Methods of vascular cell therapy and endothelial cell engraftment using mitochondria

Abstract

Described herein, inter alia, are compositions comprising and methods of making and using endothelial cells (ECs) comprising transplanted mitochondria (mitoAT-ECs). Also described herein, inter alia, are methods vascular cell therapy, promoting blood vessel formation, and increasing vascular development, angiogenesis, and cell junction via administration of a population of mitoAT-ECs.

Description

[0001] Methods of Vascular Cell Therapy and Endothelial Cell Engraftment Using Mitochondria

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 539,436, filed on September 20, 2023. The entire contents of the foregoing are incorporated herein by reference.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under Grant Number HL152133 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] BACKGROUND

[0007] Ischemic diseases, including critical limb ischemia and myocardial infarction, affect millions annually, necessitating surgical interventions such as vascular grafts to revascularize tissues distal to blockages h However, inadequate revascularization due to challenges in regenerating microvascular beds in ischemic areas persists2. While angiogenic growth factor delivery has been explored to stimulate local angiogenesis3 5, this approach is limited by the disrupted tissue microenvironment in ischemic regions6. Consequently, endothelial cell (EC) transplantation remains a priority in vascular medicine7.

[0008] A key challenge for EC therapies is the requirement for a secondary cell type to support engraftment. Although ECs can self-assemble into vascular structures, cotransplantation with perivascular cells is essential for robust engraftment and functional blood vessel formation in vivo8,9. Perivascular cell sources include smooth muscle cells10,11, pericytes12, fibroblasts13,14, and mesenchymal stromal / stem cells (MSCs)15,16. However, the use of multiple cell types complicates clinical translation and increases the complexity of phase I studies, leading to a reluctance to use two or more different cell types in clinical trials.

[0009] Further, the mechanisms by which perivascular cells facilitate EC engraftment are not fully understood. Moreover, there is a pressing need to address vascular disorders in blood vessels that can cause a range of health problems, which can be severe and even prove fatal. SUMMARY

[0010] This application is based, at least in part, on the surprising discovery of a new approach for vascular cell therapy based on a novel mechanism underlying the effects of mitochondrial transfer between mesenchymal and endothelial cells. Ischemic diseases affect millions worldwide, including critical limb ischemia and myocardial infarction. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells like mesenchymal stromal cells (MSCs), complicating clinical implementation.

[0011] This application is based, at least in part, on the surprising discovery that MSCs transfer mitochondria to ECs through tunneling nanotubes (TNTs) during transplantation and that this process is crucial for successful EC engraftment in vivo. Importantly, blocking this transfer impairs EC engraftment using traditional EC engraftment methods. Disclosed herein, inter alia, are methods of artificially transplanting mitochondria, which transiently enhanced EC bioenergetics, enabling them to form functional vessels in ischemic tissues without MSC support. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool but triggered mitophagy upon internalization. Transplanted mitochondria co-localized with autophagosomes, and PINK1 -PARKIN pathway ablation negated ECs' enhanced engraftment ability. Thus, this application reveals a novel mechanism underlying the effects of mitochondrial transfer between mesenchymal and endothelial cells and offers potential for a new vascular cell therapy approach, optionally without the use of support cells (e.g., MSCs).

[0012] Described herein, inter alia, are methods of transplanting a population of endothelial cells (ECs) in a subject comprising: contacting a population of endothelial cells with a composition comprising isolated mitochondria; and administering to the subject a therapeutically effective amount of the population of endothelial cells, optionally, wherein no support cells are administered with the endothelial cells.

[0013] Described herein, inter alia, are methods of promoting blood vessel formation in a subject, comprising: identifying a subject at in need of increased blood vessel formation; contacting a population of endothelial cells with a composition comprising isolated mitochondria; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

[0014] Described herein, inter alia, are methods of vascular cell therapy comprising: identifying a subject at in need of vascular cell therapy; contacting a population of endothelial cells with a composition comprising isolated mitochondria; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

[0015] Described herein, inter alia, are methods of increasing vascular generation or vascular regeneration in a subject comprising: identifying a subject at in need of vascular generation or vascular regeneration; contacting a population of endothelial cells with a composition comprising isolated mitochondria; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

[0016] In some embodiments, the mitochondria are not functionally active, are not respiration competent, and / or do not have mitochondrial DNA (mtDNA). In some embodiments, the population of ECs comprises human ECs. In some embodiments, the human ECs comprise any one or more of human umbilical vein endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs (e.g., white adipose tissue-derived ECs, watECs), organ-specific endothelial cells, and / or ECs derived from human induced pluripotent stem cells (iPSCs).

[0017] In some embodiments, no support cells are administered with the population of ECs. In some embodiments, the support cells are perivascular cells. In some embodiments, the perivascular cells are any one or more of (or all of): smooth muscle cells, pericytes, fibroblasts, mesenchymal stromal cells (MSCs), and / or perivascular cells derived from human induced pluripotent stem cells (iPSCs).

[0018] In some embodiments, support cells are administered with the population of ECs. In some embodiments, the support cells are perivascular cells. In some embodiments, the perivascular cells are any one or more of: smooth muscle cells, pericytes, fibroblasts, mesenchymal stromal cells (MSCs), and / or perivascular cells derived from human induced pluripotent stem cells (iPSCs). In some embodiments, administering a population of ECs contacted with the mitochondria (e.g., isolated mitochondria, exogenous mitochondria) with a population of support cells enhances the blood vessel formation, vascularization, vascular generation, and / or vascular regeneration compared to administering a population of ECs without the transplanted mitochondria with a population of the same support cells.

[0019] In some embodiments, the population of endothelial cells is administered to the subject by injecting the composition into a blood vessel of the subject. In some embodiments, the blood vessel is the hepatic portal vein of the subject, the coronary artery of the subject, the renal artery of the subject, the pulmonary artery of the subject, or the prostate artery of the subject. In some embodiments, administering is performed by intravenous, intra-articular, subcutaneous, intraperitoneal, intramuscular, intradermal, or intracardiac injection. In some embodiments, the subject is administered a single dose of the population of ECs. In some embodiments, the subject is administered multiple doses of the population of ECs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses). In some embodiments, the multiple does are administered at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 1 week, 2 weeks, 3 weeks, 1 month, 2, months, 3, months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months apart.

[0020] In some embodiments, the mitochondria are autogeneic, allogeneic, or xenogeneic. In some embodiments, the population of endothelial cells are autogeneic or allogeneic. In some embodiments, the methods further comprise, prior to the administering step, a step of collecting the isolated mitochondria from cells (e.g., donor cells).

[0021] In some embodiments, wherein the subject has (or is at risk of having) a disease or disorder of the blood vessels, aberrant vasculature, leaky blood vessels, narrow blood vessels, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, a myocardial infarction, cardiotoxicity (e.g., caused by chemotherapy), cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, valvular heart disease, cerebral cavernous malformations, hemorrhagic strokes, hereditary hemorrhagic telangiectasias, arteriovenous malformations, a metabolic disorder, diabetes, diabetic retinopathy, an ischemic injury, an IRI injury, atherosclerosis, Age-related Macular Degeneration (AMD), Pulmonary Arterial Hypertension (PAH), Hereditary Hemorrhagic Telangiectasia (HHT), peripheral artery disease (PAD), arteriovenous fistulas (e.g., dialysis patients), tumor angiogenesis, tumor metastasis, a cancer, a metabolic disease, a stroke, a wound (optionally, a chronic wound; e.g., a diabetic ulcer), and / or a mitochondrial dysfunction disorder. In some embodiments of any of the methods described herein, the subject or patient may be undergoing (or scheduled to undergo) an organ transplant or tissue transplant, selected to undergo an organ transplant or tissue transplant, or need vascularization of an organ or a tissue. In some embodiments, the organ is selected from the group consisting of skin, heart, kidney, testis, ovary, bone, lymph, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine and small intestine. In some embodiments, the organ and / or tissue is any one or more of brain, thyroid, thymus, heart, lung, liver, pancreas, kidney, bladder, pharynx, esophagus, stomach, gallbladder, pharynx, larynx, ovaries, uterus, placenta, testes, prostate, spleen, spinal cord, pancreas, small intestine, large intestine, colon, eye, skin, skin tissue, skeletal muscle, adipose tissue, facial muscle, bone marrow tissue, eye tissue, heart valves, veins, tendons, and combinations thereof.

[0022] In some embodiments of any of the methods described herein, vascularization comprises the formation of an artery, a vein, a capillary, an arteriole, a venule, or any combination thereof.

[0023] Also disclosed herein, inter alia, are populations of endothelial cells comprising isolated and / or exogenous mitochondria (mitoAT-ECs). In some embodiments, the populations of endothelial cells include a single cell type: ECs (e.g., mitoAT-ECs). In some embodiments, the ECs comprise any one or more of human umbilical vein endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs (e.g., white adipose tissue-derived ECs, watECs), organ-specific endothelial cells, and / or induced pluripotent stem cells (iPSCs)-derived ECs (iECs). In some embodiments, the organ-specific ECs are from an organ selected from: heart, muscle, kidney, testis, ovary, lymphoid, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine, and small intestine. In some embodiments, the ECs are human.

[0024] In some embodiments, the populations of endothelial cells described herein (e.g., a population of mitoAT-ECs) do not include multiple cell types. In some embodiments, the populations of endothelial cells (e.g., a population of mitoAT-ECs) do not include support cells (e.g., perivascular cells). In some embodiments, the support cells are perivascular cells; optionally, the perivascular cells can comprise any one or more (or all) of: smooth muscle cells, pericytes, fibroblast, mesenchymal stromal cells (MSCs), and / or perivascular cells derived from human induced pluripotent stem cells (iPSCs). In some embodiments, the populations of endothelial cells described herein (e.g., any of the populations of mitoAT- ECs) do not include any perivascular cells. In some embodiments, the populations of endothelial cells include multiple cell types but do not include support cells (e.g., perivascular cells). For example, a useful cell population for a transplant subject (e.g., a subject receiving or scheduled to receive an organ, tissue, or cell transplant) can include any population of endothelial cells described herein and a population of cells from the organ, tissue, or cell transplant without the presence of support cells, (e.g., perivascular cells or MSCs).

[0025] Also described herein are methods of making populations of endothelial cells (ECs) comprising transplanted or exogenous mitochondria (mitoAT-ECs). In some embodiments, the method comprises: i. obtaining a sample (e.g., a sample comprising ECs) from a subject (e.g., blood or a tissue sample); ii. isolating a population of ECs from the sample; iii. contacting the population of ECs with isolated mitochondria (optionally, exogenous mitochondria), thereby creating the population of mitoAT-ECs.

[0026] In some embodiments, the sample comprises ECs. In some embodiments, the sample comprising ECs is a human sample. In some embodiments, the sample comprising ECs is blood (e.g., human umbilical cord blood) or tissue (e.g., subcutaneous adipose tissue). In some embodiments, the sample does not contain ECs. In some embodiments, the ECs comprise any one or more of human umbilical vein endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs (e.g., white adipose tissue-derived ECs, watECs), organ-specific endothelial cells, and / or iECs. In some embodiments, the organ-specific endothelial cells are from an organ selected from: heart, muscle, kidney, testis, ovary, lymphoid, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine, and small intestine.

[0027] In some embodiments, step ii. comprises reprogramming the cells in the sample to iPSCs and differentiating the iPSCs into ECs, thereby isolating a population of ECs from the sample.

[0028] In some embodiments or any of the methods described herein and cell populations (e.g., mitoAT-ECs), the mitochondria are isolated from a population of donor cells. Useful mitochondria can be isolated or provided from any source, e.g., isolated from cultured cells or tissues. Exemplary cells include, but are not limited to, muscle tissue cells, cardiac fibroblasts, cultured cells, HeLa cells, prostate cancer cells, yeast, among others, and any mixture thereof. Exemplary tissues include, but are not limited to, liver tissue, skeletal muscle, heart, brain, kidney, and adipose tissue. Mitochondria can be isolated from cells of an autogenous source, an allogeneic source, and / or a xenogeneic source. In some instances, mitochondria are isolated from cells with a genetic modification, e.g., cells with modified mtDNA, modified nuclear DNA, or no mtDNA. In some embodiments, mitochondria are not respiratory competent or viable.

[0029] In some embodiments, the mitochondria are isolated from a number of donor cells at a ratio of about 20: 1, 19: 1, 18: 1, 17: 1, 16: 1, 15: 1 :, 14: 1 : 13: 1, 12: 1, 11 : 1, 10:, 9:1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1 3: 1, 2: 1, or 1 : 1 donor cells:ECs. For example, in some embodiments, the mitochondria are isolated from a number of donor cells that is a ratio of donor cells:ECs of 3: 1 (e.g., mitochondria from 3 donor cells (e.g., MSCs) for each recipient EC). In another example, in some embodiments, the mitochondria are isolated from a number of donor cells that is a ratio of donor cells:ECs of 10: 1 (e.g., mitochondria from 10 donor cells for each recipient EC).

[0030] In some embodiments, the isolated mitochondria (e.g., exogenous mitochondria) are incubated with the ECs in the contacting step for a time sufficient to internalize at least a portion of the mitochondria. In some embodiments, the mitochondria are incubated with the ECs about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 ,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 hours). In some embodiments, at least about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% of the ECs internalize the isolated mitochondria. In some embodiments, the population of mitoAT-ECs comprises at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% mitoAT-ECs. In some embodiments, about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% cell in the population of mitoAT-ECs comprise exogenous mitochondria.

[0031] Also disclosed herein are populations of endothelial cells (e.g., mitoAT-ECs) made using any of the methods disclosed herein and the use thereof in any of the methods disclosed herein (e.g., a methods of vascular cell therapy, increasing blood vessel formation, vascularization, vascular generation, vascular regeneration, transplantation, etc).

