii-iv) in D1 and D5) Figure 6C (shown as a red oval in the middle); ((ii-iv) in Figure 6D (The proportions are shown in black in the middle.) Figure 6E This shows the percentage of PROCR+ PDGFRA+ EPC versus mature differentiated endothelial cells in the Lin-YFP+ region of the wound between D1 and D5. p<0.05, p<0.01 (n=7).

[0090] Figures 7A-7FPROCR was shown to be expressed in human aortic scRNA-seq data and led to increased clonogenic capacity in a human full-term placenta model of endothelial colony-forming cells (ECFCs). Figure 7A Single-cell RNA sequencing data from a human control aorta are shown, with hematopoietic clusters removed and primary cell populations labeled (n=3) removed by clustering and filtering. Figure 7B The target marker for cross-cluster display is shown. Figure 7C The flow cytometry plot shown reveals PROCR from a full-term human placenta. + / - EPC gating strategy. Figure 7D Showing the PROCR culture - (i) or PROCR + (ii) Bright-field image of colonies growing in EPC (scale bar = 200 µm). Figure 7E PROCR was displayed + Immunofluorescence staining of aggregates at P6. Figure 7F Showing the PROCR culture - or PROCR + EPC results in (i) community counts and (ii) quantitative analysis of community types.

[0091] Figures 8A-8E Additional single-cell RNA sequencing analysis and flow cytometry (FMO) were shown for the human dataset. Figure 8A Clustering was shown among three samples of the aorta from a normal human. Figure 8B The SingleR unbiased labeling analysis is shown. Figure 8C and 8D EPC (shown) Figure 8C ) and MSC ( Figure 8D Scatter plot of differentially expressed genes at the top of the cluster. Figure 8E The FMO control used for FACS sorting of human full-term placenta is shown.

[0092] Figure 9 A schematic diagram of the fetal EPC and MSC cell population isolation method is shown, which involves additional selection of PROCR cells. + Biomarkers for identifying fetal EPC and by selecting PROCR - To identify fetal mesenchymal stem cells (MSCs).

[0093] Figure 10 This diagram illustrates how the PROCR+ PDGFRA+ EPC and PROCR-PDGFRA+ MSC cell population separation methods require the selection of PDGFRA and PROCR markers to identify PROCR+ PDGFRA+ EPC and PROCR-PDGFRA+ MSC.

[0094] Figure 11 The images show the experimental setup and sample data. Blood oxygen saturation, arterial blood pressure, end-tidal CO2, and rectal temperature were collected using the Marquette data acquisition system.

[0095] Figure 12 Showing from Figure 11 Schematic diagram of experimental setup and sample data Figure 13 The study demonstrated increased survival in piglets treated with a combination of hypothermia, PROCR+ / - PDGFRA+ / - EPC, and MSC cell therapy (HHS group). Statistical comparisons were performed using the Mantel-Cox test for equality of survival distributions between groups. HHS: Hypothermia and stem cell therapy group; HHV: Hypothermia and stem cell vector therapy group.

[0096] Figure 14 The mean aEEG score and background amplitude score of HI-injured piglets are shown. Statistical comparisons: Welch t-test, corrected using the Holm-Sidak method. HHS: hypothermia and stem cell treatment group; HHV: hypothermia and stem cell vector treatment group.

[0097] Figure 15A-15J The results of MRI, MRS, and neurobehavioral assessments at P8 are shown. Figure 15A Representative T2 and ADC plots are shown for the hypothermic-stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) therapy (HHS) group and the hypothermic-stem cell-mediated therapy (HHV) group at P8. White circles indicate voxel regions used for quantification. Figure 15B and Figure 15C Quantitative MRI measurements of T2 relaxation time and ADC values ​​were shown between the hypothermic-stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treatment group and the hypothermic-stem cell vector treatment group (HHV). Figure 15D Representative magnetic resonance spectra of the brain at P8 are shown. T2-weighted images, in coronal and sagittal views, show the voxel locations for proton magnetic resonance spectroscopy analysis. Figure 15E-15I The ratios of NAA / Cho, NAA / Cr, Cho / Cr, Lac / NAA, and Lac / Cr are shown respectively. Figure 15J The average neurobehavioral score over time is shown, with a significant decrease in the average neurobehavioral score at P3 post-injury. The dashed line represents the full score.

[0098] Figure 16 A-16C shows representative hematoxylin and eosin staining. Figure 16 A-16C shows a reduction in localized neuropathology within the parasagittal frontal cortex. High-power image ( Figure 16 (A'-16C') shows the presence of eosinophilic neurons (with tailed arrows), indicating acute neuronal damage that may not have been present in the hypothermic stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treatment (HHS) group or the control group. Immune cells (without tailed arrows) may also be shown. Scale bar: 50 μm on high-magnification images.

[0099] Figure 17A -17R shows altered expression of neuropathological markers in the frontal cortex of piglets with HI injury at P8. Figure 17A Figure 17C shows immunofluorescence labeling of mature neurons in the piglet brain using the neuron-specific nuclear marker NeuN. Scale bar: 250 μm. Figures 17D, 17I, and 17N show NeuN in the frontal cortex, respectively. + / mm 2 FJC + / mm 2 and C-cystase 3 + / mm 2 Quantitative analysis. Figures 17E, 17J, and 17O show NeuN in the shell and core, respectively. + / mm 2 FJC + / mm 2 and C-cystase 3 + / mm 2 Quantitative analysis. Figures 17F to 17H show representative staining of degenerated neurons with Fluoro-Jade C in the brains of patients treated with hypothermia and stem cells (PROCR+ / -PDGFRA+ / - EPC and MSC) (HHS) and those treated with hypothermia and stem cell mediators (HHV) (Scale bar: 100 μm). Regional differences in the number of degenerated neurons as shown by quantification are seen in Figures 17I and 17J. Figures 17P-17R show superimposed imaging for each group. C: Control; HHS: Hypothermia and stem cell treatment group; HHV: Hypothermia and stem cell mediator treatment group; FC: Frontal cortex; Put: Putamen.

[0100] Figure 18A -18O shows a representative image of Iba-1 markers in the frontal cortex, with controls shown (thin-tailed arrows). Figure 18A and Figure 18A ') and hypothermia and stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treatment (HHS) group ( Figure 18C and Figure 18C Microglia in a resting state in the hypothermia-transfer therapy (HHV) group and the hypothermia-transfer therapy (HHV) group Figure 18B and Figure 18B Examples of activated microglia (thick-tailed arrows) in '). In the hypothermic stem cell (PROCR+ / -PDGFRA+ / - EPC and MSC) treatment (HHS) group, the cell morphology classification of microglia in the frontal cortex ( Figure 18D Figures 18H and 18I show the total number of microglia in the frontal cortex (Figure 18F) or putamen (Figure 18J) of the stem cell-mediated therapy (HHV) group. Figures 18K to 18M show representative images of astrocytes (GFAP) in the white matter of the gyri. Figures 18K to 18M show astrocytes with long, elongated processes and large cell bodies in the HHS group animals, similar to those observed in the healthy control group (indicated by thin arrows). Figure 18N shows no significant difference in IGWM. Figure 18O shows a significant difference between HHV and HHS in PVWM. Column height represents the median, and the error bar represents the 95% CI [lower limit, upper limit]. C: Control; HHS: Hypothermia and stem cell therapy group; HHV: Hypothermia and stem cell-mediated therapy group; FC: Frontal cortex; Put: Putamen.

[0101] Figure 19 Gene expression analysis of various inflammatory markers at P8 is shown. C: Control; HHS: Hypothermia and stem cell therapy group; HHV: Hypothermia and stem cell mediator therapy group.

[0102] Figure 20 The colocalization of TNFα and IL-1β with Iba-1 is shown in HHV and HHS. Thin-tailed arrows indicate colocalization of TNFα and Iba-1. Thick-tailed arrows indicate colocalization of IL-1β and Iba-1. Scale bar: 100 μm. C: Control; HHS: Hypothermia and stem cell therapy group; HHV: Hypothermia and stem cell mediator therapy group.

[0103] Figure 21 Differential colocalization of NF-κB p65 is shown between the hypothermic-stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treatment group (HHS) and the hypothermic-stem cell-mediated treatment group (HHV). Low-magnification image scale bar: 200 μm. C: Control; HHS: Hypothermic-stem cell treatment group; HHV: Hypothermic-stem cell-mediated treatment group.

[0104] Figure 22 Representative images of CD34 markers in the parasagittal region of the frontal cortex are shown. C: Control; HHS: Hypothermia and stem cell therapy group; HHV: Hypothermia and stem cell vector therapy group.

[0105] Figure 23The results of flow cytometry analysis of human placental PROCR+ cells sorted for various expression markers are shown.

[0106] Figure 24 The gene expression levels of CD31, CD34, VE-cadherin, and PDGFRA were shown in control endothelial colony-forming cells (ECFC) (ECFC-KK and ECFC-MG), MSCs, and PROCR+ECFC.

[0107] Figure 25 Representative images of isolated PROCR+ ECFCs, isolated fetal placental mesenchymal stem cells / stromal cells (fPL-MSCs), or co-cultured in EGM2 for 2 and 5 days are shown. Arrows indicate ECFCs. Scale bar = 100 μm Figure 26 The flow cytometry results show the sorting results of CD31+ cells from PROCR+ ECFCs co-cultured with fPL-MSCs. Detailed Implementation

[0108] This technology includes pharmaceutical compositions containing or composed of isolated endothelial progenitor cell (EPC) populations, and methods for preparing and using these pharmaceutical compositions. In some embodiments, the pharmaceutical composition further comprises isolated cell populations, said isolated cell populations comprising mesenchymal stem cells (MSCs). These and additional features of the technology are described throughout the application.

[0109] definition While this technology can be implemented in various forms, the following description of several embodiments is made on the premise that this technology is to be considered as an example and is not intended to limit the technology to the specific embodiments shown. Headings are provided for convenience only and are not to be construed as limiting the technology in any way. Embodiments shown under any heading may be combined with embodiments shown under any other heading.

[0110] Unless otherwise expressly stated, the use of numerical values ​​in the various quantitative values ​​specified in this application may be described as approximate, as if the minimum and maximum values ​​within the range were preceded by the word "about". The term "about" means a quantity, level, value, number, frequency, percentage, size, size, amount, weight, or length that varies according to levels acceptable in the art. Typically, such variation may be up to 10% higher or lower than a reference quantity, level, value, quantity, frequency, percentage, size, size, amount, weight, or length, and such variation may be affected by applicable standard measurement specifications. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the indicated numerical value.

[0111] Furthermore, the scope of this technology can be contemplated as a continuous range, including every value between the listed minimum and maximum values, and any range that can be formed from such values. This technology includes any and all (and any range of such ratios) values ​​that can be formed by dividing the numerical values ​​of this technology by any other numerical value of this technology. Therefore, those skilled in the art will understand that many such ratios, ranges, and ranges of ratios can be explicitly derived from the numerical values ​​presented herein, and in all cases, such ratios, ranges, and ranges of ratios represent various embodiments of this technology.

[0112] As used herein, any reference to “endothelial progenitor cell” should be understood as a reference to any cell having the potential to develop into a cell possessing some or more of the functional or structural features that endothelial cells may have. Without limiting the invention in any way, the reference to “endothelial cell” should be understood as referring to squamous epithelial cells that form the lining of blood vessels, lymphatic vessels, or other serous cavities such as fluid-filled cavities. The phrase “endothelial cell” should also be understood as referring to cells exhibiting one or more of the morphological, phenotypic, and / or functional activities of endothelial cells, and also as mutants or variants thereof. The endothelial cells may be at any stage of differentiation and development after the endothelial progenitor stage. “Variations” include, but are not limited to, cells exhibiting some, but not all, of the morphological or phenotypic features or functional activities of endothelial cells. “Mutants” include, but are not limited to, genetically modified endothelial cells, such as endothelial cells derived from endothelial progenitor cells, which may be genetically modified after isolation by the methods of the present invention but before undergoing directed differentiation along the endothelial cell lineage. In some implementations, the subject's endothelial cells may be vascular endothelial cells (i.e., endothelial cells that form blood vessels) or immature forms of endothelial cells that will form blood vessels through proliferation and differentiation, but may still be more mature than endothelial progenitor cells.

[0113] As used herein, the term "mesenchymal stem cell" refers to any cell with the potential to develop into one or more functional or structural features that may be present in mesenchymal cells or cells of mesenchymal origin, but not cells of non-mesenchymal origin, such as cell types derived from the endoderm or mesoderm. Mesenchymal stem cells may also be referred to as "stromal stem cells," "fetal stem cells," "adult stem cells," "adipocyte-derived stem cells," "liposuction-derived stem cells," and "postnatal stem cells." Therefore, the term "mesenchymal-derived cells" should be understood to refer to cell types that can differentiate to a higher degree than pluripotent mesenchymal cells and are derived from mesenchymal stem cells. These cells will correspond to cells of tissues known to be derived from mesenchymal cells, as detailed above. For example, a subject's mesenchymal-derived cells may be irreversibly committed to differentiating along a specific cell lineage (e.g., myocyte precursor cells or adipocyte precursor cells), or they may correspond to a partially or fully differentiated form of a specific cell subtype of one of these lineages. Thus, mesenchymal stem cells exhibit the ability to differentiate into one or more cell types of mesenchymal lineages under appropriate conditions. The International Society for Cell Therapy (ISCT) has proposed a standard for defining mesenchymal stem cells (MSCs).

[0114] "Separated cell populations" generally refer to cells suspended in a culture medium and free of other components (such as carriers and excipients). "Separated cell populations" can be suspended in liquid culture media and can exist at any suitable temperature and conditions for cell survival. "Separated cell populations" can be suspended in frozen culture media and can exist in a cryopreserved state at any suitable temperature and conditions for cell survival. Cell populations can exist in growth matrices as further discussed herein or fixed on a surface. Any number of associated cells can exist in a cell population. A cell population may contain at least about 5 × 10⁵ associated cells. A cell population may contain at least about 1 × 10⁶, at least about 2 × 10⁶, at least about 5 × 10⁶, at least about 1 × 10⁷, at least about 2 × 10⁷, at least about 5 × 10⁷, at least about 1 × 10⁸, or at least about 2 × 10⁸ associated cells. In some cases, the cell population may contain at least about 1.0 × 10⁷, at least about 1.0 × 10⁸, at least about 1.0 × 10⁹, at least about 1.0 × 10¹⁰, at least about 1.0 × 10¹¹, or at least about 1.0 × 10¹² or even more related cells.

[0115] The cells of this technology (including EPCs, mature differentiated mammalian endothelial cells (D) and MSCs) can be defined according to a “marker profile.” This is the standard way to define cells (e.g., progenitor cells and stem cells) that will be obvious, identifiable, and understandable to those skilled in the art. A “marker” or “biomarker” is typically a cell surface molecule, such as a receptor or ligand or other molecule. Alternatively, a “marker” or “biomarker” may be a molecule that is not a cell surface molecule, such as an intracellular molecule. In the context of cells in this technology, a “marker” or “biomarker” can be a cell surface molecule.

[0116] In the cellular context of this technology, it should be understood that references to any specific biomarker such as “CD45,” “CD34,” “CD31,” “PROCR,” and “PDGFRA” refer to all forms of these molecules and their functional fragments, mutants, or variants. It should also be understood to include any isotype that can be produced by, for example, alternative splicing of CD45, CD34, and CD31 mRNA or by isomorphic or polymorphic forms of these molecules. References to biomarkers can be to the wild-type form of the biomarker.

[0117] References to “phenotypic profile,” “expression phenotype,” or “cells expressing the phenotype” should be understood to refer to the presence or absence of cell surface expression of the transcriptional and / or translational expression products of genes encoding subject markers. Those skilled in the art will understand that while most cells falling within the cellular scope of this technique are characterized by the presence or absence of subject markers as cell surface anchored expression products, some cells falling within the defined cell population may initially exhibit changes only at the transcriptomic level, for example, when transcription of a given marker has been upregulated but cell surface anchored expression products may not yet be produced. Typically, cells progressing to a new stage of differentiation will transiently exhibit changes in gene expression, which may not be apparent against the backdrop of changes in expression product levels. However, these cells can still be defined according to a biomarker profile, although they may not be separable by cell surface marker expression. For any biomarker used in conjunction with this technique for cell identification, the biomarker may be a “cell surface anchored expression product,” a “cell surface anchored protein,” or a cell surface anchored peptide.

[0118] Unless the context clearly specifies otherwise, references to a marker refer to the detectable expression of the marker or the level at which the marker is detected.

[0119] The terms "+" and "-" are well-known in the art and refer to the expression level of the target cell marker, since "+" corresponds to a high or intermediate expression level of the cell marker, and "-" corresponds to an none expression level of the cell marker. Cells in the top 2%, 3%, 4%, or 5% of the staining intensity can generally be designated as "hi," while those falling in the upper half of the cell population are classified as "+." Cells with fluorescence intensity below 50% can be designated as "lo" cells, and those below 1% can be designated as "-" cells.

[0120] The term "+ / -" refers to a cell population with a certain proportion of cells expressing the "+" phenotype and a certain proportion of cells expressing the "-" phenotype. For example, the PROCR+ / - EPC population includes a mixed population containing a certain proportion of cells expressing the PROCR+ phenotype and a certain proportion of cells expressing the PROCR- phenotype.

[0121] The terms “high” or “high” or “bright” are well known in the art and refer to the expression level of the target cell marker, because the expression level of the cell marker is high compared to the expression level of the cell marker in the cell population analyzed as a whole.

[0122] While most markers, such as CD45 and CD34 cell surface markers, can be defined by the presence or absence of a reference cell surface marker, CD31 expression is defined by a reference expression level, particularly a low expression level (referred to herein as "CD31lo / -"). In the cells of this technique, the "CD31lo / -" subset is defined based on FACS gating according to an isotype control. In this illustrative embodiment, only the CD31 isotype control is used, and all other antibodies can remain unchanged. Based on CD31 expression levels, three cell populations can be observed. The first cell population is CD31-negative for the generation of fetal mesenchymal stem cells. The second cell population, generating endothelial progenitor cells, is the site of positive gating initiation. Finally, there is a CD31+ cell population with limited proliferative capacity. Those skilled in the art will understand that the specific setup of the analysis and the logs that may be used can vary depending on the voltage of the FACS. However, these parameters can be determined by those skilled in the art as a routine procedure. The term "lo / -" used in connection with "CD31lo / -" is well known in the art and refers to the expression level of CD31, as the expression level of this cell surface marker is low compared to the expression level of the marker in a cell population analyzed as a whole. The term "lo" in relation to CD31lo refers to a different cell or cell population that expresses CD31 at a level lower than that of one or more other different cells or cell populations. Therefore, the terms "CD31lo / -" and "CD31lo" are used interchangeably herein to refer to endothelial progenitor cells generated by the subject isolation method. The level of CD31 expressed by CD31lo cells or CD31lo cell populations is less than 50% of the level of CD31 expressed by HUVECs or HUVEC populations (and less than 49% and more than 1% including all integer percentages in between; suitably less than 40% and more than 1% including all integer percentages in between; suitably less than 30% and more than 1% including all integer percentages in between; suitably less than 20% and more than 1% including all integer percentages in between; even more suitably less than 10% and more than 1% including all integer percentages in between).

[0123] Those skilled in the art will also understand that while the cells of this technique can be characterized by a defined phenotypic profile, these cells will express a range of other intracellular and / or cell surface markers that may be irrelevant to phenotypic characterization and isolation of the target cell population. Furthermore, to the extent that a given cell population may comprise a range of subpopulations, these subpopulations may exhibit intracellular or cell surface marker expression that differs from the profile defined herein.

[0124] Standard methods known in the art can be used to determine detectable expression, low expression, or absence of the various biomarkers discussed herein. Suitable methods include, but are not limited to, immunocytochemistry, immunoassays, flow cytometry (e.g., fluorescence-activated cell sorting (FACS)), and polymerase chain reaction (PCR) (e.g., reverse transcription PCR (RT-PCR)). Suitable immunoassays include, but are not limited to, Western blotting, enzyme-linked immunosorbent assay (ELISA), ELISPOT assay, enzyme doubling immunoassay, radiosensitive adsorbent (RAST) assay, radioimmunoassay, radiobinding assay, and immunofluorescence. Western blotting, ELISA, and RT-PCR can all be quantitative and can therefore be used to measure the expression levels of various biomarkers, if present. The use of FACS is disclosed in the examples. Antibodies and fluorescently labeled antibodies used for all the various biomarkers discussed herein can be commercially available.

[0125] The term "enrichment" should be understood to refer to increasing the ratio of cells expressing the desired phenotype to cells not expressing the desired phenotype in the starting sample. This is achieved by removing or otherwise reducing the number of cells not expressing the desired phenotype. It should be understood that the term "enrichment" is not limited to an enrichment step that removes all cells not expressing the desired phenotype from the sample / cell population. Rather, it refers to reducing the concentration of these unwanted cells in the sample / cell population. Therefore, the reduction in concentration can be of varying degrees. The method of this technique should be understood to extend to performing one or more repeated sequential enrichment steps to improve the purity of the desired subpopulation (e.g., by performing two or more consecutive enrichment steps). The decision as to whether one or more enrichment steps are necessary at any given stage can be made by a person skilled in the art on a case-by-case basis. When the number of target endothelial progenitor cells may be relatively high (e.g., in placental samples), a single enrichment step may be sufficient to enrich the desired subpopulation. However, in cases using samples such as blood (with a very low number of endothelial progenitor cells), it may be desirable to perform two or more of each enrichment step in order to maximize the purity of the desired cell population.

[0126] In any of the methods defined or described herein, the term "enrichment of cells expressing a phenotype" may also be referred to as "selection of cells expressing a phenotype" or "isolation of cells expressing a phenotype." These terms are used interchangeably. Those skilled in the art will readily understand the meaning of these terms and how to implement them.