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0033] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0034] DESCRIPTION OF DRAWINGS

[0035] FIGS. 1A-1L show MSCs facilitate EC engraftment via mitochondrial transfer. FIG. 1A, Schematic of MSCs with DsRed+ mitochondria transplanted with ECs in mice. TNTs containing DsRed+ mitochondria observed at 24 h. Scale bar, 10 pm. FIG. IB, Flow cytometry and immunofluorescence show hCD90- / hCD31+ ECs with DsRed+ mitochondria (mitoRed-ECs). Scale bars, 10 pm. FIG. 1C, Quantification of DsRed+ mitochondria- containing TNTs in MSC-EC co-culture in vitro. Effects of anti-TNFa antibody or TNFAIP2 silencing (shRNA) on TNT formation. **P < 0.01, ***P < 0.001 (n=3; ANOVA followed by Bonferroni’s post-test analysis; mean + / - s.d.). FIG. ID, Quantification of ECs receiving DsRed+ mitochondria from shTNFAIP2-MSCs or shCTR-MSCs. ***P < 0.001 (n=5; unpaired two-tailed t-test; mean + / - s.d.). FIG. IE, Time course of DsRed+ mitochondria in engrafted ECs. UEA-1+ ECs have mitoRed at day 7 but not day 14. TNFa reactivates mitoRed transfer. Scale bars, 50 pm. **P < 0.01, ***p < 0.001 (n=3; ANO VA followed by Bonferroni’s post-test analysis; mean + / - s.d.). FIG. 1F-1G, Bioluminescence imaging of grafts with lucif-ECs and MSCs. Effects of TNFAIP2 and MIR01 silencing on EC engraftment. FIG. 1G, Quantification at day 7. *P < 0.05 (n=4; paired two-tailed t-test; mean + / - s.d.). FIG. 1H, H&E staining of grafts with ECs and MSCs at day 7. Effect of silencing TNFAIP2 and MIR01 on perfused blood vessels (yellow arrowheads). Scale bar, 100 pm. FIG. II, Perfused microvessel density at day 7. *P < 0.05, ***P < 0.001 (n=4-8; ANOVA followed by Bonferroni’s post-test analysis; mean + / - s.d.). FIG. 1J, Schematic of ECs and mitoRed-MSCs co-cultured for 24 h. CD31+ / DsRed+ mitoT-ECs sorted by FACS. H&E- stained grafts show perfused blood vessels (yellow arrowheads) at day 7. Scale bar, 100 pm. FIG. IK, Perfused microvessel density at day 7. ***p < 0.001 (n=3; unpaired two-tailed t- test; mean + / - s.d.). FIG. IL, Immunofluorescent staining of human-specific (UEA-1+ h- vimentin+) vessels at day 7 in grafts with mitoT-ECs (white arrowheads and asterisk). Scale bar, 50 pm. See Figs. 5-15 for related results.

[0036] FIGS. 2A-2P show exogenous mitochondrial transplantation enhances EC engraftment. FIG. 2A, Schematic of mitochondrial isolation and transplantation. FIG. 2B, Seahorse analysis shows improved respiration in mitoAT-ECs 24 h post-transplantation, with OCR profile. Abbreviations: OM, Oligomycin; FCCP, carbonyl cyanide-p- trifluoromethoxyphenyl hydrazone; R& A, Retenone and antimycin A. FIG. 2C, ATP production calculated from the Seahorse analysis. ***P < 0.001 (n=6; unpaired two-tailed t- test; mean + / - s.d.). FIG. 2D-2F, Functional comparison of ECs and mitoAT-ECs: ATP production (FIG. 2D), migration capacity (FIG. 2E), and apoptosis upon H2O2 exposure (FIG. 2F). ***p < 0.001 (n=3; unpaired two-tailed t-test; mean + / - s.d.). FIG. 2G, Bioluminescence imaging of grafts with ECs or mitoAT-ECs at indicated times. FIG. 2H, Quantification at days 1,3 and 7. **P < 0.01, ***p < 0.001 (n=3; unpaired two-tailed t-test; mean + / - s.d.). FIG. 2I-2K, Grafts with ECs or mitoAT-ECs implanted in mice. FIG. 21, Macroscopic view at day 7. H&E staining shows perfused blood vessels (yellow arrowheads) in mitoAT-EC grafts. Scale bar, 100 pm. FIG. 2 J, Perfused microvessel density at day 7. ***p < 0.001 (n=3; unpaired two-tailed t-test; mean + / - s.d.). FIG. 2K, Immunofluorescent staining of human-specific (h-vimentin+; h-CD31+) vessels at day 7 in mitoAT-EC grafts (yellow arrowheads). Scale bars, 40 pm. FIG. 2L-2P, Hind limb ischemia in mice, with luciferase-labeled ECs or mitoAT-ECs injected. FIG. 2L, Bioluminescence images on days 1 and 7. Quantification at day 7. **P < 0.01 (n=3; unpaired two-tailed t-test; mean + / - s.d.). FIG. 2M, Immunofluorescence of human vessels in ischemic muscle with mitoAT-ECs at day 7. Yellow arrowheads indicate perfused human vessels. Scale bar, 50 pm. FIG. 2N, Laser Doppler images of ischemic hind limbs at 3 weeks. FIG. 20, Blood flow quantified by laser Doppler. ***p < 0.001, compared to ECs. #P < 0.05, ##P < 0.01, ###P < 0.001, compared to no cells. (n=5; ANOVA followed by Bonferroni’s post-test analysis; mean + / - s.d.). FIG. 2P, Mice with necrosis or intact legs in each group at 3 weeks (n=5). See Figs. 16-22 for related results.

[0037] FIGS. 3A-3K show transplanted mitochondria do not incorporate into the recipient pool. FIG. 3A, Schematic of depolarization in isolated mitochondria due to Ca2+overload and the JC-10 fluorescent assay. FIG. 3B, Mitochondrial membrane potential changes measured in MSC-isolated mitochondria in Ca2+-free C buffer and Ca2+-containing EGM2 medium. *P < 0.05 (n=2; unpaired two-tailed t-test; mean + / - s.d.). FIG. 3C, Heteroplasmy in mtDNA of ECs and MSCs. Exogenous vs. endogenous mtDNA percentage in mitoAT-ECs measured by sequencing heteroplasmic loci at days 1 and 7 after transplantation. ***p < 0.001 (n=4; unpaired two-tailed t-test; mean + / - s.d.). FIG. 3D, Schematic of split-GFP system with GFP-1-10 and GFP-11 localized in mitochondria matrix of ECs and mitoRed-MSCs, respectively. Flow cytometry shows negligible exogenous fused mitochondria (DsRed+ / GFP+) in mitoAT-ECs 24 h post-transplantation. FIG. 3E, mtDNA- free MSCs (mitoRed-pO-MSCs) engineered with Dox-inducible MTS-EcoRI-GFP complex that degrades mtDNA. qPCR evaluation of mtDNA normalized to genomic gDNA in mitoRed-pO-MSCs with / without Dox. *P < 0.05 (n=2; unpaired two-tailed t-test; mean + / - s.d.). FIG. 3F, Schematic of transplanting mtDNA-free mitochondria from mitoRed-pO- MSCs into ECs. FIG. 3G-3H, Functional comparison of ECs and mitoAT-ECs: ATP production (FIG. 3G) and migration capacity (FIG. 3H). ***p < 0.001 (n=3; unpaired two- tailed t-test; mean + / - s.d.). FIG. 31, Grafts with mtDNA-free-mitoAT-ECs implanted in mice. Macroscopic view at day 7. H&E staining shows perfused blood vessels (yellow arrowheads). Scale bar, 100 pm. FIG. 3 J, Perfused microvessel density at day 7. ***P < 0.001 (n=3-4; unpaired two-tailed t-test; mean + / - s.d.). FIG. 3K, Immunofluorescent staining of human-specific (h-vimentin+ / UEA-l+) vessels at day 7 (yellow arrowheads). DAPI stains cell nuclei. Scale bar, 50 pm. See Figs. 23-26 for related results.

[0038] FIGS. 4A-4K show enhanced engraftment ability in ECs mediated by mitophagy.

[0039] FIG. 4A, Mitochondria isolated from mitoAPEX2-MSCs transplanted into ECs. TEM micrograph shows internalized exogenous APEX2+ mitochondria co-localized with autophagosomes in mitoAT-ECs after 24 h. Scale bars, 50 nm. FIG. 4B, Immunofluorescent staining of LC3B+ autophagosomes in ECs and mitoAt-ECs. Starvation and mitochondrial transplantation increased LC3B+ autophagosomes in ECs. Inhibiting autophagy with 3- Methyladenine (3 -MA) or silencing ULK1 (shRNA) prevented LC3B+ autophagosome accumulation in mitoAT-ECs. DAPI stained cell nuclei. Scale bar, 10 pm. FIG. 4C, Immunofluorescent analysis showing co-localization of exogenous DsRed+ mitochondria and LC3B+ autophagosome in mitoAT-ECs. DAPI stains cell nuclei. Scale bars, 10 pm. FIG. 4D, Effects of silencing PINK1 or PARKIN (shRNA) on LC3B+ autophagosomes in mitoAT- ECs. ***P < 0.001 (n=30 cells / group; ANOVA, Bonferroni’s post-test; mean + / - s.d.). FIG. 4E-4G, Effects of silencing ULK1, PINK1, or PARKIN (shRNA) on engraftment ability of mitoAT-ECs. FIG. 4E, Macroscopic view of explanted grafts at day 7. FIG. 4F, H&E staining of grafts at day 7; yellow arrowheads indicate perfused blood vessels. Scale bar, 50 pm. FIG. 4G, Perfused microvessel density at day 7. **P < 0.01, ***P < 0.001 (n=4-8; ANOVA, Bonferroni’s post-test; mean + / - s.d.). FIG. 4H, Schematic: PARKIN (recipient ECs) and PINK1 (donor mitochondria) role in mitophagy activation in mitoAT-ECs. FIG. 41- 4K, Effects of silencing PINK1 and PARKIN (shRNA) on engraftment ability of ECs co- implanted with MSCs. FIG. 41, Bioluminescence imaging of grafts with lucif-ECs and MSCs at day 7. Silencing PARKIN in ECs (left) and PINK1 in MSCs (right). Quantification at day 7. *P < 0.05, ***p < 0.01 (n=4; paired two-tailed t-test; mean + / - s.d.). FIG. 4J, Macroscopic views and H&E staining of explanted grafts at day 7; yellow arrowheads indicate perfused blood vessels. Scale bar, 100 pm. FIG. 4K, Perfused microvessel density at day 7. *P < 0.05, ***p < 0.01 (n=4; paired two-tailed t-test; mean + / - s.d.). See Figs. 27-30 for related results.

[0040] FIGS. 5A-5F show engraftment of human ECs requires support from stromal cells. FIG. 5A, Schematic of the xenograft model used to examine EC engraftment. Grafts were prepared by combining human ECs with or without MSCs in a hydrogel and subcutaneously implanting them into immunodeficient nude mice. FIG. 5B, H&E staining of grafts containing human ECFCs with or without MSCs explanted at day 7. Perfused blood vessels are marked with yellow arrowheads. Insets represent macroscopic views of the explanted grafts on day 7. Scale bar, 100 pm. FIG. 5C, Perfused microvessel density at day 7 in grafts seeded with different human EC types with or without MSCs. 0.01, ***p

[0041] 0.001 (n=3; unpaired two-tailed t-test; mean + / - s.d.). FIG. 5D, Immunofluorescent staining revealed the presence of blood vessels lined by human-specific ECs (UEA-1+) and surrounded by Q-SMA+ perivascular cells at day 7 in grafts containing ECs and MSCs. Scale bars, 100 pm. FIG. 5E, Bioluminescence imaging of grafts containing lucif-ECs with or without MSCs. FIG. 5F, Quantification of bioluminescence signal at different time points. *P 0.05, 0.01, 0.001. (n=3; unpaired two-tailed t-test; mean + / - s.d.).

[0042] FIGS. 6A-6F show MSC paracrine contribution is insufficient to enable robust EC engraftment. FIG. 6A, Human ECs were implanted into nude mice with or without MSCs, MSC conditioned medium (MSC-CM), VEGF and bFGF, and PDGF receptor inhibitor (AG1296). Cells were retrieved at 24 h and analyzed for apoptosis by flow cytometry (annexin V, PI staining). Quantification of percentage apoptotic ECs at 24 h. 0.05, ***p 0.001 (n=3; unpaired two-tailed t-test; mean + / - s.d.). FIG. 6B, Detection of angiogenic factors in MSC and EC conditioned media by proteomic dot blotting arrays. Pro- angiogenic factors distinctively secreted by MSCs are labeled with indicated boxes. Relative blot intensities were measured by Imaged. FIG. 6C, H&E staining of grafts containing human ECs with or without MSCs, MSC-CM, VEGF and bFGF, and AG1296 were explanted at day 7. Perfused blood vessels are marked with yellow arrowheads. Insets represent macroscopic views of the explanted. Scale bar, 100 pm. FIG. 6D, Perfused microvessel density at day 7. ***P s£ 0.001 (n=3; unpaired two-tailed t-test; mean + / - s.d.). FIG. 6E, H&E staining of 7- day explants with multiple conditions; yellow arrowheads indicate perfused vessels. Scale bar, 100 pm. FIG. 6F, Day 7 microvessel density; ***P < 0.001 (n=3; unpaired t-test).

[0043] FIGS. 7A-7D show genetically labeling MSC mitochondria with a mitoDsRed fusion protein. FIG. 7A, Sequence of the pPB-mitoDsRed2 piggyBac transposon vector carrying a cassette encoding for a DsRed2 fluorescent protein and mitochondria-targeting sequences. FIG. 7B, MSCs transfected with the pPB-mitoDsRed2 and a transposase vector, which integrated the transposon cassette into the genomic DNA, rendering all mitochondria DsRed+. Scale bar, 10 pm. FIG. 7C, MSCs and ECs were transfected with pPB-mitoDsRed2 and costained with a MitoTracker Green fluorescence probe. In successfully transfected cells, mitoDsRed and MitoTracker Green were co-localized to their mitochondrial network (yellow arrowheads). Non-transfected cells were identified as MitoTracker Green+ but DsRed- (green arrowheads). The yellow box marks a TNT containing MitoTracker Green+ DsRed+ mitochondria in transfected MSCs. Scale bars, 10 pm. FIG. 7D, MSCs transfected with pPB- mitoDsRed2 underwent hygromycin and clonal selection, and a stable mitoRed-MSC clone was established with exceedingly high purity (>99% MSCs had DsRed+ mitochondria by flow cytometry).

[0044] FIGS. 8A-8C show formation of mitochondria-laden tunneling nanotubes (TNTs) by MSCs in the presence of ECs. FIG. 8A, Schematic of mitoRed-MSCs containing DsRed+ mitochondria subcutaneously transplanted with human ECs into nude mice. Using fluorescence microscopy, tunneling nanotubes (TNTs) containing DsRed+ mitochondria visible in explanted grafts at 24 h. Scale bar, 40 pm. FIG. 8B, Schematic of mitoRed- MSCs co-culture with human ECs in vitro. TNTs containing DsRed+ mitochondria visible after 24 h using fluorescence microscopy. Scale bar, 40 pm. FIG. 8C, Immunofluorescent staining revealed the presence of F-actin and microtubule protein in the mitochondria-laden TNTs. Scale bar, 5 pm.