[0127] These terms should be understood as referring to obtaining a highly enriched cell population. While it is desirable to isolate a pure cell population, this may not be 100% achievable because cell contamination can occur in any biological system. Therefore, a small percentage of contaminated cells may still be present. However, the inventors have determined that the potential level of contamination is very low and therefore not considered significant.

[0128] "Enrichment", "selection" or "separation" can be achieved by any suitable method known to those skilled in the art as described in more detail herein, such as sorting via FACS.

[0129] As used herein, the term "gene" refers to any and all discrete coding regions of the genome, as well as associated non-coding and regulatory regions. The term "gene" also intends to refer to an open reading frame encoding one or more specific polypeptides and optionally contains one or more introns, as well as adjacent 5' and 3' non-coding nucleotide sequences involved in expression regulation. In this respect, a gene may further contain control signals, such as promoters, enhancers, termination and / or polyadenylation signals, which may be natively associated with the specific gene or may be heterologous control signals. Therefore, the term "gene" includes and encompasses nucleic acid molecules capable of producing mRNA, antisense RNA, siRNA, shRNA, miRNA, etc. Genes may or may not be used to produce functional proteins. Genes may include coding and non-coding regions.

[0130] The term "heterogeneous gene" is used herein to describe genetic material that has been or is about to be artificially introduced into the genome of a host cell (e.g., an EPC or MSC in a cell population of the present technology) and passed on to the progeny of that host cell. Heterogeneous genes typically contain polynucleotides capable of being transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In some embodiments, they confer desired properties to the recombinant host cell into which they are introduced, or otherwise achieve a desired therapeutic or diagnostic effect. In some embodiments, they are transcribed into molecules that interfere with transcription or translation (e.g., antisense molecules) or mediate RNA interference (e.g., siRNA or shRNA).

[0131] As used herein, the term "effective amount" (which may also be used interchangeably with "therapeutic effective amount") refers to the amount or concentration of any agent (such as cells, cell compositions, isolated cell populations, or pharmaceutical compositions) described herein that effectively reduces, eliminates, treats, prevents, or controls symptoms of a symptom, disorder, or disease affecting a mammal. The term "control" means all processes that can slow, interrupt, prevent, or stop the progression of a symptom, disorder, or disease affecting a mammal. However, control does not necessarily mean the complete elimination of all symptoms of a symptom, disorder, or disease, and its purpose includes preventative treatment.

[0132] As used herein, the term "endothelial cell mitogen" refers to any protein, polypeptide, mutant protein, or part of a protein that can directly or indirectly induce 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 α and β (TGF-α and TGF-β), platelet-derived endothelial growth factor (PD-EGF), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α), 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 its entirety. Mutant proteins or fragments of mitogens may be used, provided they induce or promote EC cell growth.

[0133] Endothelial progenitor cells (EPCs) This technology includes compositions comprising isolated EPC populations, said compositions comprising pharmaceutical compositions and cellular compositions. EPCs may comprise a population of cells circulating in the blood and capable of differentiating into endothelial cells. Unisolated EPCs may be rare or present in small amounts in the blood.

[0134] The isolated EPC cluster of this technology may contain one or more isolated EPC cells. In some embodiments, the isolated EPC cluster contains two or more isolated EPC cells.

[0135] purity of the separated EPC group In some implementations, approximately 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the isolated EPC population are isolated EPCs.

[0136] In some implementations, 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.

[0137] In some implementations, 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.

[0138] Separate EPC group markers Separate EPC populations can be mixed populations of separated EPCs that can express either the PROCR+ or PROCR- phenotype, and either of these two cell types can express either the PDGFRA+ or PDGFRA- phenotype, respectively. In other words, a mixed population of PROCR+ / - PDGFRA+ / - EPCs expresses any of the following four phenotypes based on PROCR and PDGFRA: PROCR+PDGFRA+, PROCR+PDGFRA-, PROCR-PDGFRA+, or PROCR-PDGFRA-.

[0139] The isolated EPC population may contain or consist of PROCR+PDGFRA+ endothelial progenitor cells (PROCR+PDGFRA+ EPC). In some embodiments, the EPC population contains or consists of the following: PROCR + / - PDGFRA + / - Endothelial progenitor cells (PROCR) + / - PDGFRA + / - EPCs). In some implementations, the separated EPC groups contain the CD45- / CD34+ phenotype.

[0140] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the isolated EPC population are PROCR+.

[0141] In some implementations, 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+.

[0142] In some implementations, 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+.

[0143] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the isolated EPC population are PDGFRA+.

[0144] In some implementations, 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+.

[0145] In some implementations, 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+.

[0146] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated EPC populations are PROCR+PDGFRA+.

[0147] In some implementations, 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+.

[0148] In some implementations, 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 isolated EPC population are PROCR+PDGFRA+.

[0149] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated EPC populations are PROCR-.

[0150] In some implementations, 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-.

[0151] In some implementations, 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 isolated EPC population are PROCR-.

[0152] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the isolated EPC population are PDGFRA-.

[0153] In some implementations, 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-.

[0154] In some implementations, 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-.

[0155] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the isolated EPC population are PROCR-PDGFRA-.

[0156] In some implementations, 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-.

[0157] In some implementations, 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 isolated EPC population are PROCR-PDGFRA-.

[0158] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the cells in the isolated EPC population are PROCR+PDGFRA-.

[0159] In some implementations, 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-.

[0160] In some implementations, 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 isolated EPC population are PROCR+PDGFRA-.

[0161] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated EPC populations are PROCR-PDGFRA+.

[0162] In some implementations, 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+.

[0163] In some implementations, 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 isolated EPC population are PROCR-PDGFRA+.

[0164] The isolated EPC populations can express the phenotypes VE-cadherin (CDH5)+, CD34+, PROCR+, CD31lo, VEGFR2lo, lineage (lin)-, CD45-, and PDGFRA-. In some embodiments, the isolated EPC populations express the phenotypes VE-cadherin (CDH5)+, CD34+, PROCR+ / -, CD31lo, VEGFR2lo, lineage (lin)-, CD45-, and PDGFRA+ / -.

[0165] Differences relative to the unseparated EPC group Compared to the unseparated EPC clusters, the separated EPC clusters may contain changes in protein expression. For example, compared to the expression levels in the unseparated EPC clusters, the separated PROCR+PDGFRA+ EPC clusters may contain increased expression levels of PROCR (NCBI accession numbers: XP_047295786.1; XP_011526798.2; XP_047295787.1), PDGFRA (NCBI accession numbers: AAH63414.1, AAH15186.1), or VE-cadherin (NCBI accession number: CAA56306) proteins.

[0166] Separated EPC populations may contain changes in gene expression compared to non-separated EPC populations. For example, compared to the expression levels in the non-separated EPC population, the separated PROCR+PDGFRA+ EPC population may contain increased expression levels of the PROCR (NCBI gene ID: 10544), PDGFRA (NCBI gene ID: 5156), or VE-cadherin (NCBI gene ID: 1003) genes.

[0167] In some implementations, the isolated PROCR+PDGFRA+ EPC cluster contains approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 80%, 800%, 900%, or 1000% higher levels of PROCR protein or gene expression compared to the unisolated EPC cluster.

[0168] In some implementations, the isolated PROCR+PDGFRA+ EPC cluster contains 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% higher levels of PROCR protein expression or gene expression compared to the unisolated EPC cluster.

[0169] In some implementations, the isolated PROCR+PDGFRA+ EPC cluster contains 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% higher PROCR+PDGFRA+ EPC clusters compared to the unisolated EPC clusters.

[0170] In some implementations, the isolated PROCR+PDGFRA+ EPC cluster contains approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 80%, 800%, 900%, or 1000% higher PDGFRA protein expression levels or gene expression levels compared to the unisolated EPC cluster.

[0171] In some implementations, the isolated PROCR+PDGFRA+ EPC cluster contains 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% higher PDGFRA protein expression levels or gene expression levels compared to the unisolated EPC cluster.

[0172] In some implementations, the isolated PROCR+PDGFRA+ EPC cluster contains 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% higher PDGFRA protein expression levels or gene expression levels compared to the unisolated EPC cluster.

[0173] In some implementations, the isolated PROCR+PDGFRA+ EPC cluster contains approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 80%, 800%, 900%, or 1000% higher levels of VE-cadherin protein or gene expression compared to the unisolated EPC cluster.

[0174] In some implementations, the isolated PROCR+PDGFRA+ EPC cluster contains VE-cadherin protein expression levels or gene expression levels that are increased 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% compared to the unisolated EPC cluster.

[0175] In some implementations, the isolated PROCR+PDGFRA+ EPC cluster contains VE-cadherin protein expression levels or gene expression levels that are increased 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% compared to the unisolated EPC cluster.

[0176] The isolated EPC populations of this technology may further include cellular traits different from those of unisolated EPC populations. Non-limiting examples include increased proliferative capacity, increased angiogenesis capacity, increased colony-forming capacity, increased tube-forming capacity, increased implantation potential and / or capacity, increased cell elongation, and increased collagen production relative to unisolated EPC populations.

[0177] In some implementations, the isolated EPC population contains approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 80%, 700%, 800%, 900%, or 1000% increased proliferation capacity compared to the unisolated EPC population.

[0178] In some implementations, the isolated EPC population relative to the unisolated EPC population contains an increase in proliferation capacity of 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%.

[0179] In some implementations, the isolated EPC population contains 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% increased proliferation capacity compared to the non-isolated EPC population.

[0180] In some implementations, the phase-separated EPC clusters contain an increased angiogenic capacity of approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately 250%, approximately 300%, approximately 350%, approximately 400%, approximately 500%, approximately 600%, approximately 700%, approximately 800%, approximately 900%, or approximately 1000% compared to the unseparated EPC clusters.

[0181] In some implementations, the isolated EPC cluster contains an increased angiogenic capacity of 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% compared to the unisolated EPC cluster.

[0182] In some implementations, the separated EPC clusters contain angiogenic capacity that is increased 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% compared to the unseparated EPC clusters.

[0183] In some implementations, the separated EPC clusters, relative to the non-separated EPC clusters, contain approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% increased colony-forming capacity.

[0184] In some implementations, the separated EPC clusters, relative to the non-separated EPC clusters, contain a cluster-forming capacity that is 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%.

[0185] In some implementations, the separated EPC clusters, relative to the non-separated EPC clusters, contain a cluster-forming capacity that is 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%.

[0186] Colony-forming ability can be measured using an in vitro colony-forming assay. In some embodiments, isolated EPC populations are able to produce colonies that are positive for isolectin (Griffonia (Bandeiraea) Simplicifolia lectin I (GSL I, BSL I)) in an in vitro colony-forming assay.

[0187] In some implementations, isolated EPC clusters are able to produce an increased number of colonies that are positive for isolectin (Griffonia (Bandeiraea) Simplicifolia lectin I (GSL I, BSL I)) in in vitro colony formation assays compared to the number of colonies produced by unisolated EPC clusters tested in the same assay.

[0188] In some embodiments, the isolated EPC populations are able to produce at least a 39x increase in the average number of colonies 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 unisolated EPC populations tested in the same assay. In some embodiments, the increase is at least a 39.27x increase.

[0189] In some implementations, the separated EPC group contains a pipe forming capability that is approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately 250%, approximately 300%, approximately 350%, approximately 400%, approximately 500%, approximately 600%, approximately 700%, approximately 800%, approximately 900%, or approximately 1000% higher than that of the non-separated EPC group.

[0190] In some implementations, the separated EPC group contains a pipe forming capacity that is 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% greater than that of the non-separated EPC group.

[0191] In some implementations, the separated EPC group contains a pipe forming capacity that is 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% greater than that of the non-separated EPC group.

[0192] In some implementations, the separated EPC cluster contains an implantation potential increase of approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately 250%, approximately 300%, approximately 350%, approximately 400%, approximately 500%, approximately 600%, approximately 700%, approximately 800%, approximately 900%, or approximately 1000% compared to the unseparated EPC cluster.

[0193] In some implementations, the separated EPC cluster contains an implantation potential increase of 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% compared to the unseparated EPC cluster.

[0194] In some implementations, the separated EPC cluster contains an implantation potential increase of 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 the unseparated EPC cluster.

[0195] In some implementations, the separated EPC group includes one or more EPCs that are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, or about 75% longer than one or more EPCs in the unseparated EPC group.

[0196] In some implementations, the separated EPC group includes 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 least 50%, at least 60%, at least 65%, at least 70%, or at least 75% longer than one or more EPCs in the unseparated EPC group.

[0197] In some implementations, the separated EPC group includes 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 about 50%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% longer than one or more EPCs in the unseparated EPC group.

[0198] In some implementations, the separated EPC clusters, relative to the unseparated EPC clusters, contain an implantation capacity increase of approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately 250%, approximately 300%, approximately 350%, approximately 400%, approximately 500%, approximately 600%, approximately 700%, approximately 800%, approximately 900%, or approximately 1000%.

[0199] In some implementations, the separated EPC cluster contains an implantation capacity that is 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% greater than that of the unseparated EPC cluster.

[0200] In some implementations, the separated EPC cluster contains an implantation capacity that is 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% greater than that of the unseparated EPC cluster.

[0201] In some implementations, the isolated EPC clusters contain approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 900%, or 1000% increased collagen production levels compared to the unisolated EPC clusters.

[0202] In some implementations, the isolated EPC cluster contains a collagen production level that is 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% higher than that of the unisolated EPC cluster.

[0203] In some implementations, the isolated EPC cluster contains a collagen production level that is 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% higher than that of the unisolated EPC cluster.

[0204] In some embodiments, the collagen production level and / or implantation capacity of the isolated EPC clusters can be assessed using in vivo assays, including in vivo collagen plug implantation assays. In some embodiments, the isolated EPC clusters are able to generate at least a 7x or at least a 7.8x increase in the average GFP-positive area per collagen plug in an in vivo collagen plug implantation assay compared to the unisolated EPC clusters.

[0205] In some implementations, the isolated EPC populations are able to co-express CD34 and isolectins in implanted cells. The implanted cells can be cells used in in vivo co-localization assays. The expression of CD34 and / or isolectins can be increased compared to unisolated EPC populations.

[0206] In some implementations, the isolated EPC clusters do not express one or more hematopoietic proteins. Non-limiting examples of hematopoietic proteins include CD3e (NCBI accession number: NP_000724), CD11b (NCBI accession number: AAB24821.1; XP_054236247.1; XP_054236246.1; XP_054236245.1; XP_054236244.1; XP_016878705.1; 1BHO_1; 1BHO_2), CD45 (NCBI accession number: AAS46922.1; AAS46930.1; AAS46938.1; AAS46946.1; AAS46954.1; AAS46962.1; P08575.3), and B220. (NCBI Registry Numbers: XP_054193920.1; XP_054193921.1; XP_054193922.1; XP_054193924.1; XP_054193926.1; XP_054193928.1).

[0207] Differentiation The isolated EPC populations obtained using this technique can be differentiated. During or after differentiation, the isolated EPC populations may include a decrease in gene expression levels or protein expression levels compared to the undifferentiated EPC populations. Undifferentiated EPC populations may include undifferentiated, unseparated EPC populations or undifferentiated, separated EPC populations. Non-restricted examples of such proteins or genes include CD157 (NCBI accession numbers: 1ISM_A; 1ISM_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 accession numbers: NP_004818.2; NP_001244315.1; NCBI gene ID: 9429), and SOX18 (NCBI accession number: NP_060889; NCBI gene ID: 54345).

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

[0209] In some implementations, the reduction in CD157 protein expression level or CD157 gene expression level relative to the CD157 protein expression level or CD157 gene expression level in the undifferentiated EPC population 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%.

[0210] In some embodiments, the reduction in CD157 protein expression level or CD157 gene expression level relative to the CD157 protein expression level or CD157 gene expression level in the undifferentiated EPC population 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%.

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

[0212] In some implementations, the reduction in ABCG2 protein expression level or ABCG2 gene expression level relative to the ABCG2 protein expression level or ABCG2 gene expression level in the undifferentiated EPC population 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%.

[0213] In some implementations, the reduction in ABCG2 protein expression level or ABCG2 gene expression level relative to the ABCG2 protein expression level or ABCG2 gene expression level in the undifferentiated EPC population 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%.

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

[0215] In some implementations, the reduction in SOX18 protein expression level or SOX18 gene expression level relative to the SOX18 protein expression level or SOX18 gene expression level in the undifferentiated EPC population 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%.

[0216] In some implementations, the reduction in SOX18 protein expression level or SOX18 gene expression level relative to the SOX18 protein expression level or SOX18 gene expression level in the undifferentiated EPC population 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%.

[0217] Methods for generating separate EPC groups The separated EPC clusters of this technology can be generated by various methods. In some implementations, the separated EPC clusters are generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) Select cells expressing the PROCR+ and PDGFRA+ phenotypes from the CD45- / CD34+ cell population to obtain a separate EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

[0218] In some implementations, the separated EPC group is generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) Select cells expressing the PROCR+ / - and PDGFRA+ / - phenotypes from the CD45- / CD34+ cell population to obtain a separate EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

[0219] In some implementations, step (ii) includes selecting cells expressing the CD45+ phenotype, removing the cells expressing the CD45+ phenotype from the biological sample of step (i), and discarding the cells expressing the CD45+ phenotype to obtain a CD45- cell population.

[0220] In some embodiments, the step of selecting cells expressing the CD45+ phenotype includes contacting one or more cells expressing the CD45 surface protein with CD45-binding molecules to form a complex, and removing the complex from the biological sample in step (i).

[0221] In some embodiments, step (iii) includes contacting one or more cells expressing the CD34 surface protein with a CD34 binding molecule to form a complex, removing the complex from the CD45- cell population of step (ii), and retaining the complex to obtain a second cell population of CD45- / CD34+.

[0222] In some embodiments, step (iv) includes contacting one or more cells expressing the PROCR surface protein with the PROCR binding molecule to form a complex, removing the complex from the cell population, and retaining the complex.

[0223] In some implementations, the separate EPC group is generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching cells containing the PDGFRA+ phenotype in the biological sample to obtain a PDGFRA+ cell population; (iii) Select cells expressing the PROCR+ phenotype from the PDGFRA+ cell population to obtain a separate EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

[0224] In some implementations, the separate EPC group is generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PDGFRA+ / - phenotype to obtain a PDGFRA+ / - cell population; (iii) Select cells expressing the PROCR+ / - phenotype from the PDGFRA+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

[0225] In some embodiments, step (iii) includes contacting one or more cells expressing the PROCR surface protein with the PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0226] In some embodiments, step (ii) includes contacting one or more cells expressing the PDGFRA surface protein with PDGFRA binding molecules to form a complex, removing the complex from the cell population, and retaining the complex.

[0227] In some embodiments, step (iii) includes contacting one or more cells expressing the PROCR surface protein with the PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0228] In some implementations, the separate EPC group is generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PROCR+ phenotype to obtain a PROCR+ cell population; (iii) Select cells expressing the PDGFRA+ phenotype from the PROCR+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

[0229] In some implementations, the separate EPC group is generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample with cells containing the PROCR+ / - phenotype to obtain a PROCR+ / - cell population; (iii) Select cells expressing the PDGFRA+ / - phenotype from the PROCR+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

[0230] In some embodiments, step (ii) includes contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population, and retaining the complex.

[0231] In some embodiments, step (iii) includes contacting one or more cells expressing the PDGFRA surface protein with PDGFRA binding molecules to form a complex, removing the complex from the cell population, and retaining the complex.

[0232] The binding molecules in any method for generating isolated EPC clusters contain proteins. These proteins can be antibodies.

[0233] Removing the complex in any method for generating isolated EPC clusters can include microfluidic sorting. Microfluidic sorting can include bead sorting or flow cytometry. In some embodiments, flow cytometry includes fluorescence-activated cell sorting.

[0234] In some embodiments, the method for generating isolated EPC populations further includes step (iv) of culturing or contacting the isolated EPC populations with a cell population containing endothelial colony-forming cells (ECFCs) or a cell population containing MSCs, and / or step (v) of separating the isolated EPC populations from a cell population containing ECFCs or a cell population containing MSCs. Step (v) may occur at least approximately 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).

[0235] In some implementations, the separate EPC clusters and / or separate MSC clusters are generated by one or more steps disclosed in WO2014 / 138793 (which is incorporated herein by reference in its entirety).

[0236] Other methods for separating EPC and MSC groups In addition to more classic endothelial markers such as VE-cadherin or CD31, flow cytometry of CD34, VEGFR2 (KDR / FLK-1), and / or CD133 is commonly used to identify the number of circulating endothelial progenitor cells. Alternative methods for isolating endothelial progenitor cells involve partially differentiated endothelial progenitor cells after short-term culture on fibronectin, producing spindle-shaped cells capable of digesting acetylated low-density lipoprotein and staining against several specific lectins that appear within 3 days. However, both methods result in considerable contamination of hematopoietic cells.

[0237] To separate cell populations according to the methods described herein, various well-known techniques can be performed. Antibodies and other CD45, CD34, PROCR, and / or PDGFRA-specific cell surface binding molecules may be particularly useful. For example, antibodies can be attached to a solid support to allow for separation.

[0238] Flow cytometry can be used in combination with any of the methods in this technique. For example, flow cytometry can be used to gate only CD34+ cells to remove any contaminating CD45+ cells from the initial CD45- cell population.

[0239] Other techniques that provide particularly precise separation include fluorescence-activated cell sorting (FACS). FACS is a specialized form of flow cytometry based on the specific light scattering and fluorescence characteristics of each cell. FACS is also suitable for cell separation based on morphological characteristics that can be identified by forward and side light scattering.

[0240] In another instance, but specifically within the context of the CD45 negative selection step, instead of physically separating the CD45- cell subset from the CD45+ cell population, a method can be used to label CD45+ cells and then deliver targeted lysis signals (e.g., cell lysis, apoptosis, or toxicity signals) to lyse the labeled CD45+ cells. In yet another instance, the same result can be achieved by opsonizing with an antibody followed by complement administration.

[0241] Other negative selection techniques include, but are not limited to, the targeted administration of cytolytic agents, apoptotic agents, or other toxic agents. This can be most conveniently achieved by conjugating such agents to monoclonal antibodies to facilitate their targeted delivery. In another example, opsonization with an antibody followed by complement administration can achieve the same result.

[0242] Procedures for separation may include magnetic separation using antibody magnetic beads, affinity chromatography, “panning” by attaching antibodies to a solid matrix, or any other convenient technique such as laser capture microdissection. For example, CD45 antibodies labeled with magnetic beads can be used in combination with magnetic columns to provide a CD45-enriched cell population.

[0243] Mesenchymal stem cells (MSCs) This technology includes compositions comprising a pharmaceutical composition and a cell composition, said composition comprising an isolated mesenchymal stem cell (MSC) cell population. The isolated MSC population of this technology may comprise one or more isolated MSC cells. In some embodiments, the isolated MSC population comprises two or more isolated MSC cells.

[0244] Purity of isolated MSC populations In some implementations, approximately 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the isolated MSC population are isolated MSCs.

[0245] In some implementations, 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.

[0246] In some implementations, 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.

[0247] Isolated MSC colony markers In some implementations, isolated MSC populations express phenotypes CD105+, CD73+, CD90+, CD45-, CD34-, CD14- or CD11b-, CD79α- or CD19-, and HLA-DR-.

[0248] The isolated MSC population can be a mixture of isolated MSCs that can express either the PROCR+ or PROCR- phenotype, and either of these cell types can express either the PDGFRA+ or PDGFRA- phenotype, respectively. In other words, the mixture of PROCR+ / - PDGFRA+ / - EPCs expresses any one of the following four phenotypes based on PROCR and PDGFRA: PROCR+PDGFRA+, PROCR+PDGFRA-, PROCR-PDGFRA+, or PROCR-PDGFRA-.

[0249] A separate MSC population may contain + PDGFRA + Mesenchymal stem cells (PROCR)+ PDGFRA + MSCs (or MSCs) are groups of cells that are composed of or comprise MSCs. In some implementations, a group of MSCs includes PROCRs. + / - PDGFRA + / - Mesenchymal stem cells (PROCR) + / - PDGFRA + / - (MSC) or composed of them. In some embodiments, the isolated EPC group contains the CD45- / CD34+ phenotype.

[0250] In some implementations, approximately 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated MSC population are CD45- / CD34+ cells.

[0251] In some implementations, 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.

[0252] In some implementations, 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.

[0253] The isolated MSC groups can be co-formulated in the composition with the isolated EPC groups of this technology, or they can be present in a composition that does not contain isolated MSC groups.

[0254] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated MSC population are PROCR+.

[0255] In some implementations, 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+.

[0256] In some implementations, 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 isolated MSC population are PROCR+.

[0257] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated MSC population are PDGFRA+.

[0258] In some implementations, 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+.

[0259] In some implementations, 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 isolated MSC population are PDGFRA+.

[0260] In some implementations, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated MSC population are PROCR+PDGFRA+.

[0261] In some implementations, 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+.

[0262] In some implementations, 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 isolated MSC population are PROCR+PDGFRA+.

[0263] Other characteristics of isolated MSC populations The isolated MSC populations of this invention may be able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro or in vivo.

[0264] Sources of isolated EPC and MSC groups The isolated MSC populations can be isolated from biological samples. In some embodiments, the biological sample is a mammalian biological sample. The mammalian biological sample can be a human biological sample.

[0265] mammalian biological samples In some implementations, the mammalian biological sample is selected from the group consisting of mammalian placenta, mammalian umbilical cord blood, mammalian peripheral blood, and mammalian tissue-resident vascular endothelium. The mammalian placenta may include an intact mammalian placenta. The mammalian tissue-resident vascular endothelium may be selected from the group consisting of mammalian umbilical cord, mammalian pulmonary artery endothelium, mammalian aorta, and mammalian lung tissue.

[0266] Mammalian biological samples may be provided directly or may require some form of pretreatment. For example, biopsy or surgical samples may require homogenization or other forms of cell dispersion. Furthermore, where the biological sample is not in liquid form, reagents such as buffers may need to be added to mobilize the sample and produce a cell suspension. Alternatively, the biological sample may require some other form of pretreatment, such as heparinization, where the sample is whole blood, to prevent clotting. Technicians will readily understand the steps required to provide suitable and appropriate mammalian biological samples according to the specific circumstances.

[0267] Mammalian biological samples may be in the form of freshly isolated single-cell suspensions or cell aggregates from an individual (e.g., an individual who may be a treatment subject), or they may be derived from non-fresh sources, such as from cultures (e.g., where cell numbers have been expanded) or cryopreserved reserves of cells isolated from an individual or another source at an earlier point in time. It should also be understood that the initial mammalian biological sample provided may have undergone some other form of processing or manipulation, such as, but not limited to, enrichment or purification.

[0268] Initial mammalian biological samples can be obtained from intact mammalian placentas. An intact placenta should be understood as some or all of the heterogeneous cell populations that make up the placenta. In humans, the average placenta is 22 cm long and 2–2.5 cm thick, thickest at the center and thinnest at the edges. It typically weighs about 500 grams. It is deep reddish-blue or dark red and is connected to the fetus by an umbilical cord approximately 55–60 cm long. The umbilical cord contains two umbilical arteries and one umbilical vein. The cord inserts into the chorionic plate. Blood vessels branch on the surface of the placenta and further divide into a network covered by a thin layer of cells. This results in the formation of villous dendritic structures. On the maternal side, these villous dendritic structures can be grouped into leaflets called cotyledons. In humans, the placenta is typically disc-shaped, but there is considerable variation in size between different mammalian species. The placenta begins to develop after the blastocyst implants in the mother's uterine lining. The outer layer of the blastocyst becomes the trophoblast, which forms the outer layer of the placenta. This outer layer further divides into two layers: the lower cytotrophoblast and the upper syncytiotrophoblast. The syncytiotrophoblast is a continuous layer of multinucleated cells that covers the surface of the placenta. It results from the differentiation and fusion of cells in the underlying cytotrophoblast, a process that continues throughout placental development. Therefore, the syncytiotrophoblast (also known as the syncytium) helps maintain the placenta's barrier function. The placenta grows throughout pregnancy. The development of the maternal blood supply system to the placenta is completed by the end of the first trimester (approximately 12-13 weeks).

[0269] Initial mammalian biological samples can be obtained from cell populations of cotyledons. A postpartum placenta, such as an intact placenta after a cesarean section, can be used. To isolate the placental cotyledons, the decidual components can be dissected. These cotyledons can then be digested in a mixture of collagenases, dispersases, and deoxyribonucleases, followed by filtration to obtain the initial mammalian biological sample.

[0270] To obtain initial mammalian biological samples, placenta at any developmental stage can be used. While postpartum placenta is the most readily available, placenta from early pregnancy can also be used, for example, in cases of miscarriage or other termination of pregnancy. In particular, placenta and umbilical cord blood provide excellent sources of endothelial progenitor cells and mesenchymal stem cells. This provides women with the possibility of routinely isolating and storing placental / umbilical cord tissue or blood for, for example, future endothelial progenitor cell harvesting, or harvesting fresh and then freezing the endothelial progenitor cells for future use. Therefore, this provides the possibility of autologous endothelial progenitor cell therapy, or, for donor-associated individuals, more readily available MHC-matched endothelial progenitor cells than in other cases. In both cases, donor endothelial progenitor cells can be defined as tissue-compatible with the recipients of these cells.

[0271] donor Separate EPC swarms and / or separate MSC swarms may be derived from donors. In some embodiments, separate EPC swarms and / or separate MSC swarms are derived from one or more donors. In some embodiments, separate EPC swarms and / or separate MSC swarms are derived from two or more donors.

[0272] In some implementations, the isolated EPC populations and / or isolated MSC populations comprise autologous cells. These autologous cells may be derived from a subject who will receive the cells for a therapeutic therapy.

[0273] In some implementations, the isolated EPC population and / or isolated MSC population contain allogeneic cells. The allogeneic cells may be derived from a subject whose immune system is compatible with the subject to whom the cells will be administered.

[0274] The isolated EPC groups and / or isolated MSC groups may be present in a culture medium, including but not limited to liquid or frozen culture media.

[0275] Culture and maintenance of isolated EPC and MSC populations The isolated EPC populations and / or isolated MSC populations of this technique can be cultured or maintained according to any conventional methods in the art. Methods for the routine and reliable generation of isolated populations of the relevant cells in vitro on a small or large scale can be provided. Methods for culturing EPCs and MSCs have been previously established (see Chand, KK, et al. (2021) npjRegen Med 6(75), pp. 1–15), which are incorporated herein by reference in their entirety.

[0276] The methods of this technology are particularly well-suited for generating cell populations for a given individual and in the context of a specific disease. For large-scale production, one means of achieving this is through the use of bioreactors.

[0277] Bioreactors can be designed to provide culture processes that deliver culture media and oxygenation at controlled concentrations and rates that mimic the concentrations and rates of nutrients in vivo. Bioreactors have been commercialized for many years and employ a variety of culture techniques. Of the various bioreactors used for mammalian cell culture, most have been designed to allow for the production of high-density cultures of single-cell types. A typical application of these high-density systems is the production of conditioned media generated by cells as the final product. This is the case, for example, in the hybridoma production of monoclonal antibodies and packaging cell lines for the production of viral vectors. However, these applications differ from those where the therapeutic end product is the harvested cells themselves, as in this example.

[0278] Once operational, bioreactors provide automatically regulated culture medium flow rates, oxygen delivery, and temperature and pH control, and they typically allow for the production of large numbers of cells. Therefore, bioreactors offer labor savings and a minimal possibility of intermediate process contamination, and the most sophisticated bioreactors allow for setup, growth, selection, and harvesting procedures involving minimal human labor requirements and open processing steps. Such bioreactors are optimally designed for use with homogeneous cell mixtures or aggregated cell populations. Suitable bioreactors include, but are not limited to, those described in U.S. Patents 5,763,194, 5,985,653, and 6,238,908, 5,512,480, 5,459,069, 5,763,266, 5,888,807, and 5,688,687, each of which is incorporated herein by reference in its entirety.

[0279] For any large-volume cell culture, several fundamental parameters require strict control. The culture medium should be provided, under appropriate conditions, to allow stem cell maintenance, endothelial progenitor cell proliferation, endothelial progenitor cell differentiation (potentially in the context of several separate differentiation cultures and conditions), and ultimately, cell culture / preservation. Typically, various media can be delivered to the cells via a pumping mechanism within the bioreactor, with periodic feeding and media exchange. The exchange process allows for the removal of byproducts from the culture. Growing cells or tissues also require an oxygen source. Different cell types may have different oxygen requirements. Therefore, a flexible and adjustable device to provide the necessary conditions for the cells is a desirable component.

[0280] Depending on the specific culture, uniform distribution of cell populations and culture medium supply within the culture chamber can be an important process control. This control is often achieved through suspension culture designs, which can be effective when cell-cell interactions may not be significant. Examples of suspension culture systems include various tank reactor designs and breathable plastic bags. Such suspension designs can be used for cells that do not need to be assembled into a three-dimensional structure or do not require proximity to the matrix or feeder layer.

[0281] Efficient cell harvesting at the end of the culture process is a key characteristic of an effective cell culture system. One method of producing cells as a product is to culture cells in a confined space without physical barriers to recycle, allowing the cell product to be easily eluted to produce a readily manageable concentrated volume of cells suitable for final washing in a commercially available, closed-system cell washer designed for this purpose. Ideally, the system will allow for the addition of pharmaceutically acceptable carriers (with or without preservatives), or cell storage compounds, and provide efficient harvesting into appropriate aseptic packaging. Ideally, the harvesting and packaging processes can be completed without disrupting the aseptic barriers of the fluid pathways within the culture chamber.

[0282] When the product cells can be transplanted into a subject (typically when the subject is ill or immunocompromised), the absence of microorganisms is required. Once culturing begins, the culture chambers and fluid pathways within the bioreactor system should be maintained in a sterile, closed environment to preserve aseptic conditions.

[0283] Cellular modification The isolated EPC and / or MSC populations of this technology can be genetically engineered or molecularly modified to express heterologous genes. Illustrative examples include factors or proteins that directly or indirectly inhibit thrombosis, restenosis, or platelet adhesion, or enhance cell viability or possess anti-inflammatory properties. Heterologous genes can be introduced into cells using methods well-known in the art. For example, vectors (e.g., viral vectors, such as adenovirus vectors, adeno-associated virus vectors, AAV chimeric vectors, or retroviral vectors or pseudovirus vectors) can be constructed containing expression cassettes of genes, pseudogenes, mutant genes (such as dominant-negative genes), or gene silencing constructs, such as short hairpin RNA (shRNA) or microRNA (miRNA). Suitable expression cassettes can be constructed using a range of conventional cloning methods. While gene delivery via viral vectors is preferred, non-viral methods can also be used, such as plasmid or coplasmal DNA delivery via liposomal reagents, lipid complexes, or polymeric complexes, electroporation, acoustic perforation, hydrodynamic gene delivery, the use of a "gene gun," and nuclear transfection techniques and nanoparticle delivery.

[0284] Genetic engineering of subject cells is not limited to overexpressing or adding beneficial genes, but also includes suppressing, downregulating, and "knocking out" detrimental genes. This can be achieved through standard methods known in the art, such as using CRISPR and / or related gene editing technologies.

[0285] Pharmaceutical compositions and formulations This technology includes pharmaceutical compositions and cell compositions, which contain isolated EPC populations and / or isolated MSC populations of this technology. In some embodiments, the isolated EPC populations are present together with the isolated MSC populations in the composition (such as a pharmaceutical composition or cell composition).

[0286] In some implementation schemes, the ratio of isolated EPC clusters to isolated MSC clusters is approximately 1:1, 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.

[0287] In some implementations, the ratio of the separated EPC clusters to the separated MSC clusters is 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.

[0288] In some implementation schemes, the ratio of the separated EPC clusters to the separated MSC clusters is 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.

[0289] In some embodiments, isolated EPC clusters are present in a first composition (such as a pharmaceutical composition or a cell composition), and isolated MSC clusters are present in a second composition. The second composition may be formulated for delivery before, during, or after administration of the first composition.

[0290] The pharmaceutical composition may further comprise a pharmaceutically acceptable mediator. In some embodiments, the pharmaceutically acceptable mediator is phosphate-buffered saline.

[0291] Pharmaceutical compositions or cellular compositions may be formulated for administration to subjects in need. Non-limiting examples include formulations for intranasal, intrathecal, intra-arterial, intralesional, or intravenous delivery.

[0292] Medical Devices and Tissue Engineering Applications The isolated EPC clusters and / or isolated MSC clusters of this technology can be used in combination with appropriate medical devices.

[0293] The isolated EPC populations and / or isolated MSC populations of this technology can be combined with implantable cell support substrates, devices, and / or pharmaceutically acceptable carriers.

[0294] The cell-supporting substrate can be a polymer matrix. Illustrative examples include gels, such as dissolved basement membrane matrix (e.g., dissolved basement membrane matrix extracted from mouse tumors). In other embodiments, the gel can be a collagen I gel. Such a gel may also include other extracellular matrix (ECM) components, such as glycosaminoglycans, fibrins, fibronectin, proteoglycans, and glycoproteins. The gel may also include basement membrane components, such as collagen TV and laminin. Enzymes such as proteases and collagenases may be added to the gel, as may cell response regulators such as growth factors and chemokines.

[0295] Any isolated cell population and composition of this technology can be combined with a scaffold.

[0296] The stent can be seeded with isolated EPC clusters and / or isolated MSC clusters. Vessels treated with this stent can exhibit accelerated reendothelialization, thereby preventing restenosis of damaged vessels.

[0297] In some implementations, any isolated EPC populations and / or isolated MSC populations can be seeded into a polymer sheet and wrapped around the outside of a vessel that has undergone angioplasty or stent implantation. Alternatively, cells can be mixed with a gel and injected into the polymer sheet instead of being seeded directly onto the matrix.

[0298] In some embodiments, any isolated EPC populations and / or isolated MSC populations can be seeded onto a polymer matrix, such as a sponge or mesh, and then implanted into the desired tissue site. Alternatively, cells can be mixed with a gel, which is then absorbed onto the inner and outer surfaces of the matrix and can fill some of the pores in a sponge or other porous matrix. Capillary forces will retain the gel on the matrix before hardening, or may allow the gel to harden on the matrix to make it more self-supporting. Illustrative biocompatible polymer matrices include any biocompatible synthetic or semi-synthetic materials, including plastics and other polymers. In some embodiments, the biocompatible polymer matrix may be made of absorbable or non-absorbable materials. Materials that can be used to prepare biocompatible polymer matrices include, for example, poly(ethylene), polyester, poly(propylene), poly(propylene) polyesters such as poly(propylene) fumarate, polystyrene, polytetrafluoroethylene (PTFE), nylon, polypropylene / PTFE, polypropylene / cellulose, polypropylene / monocrystalline, polyester / collagen, poly(acrylate), poly(methyl methacrylate), poly(hydroxyethyl methacrylate), poly(vinyl alcohol), poly(carbonate), poly(trimethylene carbonate), poly(ethylene-co-vinyl acetate), poly(ether carbamate), poly(ester carbamate), poly(aryl ester), poly(imide), poly(anhydride-co-imide), poly(amino acid), polypeptide, poly(phosphobenzene), poly(glycolic acid), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(ε-caprolactone), and poly(p-dioxane). Poly(lactic acid-co-glycolic acid), poly(caprolactone-co-glycolic acid), poly(glycolic acid-co-trimethylene carbonate), lactide / tetramethylglycolic acid copolymer, lactide / trimethylene carbonate copolymer, lactide-5-valerol copolymer, lactide (ε-caprolactone copolymer), poly(lactide) / polyethylene oxide copolymer, asymmetric 3,6-substituted poly(1,4-dioxane-2,5-dione), poly(β-alkanonic acids) such as poly(β-hydroxybutyric acid), poly(β-hydroxybutyric acid) / (β-hydroxyvaleric acid) copolymer, poly(β-maleic acid) and poly(β-hydroxypropionic acid), poly(δ-valerol), methyl methacrylate-N-vinylpyrrolidone copolymer, polyesteramide, polyester of oxalic acid, polydihydropyran, polyalkyl-2-cyanoacrylate, composites thereof, cellulose materials, and combinations thereof.

[0299] In some embodiments, the polymer matrix is ​​biodegradable. Suitable biodegradable matrices are well known in the art and include collagen-GAG, collagen, fibrin, PLA, PGA, and PLA-PGA copolymers. Other biodegradable materials include poly(anhydrides), poly(hydroxy acids), poly(orthoesters), poly(propyl fumarate), poly(caprolactone), polyamides, polyamino acids, polyacetals, biodegradable polycyanoacrylates, biodegradable polyurethanes, and polysaccharides. Non-biodegradable polymers may also be used. Other non-biodegradable but biocompatible polymers include polypyrrole, polyaniline, polythiophene, polystyrene, polyesters, non-biodegradable polyurethanes, polyurea, poly(vinyl acetate), polypropylene, polymethacrylates, polyethylene, polycarbonate, and poly(ethylene oxide). Those skilled in the art will recognize that this is an exemplary, not an exhaustive, list of polymers suitable for tissue engineering applications.

[0300] In some implementations, the matrix can be formed with microstructures similar to the replaced ECM. Mechanical forces applied to the matrix by surrounding tissue will affect the cells on the artificial matrix and promote the regeneration of ECMs with appropriate microstructures. The mechanical properties and degradation rate of the matrix (for degradable scaffolds) can also be controlled by adjusting the crosslinking density of the matrix. The shape and size of the final implant should be appropriate for the implantation site and tissue type. The matrix can simply serve as a cell delivery medium or provide structural or mechanical functions. The matrix can be formed in any shape, such as granules, sponges, tubes, spheres, strands, coiled strands, capillary networks, membranes, fibers, meshes, or sheets.

[0301] The isolated EPC populations and / or isolated MSC cell populations of this technology can be combined with a tubular substrate. The tubular substrate can be seeded with the relevant cells. For example, the polymer matrix can be formed into tubes or networks. Such tubes can be formed from natural or synthetic ECM materials such as PLA or collagen, or can be derived from natural sources, such as decellularized tubular grafts. Cells can enclose the interior of the tubes, forming artificial channels.

[0302] Prior to implantation in an animal, cells may be allowed to proliferate on a polymer matrix or tubular substrate. During proliferation, mechanical forces may be applied to the implant to stimulate specific cellular responses or to mimic the mechanical forces the implant will experience in the animal. For example, the medium may circulate through the tubular substrate in a pulsating manner (i.e., circumferential stress) or at a sufficient speed to apply shear stress to the cells enclosed within the tube (see, Kaushall et al. (2001) Nat Med. 7(9)1035-40), which is incorporated herein by reference in its entirety. Alternatively, hydrostatic or compressive forces may be applied to the implant to be deposited within an organ such as the liver, or tensile stress may be applied to the implant to be used in tissues that will experience tensile forces.