[0045] FIGS. 9A-9D show mitochondrial transfer measured in vitro by flow cytometry. Human ECs and mitoDsRed-MSCs were co-cultured under indicated conditions for 24 h. Cells were then harvested, and the percentage of ECs (CD31+) that received mitochondrial from MSCs (mitoDsRed+) were analyzed by flow cytometry. FIG. 9A, Flow cytometric gating strategy to ensure the removal of doublets or cell aggregates. FIG. 9B, Percentage of ECs that received mitoDsRed+ mitochondria in 1 : 1 co-culture with mitoDsRed-MSCs. Indicated total cell number plated in 6-well plates. FIG. 9C, Percentage of ECs that received mitoDsRed+ mitochondria in co-culture with mitoDsRed-MSCs at indicated MSC:EC ratios. Total cell density was kept constant (4x105 cells). FIG. 9D, Percentage of ECs that received mitoDsRed+ mitochondria in 1 : 1 co-culture with mitoDsRed-MSCs (4x106 cells) in either 2D (culture plates) or 3D (collagen hydrogel) and under normoxia (21% 02) or hypoxia (1% O2).

[0046] FIGS. 10A- IOC show transfer of mitochondria from MSCs to ECs in vivo. FIG. 10A, Schematic of xenograft model to examine the MSC-to-EC mitochondrial transfer after 24 h in vivo. FIG. 10B, Immunofluorescent staining of cells retrieved from the grafts after 24 h in vivo. Viable cells were plated and stained with FITC-conjugated UEA1 lectin (for ECs) and Hochest 33342 (for nuclei). Scale bar, 100 pm. Representative images of (i) a mitoRed- MSC containing DsRed+ mitochondria, (ii) UEA1+ ECs that did not receive DsRed+ mitochondria, and (iii) UEA1+ ECs that received DsRed+ mitochondria. Scale bars, 10 pm. FIG. 10C, Additional representative images of mitoRed MSCs (UEA1-, DsRed+), ECs that did not receive DsRed+ mitochondria (UEA1+, DsRed-), and ECs that received DsRed+ mitochondria (UEA1+, DsRed+). Scale bars, 10 pm.

[0047] FIGS. 11A-11E show neglectable mitochondrial transfer from ECs to MSCs.

[0048] FIG. 11 A, Schematic of human ECs transfected with a pPB-mitoDsRed vector to render all mitochondria DsRed+. mitoRed-ECs were cultured with or without unlabeled MSCs to examine the formation of DsRed+ mitochondria-laden TNTs. FIG. 11B, FITC-phalloidin staining to visualize F-actin. Representative images illustrate the general lack of DsRed+ mitochondria-laden TNTs. Upon treatment with TNFa, some ECs exhibited structures containing aligned DsRed+ mitochondria. However, these structures were F-actin+ and thus classified as cell protrusion rather than TNTs. Scale bars, 10 pm. FIG. 11C, Quantification (fluorescent imaging) of DsRed+ mitochondria-laden TNTs originating in mitoRed-ECs under indicated conditions revealed an undetectable presence of TNTs (n=3). FIG. HD, Quantification (flow cytometry) of MSCs (gated as CD31- cells) that received DsRed+ mitochondria upon co-culture with mitoRed-ECs revealed a neglectable mitochondrial transfer (~1% of MSCs) (n=3). FIG. HE, Proportion of ECs (CD31+) with mitoDsRed+ mitochondria in 1 : 1 (ECs + mitoRed-MSCs) co-culture in the presence of angiogenic factors VEGF (10 ng / mL) or bFGF (10 ng / mL) (n=3-4; one-way ANOVA followed by Bonferroni’s post-test). All data are mean ± s.e.m. n are biological replicates. FIGS. 12A-12B show cytokine secretion by ECs in 2D culture and 3D gel implant. FIG. 12A, Human ECs were cultured in either 2D cell culture plates or encapsulated in 3D hydrogel (gel implant). Conditioned media were collected from both culture conditions and measured using inflammatory cytokines proteomic dot blotting arrays. Colored boxes mark selected cytokines that were predominately secreted from ECs in 3D. The relative blot intensities were measured by ImageJ. FIG. 12B, Impact of IL-la and IL-ip on TNT formation in MSCs. MSCs were exposed to IL-la, IL-ip, or an anti-IL-la antibody, and changes in TNT formation were observed and quantified at 24 h. *P < 0.05, **P < 0.01, ***P < 0.001 (n=17, MSCs; n=10, +IL1P; n=6, +ILla; n=ll, +ECs; n=ll, +ECs + anti-ILla; one-way ANO VA followed by Bonferroni’s post-test).

[0049] FIGS. 13A-13C show silencing TNFAIP2 impairs the transfer of mitochondrial from MSCs to ECs. FIG. 13A, TNFAIP2 was silenced in mitoRed-MSCs by shRNA (shTNFAIP2-MSCs). mitoRed-MSCs transfected with a scrambled shRNA were used as a control (shCTR-MSCs). Expression of TNFAIP2 evaluated by qPCR and normalized to GAPDH. 0.05 (n=2; unpaired two-tailed t-test; mean + / - s.d.). FIG. 13B, Fluorescence imaging of mitoRed-MSCs in cultured revealed a reduced presence of DsRed+ mitochondria- laden TNTs in shTNFAIP2- MSCs compared to shCTR-MSCs. Scale bar, 10 pm. FIG. 13C, Co-culture of ECs with mitoRed-MSCs for 24 h. Quantitative evaluation (flow cytometry) of the percentage of ECs that receive DsRed+ mitochondria from either shTNFAIP2-MSCs or shCTR-MSCs. ***P s? 0.001 (n=5; unpaired two-tailed t-test; mean + / - s.d.).

[0050] FIG. 14A-14D show in vivo temporal dynamics of mitochondrial transfer from MSCs to ECs. FIG. 14A, Schematic of genetically engineered MSCs containing DsRed+ mitochondria (mitoRed-MSCs) subcutaneously transplanted with human ECs into immunodeficient nude mice. FIG. 14B, Immunofluorescence imaging of explanted grafts after 7 days in vivo. Grafts were stained with UEA-1 lectin to visualize human ECs. Red fluorescent signal identifies DsRed+ mitochondria (white arrows). DsRed+ mitochondria were present in UEA-1+ ECs (yellow arrowhead). Grafts with unlabeled MSCs (thus no DsRed signal) were used as controls (left panels). Blood vessel lumens are marked with an asterisk. Scale bars, 50 pm. FIG. 14C, Additional immunofluorescent images of explanted grafts at different time points revealed the presence of DsRed+ mitochondria in UEA-1 + ECs (yellow arrowhead) on day 7 but not on day 14. Administration of TNFa (but not saline) on day 14 reactivated the transfer of DsRed+ mitochondria into UEA-1+ ECs, visualized on day 16. Scale bars, 50 pm. FIG. 14D, Schematic of time course presence of DsRed+ mitochondria (mitoRed) in the engrafted ECs.

[0051] FIGS. 15A-15E show silencing MIR01 impairs the transfer of mitochondrial from MSCs to ECs. FIG. 15A, MIR01 was silenced in mitoRed-MSCs by shRNA (shMIROl-MSCs). mitoRed-MSCs transfected with a scrambled shRNA were used as a control (shCTR-MSCs). Expression oiMIROl evaluated by qPCR and normalized to GAPDH. 0.05 (n=2; unpaired two-tailed t-test; mean + / - s.d.). FIG. 15B, Fluorescence imaging of mitoRed-MSCs in cultured stained with FITC-phalloidin staining to visualize F- actin. A representative image illustrates the quantification of eleven DsRed+ mitochondria within a TNT. Scale bar, 10 pm. FIG. 15C, Quantification of the number of DsRed+ mitochondria per TNT in shMIROl-MSCs compared to shCTR-MSCs. 0.001 (n=30

[0052] TNTs; unpaired two-tailed t-test; mean + / - s.d.). FIG. 15D, Co-culture of ECs with mitoRed- MSCs for 24 h. Quantitative evaluation (flow cytometry) of the percentage of ECs that receive DsRed+ mitochondria from either shMIROl-MSCs or shCTR-MSCs. ***P 0.001

[0053] (n=4; unpaired two-tailed t-test; mean + / - s.d.). FIG. 15E, Schematic diagram of grafts seeded with ECs and MSCs subcutaneously implanted into nude mice. Effect of silencing TNFAIP2 ox MIRO 1 (shRNA) in MSCs on the engraftment ability of ECs. Immunofluorescent staining revealed the presence of human-specific (h-CD31+) vessels at day 7 in grafts containing control MSCs (i.e., scrambled shRNA) but not in grafts containing either shTNFAIP2-MSCs or shMIROl-MSCs. Yellow arrowheads indicate h-CD31+ vessels. DAPI stained nuclei. Scale bars, 40 pm.

[0054] FIGS 16A-16D show artificial transplantation of exogenous mitochondria into human EC. FIG. 16A, Schematic of the artificial mitochondrial isolation and transplantation process. FIG. 16B, Micrographs of cultured ECs 4 h after being artificially transplanted with DsRed+ mitochondria (mitoAT-ECs). Scale bar, 200 pm. FIG. 16C, Flow cytometry analysis revealed a high percentage of DsRed+ mitoAT-ECs. FIG. 16D, Confocal microscopy revealing internalization of exogenous DsRed+ mitochondria in mitoAT-ECs. Scale bar, 10 p m.

[0055] FIGS. 17A-17E show genetically labeling MSC mitochondria with a mitoAPEXl fusion protein. FIG. 17A, Sequence of the pPB-mitoAPEX2 piggyBac transposon vector carrying a cassette encoding for APEX2 and mitochondria-targeting sequences. APEX2 is an engineered form of a soybean ascorbate peroxidase (APX1) that can be used as a genetically encoded tag for transmission electron microscopy (TEM). FIG. 17B, Schematic depicting the visualization of APEX2+ mitochondria by TEM. Cells expressing APEX2 are fixed and then exposed to diaminobenzidine (DAB) and hydrogen peroxide. APEX2 catalyzes the hydrogen peroxide-dependent polymerization and local deposition of DAB, which results in TEM contrast after treatment with osmium. FIG. 17C, TEM micrograph depicting APEX2 -labeled mitochondria in a mitoAPEX2 -transfected cell side by side with unlabeled mitochondria in a control cell. Scale bar, 500 nm. FIG. 17D, TEM micrograph with a high magnification view of an APEX2-labeled mitochondrion. FIG. 17E, Mitochondria were isolated from mitoAPEX2-MSCs and then transplanted into ECs. A TEM micrograph confirming internalization of the exogenous APEX2+ mitochondria in mitoATECs after 4 h. Scale bar, 500 nm.

[0056] FIGS. 18A-18D show enhanced mitochondria respiration produced by mitochondria transplantation in ECs. Mitochondria respiration was measured in mitoATECs 24 h after mitochondrial transplantation using a Seahorse XF24 Analyzer. Control ECs that did not receive mitochondria served as controls. FIG. 18A, Oxygen consumption rate (OCR) profile generated by the Seahorse analyzer with parameters of mitochondria respiration delineated in colored boxes. FIG. 18B, Oxygen consumption rate (OCR) profiles in ECs and mitoAT-ECs. Mitochondria respiration was enhanced in mitoAT-ECs (n=6 for each time point; mean + / - s.d.). FIG. 18C, ATP production, basal respiration, maximal respiration, spare respiratory capacity, proton leak, and non-mitochondrial respiration in and ECs mitoAT-ECs. ***P 0.001 (n=6; unpaired two-tailed t-test; mean + / - s.d.).

[0057] Abbreviations: FCCP, carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone. FIG. 18D, ATP production in mitoAT-ECs measured at 1 and 7 days after a one-time artificial mitochondrial transplantation. ***p < 0.001 (n=6 at day 1, n=10 at day 7; unpaired two-tailed t-test).

[0058] FIGS. 19A-19B show apoptosis resistance of human ECs transplanted with exogenous mitochondria. FIG. 19A, Evaluation of apoptosis by flow cytometry (PI / Annexin-V staining) in human ECs that were transplanted with exogenous mitochondria (mitoAT-ECs) and then incubated in 200 pM H2O2 for 12 h. Human ECs that did not receive exogenous mitochondria served as controls. FIG. 19B, Percentage of apoptotic (Annexin- V+) ECs upon exposure to H2O2. ***P 0.001 (n=3-4; ANOVA followed by Bonferroni’s post-test analysis; mean + / - s.d.). FIGS. 20A-20E show increased migration capacity of human ECs transplanted with exogenous mitochondria. FIG. 20A, Evaluation of migration capacity carried out using a standard wound closure assay, where wounds are introduced by scratching a confluent layer of plated ECs with a pipette tip. Representative phase contrast micrographs depicting wound areas at 0 h and 12 h for human ECs transplanted with exogenous mitochondria (mitoAT-ECs; right panels). Human ECs that did not receive exogenous mitochondria served as controls (left panels). Scale bar, 500 pm. FIG. 20B, Percentage of wound gap closure quantified in human ECs transplanted with exogenous mitochondria from various donor cell types. Human ECs that did not receive exogenous mitochondria served as controls. 0.001 (n=3; ANOVA followed by Bonferroni’s post-test analysis; mean + / - s.d.). FIG. 20C, Relative ATP production levels in ECs measured 24 hours after receiving different concentrations of mitochondria (mitoAT-ECs). ECs without mitochondrial transfer served as control. *P < 0.05, **P < 0.01, ***p < 0.001 compared to control (n=3-7; unpaired two-tailed t-test). FIG. 20D, Comparison of ATP production in ECs transplanted with exogenous mitochondria (mitoAT-ECs) or lysosomes (lysoAT-ECs). **P < 0.01, ***p < 0.001 (n=10; one-way ANOVA with Bonferroni’s post-test). FIG. 20E, Histological evaluations of subcutaneous grafts with lysoAT-ECs at 7 days post-transplantation. Scale bar, 100 pm. Quantitative analysis of microvascular density displaying reduced vascularization potential (n=4). All data are mean ± s.e.m. n are biological replicates.

[0059] FIGS. 21A-21C show mitochondrial transfer enhances EC engraftment in ischemic limbs. FIG. 21A, Schematic of hind limb ischemia model. Ischemia was induced in STZ-treated diabetic nude mice by ligating and excising the femoral artery and vein on day - 1. Luciferase-labeled ECs or mitoAT-ECs were injected into the ischemic sites on day 0. FIG. 21B, Blood glucose corroborating the induction of diabetes in STZ-treated nude mice. Mice were treated with a single dose of STZ (220 mg / kg), and blood glucose was measured 7 days later.. ***p 0.001 (n=5-15; unpaired two-tailed t-test; mean + / - s.d.). FIG. 21C,

[0060] Bioluminescence images of treated mice on days 1 to 21. The images depict mice injected with ECs (left) and mitoAT-ECs (right) for each time point. Each mouse was only injected in the ischemic leg.

[0061] FIGS. 22A-22C show implantation of mitoAT-ECs improves blood flow in ischemic limbs. Hind limb ischemia was induced in STZ-treated diabetic nude mice on day - 1. ECs or mitoAT-ECs were injected into the ischemic sites on day 0. FIG. 22A, Representative laser Doppler image and macroscopic photograph of an untreated ischemic hind limb at day 14. FIG. 22B, Macroscopic photographs of the ischemic legs at 3 weeks post-treatment. FIG. 22C, Laser Doppler images of ischemic hind limbs with / without cell injection over 21 days.