[0303] Medical devices that can be coated with relevant cells include “implants” or “implantable medical devices.” Implants can be introduced temporarily or permanently into mammals for the prevention, treatment, or diagnosis of medical conditions, and for wireless monitoring of physiological parameters. Such implants include, but are not limited to, vascular prostheses, vascular grafts, fixation devices for connecting prosthetic organs to the vascular circulation, stents including vascular and non-vascular stents (e.g., gastrointestinal, pulmonary, or biliary stents), covered stents, artificial heart valves, artificial hearts, heart prostheses (e.g., artificial heart valves), biological heart valve prostheses (e.g., animal-derived xenografts that can be coated with relevant cells to make them more biocompatible and less prone to thrombosis), venous valves, abdominal aortic aneurysm grafts, vascular filters (e.g., vena cava filters), catheters, guidewires, balloons, etc. Devices for preventing pulmonary embolism (e.g., embolization coils, embolization materials for vascular embolization, etc.), orthopedic implants (e.g., bone or joint prostheses), vascular sutures, stents, smooth or porous implants, endovascular devices, vascular prosthesis filters, pacemakers, pacemaker leads, electrodes, defibrillators, subcutaneous and / or intramuscular implants, vascular occluders, ventricular shunts, vascular sheaths, drug delivery devices and ports, diaphragm closure devices, sutures, neurostimulators, implantable wireless sensors (e.g., blood glucose and blood pressure monitors), artificial filtration systems or other artificial organs, insulin pumps, artificial oxygenators, etc. Other illustrative examples of suitable medical devices include MCADs (e.g., left ventricular assist devices (LVADs), including their inflow and outflow cannulas 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 levels (for glycemic control in diabetic patients), implantable insulin pumps, implantable artificial oxygenators, implantable artificial kidneys or filtration systems, artificial or tissue-engineered bladders and / or ureters, other implantable artificial organs, implantable electrical devices (e.g., pacemakers), or wireless microelectromechanical systems (MEMS). Medical devices may be made of, for example, titanium or titanium alloys, including shape memory alloys (e.g., nickel-titanium (NiTi), aluminum and vanadium alloys (Ti6A14V and (Ti6A14V ELI), and niobium alloys (Ti6A17Nb), iron alloys (Ti5A12.5Fe), including but not limited to titanium alloys containing Nb, Ta, Zr, Mo, Fe, and Si). The device can also be made of other metals, such as stainless steel.

[0304] method This technology includes methods for treating subjects in need. In some embodiments, the method includes treating a subject with hypoxic-ischemic encephalopathy (HIE) (including neonatal HIE) or brain injury. In some embodiments, brain injury includes ischemic brain injury, perinatal brain injury, and / or neurodegeneration. The method includes administering to a subject a pharmaceutical composition or cell composition comprising isolated EPC groups of this technology, wherein the isolated EPC groups comprise or consist of PROCR+ / - PDGFRA+ / - EPC. The method may further include administering to a subject a pharmaceutical composition or cell composition comprising isolated MSCs of this technology. MSCs may be co-formulated with or in different compositions from compositions comprising PROCR+ / - PDGFRA+ / - EPC.

[0305] Subjects eligible for the study may have or be at risk of developing certain diseases, selected from the following groups: myocardial infarction, congestive heart failure, peripheral vascular occlusive disease, peripheral artery disease (PAD), ischemia, myocardial ischemia, limb ischemia, ischemic retinopathy, diabetic retinopathy, stroke, transient ischemic attack, reperfusion injury, hereditary bleeding disorders, von Willebrand disease (vWD), hemophilia A, intrauterine growth retardation (IUGR), bronchopulmonary dysplasia (BPD), retinopathy of prematurity. Acute kidney injury (AKI), pulmonary arterial hypertension (PAH), bone repair and trauma, including skin trauma, diabetic foot or ulcers, gangrene, diabetic trauma, neuronal damage, neuronal damage to the brain, neuronal damage to the spinal cord, damage to peripheral nerve cells, damage to central nerve cells, brain injury, neurodegenerative diseases, 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.

[0306] In some implementations, the subjects have received or are receiving therapeutic hypothermia treatment.

[0307] Pretreatment before treatment Prior to administering cells to a subject, isolated EPC populations and / or isolated MSC populations of this technique can be cultured under endothelial cell induction conditions. Illustrative examples include inducing endothelial cell generation prior to transplantation. For example, isolated cell populations and compositions can be induced to form vascular endothelial cells on, for example, medical or surgical devices, scaffolds or matrixes or other structures (e.g., tubes), which can then be implanted into the site in which the subject requires endothelial cells. Any convenient endothelial cell generation conditions can be used in such embodiments.

[0308] Medical devices This technology includes a method for coating a blood-contact surface of an implantable medical device with isolated EPC clusters and / or isolated MSC clusters using this technology.

[0309] To increase the rate of cell diffusion, the blood-contact surface of the implantable device may be pre-coated with extracellular matrix proteins such as fibronectin, collagen, fibronectin, laminin, fibrin, or molecules, proteins, or constructs containing any of the following components: proteoglycans such as heparan sulfate, chondroitin sulfate, keratin sulfate, or molecules containing non-proteoglycan polysaccharides such as hyaluronic acid, or any combination thereof. The blood-contact surface may also be pre-coated with gelatin or gelatin matrix or gelatin foam, cellulose, microfibrillary collagen, thrombin (e.g., recombinant human thrombin (Recothrom, ZymoGenetics)), fibrin sealants (e.g., Tisseel (Baxter)) or fibrin gel, fibrin glue, fibrinolytic-inhibiting fibrin glue, adhesives or sealants, or hydrogels. Blood-contact surfaces can also be pre-coated with serum proteins or other blood components, or growth factors or hormones, such as platelet-derived growth factor BB, basic fibroblast growth factor, acidic fibroblast growth factor, or transforming growth factor β1. Pre-coating can also be achieved using synthetic polymers, such as polymers of lysine, ornithine, or arginine, or constructs treated with polymethyl methacrylate, polyacrylic acid, or L-glutamic acid, or glutaraldehyde-preserved cell matrix, or biodegradable adhesives or coatings, such as poly(DL-lactide-co-glycolic acid), or biodegradable polyesters, such as polyhydroxyalkanoates, polysorbates, or polyamino acids, such as poly-L-lysine, or chitosan, fetoglobulin, or cationic silica microbeads, or polyethylene terephthalate with or without altered microbeads or carbon-deposited surfaces modified by plasma discharge, or covalently linked avidin, biotinylated, or molecules, structures, or constructs containing RGD peptide sequences or peptides crosslinkable to RGD peptides, or molecules targeting one or more EPC-specific integrin binding sites or co-binding sites, such as the amino acid sequence DRVPHSRN, or antibodies, peptides, or aptamers targeting EPC, or any combination thereof. The aforementioned molecules can be physically adsorbed or covalently bound to surfaces such as titanium / titanium alloy surfaces. For the latter, various methods can be used, including silanization (e.g., bonding of steel to an aminosilane crosslinking agent), biotinylation, and covalent bonding with dopamine. In addition to the proteins, molecules, polymers, structures, and artificial constructs mentioned above, other cell types can be used to pre-coat the surfaces of blood contact devices to provide 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 types). Pre-coating implantable devices with autologous cells is generally desirable to avoid rejection. For example, fibroblasts can be readily harvested from a subject's skin sample for this purpose.

[0310] Biocompatible implants of this technology may contain at least one bioactive agent, representative examples of which include growth factors, analgesics / antipyretics, anti-asthmatic agents, antibiotics, antidepressants, antidiabetic agents, antifungal agents, antihypertensive agents, anti-inflammatory agents, antitumor agents, anxiolytics, immunosuppressants, antimigraine agents, sedatives / hypnotics, antipsychotics, antimanic agents, antiarrhythmic agents, antiarthritis agents, antigout agents, anticoagulants, thrombolytics, antifibrinolytics, antiplatelet agents, antibacterial agents, antiviral agents, antimicrobial agents, antiinfectives, and combinations thereof. The bioactive agent may be a cellular response regulator, such as a growth factor or chemokine. Exemplary growth factors include epidermal growth factor, bone morphogenetic protein, TGF-β, hepatocyte growth factor, platelet-derived growth factor, TGF-α, IGF-I and II, hematopoietic growth factor, heparin-binding growth factor, peptide growth factor, basic and acidic fibroblast growth factors, nerve growth factor (NGF), muscle morphogenetic factor (MMP), and vascular endothelial growth factor (VEGF). The specific growth factor used should be suited to the desired cellular activity. For example, VEGF can be used to promote EPC differentiation. Alternatively, growth factors can be selected to recruit cells to the implant or to promote or inhibit specific metabolic activities of cells recruited to the implant. The regulatory roles of the large family of growth factors are likely well known to those skilled in the art.

[0311] To further enhance angiogenesis, endothelial cell mitogens can also be administered to the subject together with or after the administration of any isolated cell populations and compositions of this technology. Endothelial cell mitogens can be administered directly, for example, intra-arterial, intramuscular, or intravenous, or a nucleic acid encoding the mitogen can be used.

[0312] Nucleic acids encoding EC mitogens can be administered via catheters (e.g., hydrogel catheters) to blood vessels perfused with ischemic tissue or to sites of vascular injury, as described, for example, in U.S. Patent 5,652,225, which is incorporated herein by reference in its entirety.

[0313] Nucleic acids can also be delivered by direct injection into ischemic tissue using the method described in U.S. Patent 6,121,246, which is incorporated herein by reference in its entirety.

[0314] Endothelial cell mitogens may contain secretion signal sequences that promote protein secretion. Proteins with native signal sequences, such as VEGF, may be required. Proteins without native signal sequences, such as bFGF, can be modified to contain such sequences using conventional genetic manipulation techniques (e.g., Nabel et al. (1993) Nature, 362, 844).

[0315] The DNA fragment encoding the desired endothelial cell mitogen can be chemically synthesized, or such DNA fragment can be obtained using conventional procedures in the art, such as PCR amplification. DNA encoding VEGF is disclosed in U.S. Patent 5,332,671, which is incorporated herein by reference in its entirety.

[0316] In some implementations, it may be necessary to use nucleic acids encoding two or more different proteins to optimize treatment outcomes. For example, DNA encoding two proteins (e.g., VEGF and bFGF) can be used to provide improvements over using bFGF alone. In some implementations, angiogenic factors can be combined with other genes or their encoded gene products to enhance the activity of target cells while inducing angiogenesis, including, for example, nitric oxide synthase, L-arginine, fibronectin, urokinase, plasminogen activator, and heparin.

[0317] The cell-seeded implants of this invention can be implanted into any tissue, including connective tissue, muscle tissue, nerve tissue, and organ tissue. For example, an implant placed in a bone defect will attract cells from the surrounding bone, which will synthesize ECM, while EPC forms blood vessels. As new ECM forms and mineralizes, it will provide a blood supply to the new bone. An implant placed in a skin defect will promote dermal formation and provide a vascular network to supply nutrients to the newly formed skin.

[0318] Cells recruited to the implant can also differentiate into other cell types. Precursor osteoblasts migrating to the bone implant can differentiate into osteoblasts. Mesenchymal stem cells migrating into blood vessels can differentiate into muscle cells. Endothelial cells forming tubular networks in the liver may induce liver tissue formation.

[0319] Any isolated cell populations and compositions of this technology can be mixed with another cell type prior to implantation. As described above, the cell mixture can be suspended in a carrier such as a culture medium or gel. Alternatively, cells can be co-seeded onto a polymer matrix or in combination with a gel absorbed into the matrix. For some applications, it may be necessary to seed one cell type directly onto the matrix and add a second cell type via a gel. Any ratio of EPC to one or more other cell types can be used. Those skilled in the art will recognize that this ratio can be readily optimized for a particular application. Exemplary ratios of EPC to other cells can 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, such as less than 10%, can also be used.

[0320] Any cell type, including connective tissue cells, nerve cells, muscle cells, organ cells, or other stem cells, can be combined with any isolated cell population and composition of this technology. For example, osteoblasts can be combined with fetal endothelial cells to promote the co-generation of bone and its vascular system in large defects. Fibroblasts combining with fetal endothelial cells and inserting into the skin will produce a fully vascularized dermis. Other exemplary cells that can be combined with fetal endothelial cells of this technology include hematopoietic cells (including hematopoietic stem cells), ligament cells, lung cells, epithelial cells, smooth muscle cells, cardiomyocytes, skeletal muscle cells, pancreatic islet cells, nerve cells, hepatocytes, kidney cells, bladder cells, and bone morphogenetic cells.

[0321] In vitro screening This technology includes isolated EPC populations and isolated MSC populations. Providing these cell populations and compositions comprising them facilitates screening systems for in vitro testing of the effectiveness and toxicity of existing or potential treatments or culture regimens. Therefore, this technology includes methods for evaluating the effect of treatments or culture regimens on the phenotypic or functional status of any isolated EPC populations and / or MSC populations of this technology, as well as pharmaceutical and cell compositions comprising them. In some embodiments, the method includes subjecting isolated EPC populations and / or MSC populations and / or pharmaceutical or cell compositions of this technology to treatment regimens and screening for altered functional or phenotypic status relative to unisolated cell populations or compositions comprising unisolated cell populations.

[0322] Example Example 1: Isolation of CD34 from human placenta + CD45 - Cell population approach.

[0323] Placental tissue was processed and single-cell suspensions were prepared as described in Patel et al., Stem Cells TransMed. 2013 Nov;2(11):839-47 and Patel et al., Placenta. 201 Nov;35(11):969-71, which are incorporated herein by reference in their entirety. The isolated placental differentiation cluster (CD)34+ single-cell suspensions were incubated with human CD34-phycoerythrin (PE), human CD45-FITC, and human CD31-V450 at 4°C for 20 min. Cells were flow-cytometrically sorted using fluorescence-activated cell sorting (FACS). Cell duplexes were removed, and dead cells were excluded using 7-aminoactinomycin D (7AAD). The target population was screened using a fluorescence minus one (FMO) control. To remove any remaining contaminating CD45+ cells from the hematopoietic lineage, CD45+ cells were selected only from CD45+ cells. - CD34 +The cell population is gated. Then all CD45 cells are... - CD34 + Cells were directly sorted into 100% fetal bovine serum via FACS (see Patel et al., Placenta. 2014 Nov;35(11):969-71, which is incorporated herein by reference in its entirety). The sorted cell fraction was considered to contain fetal EPCs and MSCs.

[0324] Example 2: PROCR + and PDGFRA + Identification of the EPC subgroup.

[0325] Materials and methods Single-cell RNA sequencing analysis Single-cell RNA sequencing was analyzed using RStudio (RStudio, MA, USA) and the Seurat software package (version 4.2.0), based on Hao et al., Cell, 2021. 184(13): 3573–3587.e29, which is incorporated herein by reference in its entirety. Data from previously performed mouse aortic fragment and publicly available human aortic scRNA-seq analyses were reanalyzed (see Shafiee et al., Stem Cell Reports, 2018. 10(3): 890–904 and Lukowski et al., Cell Reports, 2019. 27(9): 2748–2758.e3, which are incorporated herein by reference in their entirety). Data were filtered using the following criteria: >200 and <3000 gene counts / cell, <20% mitochondrial genes, and >3 cells present. The data were then normalized and integrated using the standard Seurat analysis workflow, correcting for batch effects with a scaling factor of 10,000. Principal component analysis was performed on the integrated dataset using RunPCA. The first 30 principal components were used to compute nearest neighbors and clusters using FindNeighbors and FindClusters, respectively, at a resolution of 0.4 (mouse) or 0.5 (human) to achieve optimal distinction between clusters. Cluster maps were constructed using the two-dimensional uniform manifold approximation and projection (UMAP) algorithm in Seurat. Differentially expressed (DE) genes were identified in each cluster. Cell clusters were annotated using the Bioconductor package SingleR, referencing mouse RNA sequencing data and the human primary cell atlas database (Aran et al., Nature Immunology, 2019. 20(2): p. 163-172, Mabbott et al., BMC Genomics, 2013. 14: p. 632, and Monaco et al., Cell Rep, 2019. 26(6): p. 1627-1640.e7, each incorporated herein by reference).

[0326] animal Adult C57Bl / 6 mice and NOD scid Il2rynullB2mnull (NSG) mice of mixed sexes were used in the experiment. Adult CAG-EGFP, Cdh5-CreERT2 / ROSA-EYFP, Pdgfrα-MerCreMer / ROSA-YFP, Abcg2-Ires-CreERT2 / ROSA-YFP, and Sox18-Cre / ROSA-YFP mice of mixed sexes were derived from an internal breeding population. Cdh5-CreERT2 / ROSA-EYFP, Cdh5-CreERT2 / ROSA-ZsGreen, Pdgfrα-MERCRemer / ROSA-YFP, Abcg2-Ires-CreERT2 / ROSA-YFP, and Sox18-Cre / ROSA-YFP mice were injected intraperitoneally for 5 consecutive days with 100 µL of 20 mg / mL tamoxifen (Sigma-Aldrich, MI, USA) dissolved in 90% corn oil and 10% ethanol to induce recombination of yellow fluorescent protein (YFP) in target cells prior to tissue collection (according to the standard protocol, Cdh5-CreERT2 / ROSA-ZsGreen mice only received injections for 3 days).

[0327] Slab Laying / Aortic Length Preparation The aorta was dissected and prepared for planarization IF processing, as previously described by Zhao et al., Nature Communications, 2021. 12(1): p. 2564, which is incorporated herein by reference in its entirety. The sample was sliced ​​perpendicular to the cutting plane to analyze the length of the aorta.

[0328] Immunofluorescence Tissues were prepared as described by Zhao et al., Nature Communications, 2021. 12(1): p. 2564. Primary antibodies used included: rat anti-mouse 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 rotating disk confocal microscope (Nikon, New York, USA). Bright-field imaging was performed using a Nikon Eclipse 50i bright-field microscope (Nikon, New York, USA). Image analysis was performed using Olympus Fluoview FV31S-SW software (Olympus, Shinjuku, Japan) and ImageJ (National Institutes of Health, USA).

[0330] Flow cytometry and fluorescence-activated cell sorting According to Zhao et al., Nature Communications, 2021. 12(1): p. 2564, the aortic and total skin resection wounds were digested. To compare colony formation between the thoracic and abdominal aortas, the aorta was separated at the diaphragm.

[0331] In rat aortic and total skin resection wounds, antibodies used to assess endothelial layers and subsets included: hematopoietic lineage mixture 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 and FACS were performed. A single-stain control was used to obtain cell counter voltage settings and compensation data. A fluorescence minus one (FMO) control was used to differentiate positive and negative cell populations and to set appropriate gates. All analyses were performed using FlowJo® software (FlowJo LLC, USA).

[0333] In vitro colony formation experiment Cells from fluorescently activated cell sorting and endothelial growth medium-2 (EGM2; Lonza, Basel, Switzerland) were deposited on top of crosslinked Matrigel® plates (Corning® Matrigel® basement membrane matrix, phenol red-free, LDEV-free; Corning, New York, USA). Cells were separated according to experimental conditions to allow for deposition of 10 or 100 cells per well. The plates were then incubated at 37°C with the medium changed twice weekly. Cells were intermittently imaged using a Nikon Eclipse 50i bright-field microscope (Nikon, New York, USA).

[0334] On day 12, the wells were fixed and permeabilized, then blocked in PBST. Cells were then stained overnight at 4°C with the primary antibody solution described above. The next day, cells were incubated in secondary antibody solution and stained with DAPI solution before imaging.

[0335] Angiogenesis and collagen blockage Following FACS sorting, 100 cells from the desired cell population isolated from CAG-EGFP mice were mixed with a gel solution prepared by mixing 80% collagen (3% PurCol; Advanced Biomatrix, Carlsbad, CA, USA) with 10% DMEM, 5%–7% sodium bicarbonate (to achieve pH 7.2–7.4), and water on ice. The gel + cell solution was incubated in 96-well plates at 37°C for 90–120 minutes, then topped with EGM2 and incubated overnight.

[0336] The next day, the gel was rinsed in PBS and implanted into NOD-scid Il2rynullB2mnull (NSG) mice. A lateral incision was made on each dorsal side to create a pocket beneath the skin and above the muscle, and a total of two plugs were implanted into each mouse. The incisions were sutured, and the mice were monitored daily. The plugs were collected after 7 days, and the IF was analyzed using a whole-body microscope and frozen sections. For whole-body microscopy, the gel was rinsed in PBS, then cleared in RapiClear® (SunJin Lab Co, China) for 30–60 minutes and imaged immediately. The gel was then subjected to a sucrose gradient as described above to prepare frozen sections.

[0337] Human full-term placental cells Following the protocol described in Nano et al., STAR Protoc, 2022. 3(2): p. 101354, frozen single-cell suspensions of previously processed human full-term placental samples were thawed and prepared for flow cytometry / FACS sorting. In addition to the markers outlined in Nano et al., STAR Protoc, 2022. 3(2): p. 101354, PROCR PE (1:25) was added to the group. Cells were then gated and sorted as previously described, with PROCR added. + / - Gating was performed on each of the four populations with different CD31 expression.

[0338] FACS-sorted cells were then plated onto collagen-coated plates at a density of 1000 cells per well in 48-well plates with EGM2, as previously described. Cells were cultured and expanded for up to 30 days to assess colony-forming capacity (no colonies, endothelial colonies (EC, <50 cells), low-proliferative-potential ECFCs (LPP-ECFC, <1000 cells), or high-proliferative-potential ECFCs (HPP-ECFC, >1000 cells, ability to form secondary colonies)). HPP cells were then further passaged to limiting dilution assays (1000 cells per well in 6-well plates) to assess further colony formation, either by continuous passage for expansion or by immunofluorescence expression assessment using rabbit anti-human VECAD (1:100) and mouse anti-human CD31 (1:100) staining.

[0339] Statistical analysis Data were analyzed using GraphPad Prism8 (GraphPad, United States) software. Paired t-tests, unpaired t-tests, and Mann-Whitney tests were used for individual comparisons, depending on the experimental conditions. For multiple comparisons, Friedman one-way ANOVA and Kruskal-Wallis tests were performed on the data. Results are presented as mean, with error bars representing standard deviation (SD), and a significance threshold of p < 0.05. All significance tests were performed with at least three biological replicates.