[0062] FIG. 23A-23E show depolarization of isolated mitochondria incubated in Camcontaining EGM2 medium. FIG. 23A, Schematic of the JC-10 mitochondria membrane potential fluorescent assay. FIG. 23B, Schematic depicting the depolarization of isolated mitochondria in EGM2 medium due to Ca2+overload. Of note, Ca2+presence in the EGM2 medium represents the culture conditions at which mitoAT-ECs are generated. FIG. 23C, Mitochondria membrane potential changes in Ca2+-free C buffer and Ca2+-containing (~1.6 mM) EGM2 medium were measured with JC-10 in mitochondria isolated from MSCs. The fluorescent intensities for aggregates (Red FL, 590 nm) and monomeric (Green FL, 520 nm) forms of JC-10 were measured at Ex / Em = 490 / 590 nm and 490 / 520 nm with a microplate reader. The percentage of membrane potential was represented as the FL590 / FL520 ratio. *P 0.05 (n=2; unpaired two-tailed t-test; mean + / - s.d.). FIGS. 23D-23E, Mitochondria membrane potential changes in isolated mitochondria incubated in C buffer and EGM2. FIG. 23D, Fluorescent signals for aggregates and monomeric forms of JC-10 visualized by flow cytometry as red and green, respectively. FIG. 23E, Fluorescent signals for aggregates and monomeric forms of JC-10 visualized by fluorescent microscopy. Scale bars, 50 pm.

[0063] FIGS. 24A-24C show transient presence of the transplanted exogenous mitochondria in mitoAT-ECs measured by mtDNA heteroplasmy. FIG. 24A, Sequence of mitochondrial DNA (mtDNA) at four selected loci with heteroplasmy in the hypervariable regions of ECs and MSCs, where sequences differed in a single nucleotide. Sequences depicted correspond to ECs (monoculture), MSCs (monoculture), ECs + MSCs, (1 : 1 coculture), and mitoAT-ECs (monoculture) in those four selected loci. FIG. 24B, Table summarizing the heteroplasmy at four loci in the hypervariable mtDNA regions of ECs and MSCs. FIG. 24C, ECs were transplanted with exogenous mitochondria isolated from MSCs. Percentage of exogenous vs. endogenous mtDNA in the resulting mitoAT-ECs measured by sequencing the heteroplasmic loci at days 1 and 7 after mitochondrial transplantation.

[0064] FIGS. 25A-25E show negligible exogenous mitochondria fusion in mitoAT-ECs evaluated by a genetic split-GFP system. FIG. 25A, Schematic of a genetic split-GFP system, with GFP-1-10 and GFP-11 expressed and localized in the mitochondria matrix of ECs and mitoRed-MSCs, respectively. Only when the exogenous mitochondria from MSCs fuse with the endogenous mitochondria in mitoAT-ECs, the GFP-1-10 and GFP-11 components are together, and thus GFP can render its fluorescent signal. FIG. 25B, Representative flow cytometry panels depicting the strategy to gate isolated mitochondria using microbeads of specific size as a reference. FIG. 25C, Flow cytometry evaluation of exogenous mitochondrial fusion into the endogenous mitochondrial pool in ECs. The percentage of fused exogenous mitochondria (DsRed+ / GFP+; orange box) in ECs after 24 h was neglectable after co-culture (ECs + MSCs) or transplantation (mitoAT-ECs). FIG. 25D, Fluorescent imaging evaluation of exogenous mitochondrial fusion into the endogenous mitochondrial pool in ECs. The extent of fused exogenous mitochondria (DsRed+ / GFP+) in ECs after 24 h was neglectable after co-culture (ECs + MSCs) or transplantation (mitoAT- ECs). Scale bars, 10 pm. FIG. 25E, Mitochondrial integration post-transplantation analysis.

[0065] Proportions of DsRed+ (red box) and DsRed+ / GFP+ (yellow box) in mitoAT-EC -derived mitochondria assessed at 24 hours post-transplantation, with EC-derived mitochondria as the negative control. Representative flow cytometry data showing a dose-dependent increase in DsRed+ mitochondria at 24 hours with double (x2) and quadruple (x4) transplanted mitochondria, consistently <0.1% GFP+.

[0066] FIGS. 26A-26E show transfer of mtDNA-free mitochondria enhances EC fitness.

[0067] FIG. 26A, Schematic of genetic engineering mtDNA-free cells (mitoRed- O) with a Dox- inducible MTS-EcoRI-GFP complex that degrades mtDNA. FIG. 26B, PCR gel depicting the complete degradation of mtDNA in a mitoRed- O-293T cell line upon administration of Dox for 48 h. FIG. 26C, Schematic of transplanting mtDNA-free mitochondria from mitoRed- O- MSCs into ECs. Quantification of ATP production in ECs and mitoAT-ECs. *P 0.05, ***p

[0068] 0.001 (n=3; ANOVA followed by Bonferroni’s post-test analysis). FIG. 26D,

[0069] Quantification of migration capacity by ECs and mitoAT-ECs. ***P 0.001 (n=3; ANOVA followed by Bonferroni’s post-test analysis). FIG. 26E, Transplantation of normal and mtDNA-free mitochondria into pOECs. ATP production in pOECs measured before and 24 hours after exogenous mitochondrial transplantation. ***p < 0.001; n.s. denotes not significant (n=4, ECs; n=8, pOECs; n=8, mitoAT-ECs with normal mito; n=6, mitoAT-ECs with mtDNA-fre-mito; one-way ANOVA followed by Bonferroni’s post-test). All data are mean ± s.e.m. n are biological replicates.

[0070] FIGS. 27A-27C show transplanting mitochondria transiently triggers mitophagy in ECs. FIG. 27A, Autophagic flux analysis of mitoAT-ECs, with LC3B-I to LC3B-II conversion in presence of Bafilomycin Al. FIG. 27B, Immunofluorescent staining of LC3B+ autophagosomes in mitoAT-ECs. Effect of silencing PARKIN and PINK1 (shRNA) in either the mitochondrial donor MSCs (mito) or recipient ECs. DAPI stained cell nuclei. Scale bar, 10 pm. FIG. 27C, Schematic depicting the role of PARKIN (in recipient ECs) and PINK1 (in donor mitochondria) in the activation of mitophagy in mitoAT-ECs.

[0071] FIGS. 28A-28I show transcriptional analysis of ECs upon receiving exogenous mitochondria. FIG. 28A, Schematic representation of three groups of ECs corresponding to i) ECs that received exogenous mitochondria by artificial transplantation (mitoAT-ECs), ii) ECs that received exogenous mitochondria (from MSCs) by coculture with MSCs (mitoT- ECs) and iii) ECs that were co-cultured with MSCs but did not receive exogenous mitochondria (unprimed-ECs). FIGS. 28B-28F, RNA-seq analysis across multiple EC samples, including mitoT-ECs and mitoAT-ECs. Unprimed-ECs served as controls. FIG. 28B, Number of differentially expressed genes between mitoT-EC, mitoAT-EC, and unprimed-EC samples. FIG. 28C, Principal components (PC) analysis. FIG. 28D, Heatmap and hierarchical clustering analysis of global differentially expressed genes. FIGS. 28E-28F, Gene ontology analysis between mitoT-ECs and unpaired-ECs (FIG. 28E) and mitoAT-ECs and unpaired-ECs (FIG. 28F). Analyses were carried out with all differentially expressed genes. Genes displayed correspond to positive enrichment for mitoT-ECs (FIG. 28E) and mitoAT-ECs (FIG. 28F). FIGS. 28G-28I, Mitochondrial biogenesis post-mitochondrial transplantation in mitoAT-ECs. FIG. 28G, mRN A expression heatmap of mitochondrial biogenesis genes in mitoAT-ECs versus ECs. *P < 0.05, **P < 0.01 (n=9; unpaired two-tailed t-test). FIG. 28H, Mitochondrial content in mitoAT-ECs 24h post-transplant, quantified via flow cytometry, normalized to nuclear protein, with pre-transplant ECs as control. FIG. 281, Western blot evaluation of TOM20 for mitochondrial content in mitoAT-ECs. P-Actin was used as a housekeeping protein. Lysates from regular ECs and a mixture of ECs and exogenous mitochondria pre-transplantation (ECs + mito; not transplanted) served as controls. *P < 0.05 (n=3; one-way ANOVA with Bonferroni’s post-test; mean ± s.e.m.). n are biological replicates.

[0072] FIGS. 29A-29B show upregulation of mitochondria biogenesis gene expression in mitoAT-ECs. FIG. 29A, Heatmap depicting mRNA expression (qPCR) of selected genes associated with mitochondria biogenesis in ECs that received exogenous mitochondria by artificial transplantation (mitoAT-ECs; n=9) and ECs that did not receive exogenous mitochondria (ECs; n=9). FIG. 29B, Gene expression of selected genes evaluated by qPCR in ECs vs. mitoAT-ECs. 0.05, 0.01 (n=9; unpaired two-tailed t-test; mean + / - s.d.). FIGS. 30A-30B show mitophagy regulates the engraftment of ECs with MSCs.

[0073] FIG. 30A, Schematic diagram of grafts seeded with ECs and MSCs subcutaneously implanted into nude mice. FIG. 30B, Effect of silencing PINK1 and PARKIN (shRNA) on the engraftment ability of ECs co-implanted with MSCs. Silencing was performed in ECs (shPARKIN) or MSCs (shPINKl). Immunofluorescent staining revealed the presence of human-specific (h- CD31+) vessels at day 7 in grafts containing control cells (i.e., scrambled shRNA) but not in grafts containing either shPARKIN-ECs or shPINKl-MSCs. Yellow arrowheads indicate h-CD31+ vessels. DAPI stained nuclei. Scale bars, 40 pm.

[0074] FIGS. 31A-31I. Exogenous mitochondrial transplantation induces mitophagy in mitoAT-ECs. FIG. 31A, Analysis of autophagic flux in mitoAT-ECs, showing LC3B-I to LC3B-II conversion with Bafilomycin Al. FIG. 31B, Flow cytometry analysis showing PINK1 presence on isolated mitochondria from control MSCs (shCTR-MSCs) but not from shPINKl-MSCs. Quantification of the percentage of PINK 1 -positive in isolated mitochondria from control versus shPINKl -MSCs. ***P < 0.001 (n=4; unpaired two-tailed t-test). FIG. 31C, Western blot analysis demonstrating the presence of PINK1 (63 kDa) in lysates from both MSCs and isolated mitochondria (red line box), with TOM20 (16 kDa) as a mitochondrial marker and GAPDH (36 kDa) as a cytosolic control. FIG. 31D, Immunofluorescence indicates colocalization of exogenous DsRed+ mitochondria (Red) with endogenous Parkin (Alexa 647) in mitoAT-ECs 24h post-transplantation (white arrows). Scale bar, 100 pm; insets #1-4, 10 pm. FIG. 31E-31H, Evaluation of MSC viability and functionality after PINK1 silencing (shRNA). FIG. 31E, Morphological observations of MSCs with shRNA against PINK 1 (shPINKl -MSCs) vs. control shRNA (shCTR-MSCs) show standard mesenchymal cell morphology. Scale bar, 100 pm. FIG. 31F, Flow cytometry using PI / Annexin-V highlights the high viability of shPINKl -MSCs after lentiviral transduction (n=4). FIG. 31G, qPCR analysis measures angiogenic growth factor expression (ANGPT, CXCL12, VEGF) in shCTR-MSCs vs. shPINKl-MSCs (n=4; unpaired two-tailed t-test). FIG. 31H, In vitro assay of EC vascular network formation, using conditioned medium (CM) from shPINKl-MSCs vs. shCTR-MSCs (n=4; unpaired two-tailed t-test). Scale bar, 200 pm. FIG. 311, Immunofluorescence detection of LC3B+ autophagosomes in mitoAT-ECs. Effects of Parkin and PINK1 silencing (shRNA) in either the donor MSC mitochondria (mito) or recipient ECs. DAPI denotes cell nuclei. Scale bar, 10 pm. All data are mean ± s.e.m. n are biological replicates. DETAILED DESCRIPTION

[0075] Without being bound by theory, the data presented herein demonstrates that MSCs transfer mitochondria to ECs through tunneling nanotubes (TNTs) during transplantation and that this process is crucial for successful EC engraftment in vivo.. This disclosure also shows that preemptively transplanting exogenous mitochondria into ECs to enhance engraftment in vivo. This approach induced a transient cytoprotective effect via mitophagy, enabling EC engraftment without MSC support. Our findings suggest potential for a new single-cell therapy strategy based on autologous EC engraftment, which may facilitate clinical translation.

[0076] The role of mitochondrial transfer as a form of cell-to-cell signaling in health and disease is an area of active investigation. Recent studies have shown that mitochondrial transfer between different cell types is critical for tissue repair, including the transfer from MSCs to lung alveolar epithelial cells and astrocytes to neurons. These studies reported a significant increase in ATP production in the recipient cells, suggesting functional incorporation of exogenous mitochondria into the recipient endogenous mitochondrial pool. However, the underlying mechanisms remain elusive, and reconciling how a small number of exogenous mitochondria could produce such substantial beneficial effects has been challenging.

[0077] The data in present disclosure demonstrate that mitochondrial transfer serves as a mode of cell-to-cell communication in the vascular system. Under stressful conditions, such as during cell transplantation, perivascular MSCs transiently transferred mitochondria to neighboring ECs via TNTs, enhancing their bioenergetics and overall fitness, particularly their engraftment ability. Surprisingly, the process did not depend on transferring functional mitochondria. Enhanced EC engraftment was also observed after transferring mitochondria previously rendered dysfunctional by either Ca2+overload-mediated depolarization or mtDNA degradation. Mechanistically, once internalized, the exogenous mitochondria triggered mitophagy through the canonical PINK 1 -PARKIN signaling pathway and were rapidly degraded in autophagosomes. Activation of mitophagy mediated the observed beneficial effects in recipient ECs, which were completely abrogated by blocking PINK1- P ARKIN signaling. These findings offer the first insights into a potential mechanism regulating the effects produced by mitochondrial transfer. The transferred mitochondria serve as a trigger for mitophagy and do not require functional competence, explaining how a few exogenous mitochondria could contribute significantly to ATP production in recipient cells. Further studies are needed to determine whether this mechanism, the transient activation of mitophagy, also mediates the beneficial effects described after mitochondrial transfer in other cellular systems beyond the vasculature.