[0340] Single-cell RNA sequencing reveals key biomarkers in endothelial cell populations Lin from 3 C57Bl / 6 mice - CD34 + Single-cell RNA sequencing performed on the aortic compartment was reanalyzed to elucidate genes highly expressed in the target endothelial and mesenchymal clusters. Figure 1A By increasing the resolution when re-clustering the sequencing data, more distinct subpopulations emerged, allowing for the analysis of gene expression within previously specified clusters. Figure 2A Based on the expression of known major endothelial cell markers (including Pecam1 and Cdh5), clusters 0, 10, and 12 were characterized as mature, differentiated endothelial cells (these markers are commonly used to gate endothelial cell compartments), and this was confirmed by SingleR labeling analysis. Figure 1B , Figure 2B(i-ii)). Clusters 1, 2, and 3 showed upregulation of mesenchymal markers, leading to their designation as mesenchymal (M) clusters, while cluster 4 showed expression of both mesenchymal and endothelial markers, leading to its designation as the putative endothelial progenitor (EPC) population. No hematopoietic cell markers were expressed in either group (confirmed by SingleR). Among the 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. Figure 2B (iii-v)), while Procr (adj.p = 2.07 x 10-121 in cluster 4) and Pdgfrα were expressed in EPC cluster 4, with the mesenchymal marker Pdgfrα being upregulated the most in MSC clusters (adj.p = 3.18 x 10-103 in cluster 1; adj.p = 6.38 x 10-178 in cluster 2); Figure 2B (vi-viii)). Pathway analysis and dot plotting of the target gene were performed on cluster 4 to further characterize differentially expressed (DE) genes. Figure 2C , Figure 1B-1D ).

[0341] Endothelial progenitor cells highly express PROCR and PDGFRA Flow cytometry was performed on the aorta of adult C57BL / 6 mice using the markers highlighted in the single-cell RNA sequencing analysis described above, as well as markers used to characterize the aforementioned EPC population. Endothelial layers were isolated from total aortic cells based on cell surface marker profiles and the original gating strategy outlined in Patel et al., Circulation, 2017. 135(8): p. 786-805. EPCs were identified as Lin - VE-cadherin + CD34 + CD31 lo / - Furthermore, the differentiated endothelial cells were identified as Lin - VE-cadherin + CD34 + CD31 + cell( Figure 2D Then, the expression levels of PROCR, PDGFRA, and CD157 in EPC and mature differentiated endothelial cell populations were further evaluated. Figure 2E and 2FCompared with differentiated endothelial cells, the expression frequencies of PROCR and PDGFRA in EPCs were 1.4-fold (p<0.001) and 3.3-fold (p<0.001), respectively, while the expression frequency of CD157 in differentiated endothelial cells was 5.9-fold (ns) higher than that in EPCs. Figure 2G In addition to these biomarkers, two other mouse strains were used to test further differences in gene expression between EPCs and differentiated endothelial cells, based on the above studies. Five days after tamoxifen injection, aortas from adult Abcg2-Ires-CreERT2 / ROSA-EYFP and Sox18-CreERT2 / ROSA-EYFP mice were collected for flow cytometry. Further characterization of these cell populations based on YFP expression showed that ABCG2 and SOX18 expression frequencies were 2.3-fold (p<0.05) and 4.3-fold (p<0.05), respectively, higher in differentiated endothelial cells compared to EPCs. Figure 2E-2I ).

[0342] These findings suggest that PDGFRA and PROCR are additional biomarkers for enriching progenitor cells in the already described EPC population, as demonstrated by flow cytometry and single-cell RNA sequencing. In EPC cells, PROCR was present in an average of 78.04%. + And 82.28% were PDGFRA + This indicates that these markers can allow for a more refined definition of progenitor cells. Alternative gating strategies for live aortic cells co-expressing PROCR and PDGFRA showed an average gating rate of 91.43% ( ) is Lin - VE-cadherin + And from here, another 94.97% ( ) is EPC ( Figure 2H ; p=0.0012, (p<0.0001, n=3). This demonstrates the powerful ability of individual PROCR and PDGFRA expression to label the same cell population with high confidence as the classic EPC gating strategy.

[0343] Given the significant overlap among the three different strategies for identifying endothelial progenitor cells, we will refer to this population as PROCR. +Intravascular progenitor cell populations were identified and their functions were analyzed. Single-cell RNA-seq was used to indicate that cluster 4 represents this cell population. Differential gene expression in this cluster was defined as including the expression of both major endothelial and mesenchymal genes, as seen in the analysis of top differentially expressed genes from this cluster. Figure 1B-1D ).

[0344] PROCR + EPC showed increased in vitro endothelial colony-forming capacity and increased in vivo implantation potential. To begin analyzing PROCR + The functional capacity of vascular progenitor cells (EPCs) relative to other EPCs and differentiated endothelial cell populations was assessed by collecting aortic cells and performing FACS sorting based on PROCR cell surface expression for colony formation assays. Four groups were sorted in each experiment: PROCR... + EPC, PROCR - EPC, PROCR + Differentiation of endothelial cells and PROCR - Differentiated endothelial cells ( Figure 3A Based on their morphology on day 12, two main morphologies were observed to characterize the colonies: the classic endothelial morphology (…). Figure 3B (i)), in which the endothelial marker Griffonia (Bandeiraea) Simplicifolia lectin I (GSL I, BSL I)-rhodamine (iso-lectin) in immunofluorescence (IF); Figure 3B (iii) Positive expression in, and not expressed by isolectins ( Figure 3B (iv)) Elongation morphology of the mark ( Figure 3B (ii)). From PROCR + EPC colonies showed only endothelial morphology, while all other conditions showed elongated colonies that did not express isolectins. PROCR + EPCs exhibited the highest colony-forming capacity, with an average of 17.67% of pores forming colonies per mouse, followed by PROCRs at 8.21%. - EPC orifice, 1.16% PROCR + Differentiated endothelial cell pores and 0.45% PROCR - Differentiated endothelial cell pores ( Figure 3C i-ii; (p<0.05). Differentiated endothelial cell pores (PROCR) + / - Based on the fact that isolectin positive staining never forms endothelial colonies.

[0345] To distinguish PROCR + EPC and PROCR -The efficacy of EPC was assessed, and these cell populations were then stimulated using more rigorous in vivo experiments, in which 100 PROCR cells from CAG-EGFP mice were used. + EPC, PROCR - EPC or collagen gels containing fully differentiated endothelial cells were transplanted into the subcutaneous dorsal region of NOD-scid-Il2rynullB2mnull (NSG) recipient mice, while cell-free gels served as a control. Figure 3D ). 7-day post-treatment image of the collagen gel showing PROCR + EPCs showed the highest implantation potential, with 9 out of 15 gels implanted, resulting in an average GFP+ area of ​​5.67% per plug (Fig. 1E(i), Fig. 1F), while only 2 out of 10 PROCR-EPC gels were implanted, with an average GFP+ area of ​​0.79% per plug. Figure 3E (ii) Figure 3F (p<0.05). Differentiated endothelial cells could never implant (0 / 6 gel), showing 0% GFP+ area, consistent with the results of cell-free gels. Furthermore, IF staining was performed on sections from these gels for further characterization of implanted cells. GFP+PROCR + EPCS co-expresses the endothelial marker CD34 and isolectins, and these endothelial markers are associated with those from sources containing PROCR. - GFP+ cells in the collagen gel of EPC did not overlap. Figure 3G These findings more strongly point to key functional differences between EPCs and differentiated endothelial cells, as previously reported, but even more significantly to key functional differences between PROCR-expressing EPCs and PROCR-deficient EPCs.

[0346] PROCR + EPC forms a niche in the thoracic aorta and exhibits increased homology. To confirm the expression of PROCR in aortic endothelial cells and to discover its in situ anatomical distribution, derived from Cdh5... - The aorta of CreERT2 / ROSA-EYFP mice (where endothelial cells could be labeled with YFP after administration of tamoxifen) was used for in vitro analysis. IF staining showed that, compared with the abdominal aorta (21.10%), [the aorta was significantly different in size and shape]. Figure 4B and 4D ; Compared to the thoracic aorta (p=0.005, n=5), the aorta (68.18%)... Figure 4A and 4C Co-expression of PROCR and YFP was higher in the aorta. Following the discovery of spatial differences in PROCR expression in the aorta, further research is needed to confirm whether this is also related to functional spatial differences in clonogenic capacity. For Cdh5...- YFP+EPCs from the thoracic and abdominal aortas of CreERT2 / ROSA-ZsGreen mice were sorted by FACS and seeded in Matrigel® to compare the colony-forming ability of the two cell populations. Interestingly, 7 / 13 pores containing YFP+EPCs from the thoracic aorta formed branched endothelial colonies, while 0 / 13 pores from the abdominal aorta formed colonies. Figure 4E and 4F ; (p=0.0052, n=3).

[0347] PROCR+EPC forms differentiated endothelial cells during in vivo homeostasis and injury. Because PROCR and the mesenchymal marker PDGFRA are significantly different in PROCR results from flow cytometry and scRNA sequencing. + High overlap was observed in CEPCs, and Pdgfrα-MerCreMer / Rosa-EYFP mice were used to track CEPC fate. Tamoxifen was administered to animals to permanently label PDGFRA-expressing cells with YFP, and this cell population was tracked in the target tissue over time. Flow cytometry was used for the first time to confirm the presence of PDGFRA-expressing cells in the aorta of adult steady-state mice following short-term tamoxifen administration. - The PDGFRA(YFP)+ subset is mainly composed of EPCs rather than differentiated endothelial cells. Figure 5A (90.85% vs. 2.87%, n=4, p<0.0001). To confirm that the cell population tracked in this model was indeed the same endothelial cell population as previously studied, the endothelial cells from Cdh5 were compared. - A comparison was made between YFP+PROCR+EPC colonies cultured in the aorta of CreERT2 / ROSA-EYFP and Pdgfrα-MerCreMer / ROSA-EYFP, and no differences were found in morphology or phenotype. However, both endothelial markers, isolectin and ERG, were expressed in immunofluorescence analysis. Figure 5B ).

[0348] Previous studies have rarely been able to identify a single Cre reporter system that distinguishes between progenitor and differentiated cells. This presents a unique opportunity to demonstrate the effectiveness of PROCR. + Lineage relationships between EPCs and differentiated endothelial cells. Following tamoxifen induction at 4 weeks of age, lineage tracing was performed in the steady-state aorta to label cells expressing PDGFRA. The aorta was then assessed from juvenile to adult to trace the fate of YFP+ cells. IF staining of the aorta collected from day 1 (D1) after tamoxifen treatment showed no co-expression of any mature endothelial markers in YFP+ cells in the intima. Figure 5C(i)). This further indicates that at least a portion of the YFP-labeled PDGFRA-expressing cells can be endothelial, such as in the intima, and not just in the mesenchymal layer of the aorta. When examining the fate of these PDGFRA-expressing cells labeled on D1, IF at D84 demonstrated co-expression of endogenous YFP with CD31 and ERG in the intima, confirming the endothelial fate of these cells (i). Figure 5C (ii)). This result was quantitatively confirmed using flow cytometry, where the percentage of CEPCs observed between D1 and D84 ranged from Lin. - The average percentage of YFP+ compartments ranged from 69.58% to 88.24%, while the percentage of differentiated endothelial cells significantly increased from 0.74% at D0 to 4.67% at D84. Figure 5D and 5E n=5, p<0.01, p<0.001).

[0349] To analyze this mechanism in the context of injury, we similarly administered one course of tamoxifen injection to adult Pdgfra-MerCreMer / Rosa-EYFP mice, followed by total skin excision of the wound on day 0 and collection of wounds within the wound healing timeframe. IF staining on day 1 showed that YFP expression was primarily concentrated in a fibroblast-like cell population and did not overlap with mature endothelial markers. Figure 6A ), while the IF at D5 showed co-expression of endogenous YFP with differentiation endothelial markers CD31 and ERG (). Figure 6B Flow cytometry at each time point confirmed the PROCR between D1 and D5. + The percentage range of EPC is Lin - The average percentage of YFP+ compartments increased from 29.43% to 43.29%, while the percentage of differentiated endothelial cells significantly increased from an average of 0.04% on day 1 to 1.19% on day 5. Figure 6C-6E n=7 p<0.05; p<0.01). In summary, using PDGFRA expression as a PROCR... + EPC reporter factors allow for tracking the fate of this cell population to demonstrate its contribution to differentiated endothelial cells in the homeostatic aorta and skin wounds.

[0350] PROCR was expressed in human aortic scRNA-seq data and caused clonogenicity in a human full-term placental model of ECFC. Increased ability To begin investigating whether this characterization also applies to human tissues, publicly available single-cell RNA sequencing data from normal human aortas were reanalyzed to investigate the biomarkers for this study (see Li et al., Circulation, 2020.142(14): p. 1374-1388, which is incorporated herein by reference in its entirety). Data from three normal aortic samples were re-clustered before removing hematopoietic clusters based on known biomarker expression and SingleR labeling analysis (…). Figure 7A , Figure 8A and 8B The remaining clusters were identified as predominantly mesenchymal (M) based on the SingleR marker; however, based on the high overlap between the leading differentially expressed (DE) genes in these clusters and EPC cluster 4 in the mouse scRNA seq dataset, the human counterparts of the EPC clusters were specifically identified as clusters 3 and 12 (Table 1 and 12). Figure 8B ).

[0351] Table 1: Overlapping genes of the top 100 differentially expressed genes from clusters 3 and 12 of the human normal aorta dataset and the mouse aorta sc-RNA seq PROCR+ EPC cluster. These overlapping genes were found to be associated with endothelium, mesenchyme, extracellular matrix, and cell cycle pathways, indicating genes that maintain populations between endothelial and mesenchymal states. Genes such as ACKR3 (or CXCR7) may be downstream of PDGFRa signaling and contribute to angiogenesis, potentially highlighting their importance in the putative endothelial progenitor cell population. Other listed genes play important roles in endothelial identification or regulation of mesenchymal transition and fibrosis. The aforementioned target biomarkers were then analyzed in the remaining endothelial, MSC, and EPC-like clusters using feature mapping, dot mapping of leading DE genes, and pathway analysis. Figure 7C , Figure 8C and 8D The major endothelial genes PECAM1 and CDH5, along with ABCG2 and SOX18, were most highly expressed in differentiated endothelial cluster 8. Figure 7C (i-iv)). CD34, PROCR, and PDGFRA were expressed in EPC clusters 3 and 12, as well as differentiated endothelial cell cluster 8. Figure 7B (v-vii)).

[0352] After confirming that the expression of the target biomarkers in the human control aorta using scRNA-seq was similar to that seen in the previously described mouse model, functional assays were performed to investigate whether progenitor cell capacity in human cells expressing these biomarkers increased as seen in the mouse studies. A human full-term placental cell model was used, consistent with the previously described EPC population known as ECFC. Placental cells were isolated and FACS sorted as previously described (see Nano et al., STAR Protoc, 2022. 3(2): p. 101354, which is incorporated herein by reference in its entirety), and PROCR was performed on each population with different CD31 expression levels (negative, low, intermediate, and high). + / - Additional gating ( Figure 7C , Figure 8E These groups were then cultured on collagen-coated plates until colonies formed. Among all donors, those containing CD31... int program - Only 1 / 13 of the cells formed colonies, and they grew to less than 50 cells before death, therefore they were classified as endothelial clusters (ECs). Figure 7D (i), Figure 7F (i-ii), n=3). Conversely, 5 / 7 wells containing CD31intPROCR+ cells grew colonies in all donors, and 3 / 5 of these colonies continued to expand and reach high proliferation potential (HPP; >1000 cells) colony size. Figure 7D (ii), Figure 7F (i-ii)). These HPP colonies were then passaged and showed the ability to continuously expand via P6, as well as the ability to form additional LPP and EC at limiting dilutions. Furthermore, IF staining of these colonies confirmed the endothelial nature of this population, which exhibits positive expression of CD31 and VE-cadherin. Figure 7E The remaining 2 / 5 of the settlements form EC (EC). Figure 7F (i-ii)).

[0353] In addition to expression confirmation, shared EPCs were functionally tested in a series of assays. Compared to PROCR-EPCs or endothelial differentiated cell populations, shared EPCs showed increased functional progenitor capacity, regardless of whether they expressed PROCR. Shared EPCs consistently formed more colonies in vitro than all other cell populations and were the only population to form strictly endothelial colonies morphologically and based on positive staining for endothelial marker isolectins. PROCR+ mature differentiated endothelial cells formed 15-fold fewer colonies, none of which showed an endothelial phenotype, summarizing the overlap in PROCR and EPC marker expression (Lin). - CD34 + VE-cadherin + CD31 loThis may require the ability to enrich functional progenitor cells in the endothelium. Furthermore, EPCs derived from the mouse aorta showed increased implantation potential in the in vivo 3D collagen matrix, forming vascular networks embedded in the gel in hosts stained positive for mature endothelial markers to a significantly greater extent than PROCRs, even after 7 days of implantation. - EPCs, on the other hand, cannot engraft mature, differentiated endothelial cells regardless of their PROCR expression. These experiments clearly highlight the remarkable self-renewal capacity of the CEPC population, which goes beyond the definition of EPCs or PROCR staining alone.

[0354] Fate tracking of PROCR+PDGFRA+ EPC cells from time D0 in Pdgfrα-MerCreMer / ROSA-EYFP mice in both steady-state aorta and total skin resection wounds demonstrated their ability to differentiate into mature endothelial cells via IF and flow cytometry. This is the first formal demonstration that the mesenchymal marker PDGFRA can be used to label cell populations with endothelial fate in both steady-state and injury conditions. In particular, few reporter genes can distinguish between progenitor and differentiated cells in the endothelium during steady-state aorta. The stained intima location, flow cytometry gating, and final endothelial fate of YFP-labeled cells expressing PDGFRA in the steady-state aorta clearly indicate their endothelial capacity. Finally, translation into a human model using scRNA-seq showed that PROCR and PDGFRA can be expressed in EPC-like cell populations in normal human aorta. Furthermore, when further gated to positive PROCR expression, previously defined human term placental ECFCs showed increased colony-forming capacity, forming higher yields of colonies and secondary colonies, and self-renewing to at least P6.

[0355] These data demonstrate that, in mouse and human models, combining EPC expression requirements with PROCR and PDGFRA expression characterizes a more specific progenitor cell population with enhanced functional capabilities within the endothelial compartments of various tissue beds. The stringent functional requirements, along with the addition of a broader array of cell surface markers, allow for more specific targeting of EPCs, potentially opening up possibilities for the clinical application of angiogenic agents such as pro-angiogenic and anti-angiogenic agents, as well as advancements in tissue engineering and bioengineering.

[0356] Example 3: Used for separating PROCR + and PDGFRA + Cellular approach Placental tissue was processed and single-cell suspensions were prepared as described below: Patel et al., 2013 Stem CellsTransl Med. 2013 Nov;2(11):839-47 and Patel et al., Placenta. 2014 Nov;35(11):969-71. The isolated placental differentiation cluster (CD)34+ single-cell suspensions were incubated at 4 °C for 20 min 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). In addition to the markers outlined in Example 1, PROCR PE (1:25) and PDGFRA antibodies were added to the groups. Cells were sorted using flow cytometry. Cell duplexes were removed, and dead cells were excluded using 7-aminoactinomycin D (7AAD). The target population was screened using a fluorescence minus one (FMO) control. Cells were then gated and sorted as previously described in Example 1, with the addition of PROCR. + / - and PDGFRA + / - Door.

[0357] Example 4: Used for separating PROCR + and PDGFRA + Cellular approach Placental tissue was processed to prepare a single-cell suspension for PDGFRA isolation using PDGFRA antibody. + Single-cell population.

[0358] Separated placental PDGFRA + Single-cell suspensions were incubated with human PROCR PE. Cells were sorted using flow cytometry. Cell duplexes were removed, and dead cells were excluded using 7AAD. An FMO control was used to gate the target population. Cells were then gated and sorted using PROCR+ / - gates. Figure 9 and 10 ).

[0359] Example 5: Used for separating PROCR + and PDGFRA + Cellular approach Process placental tissue to prepare single-cell suspensions for isolation of PROCR using human PROCR PE antibody. + Single-cell population.

[0360] PROCR of the separated placenta +Single-cell suspensions were incubated with human PDGFRA antibody. Cells were sorted using FACS flow cytometry, cell duplexes were removed, and dead cells were excluded using 7AAD. An FMO control was used to gate the target population. Cells were then gated and sorted using PDGFRA + / -. Figure 9 and 10 ).

[0361] Example 6: Placental stem cell enhancement for hypothermia therapy in hypoxic-ischemic encephalopathy Animal and human tissues Human placenta is obtained from healthy women who undergo cesarean section at full term (38-39 weeks of gestation), allowing for the isolation and use of any stem cell population derived from placental tissue.

[0362] This project used Large White piglets (n=23) that were <24 hours old (day 1 after birth, P) and weighed between 1.32 kg and 2.18 kg. Five additional P8 control animals weighing between 2.25 kg and 2.90 kg were used in this project.

[0363] Experimental setup Piglets were anesthetized, intubated, and ventilated, as detailed elsewhere (Miller et al., Journal of Neurochemistry. 2016;139(3):471-84, which is incorporated herein by reference in its entirety). All piglets received intramuscular (im) doses of vitamin K (Konakion, 2.0 mg, im) and prophylactic intravenous (iv) doses of cephalosporin (DBL™ cephalosporin sodium, 0.2 mg / kg iv), gentamicin (gentamicin sulfate, 0.25 mg / kg iv), and penicillin (penicillin G, 12 mg / kg iv). Figure 11 and Figure 12Rectal temperature was maintained at 38.5 ± 0.2 °C using a top-mounted radiant heater until HTH began. Arterial pressure was recorded via an umbilical artery catheter, and temperature was monitored using a rectal thermometer (Marquette). Dual-channel EEG (Unique CFM 6.0, Inspiration Healthcare, United Kingdom) and bipolar ECG were continuously recorded at 256 samples per second. Arterial blood gas analysis was performed throughout the intensive monitoring period to provide information on active titration of pH, HCO3, and glucose (glu). Ventilator settings were adjusted during anesthesia to maintain arterial oxygen saturation >96% and end-expiratory carbon dioxide pressure at 35–45 mmHg. Intravenous glucose (10%, 3 mL / kg / h) was titrated to maintain blood glucose levels within the normal range (2.6–8.3 mmol / L). Piglets were randomly assigned to hypoxia-ischemia, hypothermia, and progeny stem cell (PROCR) groups. + / - PDGFRA + EPC and MSC treatment group (HHS; n=7) or hypoxic-ischemic, hypothermic, and stem cell-mediated treatment group (HHV; n=4) (control group n=5) Figure 11 and Figure 12 ).

[0364] A brain function monitoring system acquired video, a 2-channel electroencephalogram (EEG), and a 1-channel electrocardiogram (ECG). Glucose and dopamine were infused via the mammary vein, and propofol / alfentanil was infused via the great auricular vein. Arterial blood was collected via the umbilical artery for blood gas analysis. Rectal temperature was controlled by an overhead heater until the hypothermia protocol was initiated, with the Tecotherm Neo temperature control system used (top right image). Hypotherm was induced and maintained using the Tecotherm Neo temperature control system, and the animals were brought back to normal body temperature. Figure 11 and 12 ).

[0365] Two hours after anesthesia induction, hypoxic-ischemic (HI) injury was induced by reducing the fraction of inhaled oxygen (FiO2) to 4%. FiO2 was manipulated as needed (2%–10%) to maintain low-amplitude EEG (<5 μV) and induce hypotension (<30 mmHg) (Bjorkman et al., Brain Res. 2006 July 19;1100(1):110–7). After the HI period, FiO2 was restored to 21%, and piglets were kept under mild sedation.

[0366] Cell sorting According to Example 1 (Patel et al., Stem Cells Transl Med. 2013 Nov; 2(11): 839-47), the cells were freshly isolated directly from human full-term placenta (SLS).

[0367] Stem cell administration Two hours after injury, animals received a single dose of freshly isolated human placental stem cells (250,000-750,000 cells, 1:1 MSC:PROCR) suspended in 1 mL of phosphate-buffered saline (PBS, pH 7.4). + / - Animals in the excipient treatment group received an intravenous injection of 1 mL PBS. Animal treatment was randomized using a coin toss method, and researchers blinded the animals until the data analysis conclusions.

[0368] Hypothermia therapy (HTH) Following stem cell administration, whole-body warming therapy (HTH) was initiated immediately 2 hours post-injury using a servo-controlled cooling device (Tecotherm, Inspiration Healthcare, Leicester, UK). Animals were wrapped in pre-cooled mattresses, and their body temperature was lowered from the average piglet temperature (approximately 38.5°C) to 33.5°C. HTH was maintained for 24 hours (2–26 hours post-HI). Animals were then rewarmed at 0.5°C / hour for 10 hours (26–36 hours post-injury).

[0369] Post-injury recovery and monitoring Anesthesia was stopped / terminated after rewarming, weaning from ventilation, and extubation. After recovery from anesthesia, animals were housed in pairs and fed artificial pig milk (Woombaroo pig milk substitute, ProviCo, Australia) every 3-4 hours via oropharyngeal tube or bottle.

[0370] Neurobehavioral score After recovery from anesthesia, animals were assessed daily according to neurobehavioral criteria until the end of the experiment. Figure 15A-15J Neurobehavioral scoring criteria include assessment of breathing, consciousness, walking and limb control, general activity and presence of clinical seizures (Bjorkman et al., Neuroscience. 2010;166(1):157-67).

[0371] Magnetic resonance (MR) method At P8, piglets were anesthetized with isoflurane (1–3%) mixed with oxygen for the duration of the scanning protocol. After induction, the animals were placed in a prone position within the magnet aperture. Sagittal, coronal, and axial sections were obtained using a 150 mm volume head coil. Images were acquired at TR / TE 8500 / 60 ms, 1.6 mm section thickness, and a 64x64 acquisition matrix. Bilateral target regions above the frontal cortex were defined on T2 maps and applied to the apparent diffusion coefficient (ADC) map and the extracted raw values ​​(see Björkman et al., Neuroscience. 2010 / 03 / 10 / ;166(1):157–67, which is incorporated herein by reference in its entirety).

[0372] 1H-MR spectra were obtained on a 7T Bruker / Siemens whole-body scanner. Single spectra were obtained from 10 mm³ voxels in the frontoparietal region of the brain at P8 using single-voxel spectroscopy with the following parameters: TR = 6000 ms, TE = 60 ms, and 128 averages. Metabolite spectra were exported, processed, and analyzed using the AMARES tool within jMURI 149 (see Vanhamme et al., Journal of Magnetic Resonance. 1997 1997 / 11 / 01 / ;129(1):35-43, which is incorporated herein by reference in its entirety). The spectra were manually phased, with notches (10 Hz) applied to the spectra and Lorentz peaks fitted to regions corresponding to the peaks of N-acetylaspartate (NAA), creatine (Cr), choline (Cho), and lactate (lac). Calculate the peak area ratios of NAA / Lac, NAA / Cho, NAA / Cr, Lac / Cr, Lac / Cho, Lac / NAA, and Cho / Cr.

[0373] Euthanasia and Organ Treatment Following MRI / S, animals were given an overdose of sodium pentobarbital (325 mg / ml, 120 mg / kg) via intraperitoneal injection. The brain was perfused with 0.9% saline via the heart, removed, and coronally sectioned into 3 mm sections, then halved. The right hemisphere and brainstem were fixed overnight in 4% paraformaldehyde (PFA) / 0.1 M PBS (pH 7.4). Brain regions from the left hemisphere were isolated, flash-frozen in liquid nitrogen, and stored at -80°C. Before fixation in 2% PFA / 0.1% PBS (pH 7.4), the lungs, heart, liver, spleen, and kidneys were removed for weighing and gross pathological examination (see Björkman et al., Neuroscience. 2010 / 03 / 10 / ;166(1):157-67).

[0374] Sappanwood and Eosin Tissue sections were dewaxed in xylene and rehydrated with fractionating alcohol. The sections were stained with hematoxylin and eosin using an automated system (Leica ST5010 Autostainer XL, Leica Biosystems North Ryde, NSW, AUS). Histological damage on three slides spaced 48 μm apart for each brain section was examined and qualitatively described.

[0375] Immunomarkers Paraffin-embedded coronal sections were sectioned at 6 μm. Sections were dewaxed with xylene using an automated system (Leica ST5010 Autostainer XL, Leica Biosystems North Ryde, NSW, Australia) and rehydrated via a gradient of alcohols. Antigen retrieval was performed for 20 min at 90°C in a keratolytic chamber (Biocare Medical) with either 10 mM citrate buffer (pH 6) or TRIS-EDTA buffer (pH 9) before cooling to room temperature (RT). A hydrophobic barrier was drawn around the tissue, followed by nonspecific blocking for 1 h at room temperature with 5% donkey serum in PBS containing 0.5% Triton-X 100 and 0.05% Tween-20. Primary antibodies (C-Cas3, GFAP, Iba-1, IL-1β, NeuN, NFκB-p65, TNFα) were incubated according to the conditions in Table 2. Slides were washed in PBS and then incubated with species-specific secondary fluorophores at room temperature for 1 h. Sections were washed with PBS, counterstained with 4',6-diamidinyl-2-phenylindole (DAPI), and fixed with Prolong Gold antifade (Molecular Probes, Invitrogen Australia, Victoria, Australia). A Zeiss Axio microscope (Axioscope 5; Zeiss Microscopy, Australia) (equipped with a Plan-Apochromat 10x / 0.45 M27 objective (878.94 μm x 662.84 μm), an EC Plan-Neofluar 20x / 0.50 M27 objective (439.47 μm x 331.42 μm), or an EC Plan-Neofluar 40x / 0.75 M27 objective (219.74 μm x 165.71 μm)) was used to observe the stained and labeled sides. Photographs were taken using an Axiocam 503 camera (Zeiss Microscopy, Australia). A secondary antibody-only (negative) control was also run to exclude nonspecific binding (data not shown).

[0376] Table 2: Primary Antibodies

[0377] Fluoro-Jade C staining (FJC) Sections were dewaxed with xylene using an automated system (Leica ST5010 Autostainer XL, Leica Biosystems NorthRyde, NSW, Australia) and rehydrated with gradient ethanol. Degenerated neurons were observed using Fluoro-Jade C (FJC). Slides were transferred to FJC (Merck Millipore, Germany) solution dissolved in acetic acid medium containing DAPI to counterstain cell nuclei. FJC-positive cells were counted for each piglet and averaged before visualization.

[0378] Gene expression analysis Frozen brain tissue (frontal cortex and basal ganglia) was destroyed using needles and syringes. Total RNA was extracted and cDNA was synthesized using random hexamers.

[0379] Quantitative polymerase chain reaction (qPCR) was performed. The results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analyzed using the ΔΔCT method and expressed as logarithmic fold change (log(2)2^(ΔΔCT)) and 95% CI [upper limit, lower limit] Livak et al., Methods. Dec 2001;25(4):402-8).

[0380] Statistical methods Preliminary results using the same injury model resulted in an effect size of f=0.62 for the number of NeuN-positive cells, suggesting that 10 animals per group are required (efficacy 80%; p=0.05). In G Power analysis was performed in Power version 3.1.9.4. All analyses were completed using R software version 5.3.2. The Mantel-Cox log-rank test was used to examine differences in survival rates between the two groups. Animals that did not reach the P8 endpoint were excluded from further analysis. Initial data exploration was performed using the Shapiro-Wilks 223 normality test and the Levene test, assuming equal variances. No deviations from normal values ​​were observed, but occasional deviations from the isovariance assumption were observed between physiological EEG measurements and neurobehavioral measurements. The Welches T-test was used to compare group means among physiological measurements, EEG measurements, and neurobehavioral scores; multiple comparisons were controlled using the Holm-Sidak method. These data can be expressed as mean ± standard deviation (SD) because they can be parametric comparisons. Significant deviations from the normality and isovariance assumptions were observed in MRI, MRS, immunofluorescence, and qPCR data. Because this reduces the validity of parametric tests, these measurements were analyzed using the Mann-Whitney U test or the Kruskal-Wallis H test, along with the two-stage linearly increasing procedures of Benjamini, Krieger, and Yekutieli. Since these results may be nonparametric, they may be presented as medians [CI 95% lower limit, upper limit]. Statistical significance is accepted when q < 0.05 or p < 0.05 (if applicable). Graphs were generated using Graph Pad Prism 9 or IBM SPSS Statistics for Windows version 27.0.

[0381] Animal research group characteristics Twenty-three piglets were used in this study. Two male piglets were excluded prior to stem cell administration because they died from HI injury. Three other males in the HHV group were excluded because they died before the experimental endpoint. No animals died in the HHS group. Statistical analysis of survival outcomes did not reach statistical significance. Figure 13Therefore, for the final analysis, all exclusions were applied to the 4 animals (4 males) in the HHV group and the 7 animals (3 males, 4 females) in the HHS group. To meet the requirements for health biology comparisons, brains were obtained from five healthy piglets (2 males, 3 females) and removed at P8(C). No significant differences in P1 body weight were observed between the HHV and HHS groups. Significant differences were observed between the groups in body weight, brain weight, heart weight, or liver weight (Table 3). Post-hoc analysis showed that the control group was significantly different from the HHV group in all parameters. The control group also showed significant differences from the HHS group in P8 body weight, heart weight, and liver weight. The liver weight of the HHS group was significantly higher than that of the HHV group. No other significant differences were observed between the HHV and HHS groups. Measurements of heart rate and blood pressure (Table 4), as well as arterial blood pH, lactate concentration (lac), arterial alkali excess (ABE), HCO3 concentration, and glucose (Glu) concentration were also significantly different. Figure 14 No significant differences were observed at any time point. In all animals, the aEEG score decreased to 0 after HI. Two hours after HI, there was no significant difference between the group means and baseline. There were no differences in aEEG scores between groups at any time point. At 0 hours (adjusted p=0.00045) and 1 hour (adjusted p=0.012) after HI, EEG background was significantly reduced in hypothermic and stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treated animals and hypothermic and stem cell vector treated animals (HHV), but not at any other time point.

[0382] Table 3: Tissue Weight

[0383] Table 4: Heart Rate and Blood Pressure Indicators

[0384] EEG results after HI injury HI injury resulted in a decrease in background amplitude from baseline of 9.00 μV ± 0.70 μV in the HHV group and 10.76 μV ± 1.81 μV in the HHS group to 1.37 μV ± 0.20 μV and 1.42 μV ± 0.36 μV, respectively. No significant difference in background amplitude was observed at any time point. One electrographically recorded seizure lasting 12 seconds was observed in the HHS group. Three animals in the HHV group had electrographically recorded seizures, ranging from 2 to 22, with a mean seizure load of 00:07:14, ranging from 11 to 22 minutes. A burst suppression pattern following injury was observed in two of the three piglets that experienced seizures in the HHV group, but not in the HHS group.

[0385] MRI and MRS results after HI injury MRI scans were collected from 11 piglets. Due to motion artifacts, one piglet from each group was excluded, leaving three piglets in the HHV group and six piglets in the HHS group. Figure 15A Since the number of groups in the HHV group was low and the true outlier was 269, the Mann-Whitney U test was used. The mean T2 relaxation time 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]) was (Mann-Whitney U = 8, n1 = 4, n2 = 6, p = 0.47). Figure 15B ) or ADC value (Mann-Whitney U=4, n1=4, n2=6, p=0.11; see Figure 15C There were no significant differences. The MR spectra of 8 piglets were measured and analyzed, including 3 from the HHV group and 5 from the HHS group. Figure 15D Due to the low number of groups and large true outliers in the HHV group, the Welch T test was not suitable, and the Mann-Whitney U test was more appropriate. Significant differences were observed in the 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]). Figure 15E No significant differences were found in the ratios for any other measurement. Figure 15F-15I A significant difference in the NAA / Cho ratio was observed only between the hypothermia-stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treatment group (HHS) and the hypothermia-stem cell vector treatment group (HHV). The hypothermia-stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC) treatment group had significantly higher neurobehavioral scores at P4 and P5, but not at any subsequent time points. A full description of the results is shown in Table 5.

[0386] Table 5: Experimental Results

[0387] Post-HI functional recovery and improvement At P3, neurobehavioral scores decreased from 22±1 in the pre-HI assessment in the HHV and HHS groups to 6±8 and 17±4, respectively (see [reference]). Figure 15JThe HHS group had significantly higher neurobehavioral scores at 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 points.

[0388] Histological neuropathology after stem cell administration reduces HI Trends and differences in EEG, MRI, and neurobehavioral scores were reflected in brain tissue pathology. Age-matched controls were used for biological comparisons of healthy individuals (C). No instances of HI-related neuropathology were observed in the control group (C). Figure 16 A and 16A'). Animals in the HHV group had multiple cases of significant tissue damage in all examined brain regions. Diffusely coagulated ischemic neurons were observed in the frontal cortex, which were absent 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 (A and 16A'). Figure 16 The neuropathology in this area becomes more severe in deeper regions of the gyri. This area did not show neuropathology in the HHS group. Figure 16 C and 16C').

[0389] Stem cell administration reduced neuropathological indicators after HI. Trends and differences in MRI / S and neurobehavioral scores were reflected in brain immunofluorescence analysis, which examined mature neurons with NeuN ( Figure 17AThe number of degenerated neurons with FJC (Fig. 17F-17H) and apoptotic cells labeled with cleaved caspase 3 (Fig. 17K-17JM) was assessed. The frontal cortex (FC) (Fig. 17D, 17I, 17N) and putamen (OUT) (Fig. 17E, 17J, 17O) were assessed. Exploratory analysis of these immunofluorescence data revealed significant biases to normality and sphericity that could not be corrected by logarithmic or Box-Cox transformations. Therefore, the Kruskal-Wallis test was used to analyze the data. A significant difference in the number of NeuN-positive cells was observed in the frontal cortex between the two groups, H(2) = 11.25, p = 0.0002. Post-hoc comparisons showed that the median NeuN number in the control group (979 [893, 1021]) was significantly higher than that in the HHV group (703 [-53, 1163]; C vs. HHV, q = 0.010) and the HHS group (849 [788, 874]; C vs. HHV, q = 0.0152). Similarly, a significant difference was found between the HHV and HHS groups (HHV vs. HHS, q = 0.0471). No significant difference was observed in the putamen (H2) = 2.757, p = 0.26. FJC staining analysis revealed a significant difference in the number of degenerated neurons in the frontal cortex (H2) = 6.285, p = 0.041. Post-hoc analysis showed no significant differences 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, q = 0.057; C 307 vs. HHS, q = 0.574). Furthermore, no significant differences were observed between the HHV and HHS groups (HHV vs. HHS, q = 0.84). No significant differences were observed in the putamen (H(2) = 309 3.359, p = 0.1753). No significant differences were observed in the number of apoptotic cells (cleaved casp-3) in the frontal cortex (H(2) = 0.9274, p = 0.656) or in the putamen (H(2) = 2.70, p = 0.2774). Colocalization studies showed that in all experimental groups, lysed caspase-3 positive cells mainly colocalized with astrocytes (Fig. 17P-17R).

[0390] Hypothermic and stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC)-treated (HHS) brains showed similar levels of mature neurons throughout the frontal cortex, while HHV consistently showed fewer neurons. Significantly fewer NeuN-positive cells were observed in the HHV group. This reduction occurred in the frontal cortex of the hypothermic and stem cell (PROCR+ / - PDGFRA+ / - EPC and MSC)-treated (HHS) group, but not in the putamen. FJC staining was not observed in the frontal cortex of the control group.

[0391] Stem cell therapy is associated with changes in glial cell activation. Increased neuropathological findings may be related to glial cell activation induced by localized neuroinflammation; therefore, the morphology of microglia and astrocytes was examined. Iba-1-positive microglia in the control brain showed a stellate morphology, with light cell bodies indicating a resting state and finely extended processes. Figure 18A and 18A In contrast, many Iba-1 positive microglia in the HHV brain resemble the morphology of activated microglia, with darker cell bodies and thickened retracted processes. Figure 18B and 18B Conversely, microglia in the HHS brain were similar to those in the control group. Figure 18C , 18C ').

[0392] Quantitative analysis of the number of resting and activated microglia revealed that in the frontal cortex, Hresting(2) = 12.21, p < 0.0001; Hactivated(2) = 10.27, p < 0.0013. Figure 18DSignificant differences were found in the resting (2) values ​​of H1F, 18E and H2H (2) = 10.33, p = 0.0013, and H2H activation (2) = 10.64, p = 0.0009 (Fig. 18F, 18H, 18I). The number of resting microglia in the control group (237 [218, 249]) was significantly higher than that in the HHV group (95 [6, 151]; C vs. HHV, q rest = 0.0013) and the HHS group (140 [114, 156]; C vs. HHS, q rest = 0.038). Based on the observations, the number of activated microglia in the control group (40 [32, 56]) was significantly lower than that in the HHV group (230 [77, 474]; C, HHV, q = 0.004) and the HHS group (222 [162, 241]; C vs. HHS, q = 0.179). Similarly, in the putamen, post-hoc analysis of microglia morphology showed that the number of resting microglia in the control group (234 [188, 252]; C vs. HHV, q = 0.004) was significantly higher than that in the HHV group (114 [-5.9, 207]; C vs. HHV, q = 0.004) and the HHS group (138 [115, 152]; C vs. HHS, q = 332 0.0043). No significant difference in the number of resting microglia was observed between the HHV and HHS groups (HHV vs. HHS, q = 0.27). Post-hoc analysis showed that the number of activated microglia in the control group (47 [39, 55]) was significantly less than that in the 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 a significant difference between the total number of HFCs in the frontal cortex (2) = 12.11, p < 0.0001 and the total number of HPUTs in the putamen (2) = 8.153, p = 0.0083. Post-hoc analysis showed that the HI-damaged group (HHV, [330, 503]; HHS, 328 [314, 371]) had significantly more microglia than the control group (276 [263, 288]; C vs. HHV, q = 0.0011; C vs. HHS, q = 0.039), and there was no significant difference between the HHV and HHS groups (HHV vs. HHS, q = 0.055) (Fig. 18F). A similar relationship was observed in the putamen, where the control group (282 [238, 295]) had significantly fewer microglia than the HHV group (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 HHV and HHS (HHV vs. HHS, q = 0.18; see Figure 18J).

[0393] GFAP-positive astrocytes were observed throughout the white matter region. In C and HHS brains, GFAP-positive cells exhibited the typical morphology of normal astrocytes, characterized by multiple long branching processes from the cell body (Fig. 18K', 18M'). In HHV brains, many GFAP-positive astrocytes showed a reactive morphology with retracted processes and large cell bodies. Figure 20 L'). Astrocytes in HHV white matter exhibited the morphological characteristics of activated astrocytes, with enlarged cell bodies and short, thickened processes. Differences were observed between groups in astrocyte coverage levels. These qualitative descriptions were quantitatively captured by measuring the mean astrocyte coverage in each region. A significant difference in GFAP coverage was observed between the HHV and HHS groups (HHV vs. HHS, q = 0.045). No significant difference in GFAP coverage was observed in IGWM, H(2) = 2.586, p = 0.116 (Fig. 18N). A significant difference in GFAP coverage was observed in PVWM, H(2) = 6.659, p = 0.01 (Fig. 18O). Post-hoc analysis showed that the control group (17.25 [15.62, 19.53]) was significantly different from the HHV group (13.84 [11.93, 16.33]), but not significantly different from the HHS group (15.88 [14.90, 17.30]) (C vs. HHV, q = 0.006; C vs. HHS, q = 0.0683). Figure 18A -18O).