[0078] In addition to natural transfer from MSCs, the data presented herein showed that artificial transplantation of exogenous mitochondria could also trigger mitophagy in ECs, resulting in similar cytoprotective effects, including enhanced engraftment capability. Recently, mitochondrial transplantation has gained relevance as a potential treatment for multiple diseases, including cancer35, Parkinson36, and myocardial infarction37,38. The beneficial effects observed in preclinical models led to a recent first-in-human study in pediatric patients with myocardial ischemia, showing promising results39. However, the mechanisms of action by which the in vivo transplanted mitochondria amplify bioenergetics in recipient cells remain debated40,41, and the specific effects of mitochondrial transplantation in ECs have not been examined.

[0079] In previous studies, there were two main points of contention concerning the mechanism underlying mitochondrial transplantation, including 1) uncertainty on whether mitochondria could survive and retain activity throughout the transplantation process and 2) skepticism on how a low number of transplanted mitochondria could significantly increase ATP in the recipient cells. Nevertheless, our findings suggest a possible mechanism that could reconcile these concerns.

[0080] First, the data presented herein demonstrated that the exogenous mitochondria did not need to remain functional and merely served as a trigger of mitophagy once internalized into the recipient cells. Indeed, all depolarized, Ca2+overloaded, and even mtDNA-free mitochondria could effectively trigger mitophagy in ECs. Without being bound by theory, upon internalization, the exogenous mitochondria were always rapidly degraded in autophagosomes and thus did not incorporate into the endogenous mitochondrial pool in any significant way.

[0081] Second, because they act as triggering agents, the number of mitochondria required to activate mitophagy can be minuscule. Moderate mitophagy can benefit cells by virtue of degrading dysfunctional mitochondria and replacing them with new organelles via mitochondrial biogenesis, which ultimately translates into a meaningful increase in ATP. Indeed, the data presented herein showed that once mitophagy was triggered in ECs, expression of genes mediating mitochondrial biogenesis (e.g., PPARGC1B, TMEM59, GFM1, TEAD4) was significantly upregulated, followed by a marked increase in ATP production. In short, we demonstrated that the internalization of a small number of nonfunctional mitochondria could effectively trigger mitophagy and produce significant beneficial effects on the receiving ECs.

[0082] The data presented herein have direct translational implications. Importantly, the ex vivo artificial mitochondrial transplantation effectively improves the engraftment capacity of ECs in ischemic tissues, where they form new blood vessels and provide robust therapeutic effects, without the need for additional cell types. The reliance on a single cell type simplifies clinical translation enormously. We envision our mitochondrial transplantation approach could become the basis for a new strategy in vascular cell therapies. Whether this strategy could benefit other forms of cell therapies remains to be determined.

[0083] Populations of Endothelial Cells (ECs) and Methods of Making EC Populations

[0084] Compositions described herein and useful in any of the methods herein include populations of endothelial cells that have been contacted with a composition comprising isolated mitochondria (optionally exogenous mitochondria). Generally speaking, these cell populations do not require (and in some embodiments, do not include) multiple cell types, such as support cells (e.g., perivascular cells). In some embodiments, the populations of endothelial cells described herein include a single cell type: ECs (e.g., endothelial cells comprising isolated and / or exogenous mitochondria, mitoAT-ECs).

[0085] Populations of mitoAT-ECs can comprise ECs from a number of sources. In some embodiments, the ECs comprise any one or more of human umbilical vein endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs (e.g., white adipose tissue-derived ECs, watECs), organ-specific endothelial cells, and / or induced pluripotent stem cells (iPSCs)-derived ECs (iECs). In some embodiments, the organ-specific ECs are from an organ selected from: heart, muscle, kidney, testis, ovary, lymphoid, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine, and small intestine. In some embodiments, the ECs are isolated from a human.

[0086] In some embodiments, the populations of endothelial cells described herein (e.g., a population of mitoAT-ECs) do not include multiple cell types. In some embodiments, the populations of endothelial cells (e.g., a population of mitoAT-ECs) do not include support cells (e.g., perivascular cells). In some embodiments, the support cells are perivascular cells; optionally, the perivascular cells can comprise any one or more (or all) of: smooth muscle cells, pericytes, fibroblast, mesenchymal stromal cells (MSCs), and / or perivascular cells derived from human induced pluripotent stem cells (iPSCs). In some embodiments, the populations of endothelial cells described herein (e.g., any of the populations of mitoAT- ECs) do not include any perivascular cells.

[0087] In some embodiments, the populations of endothelial cells include multiple cell types but do not include support cells (e.g., perivascular cells). For example, a useful cell population for a transplant subject (e.g., a subject receiving or scheduled to receive an organ, tissue, or cell transplant) can include any population of endothelial cells described herein and a population of cells from the organ, tissue, or cell transplant without the presence of support cells, (e.g., perivascular cells or MSCs).

[0088] Also described herein are methods of making populations of endothelial cells (ECs) comprising transplanted mitochondria (mitoAT-ECs). In some embodiments, the method comprises: iv. obtaining a sample (e.g., a sample comprising ECs) from a subject (e.g., blood or a tissue sample); v. isolating a population of ECs from the sample; vi. contacting the population of ECs with isolated mitochondria (optionally, exogenous mitochondria), thereby creating the population of mitoAT-ECs.

[0089] In some embodiments, the sample comprising ECs is a human sample. In some embodiments, the sample comprising ECs is blood (e.g., human umbilical cord blood) or tissue (e.g., subcutaneous adipose tissue). In some embodiments, the sample need not contain ECs. In some embodiments, human induced pluripotent stem cells (iPSC) technology can be used and cells from any donor to be reprogrammed into a pluripotent, self-renewing state and thus allow the expansion of a homogeneous population of cells (e.g., ECs) from any genetic background. iPSCs may ultimately result in cell therapies generated from the patient's own cells in an autologous transplantation that may prevent graft rejection. Briefly, iPSCs have been generated by expression of several key genes shown to be required for full reprogramming, namely combinations of: Oct4, Sox2, Klf4, c-Myc, 1-Myc, Lin28, and / or Nanog. Thus, in some embodiments, step ii. comprises reprogramming the cells in the sample to iPSCs and differentiating the iPSCs into ECs, thereby isolating a population of ECs from the sample. Additional information, including additional methods of isolating, making, and differentiating iPSCs are known in the art (e.g., US 20240228951 Al; US 20240050483 Al; US 2022 / 0243174; US 20200385685 Al; US 20200182861 Al; US 2018 / 0371422; WO 2015 / 073625; US 2016 / 0002604; US 2014 / 0199274; US 2013 / 0052268; US 2012 / 0128655; and US 2009 / 0226401; as well as US 11,898,169; US 11,001,809; US 10,844,356; US 10,676,165; US 9,657,273; US 9,750,768; US 9,580,689; and US 9,376,664), each of the foregoing is incorporated herein by reference in its entirety.

[0090] In some embodiments, the ECs comprise any one or more of human umbilical vein endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue- derived ECs (e.g., white adipose tissue-derived ECs, watECs), organ-specific endothelial cells, and / or iECs. In some embodiments, the organ-specific endothelial cells are from an organ selected from: heart, muscle, kidney, testis, ovary, lymphoid, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine, and small intestine.

[0091] In some embodiments, a population of mitoAT-ECs described herein can be made using mitochondria isolated from a population of donor cells. In some embodiments, the donor cells are from the same subject as the sample (i.e., autologous donor cells). In some embodiments, the donor cells are from a subject different from the subject from which the sample was obtained in step (i) (i.e., allogenic donor cells). In some embodiments, the donor cells are any cell type with mitochondria. In some embodiments, the donor cells are human. In some embodiments, the donor cells are perivascular cells (e.g., mesenchymal stromal / stem cells (MSCs)) or any cell type such as described below.

[0092] Mitochondria for use in the present application can be isolated or provided from any source, e.g., isolated from cultured cells or tissues. Exemplary cells include, but are not limited to, muscle tissue cells, cardiac fibroblasts, cultured cells, HeLa cells, prostate cancer cells, yeast, among others, and any mixture thereof. Exemplary tissues include, but are not limited to, liver tissue, skeletal muscle, heart, brain, kidney, and adipose tissue. Mitochondria can be isolated from cells of an autogenous source, an allogeneic source, and / or a xenogeneic source. In some instances, mitochondria are isolated from cells with a genetic modification, e.g., cells with modified mtDNA or modified nuclear DNA. In some embodiments, mitochondria need not be respiratory competent.

[0093] Mitochondria can be isolated from cells or tissues by any means known to those of skill in the art. In one embodiment, tissue samples or cell samples are collected and then homogenized. Following homogenization, mitochondria are isolated by repetitive centrifugation. Alternatively, the cell homogenate can be filtered through nylon mesh filters. Additional methods of isolating mitochondria are described, for example, in McCully JD, Cowan DB, Pacak CA, Toumpoulis IK, Dayalan H and Levitsky S, Injection of isolated mitochondria during early reperfusion for cardioprotection, Am J Physiol 296, H94-H105. PMC2637784 (2009); Frezza, C., Cipolat, S., & Scorrano, L, Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts. Nature protocols, 2(2), 287- 295 (2007); and a PCT application entitled “Products and Methods to Isolate Mitochondria” (PCT / US2015 / 035584; WO 2015192020); each of which is incorporated by reference in their entirety for any and all purposes.

[0094] In some embodiments, the mitochondria are isolated from a number of donor cells at a ratio of about 20: 1, 19: 1, 18: 1, 17: 1, 16: 1, 15: 1 :, 14: 1 : 13:1, 12: 1, 11 : 1, 10:, 9: 1, 8: 1, 7:1, 6: 1, 5: 1, 4: 1 3: 1, 2: 1, or 1 : 1 donor cells:ECs. For example, in some embodiments, the mitochondria are isolated from a number of donor cells that is a ratio of donor cells:ECs of 3: 1 (e.g., mitochondria from 3 donor cells (e.g., MSCs) for each recipient EC). In another example, in some embodiments, the mitochondria are isolated from a number of donor cells that is a ratio of donor cells:ECs of 10: 1 (e.g., mitochondria from 10 donor cells (e.g., MSCs) for each recipient EC).

[0095] In some embodiments, the isolated mitochondria (e.g., exogenous mitochondria) are incubated with the ECs in the contacting step for a time sufficient to internalize at least a portion of the mitochondria. In some embodiments, the mitochondria are incubated with the ECs about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 ,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 hours). In some embodiments, at least about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% of the ECs internalize the isolated mitochondria. In some embodiments, the population of mitoAT-ECs comprises at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% mitoAT-ECs. In some embodiments, about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% cell in the population of mitoAT-ECs comprise exogenous mitochondria. In some embodiments, the mitochondria are not respiratory competent. Additional information about making cell populations, including 3D cell cultures is known in the art (See, e.g., US 2024 / 0287463 Al; US 2024 / 0287463 Al; US 2020 / 0182861 Al; US 2019 / 0376044 Al; US 2020 / 0199541 Al; US 2023 / 0287357 Al; US 2023 / 0174949 Al; US 2023 / 0364267 Al; US Pat. No. 11,214,768; WO 2024 / 133285; WO 2022 / 226337; and WO 2023 / 196683), each of the foregoing is incorporated herein by reference in its entirety.

[0096] In some embodiments, disclosed herein are populations of mitoAT-ECs made using any of the methods disclosed herein. Cell Compositions and Formulations

[0097] Also disclosed herein are compositions comprising populations of ECs comprising transplanted mitochondria (e.g., exogenous mitochondria) made using any of the methods described herein (see, e.g., above section). In some embodiments, described herein, inter alia, are populations of mitoAT-ECs assembled into a network of CD31+ vascular structures.

[0098] In some embodiments, also described herein vascular organoids (VOs) and 3D cell cultures comprising the CD31+ vascular structures comprising a population of mitoAT-ECs. In some embodiments, the VOs or 3D cell cultures comprise a network of lumenized vessels with apical-basal polarization. In some embodiments, the VOs or 3D cell cultures comprise a network of lumenized vessels comprising arterial, venous, and / or capillary ECs.

[0099] Also described herein, inter alia, are compositions comprising any of the populations of cells described herein (e.g., mitoAT-ECs). In some embodiments, the composition may further comprise one or more of an agent, an excipient, a matrix, or a gel. In some embodiments, any composition may further comprise a gel or matrix comprising a hydrogel. In some embodiments, any composition may further comprise a gel or matrix comprising gelatin, collagen, fibrinogen, thrombin, fibrin, or any combinations thereof. In some embodiments, any composition may further comprise a gel or matrix comprising about 1.5 mg / mL collagen, about 30 pg / mL fibrinogen, and about 1 mg / mL human fibronectin. In some embodiments, the matrix can contain collagen and / or fibrin. In some embodiments, fibrin is formed with fibrinogen and thrombin, (optionally, about 50 pg / mL thrombin). In some embodiments, any composition may further comprise a gel or matrix comprising any one or more of gelatin, collagen, fibrinogen, laminin, entactin, or combinations thereof. In some embodiments, any composition may further comprise a gel or matrix comprising laminin, entactin, and collagen. In some embodiments, any composition may further comprise a gel or matrix comprising about 5.25 mg / mL laminin, about 5.25 mg / mL entactin, and about 0.2 mg / mL collagen IV. In some embodiments, any composition may further comprise a gel or matrix is Matrigel™. Matrigel is known in the art (U.S. Pat. No. 4,829,000).

[0100] In some embodiments, any of the populations of cells, vascular organoids, or compositions can formulated to be administered to the subject (e.g., subcutaneous, intradermal, intramuscular, intranodal, intravenous, intraprostatic, intratumor, intralymphatic, intraarticular, intracardiac, and intraperitoneal injection). In some embodiments, any of the populations of cells, vascular organoids, or compositions can be used to vascularize a tissue or organ prior to transplantation to the patient, and accordingly, composition described herein can also comprise cells and tissue from any one or more of the following skin, heart, kidney, testis, ovary, bone, lymph, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine and small intestine.