[0394] Analysis of pro-inflammatory cytokine mRNA expression For all biomarkers except CCR5, there were significant differences in the expression of several inflammatory biomarkers in the frontal cortex between the experimental groups. Figure 19 Post-hoc analysis revealed no significant differences between HHV and HHS for any inflammatory markers (see Table 6). To identify the cellular origin of pro-inflammatory cytokines, co-localization studies were performed using antibodies against TNFα, IL-1β, and NF-κB with Iba-1 and GFAP. In both the HHV and HHS groups, TNFα and IL-1β co-localized with Iba-1. Figure 20 NF-κB co-localizes with Iba-1 in the HHV group, but not in the HHS group. Figure 21 ).like Figure 21As shown in the image above, NF-κB is preferentially expressed in microglia of the HHV group in the frontal cortex (triangles). HHS-treated animals primarily express activated NF-κB in neurons (small-tailed arrows). High-magnification image scale bar = 50 μm. Lower NF-κB expression is generally higher in the deep cortical regions of the HHV group (white arrow area) than in the HHS group (right). The primary source of NF-κB in HHS is neurons.

[0395] Compared with the control group, hypoxic-ischemic injury was associated with a significant increase in the transcription of inflammatory markers. No significant differences in any inflammatory markers were observed between the HS group and the HHV group.

[0396] Table 6: Inflammatory Markers

[0397] Stem cell therapy restores reduced vascular coverage Vascular coverage was measured to provide an indication of vascular protection. Significant differences in vascular coverage were found in FC [χ²=8.250, p=0.0073] and IGWM [χ²=10.89, p=0.0002] (6.21). Post-hoc analysis in FC showed that CD34 coverage in HHV (2.356 [2.142, 2.582]) was significantly lower than that in C (2.881 [2.551, 9.974]) (C vs. HHV; q=0.0065). CD34 coverage in HHS brain (2.688 [2.563, 2.947]) was significantly higher than that in HHV brain (q=0.0065). No significant difference was observed between C and HHS (q=0.35). Post-hoc analysis by IGWM showed that CD34 coverage in the HHV group (1.922 [1.149, 2.731]) and the HHS group (2.695 [2.031, 3.169]) was significantly lower than that in the C group (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). Figure 22 ).like Figure 22 As shown, quantitative analysis of CD34 coverage revealed that the HHS group had significantly higher coverage in the frontal cortex than the HHV group, with no significant difference compared to C. Comparison of white matter within the gyri showed that the control group had significantly higher coverage than both the HHV and HHS groups. There was no significant difference between HHV and HHS. Values ​​are shown as median [lower limit, upper limit].

[0398] Example 7: Human placental cells Human placental tissue was obtained from healthy women undergoing cesarean section at full term (38–39 weeks of gestation), allowing for the isolation and use of any stem cell population derived from the placental tissue. PROCR+ cells were isolated from each sample, producing single-cell suspensions. Isolation was performed as described below: see Shafiee, A. et al. Meso-endothelial bipotent progenitors from human placenta display distinct molecular and cellular identity. Stem cellreports, 2018. 10(3): p. 890–904, which is incorporated herein by reference in its entirety.

[0399] PROCR of the separated placenta + Single-cell suspensions were incubated with human PDGFRA antibody. Cells were sorted using FACS flow cytometry, cell duplexes were removed, and dead cells were excluded using 7AAD. An FMO control was used to gate the target population. Cells were then gated and sorted using PDGFRA+ / - gates. Figure 23 Cell selection was based on the CD45-CD34+CD31Int phenotype.

[0400] Colony formation in ECFCs was assessed using FACS. Gene expression in PROCR+ ECFCs was measured compared to MSCs and PROCR+ cells, specifically CD31, CD34, VE-cadherin, and PDGFRA expression in control ECFCs (ECFC-KK and ECFC-MG). Figure 24 PROCR+ ECFCs and an equal volume of FPL-MSCs were co-cultured in endothelial growth medium for 5 days, and live and dead cells were assessed. Figure 25 The control ECFC was isolated into a CD45-CD34+CD31Int cell population.

[0401] According to Shafiee, A. et al., Priming of endothelial colony-forming cells in amesenchymal niche improves engraftment and vasculogenic potential by initiating mesenchymal transition orchestrated by NOTCH signaling, The FASEB Journal, 2017. 31(2): p. 610-624 (which is incorporated herein by reference in its entirety). fPL-MSCs and PROCR+ ECFCs were co-cultured at a 1:1 ratio (5 × 10⁵ cells: 5 × 10⁵ cells) in collagen-coated flasks in endothelial growth medium (EGM2) at a final concentration of 10 × 10⁵ cells. In the control group, ECFCs and fPL-MSCs were cultured separately in collagen-coated flasks in EGM2 (final concentration 5 × 10⁵ cells). Cultures were maintained in EGM2 during the experiment and the medium was changed after 3 days.

[0402] After 5 days of co-culture (or monoculture in the control group), cells were isolated using a dissociation reagent, washed, resuspended in FACS buffer, and transferred to 2 ml microtubes. 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 minutes, 1 ml of FACS buffer was added to each microtube, and cells were centrifuged at 400 × g for 4 minutes and resuspended in 100 μl of ice-cold FACS buffer. Cells were filtered through a 40 μm cell filter to reduce cell aggregates before running the samples through the FACS machine (BD Biosciences, USA). CD31+ cells from PROCR+ ECFCs co-cultured with fPL-MSCs were FACS sorted according to the PDGFRA+ / - gate.

[0403] The results showed a significant change in cell population between the ECFC-only group and the induced ECFC group. In the ECFC-only group, there were 49,831 CD31+CD90- cells and 2,424 CD31+CD90+ cells. Conversely, the induced ECFC group showed 19,066 CD31+CD90- cells and 8,146 CD31+CD90+ cells. Notably, only 4.6% of the CD31+ cells in the ECFC-only group were CD90+, while a significant 30% of the CD31+ cells in the induced ECFC group were CD90+. These findings suggest that induced ECFC significantly increases the proportion of CD31+CD90+ cells. The expression of this mesenchymal marker is an indicator of cell fluidity and potential for better angiogenesis. Induced ECFC significantly increased the proportion of CD31+CD90+ cells. MSCs and ECFCs were cultured alone or co-cultured (induced ECFC) for 5 days (…). Figure 26 ).

[0404] Additional Implementation Plan This technology includes, but is not limited to, the following specific implementation schemes described in paragraphs

[0400] -

[0514] : 1. A pharmaceutical composition comprising or consisting of: isolated endothelial progenitor cell (EPC) populations, said isolated endothelial progenitor cell (EPC) populations comprising PROCR+PDGFRA+EPC.

[0405] 2. A pharmaceutical composition comprising or consisting of: isolated EPC groups, said isolated EPC groups comprising PROCR+ / - PDGFRA+ / - EPC, said pharmaceutical composition for use in treating neonatal hypoxic-ischemic encephalopathy (HIE) in a subject of need.

[0406] 3. A pharmaceutical composition comprising or consisting of: a group of isolated EPCs, said isolated EPCs comprising PROCR+ / - PDGFRA+ / - EPCs.

[0407] 4. A pharmaceutical composition comprising or consisting of: isolated EPC clusters, said isolated EPC clusters comprising PROCR+ / - PDGFRA+ / - EPC, said pharmaceutical composition for use in treating brain injury in a subject of need.

[0408] 5. The pharmaceutical composition according to embodiment 1, wherein, relative to the expression levels in the unseparated EPC group, the separated EPC group comprises an increase in the expression levels of PROCR protein, PDGFRA protein, or VE-cadherin protein.

[0409] 6. The pharmaceutical composition according to embodiment 5, wherein the increase in the PROCR protein expression level relative to the PROCR protein expression level in the unseparated EPC 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%.

[0410] 7. The pharmaceutical composition according to embodiment 5, wherein the increase in the PDGFRA protein expression level relative to the PDGFRA protein expression level in the unseparated EPC 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%.

[0411] 8. The pharmaceutical composition according to any one of embodiments 1-7, wherein, relative to the expression levels in the unseparated EPC group, the separated EPC group comprises an increase in the expression levels of the PROCR gene, the PDGFRA gene, or the VE-cadherin gene.

[0412] 9. The pharmaceutical composition according to embodiment 8, wherein the increase in the PROCR gene expression level relative to the PROCR gene expression level in the unseparated EPC 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%.

[0413] 10. The pharmaceutical composition according to embodiment 8, wherein the increase in the PDGFRA gene expression level relative to the PDGFRA gene expression level in the unseparated EPC 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%.

[0414] 11. The pharmaceutical composition according to embodiment 8, wherein the increase in the VE-cadherin gene expression level relative to the VE-cadherin gene expression level in the unseparated EPC 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%.

[0415] 12. The pharmaceutical composition according to any one of embodiments 1-11, wherein the isolated EPC group contains increased proliferative capacity relative to the unisolated EPC group.

[0416] 13. The pharmaceutical composition according to any one of embodiments 1-12, wherein the isolated EPC group contains increased angiogenic capacity relative to the unisolated EPC group.

[0417] 14. The pharmaceutical composition according to embodiment 13, wherein the increase in angiogenesis capacity relative to the level of angiogenesis capacity in the unseparated EPC 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%.

[0418] 15. The pharmaceutical composition according to any one of embodiments 1-14, wherein the separated EPC group comprises an increased colony-forming ability or tube-forming ability relative to the unseparated EPC group.

[0419] 16. The pharmaceutical composition according to embodiment 15, wherein the increase in colony-forming ability relative to the level of colony-forming ability in the unseparated EPC 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%.

[0420] 17. The pharmaceutical composition according to embodiment 15, wherein the increase in tube-forming ability relative to the tube-forming ability level in the unseparated EPC 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%.

[0421] 18. The pharmaceutical composition according to any one of embodiments 1-17, wherein the separated EPC group contains increased implantation potential relative to the unseparated EPC group.

[0422] 19. The pharmaceutical composition according to embodiment 18, wherein the increase in implantation potential relative to the implantation potential level in the unseparated EPC 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%.

[0423] 20. The pharmaceutical composition according to any one of embodiments 1-19, wherein, relative to one or more EPCs in the unseparated EPC group, the separated EPC group comprises one or more EPCs having a more elongated cell shape.

[0424] 21. The pharmaceutical composition according to embodiment 20, wherein the one or more EPCs are elongated by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75% compared to the unseparated EPC group.

[0425] 22. The pharmaceutical composition according to any one of embodiments 1-21, wherein the isolated EPC group comprises the CD45- / CD34+ phenotype.

[0426] 23. The pharmaceutical composition according to any one of embodiments 3-22, wherein the pharmaceutical composition is a first composition, the first composition being formulated for administration before, during or after administration to a subject in need of a second composition comprising an isolated population of mesenchymal stem cells (MSCs).

[0427] 24. A pharmaceutical composition comprising... (a) The first isolated cell population, containing PROCR+ PDGFRA+ EPC; and (b) The second isolated cell population, which contains MSCs.

[0428] 25. A pharmaceutical composition comprising... (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains MSCs.

[0429] 26. A pharmaceutical composition for use in treating HIE in a subject of need, said pharmaceutical composition comprising... (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains MSCs.

[0430] 27. The pharmaceutical composition according to embodiment 26, wherein the HIE is neonatal HIE.

[0431] 28. A pharmaceutical composition for use in treating brain injury in a subject of need, said pharmaceutical composition comprising... (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains MSCs.

[0432] 29. The pharmaceutical composition according to embodiment 28, wherein the brain injury comprises neurodegeneration.

[0433] 30. The pharmaceutical composition according to any one of embodiments 1-29, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0434] 31. The pharmaceutical composition according to embodiment 30, wherein the pharmaceutically acceptable medium is phosphate-buffered saline.

[0435] 32. The pharmaceutical composition according to any one of embodiments 3-31, wherein the pharmaceutical composition is formulated for intranasal delivery, intrathecal delivery, intra-arterial delivery, intralesional delivery or intravenous delivery to a subject in need.

[0436] 33. The pharmaceutical composition according to embodiment 28, wherein the brain injury comprises ischemic brain injury.

[0437] 34. The pharmaceutical composition according to any one of embodiments 26-33, wherein the subject has received or is receiving therapeutic hypothermia treatment.

[0438] 35. A method of treating HIE in a subject in need, the method comprising administering to the subject a pharmaceutical composition comprising: (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains MSCs.

[0439] 36. The method according to embodiment 35, wherein the HIE is neonatal HIE.

[0440] 37. A method of treating brain injury in a subject in need, the method comprising administering to the subject a pharmaceutical composition comprising: (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains MSCs.

[0441] 38. The method according to embodiment 37, wherein the brain injury includes neurodegeneration.

[0442] 39. The method according to embodiment 37, wherein the brain injury includes ischemic brain injury.

[0443] 40. A composition for use in the treatment of neonatal HIE, said composition comprising a mixture of two isolated cell populations, wherein the first isolated cell population consists of PROCR+ / - PDGFRA+ / - EPC and the second isolated cell population consists of MSCs.

[0444] 41. A method for treating neonatal HIE in a subject in need, the method comprising administering to the subject a cell composition comprising a mixture of two separate cell populations, wherein the first separate cell population consists of PROCR+ / -PDGFRA+ / - EPC and the second separate cell population consists of MSCs.

[0445] 42. The method according to any one of embodiments 35-41, wherein the subject has received or is receiving therapeutic hypothermia treatment.

[0446] 43. A pharmaceutical composition comprising a cell composition and a pharmaceutically acceptable mediator for use in the treatment of HIE, wherein the cell composition comprises a mixture of two separate cell populations, wherein a first separate cell population consists of mammalian PROCR+ / -PDGFRA+ / -PROCR+ / - PDGFRA+ / - EPCs, and a second separate cell population consists of MSCs.

[0447] 44. The pharmaceutical composition according to embodiment 43, wherein the pharmaceutically acceptable medium is phosphate-buffered saline.

[0448] 45. The isolated cell population, cell composition, pharmaceutical composition, or cell composition for use according to any one of embodiments 26, 27, 35, 36, or 41, wherein use in the treatment of HIE or the treatment of neonatal HIE comprises providing therapeutic hypothermia treatment to a subject in need, and further 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.

[0449] 46. ​​The pharmaceutical composition, method, or cell composition according to 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.

[0450] 47. The pharmaceutical composition, method, or cell composition according to 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.

[0451] 48. The pharmaceutical composition, method, or cell composition according to any one of embodiments 24-47, wherein the MSC is a CD45- / CD34+ cell.

[0452] 49. The pharmaceutical composition, method, or cell composition according to 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 are CD34+ / CD45- isolated cell populations.

[0453] 50. A pharmaceutical composition, method, or cell composition according to any one of embodiments 24-49, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated cell population or the second isolated cell population expresses PROCR and PDGFRA.

[0454] 51. An isolated population of EPC cells comprising PROCR+PDGFRA+EPC, said isolated population of EPC cells being generated by the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) Select cells expressing the PROCR+ and PDGFRA+ phenotypes from the CD45- / CD34+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

[0455] 52. An isolated population of EPC cells comprising PROCR+ / - PDGFRA+ / - EPC cells, said isolated population of EPC cells being generated by the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) Select cells expressing the PROCR+ / - and PDGFRA+ / - phenotypes from the CD45- / CD34+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

[0456] 53. The separated EPC group according to embodiment 51 or 52, wherein step (ii) includes selecting the expression CD45. + Cells with the phenotype removed from the biological sample of step (i) expressing CD45. + Cells with the specified phenotype were discarded, and those expressing CD45 were removed. + Cells with this phenotype acquire CD45. -Cell population.

[0457] 54. The separated EPC group according to embodiment 53, wherein the expression CD45 is selected. + The steps of phenotyped cells include contacting one or more cells expressing the CD45 surface protein with CD45 binding molecules to form a complex, and removing the complex from the biological sample in step (i).

[0458] 55. An isolated EPC cluster according to any one of embodiments 51-54, wherein step (iii) comprises contacting one or more cells expressing CD34 surface protein with CD34 binding molecules to form a complex from the CD45 in step (ii). - The complex is removed from the cell population, while the complex is retained, thereby obtaining a CD45- / CD34+ cell population.

[0459] 56. An isolated EPC population according to any one of embodiments 51-55, wherein step (iv) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0460] 57. A method for separating an EPC group containing PROCR+PDGFRA+EPC, the method comprising the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) Select cells expressing the PROCR+ and PDGFRA+ phenotypes from the CD45- / CD34+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

[0461] 58. A method for separating an EPC group containing PROCR+ / - PDGFRA+ / - EPCs, the method comprising the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) Select cells expressing the PROCR+ / - and PDGFRA+ / - phenotypes from the CD45- / CD34+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

[0462] 59. The method according to embodiment 57 or 58, wherein step (ii) includes selecting an expression CD45. + Cells with the phenotype removed from the biological sample of step (i) expressing CD45. + Cells with the specified phenotype were discarded, and those expressing CD45 were removed. + Cells with this phenotype acquire CD45. - Cell population.

[0463] 60. The method according to embodiment 59, wherein the expression CD45 is selected. + The steps of phenotyped cells include contacting one or more cells expressing the CD45 surface protein with CD45 binding molecules to form a complex, and removing the complex from the biological sample in step (i).

[0464] 61. The method according to any one of embodiments 57-60, wherein step (iii) comprises contacting one or more cells expressing CD34 surface protein with CD34 binding molecules to form a complex from the CD45 in step (ii). - The complex is removed from the cell population, while the complex is retained, thereby obtaining a CD45- / CD34+ cell population.

[0465] 62. The method according to any one of embodiments 57-61, wherein step (iv) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0466] 63. A segregated population of EPCs comprising or consisting of: PROCR + PDGFRA + EPCs, said segregated population of cells being generated by the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching cells containing the PDGFRA+ phenotype in the biological sample to obtain a PDGFRA+ cell population; (iii) Select cells expressing the PROCR+ phenotype from the PDGFRA+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

[0467] 64. A separate EPC group comprising or consisting of: PROCR+ / - PDGFRA+ / - EPC, said separate EPC group being generated by the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PDGFRA+ / - phenotype to obtain a PDGFRA+ / - cell population; (iii) Select cells expressing the PROCR+ / - phenotype from the PDGFRA+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

[0468] 65. The isolated EPC population according to embodiment 63 or 64, wherein step (iii) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0469] 66. A method for separating an EPC group, the EPC group comprising or consisting of the following: PROCR + PDGFRA + EPC, the method comprising the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching cells containing the PDGFRA+ phenotype in the biological sample to obtain a PDGFRA+ cell population; (iii) Select cells expressing the PROCR+ phenotype from the PDGFRA+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

[0470] 67. A method for separating an EPC group, the EPC group comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC, the method comprising the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PDGFRA+ / - phenotype to obtain a PDGFRA+ / - cell population; (iii) Select cells expressing the PROCR+ / - phenotype from the PDGFRA+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

[0471] 68. The method according to embodiment 66 or 67, wherein step (ii) comprises contacting one or more cells expressing the PDGFRA surface protein with a PDGFRA binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0472] 69. The method according to any one of embodiments 66-68, wherein step (iii) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0473] 70. A separate EPC group comprising or consisting of: PROCR + PDGFRA + EPC, said separate EPC group being generated by the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PROCR+ phenotype to obtain a PROCR+ cell population; (iii) Select cells expressing the PDGFRA+ phenotype from the PROCR+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

[0474] 71. A separate EPC group comprising or consisting of: PROCR+ / - PDGFRA+ / - EPC, said separate EPC group being generated by the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample with cells containing the PROCR+ / - phenotype to obtain a PROCR+ / - cell population; (iii) Select cells expressing the PDGFRA+ / - phenotype from the PROCR+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

[0475] 72. The isolated EPC population according to embodiment 70 or 71, wherein step (ii) includes contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0476] 73. An isolated EPC population according to any one of embodiments 70-72, wherein step (iii) comprises contacting one or more cells expressing the PDGFRA surface protein with PDGFRA binding molecules to form a complex, removing the complex from the cell population and retaining the complex.

[0477] 74. A method for separating an EPC group, the EPC group comprising or consisting of the following: PROCR + PDGFRA + EPC, the method comprising the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PROCR+ phenotype to obtain a PROCR+ cell population; (iii) Select cells expressing the PDGFRA+ phenotype from the PROCR+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

[0478] 75. A method for separating an EPC group, the EPC group comprising or consisting of the following: PROCR+ / -PDGFRA+ / - EPC, the method comprising the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample with cells containing the PROCR+ / - phenotype to obtain a PROCR+ / - cell population; (iii) Select cells expressing the PDGFRA+ / - phenotype from the PROCR+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

[0479] 76. The method according to embodiment 74 or 75, wherein step (ii) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0480] 77. The method according to any one of embodiments 74-76, wherein step (iii) comprises contacting one or more cells expressing the PDGFRA surface protein with a PDGFRA binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

[0481] 78. The isolated EPC group or method according to any one of embodiments 54-56, 60-62, 65, 68, 69, 72, 73, 76 or 77, wherein the binding molecule comprises a protein.

[0482] 79. The isolated EPC group or method according to embodiment 78, wherein the protein comprises an antibody.

[0483] 80. The isolated EPC group or method according to any one of embodiments 54-56, 60-62, 65, 68, 69, 72, 73, 76 or 77-79, wherein the removal of the complex comprises microfluidic sorting.

[0484] 81. The isolated EPC group or method according to embodiment 80, wherein the microfluidic sorting includes microbead sorting or flow cytometry.

[0485] 82. The isolated EPC cluster or method according to embodiment 81, wherein the flow cytometry includes fluorescence-activated cell sorting.

[0486] 83. The isolated EPC population or method according to any one of embodiments 51-82, wherein the step further includes step (iv) of culturing or contacting the isolated EPC population with a cell population containing endothelial colony-forming cells (ECFCs) or a cell population containing MSCs.

[0487] 84. The isolated EPC population or method according to embodiment 83, wherein the step further includes step (v), separating the isolated EPC population from the cell population containing ECFCs or the cell population containing MSCs.

[0488] 85. The separated EPC group or method according to 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).

[0489] 86. The isolated EPC group or method according to any one of embodiments 51-85, wherein the biological sample is a mammalian biological sample.