[0101] A pharmaceutical composition can be formulated for various clinical uses, e.g., imaging, treating wounds, treating injuries, preserving organs, improving mitochondrial functions in organs or tissues, and skin care. In some cases, the pharmaceutically acceptable carrier is a contrast agent for imaging purpose. In some embodiments, the pharmaceutical composition may include antiseptic agents, antibacterial agents (e.g., antibiotics), antifungal agents, disinfectants, analgesic agents, anesthetic agents, steroids, nutritional supplements, ethereal oils, etc. An anesthetic agent is a drug that can prevent pain during surgery or treatment. Exemplary analgesic agents include, without limitation, paracetamol, nonsteroid anti-inflammatory drugs, salicylates, ibuprofen and lidocaine. Exemplary antibacterial agents include, without limitation, di chlorobenzyl alcohol, amylmetacresol and antibiotics. Exemplary antibiotics include penicillins carbapenems, cephalosporins aminoglycosides, bacitracin, gramicidin, mupirocin, chloramphenicol, thi amphenicol, lincomycin, clindamycin, macrolides, novobiocin, polymyxins, rifamycins, spectinomycin, tetracyclines, vancomycin, teicoplanin, streptogramins, anti- folate agents, sulfonamides, trimethoprim, pyrimethamine, nitrofurans, methenamine mandelate, methenamine hippurate, nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acid, cycloserine, capreomycin, ethionamide, prothionamide, thiacetazone and viomycin. Antiseptic agents are antimicrobial substances that can be applied to living tissue / skin to reduce the possibility of infection, sepsis, or putrefaction. Exemplary antiseptics include, without limitation, chlorhexidine and salts thereof, benzalkonium and salts thereof, triclosan and cetylpyridium chloride. Exemplary antifungal agents include, without limitation, tolnaftate, miconazole, fluconazole, clotrimazole, econazole, ketoconazole, itraconazole, terbinafine, amphotericin, nystatin and natamycin. Exemplary steroids include, without limitation, prednisone acetate, prednisone valerate, prednisolone, alclometasone dipropionate, fluocinolone acetonide, dexamethasone, methylprednisolone, desonide, pivolate, clocortolone pivolate, triamcinolone acetonide, prednicarbate, fluticasone propionate, flurandrenolide, mometasone furoate, desoximetasone, betamethasone, betamethasone dipropionate, betamethasone valerate, betamethasone propionate, betamethasone benzoate, diflorasone diacetate, fluocinonide, halcinonide, amcinonide, halobetasol propionate, and clobetasol propionate. Exemplary nutritional supplements include, without limitation, vitamins, minerals, herbal products and amino acids. Vitamins include without limitation, vitamin A, those in the vitamin B family, vitamin C, those in the vitamin D family, vitamin E and vitamin K. Ethereal oils include without limitation, those derived from mint, sage, fir, lavender, basil, lemon, juniper, rosemary, eucalyptus, marigold, chamomile, orange and the like. Many of these agents are described, e.g., in WO 2008152626, which is incorporated by reference in its entirety for any and all purposes.

[0102] Additional information and methods of formulating populations of cells, vascular organoids, or compositions are known in the art (See, e.g., US 2024 / 0287463 Al; US 2024 / 0287463 Al; US 2020 / 0182861 Al; US 2019 / 0376044 Al; US 2020 / 0199541 Al; US 2023 / 0287357 Al; US 2023 / 0174949 Al; US 2023 / 0364267 Al; US Pat. No. 11,214,768; WO 2024 / 133285; WO 2022 / 226337; and WO 2023 / 196683), each of the foregoing is incorporated herein by reference in its entirety.

[0103] Methods of Using the Populations of ECs and Compositions Comprising the ECs

[0104] Described herein, inter alia, are methods of administering any of the populations of cells (e.g., mitoAT-ECs) and / or VOs described herein to a subject. Useful populations of cells and VOs are described throughout, for example, in the above two sections.

[0105] In some embodiments, the subject has, or is at risk of having or developing, a disease or disorder associated with a blood vessel disorder (e.g., leaky blood vessels, narrow blood vessels, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias and valvular heart disease cerebral cavernous malformations, hemorrhagic strokes, hereditary hemorrhagic telangiectasias, or arteriovenous malformations). In some embodiments of any of the methods described herein, the subject has (or is at risk of having or developing) any one or more of the following: a metabolic disorder, a myocardial infarction, diabetes, diabetic retinopathy, an ischemic injury, a disease or disorder of the blood vessels, atherosclerosis, Age-related Macular Degeneration (AMD), Pulmonary Arterial Hypertension (PAH), Hereditary Hemorrhagic Telangiectasia (HHT), peripheral artery disease (PAD), arteriovenous fistulas (e.g., dialysis patients), tumor angiogenesis, tumor metastasis, a cancer, a metabolic disease, an immunological disease, a mitochondrial dysfunction disorder, a stroke, and / or a wound (optionally, a chronic wound; e.g., a diabetic ulcer). In some embodiments, described herein are methods to treat, or reduce the risk of developing, any one or more of the following: a disease or disorder of the blood vessels, aberrant vasculature, leaky blood vessels, narrow blood vessels, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, a myocardial infarction, cardiotoxicity (e.g., caused by chemotherapy), cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, valvular heart disease cerebral cavernous malformations, hemorrhagic strokes, hereditary hemorrhagic telangiectasias, arteriovenous malformations, a metabolic disorder, diabetes, diabetic retinopathy, an ischemic injury, an IRI injury, atherosclerosis, Age-related Macular Degeneration (AMD), Pulmonary Arterial Hypertension (PAH), Hereditary Hemorrhagic Telangiectasia (HHT), peripheral artery disease (PAD), arteriovenous fistulas (e.g., dialysis patients), tumor angiogenesis, tumor metastasis, a cancer, a metabolic disease, a stroke, and / or a wound (optionally, a chronic wound; e.g., a diabetic ulcer).

[0106] Also described herein are methods of increasing blood vessel formation, methods of vascular cell therapy, and / or methods of vascular generation or regeneration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a population of mitoAT-ECs. In some embodiments, the methods further comprising identifying a subject in need or increasing blood vessel formation, vascular cell therapy, vascular generation, and / or vascular regeneration.

[0107] Described herein, inter alia, are methods of increasing vascular development, angiogenesis, and cell junction via administering a population of mitoAT-ECs to a subject. In some embodiments, described herein are methods of administering a population of mitoAT- ECs (e.g., therapeutic vascularization) and methods of modeling vascular diseases (e.g., a 3D vascular organoid (VO)) are also described herein.

[0108] Also described herein are methods of tissue engineering (e.g., small-diameter vascular grafts). Tissue-engineered small-diameter vascular grafts are bioengineered constructs designed to replace damaged or diseased blood vessels. Small-diameter vascular grafts are particularly relevant for clinical applications such as coronary artery bypass grafting (CABG), peripheral artery disease (PAD), and arteriovenous fistulas for dialysis patients. In some embodiments, any of the populations of cells described herein, any of the VOs or 3D cell cultures described herein, and / or any of the compositions described herein (e.g., a composition comprising mitoAT-ECs) are used as a cell source for tissue engineering applications, particularly in the development of small-diameter vascular grafts. In some embodiments, described herein are methods of transplanting any of the populations of cells described herein, any of the VOs or 3D cell cultures described herein, and / or any of the compositions described herein. In some embodiments, the method comprises administering to the subject an effective amount of the population of cells, vascular organoids, or composition.

[0109] In some embodiments, described herein are methods of increasing blood vessel formation comprising administering to a subject in need thereof an effective amount of any of the populations of cells described herein, any of the VOs or 3D cell cultures described herein, and / or any of the compositions described herein. In some embodiments, the method comprises: identifying a subject in need of increased blood vessel formation; and administering to the subject an effective amount of any of the populations of cells described herein, any of the VOs or 3D cell cultures described herein, and / or any of the compositions described herein.

[0110] Also described herein, inter alia, are methods of increasing vascular generation or vascular regeneration comprising administering to a subject in need thereof an effective amount of any of the populations of cells described herein, any of the VOs or 3D cell cultures described herein, and / or any of the compositions described herein. In some embodiments, the method comprises: identifying a subject at in need of vascular generation or vascular regeneration; administering to the subject an effective amount of any of the populations of cells described herein, any of the VOs or 3D cell cultures described herein, and / or any of the compositions described herein.

[0111] Also described herein, inter alia, are methods of vascular cell therapy comprising administering to a subject in need thereof an effective amount of any of the populations of cells described herein, any of the VOs or 3D cell cultures described herein, and / or any of the compositions described herein. In some embodiments, the method comprises: identifying a subject in need of vascular cell therapy; administering to the subject an effective amount of any of the populations of cells described herein, any of the VOs or 3D cell cultures described herein, and / or any of the compositions described herein.

[0112] Methods described herein can also be used to increase blood flow and / or oxygen delivery for various organs or tissues (e.g., heart, lung, kidney, brain, skeletal muscle). In some instances, methods described herein can be used to treat peripheral vascular disease PVD). PVD is a blood circulation disorder that causes the blood vessels outside of the heart and brain to narrow, block, or spasm. This can happen in the arteries or veins. PVD typically causes pain and fatigue, often in the legs, and especially during exercise.

[0113] In some embodiments of any of the methods described herein, the subject or patient may be undergoing an organ transplant, selected to undergo an organ transplant, or need vascularization of an organ. In some embodiments, the organ is selected from the group consisting of skin, heart, kidney, testis, ovary, bone, lymph, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine and small intestine. In some embodiments of any of the methods described herein, the population of cells, vascular organoid, or composition is administered to the subject, before, during, or after a cell transplant, tissue transplant, or organ transplant.

[0114] The disclosure also provides methods of improving transplanted organ, tissue and / or cell integration. In some embodiments, the methods comprise contacting the ECs of an organ or tissue with isolated mitochondria (e.g., exogenous mitochondria). In some embodiments, the ECs are contacted with the mitochondria before the organ or tissue is transplanted in the subject. In some embodiments, the ECs are contacted with the mitochondria during the transplantation surgery. In some embodiments, the ECs are contacted with the mitochondria after the organ or tissue is transplanted in the subject. The contacting can be performed ex vivo and / or in situ by any method known in the art. For example, the exposure may be performed ex vivo in any chamber or space having sufficient volume for submerging the ECs, completely or partially, in a composition comprising the isolated mitochondria. In some embodiments, the ECs of an organ may be perfused with a composition comprising the mitochondria. The term “perfusion” is an art recognized term, and relates to the passage of a liquid, e.g., a composition comprising exogenous mitochondria, through the ECs of an organ or tissue. Optionally, in in situ or ex vivo perfusions, the ECs can be perfused with a wash solution, e.g., UW solution, prior to perfusion with a composition comprising the mitochondria, to remove the donor's blood from the organ. As another option, the UW solution can include the mitochondria. Additional in situ exposures can be performed by any method known in the art (see e.g., Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press, 1994). In some embodiments, the tissue is skin tissue or bone marrow. In some embodiments, the cells are stem cells. In these cases, population of cells, vascular organoid, or composition can improve the integration of the transplanted organ, tissue and cells in the recipient’s body. In some embodiments, these methods can be used to control, prevent, reduce, and / or treat IRI damage for transplanted organs and tissues, including but not limited to heart, brain, liver, kidney, lung, pancreas, eye, skeletal muscle, and skin. In some embodiments, the organ or tissue is selected from the group consisting of brain, thyroid, thymus, heart, lung, liver, pancreas, kidney, bladder, pharynx, esophagus, stomach, gallbladder, pharynx, larynx, ovaries, uterus, placenta, testes, prostate, spleen, spinal cord, pancreas, small intestine, large intestine, colon, eye, skin, skin tissue, skeletal muscle, adipose tissue, facial muscle, bone marrow tissue, eye tissue, heart valves, veins, tendons, and combinations thereof.

[0115] In some embodiments of any of the methods described herein, vascularization comprises the formation of an artery, a vein, a capillary, an arteriole, a venule, or any combination thereof.

[0116] In some embodiments of any of the methods described herein, the methods normalize and / or correct aberrant vasculature; where the vasculature lacks stable structure and / or function; and / or where there is leaking or narrowing of a blood vessel, etc. In some embodiments of any of the methods described herein, the subject has a disorder characterized by aberrant vasculature (e.g., diabetic retinopathy, tumor angiogenesis, tumor metastasis, stroke, ischemic injury, reperfusion injury, atherosclerosis, Age-related Macular Degeneration (AMD), Pulmonary Arterial Hypertension (PAH), Hereditary Hemorrhagic Telangiectasia (HHT), peripheral artery disease (PAD), arteriovenous fistulas (e.g., dialysis patients), and / or a wound (e.g., a chronic wound; e.g., a diabetic ulcer).

[0117] Without being bound by theory, narrowing of blood vessels result from plaque buildup on the walls of the vessels and / or chronic inflammation, which can include conditions such as ischemic disease, peripheral artery disease, angina, heart attack, stroke, Reynaud's disease, Brueger's disease, hypertension, chemotherapeutic compromise, and erectile dysfunction. Furthermore, each condition and blood vessel disease frequently results in distal vessel injury and / or dysfunction that, in turn, complicates and, in many cases, exacerbates revascularization strategies and recovery from ischemic injury. The mtioAT-ECs, cell populations, VOs, and / or compositions described herein can potentially restore the integrity of the vasculature, help stabilize these vessels, reduce the severity of any injury caused by these conditions, and / or prevent further complications from arising as a result of the condition(s).

[0118] Without being bound by theory, tumors often exhibit abnormal vasculature characterized by a lack of proper mural cell coverage, leading to leaky and dysfunctional blood vessels. Introducing any of the mitoAT-ECs, cell populations, VOs, and compositions herein could help stabilize these vessels, improving the delivery of therapeutics, and / or reduce metastasis.

[0119] Without being bound by theory, in diabetic retinopathy, pericyte loss leads to weakened blood-retinal barriers, resulting in retinal ischemia and neovascularization. The mitoAT-ECs, cell populations, VOs, and / or compositions described herein can potentially restore the integrity of retinal vasculature and / or reduce the progression of the disease.

[0120] Without being bound by theory, after a stroke or ischemic injury, there is often a loss of vascular integrity and a need for vascular repair. The mitoAT-ECs, cell populations, VOs, and / or compositions described herein can aid in re-establishing stable blood vessels and / or promoting recovery of the affected tissue.

[0121] Without being bound by theory, in atherosclerosis, the stability of blood vessels is compromised due to inflammatory processes and endothelial dysfunction. The mitoAT-ECs, cell populations, VOs, and / or compositions described herein can help reinforce vascular walls and / or mitigate the progression of atherosclerotic plaques.

[0122] Without being bound by theory, in wound healing, chronic wounds, such as diabetic ulcers, often suffer from poor vascularization and deficient mural cell coverage. The mitoAT- ECs, cell populations, VOs, and / or compositions described herein can enhance angiogenesis and vascular stability, promoting better wound healing outcomes.

[0123] Without being bound by theory, in Age-related Macular Degeneration (AMD), especially the wet form, choroidal neovascularization occurs with deficient pericyte support, leading to fragile and leaky vessels. The mitoAT-ECs, cell populations, VOs, and / or compositions described herein can help in providing the necessary support to these new vessels, reducing leakage, and / or reducing vision loss.

[0124] Without being bound by theory, Pulmonary Arterial Hypertension (PAH) is characterized by abnormal proliferation of pulmonary vascular cells and deficient pericyte coverage, leading to vascular remodeling and hypertension. The mitoAT-ECs, cell populations, VOs, and / or compositions described herein could stabilize these blood vessels and / or alleviate one or more symptoms (e.g., hypertension).

[0125] Without being bound by theory, Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic disorder leading to abnormal blood vessel formation with deficient mural cell coverage, resulting in bleeding and arteriovenous malformations. The mitoAT-ECs, cell populations, VOs, and / or compositions described herein can potentially normalize these vessels and / or reduce bleeding episodes. In some embodiments of any of the methods described herein, the population of cells, vascular organoid, or composition is administered to the subject by direct injection into a blood vessel or subcutaneous, intradermal, intramuscular, intranodal, intravenous, intraprostatic, intratumor, intralymphatic, intraarticular, intracardiac, and intraperitoneal injection.