[0490] 87. The isolated EPC group or method according to embodiment 86, wherein the mammalian biological sample is selected from the group consisting of mammalian placenta, mammalian umbilical cord blood, mammalian peripheral blood, and mammalian tissue-resident vascular endothelium.

[0491] 88. The isolated EPC group or method according to embodiment 87, wherein the mammalian placenta is an intact mammalian placenta.

[0492] 89. The isolated EPC group or method according to embodiment 87, wherein the vascular endothelium residing in the mammalian tissue is selected from the group consisting of: mammalian umbilical cord, mammalian pulmonary artery endothelium, mammalian aorta, and mammalian lung tissue.

[0493] 90. The isolated EPC group or method according to any one of embodiments 51-89, wherein the isolated EPC group comprises an increase in the expression level of PROCR protein, PDGFRA protein, or VE-cadherin protein, relative to the expression level in the unisolated EPC group.

[0494] 91. The isolated EPC population or method according to any one of embodiments 51-90, wherein the isolated EPC population contains increased proliferation capacity relative to the unisolated EPC population.

[0495] 92. The isolated EPC cluster or method according to any one of embodiments 51-91, wherein the isolated EPC cluster contains increased angiogenic capacity relative to the unisolated EPC cluster.

[0496] 93. A separated EPC cluster or method according to any one of embodiments 51-92, wherein the separated EPC cluster includes an increase in cluster forming capability or tube forming capability relative to the unseparated EPC cluster.

[0497] 94. The separated EPC cluster or method according to any one of embodiments 51-93, wherein the separated EPC cluster contains increased implantation potential relative to the unseparated EPC cluster.

[0498] 95. The isolated EPC group or method according to any one of embodiments 51-94, wherein the isolated EPC group comprises one or more EPCs having a more elongated cell shape relative to one or more EPCs in the unisolated EPC group.

[0499] 96. The separated EPC group or method according to embodiment 95, wherein the one or more EPCs are elongated by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, or 75%.

[0500] 97. A pharmaceutical composition, method, isolated EPC group or cell composition according to any one of embodiments 1-96, wherein the PROCR+ PDGFRA+ EPC or the PROCR+ / - PDGFRA+ / - EPC expresses one or more proteins selected from the group consisting of: CD32, CDH5, CD34, CD31, VEGFR2, VE-cadherin and CD157.

[0501] 98. The pharmaceutical composition, method, isolated EPC group or cell composition according to any one of embodiments 1-97, wherein the PROCR+PDGFRA+EPC or the PROCR+PDGFRA+EPC does not express one or more hematopoietic proteins.

[0502] 99. The pharmaceutical composition, method, isolated EPC group or cell composition according to embodiment 98, wherein one or more hematopoietic proteins are selected from the group consisting of: CD3e, CD11b, CD45 and B220.

[0503] 100. The pharmaceutical composition, method, isolated EPC population or cell composition according to any one of embodiments 1-99, wherein, relative to undifferentiated PROCR+ PDGFRA+ EPC or undifferentiated PROCR+ / - PDGFRA+ / - EPC, the PROCR+ PDGFRA+ EPC or the PROCR / -+ PDGFRA+ / - EPC reduces the expression level of one or more genes of CD157, ABCG2 or SOX18 upon differentiation.

[0504] 101. The pharmaceutical composition, method, isolated EPC population, or cell composition according to embodiment 100, wherein the reduction in CD157 gene expression level relative to the CD157 gene expression level in the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0505] 102. The pharmaceutical composition, method, isolated EPC population or cell composition according to embodiment 100, wherein the reduction in the gene expression level of ABCG2 relative to the gene expression level of the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0506] 103. The pharmaceutical composition, method, isolated EPC population, or cell composition according to embodiment 100, wherein the reduction in the gene expression level of SOX18 relative to the gene expression level of the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0507] 104. The pharmaceutical composition, method, isolated EPC population or cell composition according to any one of embodiments 1-99, wherein, relative to undifferentiated PROCR+ PDGFRA+ EPC or undifferentiated PROCR+ / - PDGFRA+ / - EPC, the PROCR+ PDGFRA+ EPC or the PROCR / -+ PDGFRA+ / - EPC reduces the expression level of one or more proteins of CD157, ABCG2 or SOX18 upon differentiation.

[0508] 105. The pharmaceutical composition, method, isolated EPC population, or cell composition according to embodiment 104, wherein the reduction in CD157 protein expression level relative to the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0509] 106. The pharmaceutical composition, method, isolated EPC population, or cell composition according to embodiment 104, wherein the reduction in the protein expression level of ABCG2 relative to the protein expression level of the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0510] 107. The pharmaceutical composition, method, isolated EPC population or cell composition according to embodiment 100, wherein the reduction in the protein expression level of SOX18 relative to the protein expression level of the undifferentiated PROCR+ PDGFRA+ EPC or the undifferentiated PROCR+ / - PDGFRA+ / - EPC is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0511] 108. The pharmaceutical composition, method, isolated EPC population or cell composition according to any one of embodiments 1-107, wherein the isolated cell population, the first isolated cell population or the second isolated cell population comprises mammalian cells.

[0512] 109. The pharmaceutical composition, method, isolated EPC group, or cell composition according to embodiment 108, wherein the mammalian cell is a human cell.

[0513] 110. The pharmaceutical composition, method, isolated EPC group, or cell composition according to embodiment 108 or 109, wherein the mammalian cell is a placental cell.

[0514] 111. The pharmaceutical composition, method, isolated EPC population or cell composition according to any one of embodiments 1-110, wherein the isolated cell population, the first isolated cell population or the second isolated cell population is derived from a donor.

[0515] 112. The pharmaceutical composition, method, isolated EPC population or cell composition according to any one of embodiments 1-111, wherein the isolated cell population, the first isolated cell population or the second isolated cell population is derived from two or more donors.

[0516] 113. The pharmaceutical composition, method, isolated EPC population or cell composition according to embodiment 111 or 112, wherein the isolated cell population, the first isolated cell population or the second isolated cell population comprises autologous cells or allogeneic cells.

[0517] 114. The pharmaceutical composition, method, isolated EPC population or cell composition according to any one of embodiments 1-113, wherein the isolated cell population, the first isolated cell population or the second isolated cell population is present in a culture medium.

[0518] 115. The pharmaceutical composition, method, isolated EPC group or cell composition according to embodiment 114, wherein the culture medium comprises liquid culture medium or frozen culture medium.

Claims

1. A pharmaceutical composition comprising or consisting of the following: An isolated population of endothelial progenitor cells (EPCs), the isolated population of endothelial progenitor cells (EPCs) comprising PROCR+ PDGFRA+ EPCs.

2. A pharmaceutical composition comprising or consisting of the following: A group of isolated EPCs comprising PROCR+ / - PDGFRA+ / - EPCs, the pharmaceutical composition being used in the treatment of neonatal hypoxic-ischemic encephalopathy (HIE) in subjects of need.

3. A pharmaceutical composition comprising or consisting of the following: A separate EPC group, wherein the separate EPC group comprises PROCR+ / - PDGFRA+ / - EPC.

4. A pharmaceutical composition comprising or consisting of the following: A group of isolated EPCs, the isolated group of isolated EPCs comprising PROCR+ / - PDGFRA+ / - EPCs, the pharmaceutical composition being used in the treatment of brain injury in subjects of need.

5. The pharmaceutical composition of claim 1, wherein, relative to the expression levels in the unseparated EPC group, the separated EPC group comprises an increase in the expression levels of PROCR protein, PDGFRA protein, or VE-cadherin protein.

6. The pharmaceutical composition according to claim 5, wherein the increase in the PROCR protein expression level relative to the PROCR protein expression level in the unseparated EPC 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%.

7. The pharmaceutical composition according to claim 5, wherein the increase in PDGFRA protein expression level relative to the PDGFRA protein expression level in the unseparated EPC 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%.

8. The pharmaceutical composition according to any one of claims 1-7, wherein, relative to the expression levels in the unseparated EPC group, the separated EPC group comprises an increase in the expression levels of the PROCR gene, the PDGFRA gene, or the VE-cadherin gene.

9. The pharmaceutical composition according to claim 8, wherein the increase in the PROCR gene expression level relative to the PROCR gene expression level in the unseparated EPC 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%.

10. The pharmaceutical composition of claim 8, wherein the increase in PDGFRA gene expression level relative to the PDGFRA gene expression level in the unseparated EPC 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%.

11. The pharmaceutical composition according to claim 8, wherein the increase in the VE-cadherin gene expression level relative to the VE-cadherin gene expression level in the unseparated EPC 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%.

12. The pharmaceutical composition according to any one of claims 1-11, wherein the isolated EPC group contains increased proliferative capacity relative to the unisolated EPC group.

13. The pharmaceutical composition according to any one of claims 1-12, wherein the isolated EPC group comprises increased angiogenic capacity relative to the unisolated EPC group.

14. The pharmaceutical composition of claim 13, wherein the increase in angiogenesis capacity relative to the level of angiogenesis capacity in the unseparated EPC 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%.

15. The pharmaceutical composition according to any one of claims 1-14, wherein the separated EPC group comprises an increased colony-forming ability or tube-forming ability relative to the unseparated EPC group.

16. The pharmaceutical composition of claim 15, wherein the increase in colony-forming ability relative to the level of colony-forming ability in the unseparated EPC 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%.

17. The pharmaceutical composition of claim 15, wherein the increase in tube-forming ability relative to the tube-forming ability level in the unseparated EPC 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%.

18. The pharmaceutical composition according to any one of claims 1-17, wherein the separated EPC group contains increased implantation potential relative to the unseparated EPC group.

19. The pharmaceutical composition of claim 18, wherein the increase in implantation potential relative to the implantation potential level in the unseparated EPC 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%.

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

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

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

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

24. A pharmaceutical composition comprising... (a) The first isolated cell population, containing PROCR+ PDGFRA+ EPC; and (b) The second isolated cell population, which contains MSCs.

25. A pharmaceutical composition comprising... (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains MSCs.

26. A pharmaceutical composition for use in treating HIE in a subject of need, said pharmaceutical composition comprising... (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains MSCs.

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

28. A pharmaceutical composition for use in treating brain injury in a subject of need, said pharmaceutical composition comprising... (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains MSCs.

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

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

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

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

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

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

35. A method of treating HIE in a subject in need, the method comprising administering to the subject a pharmaceutical composition comprising: (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains 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, the method comprising administering to the subject a pharmaceutical composition comprising: (a) The first isolated cell population, comprising PROCR+ / - PDGFRA+ / - EPC; and (b) The second isolated cell population, which contains 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 the treatment of neonatal HIE, said composition comprising a mixture of two isolated cell populations, wherein the first isolated cell population consists of PROCR+ / - PDGFRA+ / - EPC and the second isolated cell population consists of MSCs.

41. A method for treating neonatal HIE in a subject in need, the method comprising administering to the subject a cell composition comprising a mixture of two separate cell populations, wherein the first separate cell population consists of PROCR+ / -PDGFRA+ / - EPC and the second separate cell population consists of MSCs.

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

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

44. The pharmaceutical composition for use according to claim 43, wherein the pharmaceutically acceptable medium is a phosphate-buffered saline solution.

45. The isolated cell population, cell composition, pharmaceutical composition, or cell composition for use according to any one of claims 26, 27, 35, 36, or 41, wherein use in the treatment of HIE or the treatment of neonatal HIE comprises providing therapeutic hypothermia treatment to a subject in need, and further 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, method, or cell composition according to 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, method, or cell composition according to 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, method, or cell composition according to any one of claims 24-47, wherein the MSC is a CD45- / CD34+ cell.

49. The pharmaceutical composition, method, or cell composition according to 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 are CD34+ / CD45- isolated cell populations.

50. The pharmaceutical composition, method, or cell composition according to any one of claims 24-49, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the isolated cell population or the second isolated cell population expresses PROCR and PDGFRA.

51. An isolated population of EPC cells comprising PROCR+PDGFRA+EPC, said isolated population of EPC cells being generated by the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) Select cells expressing the PROCR+ and PDGFRA+ phenotypes from the CD45- / CD34+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

52. An isolated population of EPC cells comprising PROCR+ / - PDGFRA+ / - EPC cells, said isolated population of EPC cells being generated by the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Selecting cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; and (iv) Select cells expressing the PROCR+ / - and PDGFRA+ / - phenotypes from the CD45- / CD34+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

53. The isolated EPC group according to claim 51 or 52, wherein step (ii) includes selecting the expression CD45. + Cells with the phenotype removed from the biological sample of step (i) expressing CD45. + Cells with the specified phenotype were discarded, and those expressing CD45 were removed. + Cells with this phenotype acquire CD45. - Cell population.

54. The separated EPC group according to claim 53, wherein the expression CD45 is selected. + The steps of phenotyped cells include contacting one or more cells expressing the CD45 surface protein with CD45 binding molecules to form a complex, and removing the complex from the biological sample in step (i).

55. The isolated EPC cluster according to any one of claims 51-54, wherein step (iii) comprises contacting one or more cells expressing CD34 surface protein with CD34 binding molecules to form a complex from the CD45 in step (ii). - The complex is removed from the cell population, while the complex is retained, thereby obtaining a CD45- / CD34+ cell population.

56. The isolated EPC population according to any one of claims 51-55, wherein step (iv) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

57. A method for separating an EPC group containing PROCR+PDGFRA+EPC, the method comprising the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Select cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; as well as (iv) Select cells expressing the PROCR+ and PDGFRA+ phenotypes from the CD45- / CD34+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

58. A method for separating an EPC group containing PROCR+ / - PDGFRA+ / - EPCs, the method comprising the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching the biological sample with cells containing the CD45- phenotype to obtain a CD45- cell population; (iii) Select cells containing the CD34+ phenotype from the CD45- cell population to obtain a CD45- / CD34+ cell population; as well as (iv) Select cells expressing the PROCR+ / - and PDGFRA+ / - phenotypes from the CD45- / CD34+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

59. The method according to claim 57 or 58, wherein step (ii) comprises selecting an expression CD45. + Cells with the phenotype removed from the biological sample of step (i) expressing CD45. + Cells with the specified phenotype were discarded, and those expressing CD45 were removed. + Cells with this phenotype acquire CD45. - Cell population.

60. The method of claim 59, wherein the expression CD45 is selected. + The steps of phenotyped cells include contacting one or more cells expressing the CD45 surface protein with CD45 binding molecules to form a complex, and removing the complex from the biological sample in step (i).

61. The method according to any one of claims 57-60, wherein step (iii) comprises contacting one or more cells expressing CD34 surface protein with CD34 binding molecules to form a complex from the CD45 in step (ii). - The complex is removed from the cell population, while the complex is retained, thereby obtaining a CD45- / CD34+ cell population.

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

63. A separate group of EPCs, comprising or consisting of the following: PROCR + PDGFRA + EPC, the isolated cell population is generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching cells containing the PDGFRA+ phenotype in the biological sample to obtain a PDGFRA+ cell population; (iii) Select cells expressing the PROCR+ phenotype from the PDGFRA+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

64. A separate group of EPCs, comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC, the separated EPC group is generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PDGFRA+ / - phenotype to obtain a PDGFRA+ / - cell population; (iii) Select cells expressing the PROCR+ / - phenotype from the PDGFRA+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

65. The isolated EPC population according to claim 63 or 64, wherein step (iii) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

66. A method for separating an EPC group, said EPC group comprising or consisting of the following: PROCR+ PDGFRA+ EPC, the method includes the following steps: (i) Obtaining biological samples from subjects; (ii) Enriching cells containing the PDGFRA+ phenotype in the biological sample to obtain a PDGFRA+ cell population; (iii) Select cells expressing the PROCR+ phenotype from the PDGFRA+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

67. A method for separating an EPC group, said EPC group comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC, the method includes the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PDGFRA+ / - phenotype to obtain a PDGFRA+ / - cell population; (iii) Select cells expressing the PROCR+ / - phenotype from the PDGFRA+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

68. The method of claim 66 or 67, wherein step (ii) comprises contacting one or more cells expressing the PDGFRA surface protein with the PDGFRA binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

69. The method according to any one of claims 66-68, wherein step (iii) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

70. A separate group of EPCs, comprising or consisting of the following: PROCR+ PDGFRA+ EPC, the separated EPC group is generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PROCR+ phenotype to obtain a PROCR+ cell population; (iii) Select cells expressing the PDGFRA+ phenotype from the PROCR+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

71. A separate group of EPCs, comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC, the separated EPC group is generated through the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample with cells containing the PROCR+ / - phenotype to obtain a PROCR+ / - cell population; (iii) Select cells expressing the PDGFRA+ / - phenotype from the PROCR+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

72. The isolated EPC population according to claim 70 or 71, wherein step (ii) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

73. The isolated EPC population according to any one of claims 70-72, wherein step (iii) comprises contacting one or more cells expressing the PDGFRA surface protein with PDGFRA binding molecules to form a complex, removing the complex from the cell population and retaining the complex.

74. A method for separating an EPC group, said EPC group comprising or consisting of the following: PROCR+ PDGFRA+ EPC, the method includes the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample containing the PROCR+ phenotype to obtain a PROCR+ cell population; (iii) Select cells expressing the PDGFRA+ phenotype from the PROCR+ cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ PDGFRA+ EPC.

75. A method for separating an EPC group, said EPC group comprising or consisting of the following: PROCR+ / -PDGFRA+ / -EPC, the method includes the following steps: (i) Obtaining biological samples from subjects; (ii) Enrich the biological sample with cells containing the PROCR+ / - phenotype to obtain a PROCR+ / - cell population; (iii) Select cells expressing the PDGFRA+ / - phenotype from the PROCR+ / - cell population to obtain the isolated EPC population comprising or consisting of the following: PROCR+ / - PDGFRA+ / - EPC.

76. The method of claim 74 or 75, wherein step (ii) comprises contacting one or more cells expressing the PROCR surface protein with a PROCR binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

77. The method according to any one of claims 74-76, wherein step (iii) comprises contacting one or more cells expressing the PDGFRA surface protein with a PDGFRA binding molecule to form a complex, removing the complex from the cell population and retaining the complex.

78. The isolated EPC group or method according to 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 group or method according to claim 78, wherein the protein comprises an antibody.

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

81. The isolated EPC cluster or method according to claim 80, wherein the microfluidic sorting comprises microbead sorting or flow cytometry.

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

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

84. The isolated EPC population or method according to claim 83, wherein the step further comprises step (v), separating the isolated EPC population from the cell population containing ECFCs or the cell population containing MSCs.

85. The separated EPC group or method according to 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 group or method according to any one of claims 51-85, wherein the biological sample is a mammalian biological sample.

87. The isolated EPC group or method according to claim 86, wherein the mammalian biological sample is selected from the group consisting of mammalian placenta, mammalian umbilical cord blood, mammalian peripheral blood, and vascular endothelium residing in mammalian tissues.

88. The isolated EPC group or method according to claim 87, wherein the mammalian placenta is an intact mammalian placenta.

89. The isolated EPC group or method according to claim 87, wherein the vascular endothelium residing in the mammalian tissue is selected from the group consisting of: mammalian umbilical cord, mammalian pulmonary artery endothelium, mammalian aorta, and mammalian lung tissue.

90. The isolated EPC group or method according to any one of claims 51-89, wherein the isolated EPC group comprises an increase in the expression levels of PROCR protein, PDGFRA protein, or VE-cadherin protein, relative to the expression levels in the unisolated EPC group.

91. The isolated EPC swarm or method according to any one of claims 51-90, wherein the isolated EPC swarm comprises increased proliferation capacity relative to the unisolated EPC swarm.

92. The isolated EPC cluster or method according to any one of claims 51-91, wherein the isolated EPC cluster contains increased angiogenesis capacity relative to the unisolated EPC cluster.

93. The separated EPC cluster or method according to any one of claims 51-92, wherein the separated EPC cluster includes an increase in cluster-forming capability or tube-forming capability relative to the unseparated EPC cluster.

94. The separated EPC cluster or method according to any one of claims 51-93, wherein the separated EPC cluster contains increased implantation potential relative to the unseparated EPC cluster.

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

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

97. The pharmaceutical composition, method, isolated EPC group, or cellular composition according to any one of claims 1-96, wherein the PROCR+ PDGFRA+ EPC or the PROCR+ / - PDGFRA+ / - EPC expresses one or more proteins selected from the group consisting of: CD32, CDH5, CD34, CD31, VEGFR2, VE-cadherin, and CD157.

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

99. The pharmaceutical composition, method, isolated EPC group or cellular composition according to claim 98, wherein the one or more hematopoietic proteins are selected from the group consisting of: CD3e, CD11b, CD45 and B220.

100. The pharmaceutical composition, method, isolated EPC population or cell composition according to any one of claims 1-99, wherein, relative to undifferentiated PROCR+ PDGFRA+ EPC or undifferentiated PROCR+ / - PDGFRA+ / - EPC, the PROCR+ PDGFRA+ EPC or the PROCR / -+ PDGFRA+ / - EPC reduces the expression level of one or more genes of CD157, ABCG2 or SOX18 upon differentiation.

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

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

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

104. The pharmaceutical composition, method, isolated EPC population, or cell composition according to any one of claims 1-99, wherein, relative to undifferentiated PROCR+ PDGFRA+ EPC or undifferentiated PROCR+ / - PDGFRA+ / - EPC, the PROCR+ PDGFRA+ EPC or the PROCR / -+ PDGFRA+ / - EPC reduces the expression level of one or more proteins of CD157, ABCG2, or SOX18 upon differentiation.

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

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

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

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

109. The pharmaceutical composition, method, isolated EPC group, or cell composition according to claim 108, wherein the mammalian cell is a human cell.

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

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

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

113. The pharmaceutical composition, method, isolated EPC population or cell composition according to claim 111 or 112, wherein the isolated cell population, the first isolated cell population or the second isolated cell population comprises autologous cells or allogeneic cells.

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

115. The pharmaceutical composition, method, isolated EPC group, or cell composition according to claim 114, wherein the culture medium comprises a liquid culture medium or a frozen culture medium.

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