[0126] In some embodiments of any of the methods described herein, the population of ECs (e.g., mitoAT-ECs) does not comprise support cells. In some embodiments of any of the methods described herein, a population of support cells (e.g., perivascular cells) is not administered before, with, or after the administration of a population of ECs (e.g., a population of mitoAT-Ecs). In some embodiments of any of the methods described herein, the population of cells comprises a single cell type (e.g., ECs, e.g., mitoAT-ECs).

[0127] An effective amount can be administered in one or more administrations, applications or dosages. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of any therapeutic population of cells, VO, 3D cell culture, composition described herein can include a single treatment or a series of treatments.

[0128] A skilled artisan will be able to determine and identify a subject or patient suitable for any of the methods described herein (e.g., a subject suffering from or at risk of having a stroke, a subject suffering from or at risk of having diabetes, a subject suffering from or at risk of having diabetic retinopathy, a subject suffering from or at risk of having an ischemic injury, a subject suffering from or at risk of having a disease or disorder of the blood vessels, and / or a subject having or selected to have a transplant).

[0129] Additional information about methods of formulating and administering cell populations, VOs and 3D cell cultures are known in the art See, e.g., US 2024 / 0287463 Al; US 2024 / 0287463 Al; US 2020 / 0182861 Al; US 2019 / 0376044 Al; US 2020 / 0199541 Al; US 2023 / 0287357 Al; US 2023 / 0174949 Al; US 2023 / 0364267 Al; US Pat. No.

[0130] 11,214,768; WO 2024 / 133285; WO 2022 / 226337; and WO 2023 / 196683; each of which is incorporated herein by reference in its entirety). EXAMPLES

[0131] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0132] Methods and Materials

[0133] Cell culture

[0134] Human endothelial colony-forming cells (ECFCs; referred to herein as ECs) and mesenchymal stem cells (MSCs) were isolated from human umbilical cord blood and subcutaneous adipose tissue, respectively, following established protocols10,19. ECs were cultured on plates coated with 1% (w / v) gelatin and maintained in EC medium consisting of EGM-2 (minus hydrocortisone; PromoCell) supplemented with 20% fetal bovine serum (FBS; Hyclone) and IX glutamine-penicillin-streptomycin (GPS; Invitrogen). MSCs were cultured on uncoated plates in mesenchymal stem cell growth medium (MSCGM; ATCC) containing MSC growth supplement (ATCC) and IX GPS. ECs and MSCs from passages 6 to 12 were used in all experiments.

[0135] Generation of mitoDsRed-labeled cells

[0136] MitoDsRed-labeled cells were generated by transfecting cells with a piggyBac transposon vector containing a CMV promoter-driven DsRed reporter gene fused to a mitochondrial targeting sequence from cytochrome C oxidase (mitoDsRed) and a super piggyBac transposase expression vector (System Biosciences). A 5: 1 ratio of transposon to transposase vectors was employed, using a total of 2.4 pg of DNA to transfect IxlO6ECs or MSCs. Following hygromycin selection, the transposon system facilitated stable expression of the mitoDsRed reporter gene, allowing mitochondrial visualization under a fluorescence microscope. MitoTracker™ Green FM Special Packaging (Thermo fisher; Cat.# M7514) was used to visualize total mitochondria in indicated experiments.

[0137] Generation of mitoAPEXl-labeled MSCs

[0138] Mitochondrial-specific APEX2 -labeled cells were generated by transfecting a PiggyBac vector carrying a CMV promoter-driven APEX2 gene fused with a mitochondrial targeting sequence from cytochrome C oxidase (mitoAPEX2) and a super PiggyBac transposase expression vector (System Biosciences). The ratio between transposon and transposase vectors was 5: 1, and 2.4 pg of DNA was used to transfect IxlO6ECs or MSCs. After hygromycin selection, stable mitoAPEX2 reporter gene expression was achieved. To visualize APEX2 activity under TEM, cells were chemically fixed, stained with DAB and H2O2 for 15 min, and processed for TEM using heavy -metal staining. DAB staining provided strong contrast of the mitochondrial matrix in transfected cells.

[0139] In vitro mitochondrial transfer via co-culture

[0140] Co-culture experiments involved human ECs and mitoDsRed-labeled MSCs on 1% gelatin-coated plates in EC-medium. Cell density impact on mitochondrial transfer was assessed by co-culturing mitoDsRed-MSCs and ECs in a 1 : 1 ratio, with total cell numbers ranging from 0.05 to 0.8xl06per well. Different donor-to-recipient ratios, from 1 : 10 to 10: 1, were also evaluated, keeping the total cell count at 4xl05per well. The effects of environmental conditions were studied by co-culturing mitoDsRed-MSCs and ECs at a 1 : 1 ratio, with 4xl05total cells, in either 2D or 3D (collagen hydrogel) conditions, under normoxia (21% O2) or hypoxia (1% O2). After 24 hours, analysis was conducted via microscopy and flow cytometry. Selected experiments examined the role of VEGF (10 ng / mL), bFGF (10 ng / mL), TNFa (10 ng / mL), anti-TNFa antibody (10 ng / mL), IL-la (20 ng / mL), IL-ip (20 ng / mL), and anti-IL-la antibody (10 ng / mL) on mitochondrial transfer and TNT formation.

[0141] Immunofluorescence staining

[0142] Cells were seeded at a density of 60,000 cells / cm2in eight-well LAB-TEK chamber slides. Upon reaching confluency, cells were fixed using 4% paraformaldehyde (PF A), permeabilized with 0.1% Triton X-100 in PBS, and then blocked for 30 minutes with 5% horse serum (Vector Laboratories, catalog no. S-2000). Cells were then incubated with primary antibodies for 30 minutes at room temperature. Primary antibodies included antibodies against LC3B (Cell Signaling, Cat.# 2775), beta-actin (Abeam, Cat.# 8226), and alpha-tubulin (EP1332Y; Abeam, Cat.# ab52866). Following three washes with PBS, cells were incubated with secondary antibodies for 30 minutes at room temperature. Cells were washed three times with PBS and stained with 4',6-diamidino-2-phenylindole (DAPI; 0.5 pg / ml) for 5 minutes. Slides were mounted using DAKO fluorescence mounting medium (Agilent, catalog no. S302380-2). In vivo cell engraftment

[0143] Animal experiments were approved by the Institutional Animal Care and Use Committee at Boston Children’s Hospital and carried out in an AAALAC-approved facility. Human ECs (8xl05cells) were resuspended in 200 pL ice-cold collagen-fibrin-laminin hydrogel solution containing bovine collagen I (1.5 mg / ml; Trevigen), fibrinogen (3 mg / ml), laminin-1 (2 mg / mL), FGF-2 (1 pg / ml; PeproTech), and erythropoietin (5 pg / ml; ProSpec), with or without MSCs (1.2xl06cells). The mixture was subcutaneously injected into 6-week- old male athymic nu / nu mice (Massachusetts General Hospital, Boston, MA). During anesthesia, mice received 50 pl of thrombin (10 U / ml; Sigma-Aldrich) subcutaneously before 200 pl of cell-laden hydrogel solution. Supplements added to the cell-hydrogel mixture as needed included VEGF-A (R&D Systems; 100 ng / implant), bFGF (50 ng / implant), PDGFR inhibitor Tyrphostin AG1296 (Sigma; 1.33 pg / implant), or concentrated MSC-conditioned medium (100 pL / implants). Conditioned media were generated by 1.2xl06MSCs in EBM-2, 5% FBS, and concentrated 10-fold. Mice were euthanized, and grafts explanted at different time points (e.g., after 7 or 14 days). In some experiments, rhodamine-conjugated UEA-1 lectin (Vector Laboratories; 100 pL; 1 mg / mL in saline) was injected intravenously before harvesting. In others, TNFa or saline were injected into EC-MSC implants on day 14, with grafts explanted on day 16.

[0144] Flow cytometry and cell sorting

[0145] Cells were stained for flow cytometry and analyzed with a Guava easyCyte 6HT / 2L flow cytometer (Millipore Corporation) and FlowJo software (Tree Star Inc.). Antibody labeling was performed on ice for 20 min, followed by 3 washes with PBS (1% BSA, 0.2 mM EDTA) and fixation with 1% paraformaldehyde. Antibodies included PE-conjugated anti-h- CD90 (BD Biosciences, Cat.# 555596), APC-conjugated anti-h-CD90 (eBioscience, Cat.# 17-0909-41), and FITC-conjugated anti-h-CD31 (BD Biosciences, Cat.# 555445). In indicated experiments, cells were retrieved from explanted grafts before flow cytometry analysis. Grafts were removed from euthanized mice, and cells were retrieved via enzymatic digestion (1 mg / mL collagenase, 2.5 U / mL dispase) for 1 h at 37 °C. Cells were prepared into single-cell suspensions. When indicated, cells were sorted into hCD31+ and hCD31- cells using magnetic-activated cell sorting (MACS) with anti -human CD31 antibody-coated magnetic beads (DynaBead). In indicated experiments, cells were sorted into hCD31+mitoDsRed+ (mitoT-ECs) and hCD31+mitoDsRed- (unprimed-ECs) using fluorescence-activated cell sorting (FACS) with a FACSAria II 5-LASER sorter system (BD Bioscience). Rabbit anti-PINKl (Thermo Fisher, PA5-86941) was used for the detection of PINK1 proteins in the presence of isolated mitochondria.

[0146] Animal Experiments and Mouse hind limb ischemia model

[0147] All animal experiments and procedures adhered to a protocol approved by the Institutional Animal Care and Use Committee at Boston Children's Hospital in an Association for Assessment and Accreditation of Laboratory Animal Care-approved facility. Mouse methods were performed according to the guidelines and regulations in the approved protocol. Mice were housed under a 12-hour dark / light cycle with ambient temperature maintained at 68-74 degrees F and relative humidity at 30-70%. Sample size, including the number of mice per group, was determined to ensure adequate power and was based on the means and variations observed in our pilot experiments and historical data. No data were excluded. No exclusion criteria were applied for all analyses. Mice were randomly selected and allocated groups for each type of experiment. There was no expected outcome prior to the analysis, and blinding was not relevant.

[0148] Immune-deficient athymic nu / nu nude mice (10 weeks old) were induced diabetic with a single intraperitoneal injection of streptozotocin (STZ; 220 mg / kg) and had their blood glucose levels monitored after 2 days. Seven days after STZ injection, diabetic mice were anesthetized using isoflurane. Once anesthesia was achieved, 7-0 silk sutures were tied around the proximal and deep femoral artery and vein, and the vessels between the ties were cut to completely block blood flow. ECs (2xl06) were resuspended in 50 pL of ice-cold Phenol Red-free Matrigel (BD Bioscience, San Jose, CA) and injected into the muscle where the femoral artery and vein ligation occurred.

[0149] Laser Doppler imaging

[0150] Blood flow in hind limbs was analyzed using the LDI2-IR laser Doppler blood flow imager (Moor Instruments). Imaging was performed prior to surgery and on post-operative days 0, 1, 3, 5, 7, 10, 14, and 28. The relative changes in blood flow in the hind limbs were expressed as the ratio of the operated hind limb to the blood flow in the contra-lateral (opposite) hind limb, using the company's software. Bioluminescence imaging

[0151] Luciferase-expressing ECs (luc-ECs) were generated by transfection with a PiggyBac vector carrying a CMV promoter-driven firefly luciferase reporter gene and a super PiggyBac transposase expression vector (System Biosciences) or lentivirus transduction. A 5: 1 ratio between transposon and transposase vectors was used, with 2.4 pg of DNA for IxlO6ECs. Puromycin-selected luc-ECs demonstrated stable luciferase expression. Luc-ECs (8xl05cells) were resuspended in 200 pL ice-cold collagen-fibrin-laminin hydrogel, with or without MSCs (1.2xl06cells), and subcutaneously injected into 6-week-old male athymic nu / nu mice. Mice were imaged at 0.5 hours and on days 1, 3, 7, 14, and 28 post-injection using an IVIS 200 Imaging System (Xenogen Corporation). Mice were anesthetized, and luciferin (Promega) was injected intraperitoneally (125 mg / kg). Bioluminescence was detected 5 minutes post-luciferin administration, and data were analyzed using Live Image 3.0 (Xenogen Corporation).

[0152] Histology and immunofluorescence staining

[0153] Explanted grafts were fixed overnight in 10% buffered formalin, paraffin-embedded, and sectioned (7 pm). Hematoxylin and eosin (H&E)-stained sections were examined for erythrocyte-filled blood vessels. For immunostaining, sections underwent deparaffinization, antigen retrieval with tris-EDTA buffer (10 mM Tris-Base, 2 mM EDTA, 0.05% Tween-20, pH 9.0) or citric buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0), blocking with 5- 10% serum, and overnight incubation at 4°C with primary antibodies. Fluorescent staining used fluorescently-conjugated secondary antibodies (1 :200) and DAPI counterstaining (Vector Laboratories). Human-specific anti-CD31 antibody (Agilent; M082329-2 J; Clone JC70A), human-specific vimentin antibody (Abeam; Ab8069; clone V9), and Ulex Europaeus Agglutinin I (UEA-I; Vector Laboratories) stained human blood vessels. Perivascular cells were stained by anti-a-SMA antibody (Sigma; A2547; Clone 1 A4).

[0154] Microvessels density

[0155] Microvessel density was calculated as the average number of erythrocyte-filled vessels (vessels / mm2) in H&E-stained sections from implant centers. The entire area of each section was analyzed. Human-specific microvessel density was quantified in indicated experiments using human-specific CD31 (h-CD31) immunostained slides. Gene expression silencing by shRNA shRNA lentiviral particles targeting human TNFAIP2, MIR01, PARKIN, and PINK1, along with negative control shRNA constructs, were obtained from Santa Cruz Biotechnology. Cells (5xl05) were transduced with shRNA lentiviral particles (MOI 10:1) and Polybrene (5 pg / ml). After 48 h, cells were selected with 1 pg / mL puromycin. mRNA was isolated from transfected cells, and qRT-PCR was performed to validate shRNA silencing efficiency.

[0156] Mitochondria isolation and artificial transfer

[0157] Mitochondria were isolated from donor cells using the Mitochondria Isolation Kit for Cultured Cells (Thermo Scientific). Cells were harvested with trypsin, washed twice with cold PBS, and resuspended in 800 pL of buffer A with lOpL of Mitochondria Isolation Reagent B. After a 5-minute incubation on ice with intermittent vortexing, 800 pL of buffer C was added, and the cell suspension was homogenized with a Dounce Tissue Grinder. Following centrifugation at 700 * g for 10 minutes at 4°C, the supernatant was transferred to a new tube and centrifuged at 12,000 * g for 15 minutes at 4°C. The isolated mitochondrial pellet was resuspended in 500 pL buffer C with 1% BSA and then centrifuged at 3,000 x g for final purification. For artificial mitochondrial transfer, isolated mitochondria were mixed with EC-medium and added to ECs in culture at a 3: 1 donor-to-recipient cell ratio. For titration experiments, the number of isolated mitochondria was quantified using flow cytometry (FACSAria II 5-LASER sorter system) and then transferred at various mitochondria-to-cell ratios, including lx, 2x, 4x, 8x, 16x, 32x, and 64x mitochondria per cell. After 12 hours of mitochondrial transplantation, the plates were washed twice with PBS, and cells were maintained in culture in EC -medium.

[0158] Lysosome isolation and artificial transfer

[0159] Lysosomes were isolated utilizing differential sedimentation from supernatants derived post-mitochondrial isolation and purification (3,000 x g). To eliminate residual mitochondria, these supernatants were sequentially filtered through 0.8 pm and 1.2 pm pore size sterile syringe filters (Millipore Sigma). Lysosomes were then collected via centrifugation at 20,000 x g. For the final purification step, the isolated lysosomes were resuspended in 500 pL of buffer C containing 1% BSA and subjected to a centrifugation at 3,000 x g. The artificial transfer of lysosomes to ECs was conducted by mixing isolated lysosomes with EC culture medium, adding them to EC cultures at a 3 : 1 donor-to-recipient cell ratio. Post a 12-hour lysosomal transplantation period, the cell cultures were washed twice with PBS and subsequently maintained in EC-medium.

[0160] Quantitative analysis of exogenous mitochondria and Parkin co-localization

[0161] ECs, post 12-hour DsRed-mitochondrial transplantation, were fixed using ice-cold methanol at 4°C. Permeabilization was achieved with 0.1% Triton X-100 in PBS, followed by blocking in 5% horse serum (Vector Laboratories) for 1 hour. Overnight incubation at 4°C was then conducted with the anti-Parkin antibody (Abeam, Cat. No. ab77924). Subsequent to three PBS washes, cells were incubated with appropriate secondary antibodies at room temperature for 1 hour. Following an additional trio of PBS washes, nuclear staining was performed using DAPI for 15 minutes. A quantitative assessment of Parkin and mitochondrial fluorescence was conducted with ImageJ based on pixel coloration metrics: Parkin (yellow), Mitochondria (red), and their co-localization (orange).

[0162] JC-10 mitochondria membrane potential fluorescent assay

[0163] Depolarization of isolated mitochondria was evaluated with the JC-10 mitochondria membrane potential fluorescent assay (Enzo Life Sciences, Cat.# ENZ-52305) according to the manufacturer's instructions. Isolated mitochondria were assessed immediately after isolation in Ca2+-containing (~1.6 mM) EGM2 medium and Ca2+-free C buffer. The fluorescent intensities for aggregates (Red FL, 590 nm) and monomeric (Green FL, 520 nm) forms of JC-10 were measured at Ex / Em = 490 / 590 nm and 490 / 520 nm with a microplate reader. The percentage of membrane potential was represented as the FL590 / FL520 ratio.

[0164] Migration assay

[0165] Migration Assay The scratch assay was conducted on confluent ECs in a 6-well plate. Scratch wounds were created using a 1000-pL pipette tip, and artificial mitochondrial transfer was performed immediately. The scratch size was measured after 12 hours for each condition.

[0166] Apoptosis assay

[0167] Confluent ECs were treated with 0.2 mM H2O2 for 12 hours, with or without artificial mitochondrial transfer. Apoptosis was assessed using APC-conjugated Annexin V and propidium iodide (PI) staining (Annexin V Apoptosis Detection Kit; eBioscience) via flow cytometry, following the manufacturer's instructions.

[0168] ATP measurement

[0169] Confluent ECs were treated with artificial mitochondrial transfer or control for 24 hours. Total cellular ATP was measured using the Luminescent ATP Detection Assay Kit (Abeam, abl 13849) according to the manufacturer's instructions.

[0170] Bioenergetics assessment

[0171] Bioenergetics changes resulting from artificial mitochondrial transfer in ECs were investigated by measuring oxygen consumption rate (OCR) using a Seahorse XFe96 Analyzer. ECs were plated at various seeding densities to determine the optimal density of 30,000 cells / well. Optimal drug concentrations were 1.5 pmol / L for oligomycin, 1 pmol / L for FCCP, and 1.25 pmol / L of rotenone + 2.5 pmol / L of antimycin A (R+A). Transferred mitochondria were co-incubated with ECs for 24 hours before removing unintemalized mitochondria by changing the medium. Seahorse assays were conducted in Seahorse XF DMEM supplemented with 10 mmol / L glucose, 1 mmol / L sodium pyruvate, and 2 mmol / L L-glutamine. OCR measurements were performed with the sequential treatment of Oligomycin, FCCP, and R+A following the Seahorse XFe96 Analyzer program.

[0172] RNAseq analysis

[0173] The following groups were analyzed: unprimed-ECs, mitoT-ECs, and mitoAT-ECs. Each group consists of at least two biological replicates. Total RNA was extracted using RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. RNA quantity and quality were checked with NanoDrop and Agilent Bioanalyzer. Libraries were prepared and sequenced by Genewiz (NJ, USA) using the Illumina HiSeq 2500 platform (Illumina, CA) with 2 x 150 paired-end configurations. The raw reads were quality controlled (FastQC) and trimmed. Reads were then aligned to UCSC (University of California, Santa Cruz) hg38 genome (STAR aligner), and transcript expression was calculated by Salmon. Pairwise comparison differentially expressed (DE) genes were called by DESeq2 (version 1.38.1, threshold used: fold change > 2, P <0.05). Intersected combined DE genes were subjected to heatmap and principal component analysis (PCA) plotting (R version 4.2.1). DE genes upregulated in mitoT-ECs, and mitoAT-ECs groups against unprimed-ECs were annotated for gene ontology (GO) analysis with R package ClusterProfiler (version 4.6.2).

[0174] Quantitative Real-Time PCR

[0175] Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. RNA concentration and purity were measured using a NanoDrop 8000 spectrophotometer (Thermo Fisher) based on the absorbance ratio at 260 and 280 nm. cDNA synthesis was performed using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher). Quantitative real-time PCRs were conducted on the QuantStudio 6 Flex Real-Time PCR System with PowerUp SYBR Green Master Mix (Thermo Fisher), using GAPDH as the housekeeping gene.

[0176] Human cytokine protein array

[0177] Selected cytokines were evaluated in conditioned medium samples using the Proteome Profiler Human Angiogenesis Array (R&D Systems; ARY007) as per the manufacturer's instructions. Antigen-antibody reactions were visualized with LumiGLO substrate (Kirkegaard & Perry Laboratories, Inc.) and chemiluminescent-sensitive film (Kodak). Densitometry was carried out using image analysis (ImageJ) to estimate protein amounts in each sample.

[0178] Western Blot analysis

[0179] Mitochondrial and cellular lysates were prepared using radioimmunoprecipitation assay (RIP A) buffer (Millipore Sigma, Cat. No. R278). Protein concentrations were quantified employing a bicinchoninic acid (BCA) assay (Thermo Scientific, Cat. No. 23227). Equal amounts of protein from each sample were resolved on Novex™ 4-12% Tris-Glycine gels (Thermo Fisher) and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad) utilizing the Novex -mini cell Xcell Surelock system (Invitrogen). Membrane blocking was performed with 5% non-fat milk powder in TBS-T for 1 hour at room temperature, followed by incubation at 4 °C overnight with primary antibodies: rabbit anti-GAPDH (Sigma, Cat. No. G9545), rabbit anti-P-Actin (ABclonal, Cat. No. AC038), rabbit anti-PINKl (NOVUS Biological Inc, Cat. No. BC100-494), and mouse anti-TOM20 (Abeam, Cat. No. ab56783). After thorough washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands were visualized using HRP-enhanced chemiluminescence (ECL) substrate (Kindle Biosciences, western blot detection kit, Cat. No. R1004).

[0180] Mitochondria DNA heteroplasmy

[0181] To assess the persistence of transferred mitochondria in recipient ECs over time, we used a mitochondrial DNA (mtDNA) genotyping approach that relies on detecting heteroplasmy. mtDNA was isolated from cells using the Mitochondrial DNA Isolation Kit (Abeam; ab65321). PCR amplification of the hypervariable regions (HVR1 & HVR2) of mtDNA was performed using the following primers: 1) HVR1, Primer name: Al mitoPrimer™ (LI 5997) (Amplicon Size: 342 bps; Primer sequence: 5' CAC CAT TAG CAC CCA AAG CT 3') and Primer name: Bl mitoPrimer™ (H16391) (Amplicon Size: 342 bps; Primer sequence: 5' GAG GAT GGT GGT CAA GGG AC 3') and 2) HVR2, Primer name: Cl mitoPrimer™ (L048) (Amplicon Size: 268 bps; Primer sequence: 5' CTC ACG GGA GCT CTC CAT GC 3') and Primer name: DI mitoPrimer™ (H408) (Amplicon Size: 268 bps; Primer sequence: 5' CTG TTA AAA GTG CAT ACC GCC A 3'). Then, Sanger sequencing was performed using the same primers to read the mtDNA sequences.

[0182] Split-GFP mitochondrial fusion assay

[0183] A split-GFP system strategy was adopted to determine if transferred mitochondria fuse with the recipient's mitochondrial pool. In this system, GFP is split into two components, which only produce a fluorescent signal when both components are present. Two piggyBac transposon vectors were created, each encoding one of the two GFP components (components GFP-1-10 and GFP-11) fused with a mitochondrial targeting sequence from cytochrome C oxidase (referred to as mtGFP-1-10 and mtGFP-11). Mitochondrial donor mitoDsRed-MSCs were transduced with mtGFP-11, and recipient ECs were transduced with mtGFP-1-10. After natural or artificial mitochondrial transfer, green fluorescence is only observed if donor mitochondria fuse with the endogenous mitochondrial pool (i.e., mtGFP-1- 11). The fluorescence of isolated mitochondria was analyzed by flow cytometry (a BD FACS LSRFortessa).

[0184] Mitochondria DNA-free human MSC-p° cells

[0185] A piggyBac transposon vector carrying a Dox-inducible mitochondrial targeting GFP- EcoRI fusion protein was constructed. This vector was co-transfected with a super piggyBac transposase vector into human mitoDsRed-MSCs and selected with 2 pg / mL puromycin. Stable engineered MSC lines were maintained in normal MSC medium. To induce mtDNA depletion, Dox (0.5 pg / mL) was added to activate the expression of the mitochondrial targeting GFP-EcoRI fusion protein, which led to the digestion of mtDNA. Depletion of mtDNA was confirmed after 3 days by qPCR. The resulting mtDNA-free human MSC-pO cells were maintained in DMEM high glucose supplemented with GlutaMAX, pyruvate (110 pg / mL), 50 pg / mL uridine, and 10% FBS. Control mtDNA-free EC-p° cells were similarly generated and maintained in EGM-2 supplemented with 20% fetal bovine serum, GlutaMAX, 110 pg / mL pyruvate, and 50 pg / mL uridine.

[0186] Autophagic flux analysis

[0187] MitoAT-ECs were cultured until they reached 80% confluency. Cells were then treated with 100 nM Bafilomycin Al for 3 hours to inhibit autophagosome-lysosome fusion. Cells were lysed with RIPA buffer containing protease inhibitors, and protein concentration was determined using a BCA assay. A total of 30 pg of protein was separated on a 12% SDS- PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in TBST buffer for 1 hour at room temperature and probed overnight at 4°C with the primary antibody against LC3B (Abeam, Cat.# ab51520) at a dilution of 1 : 1000 in TBST buffer. The membrane was then washed three times with TBST buffer and incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody. The immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection system.

[0188] Microscopy

[0189] Images were captured using the Axio Observer Z1 inverted microscope (Carl Zeiss) and AxioVision Rel. 4.8 software. Fluorescent images were taken with an ApoTome.2 Optical sectioning system (Carl Zeiss) and 20X or 40X objective lens. Non-fluorescent images were taken with an AxioCam MRc5 camera using a 10X or 20X objective lens.

[0190] Statistical analyses

[0191] All statistical analyses were performed using GraphPad Prism v.7 software (GraphPad Software Inc.). The sample size, including the number of mice per group, was chosen to ensure adequate power and based on historical laboratory data. No exclusion criteria were applied for all analyses. All data were expressed as mean ± standard error of the mean (s.e.m.). Comparisons between multiple groups were performed by ANOVA followed by Bonferroni's post-test analysis. Unpaired two-tailed Student's t-test was used for comparisons between two groups. A value of P<0.05 was considered to be statistically significant.

[0192] Example 1: Endothelial cell engraftment requires support from stromal cells

[0193] To investigate engraftment, we employed a xenograft model where human ECs were combined with or without supporting MSCs in a collagen-based hydrogel, and the mixture was subcutaneously implanted into immunodeficient nude mice (Fig.5a). Implants containing only ECs failed to produce perfused grafts by day 7, while co-implanting ECs with MSCs generated an extensive network of microvessels connected to the host circulatory system (Fig.5b-5d). The marked difference in microvessel density due to MSC presence was evident in histological analysis (Fig.5b,5c). Engraftment disparity was confirmed using bioluminescence imaging of luciferase-expressing ECs (luc-ECs) for up to 4 weeks (Fig.5e,5f). MSC dependency for engraftment and functional blood vessel formation in vivo was further substantiated with various types of primary human ECs, including HUVECs, endothelial colony-forming cells (ECFCs), and adipose tissue-derived ECs (Fig.5b,5c).

[0194] Perivascular cells play a crucial role in modulating blood vessel stability and function. However, the mechanisms by which perivascular cells facilitate EC engraftment remain partially understood. MSCs secrete a range of angiogenic factors that are absent in ECs (Fig.6b). Nonetheless, supplementing MSC-secreted factors could not replace MSCs and was insufficient to support early survival (24 h post-implantation; Fig.6a) or blood vessel-forming ability of ECs at day 7 (Fig.6c,6e), indicating that MSCs' supportive role is not exclusively paracrine. Moreover, inhibiting PDGFR signaling (tyrphostin AG1295), which delays MSC- EC juxtaposition25, did not affect EC engraftment (Fig.6a,6c-6f), suggesting that the direct perivascular contact is not essential for maintaining EC viability under immediate postimplantation stress.

[0195] Example 2: Mitochondrial transfer from MSCs to ECs via tunneling nanotubes

[0196] Recent studies have demonstrated that MSCs can naturally transfer mitochondria to other parenchymal cells, including lung alveolar epithelial cells and neurons, during regenerative processes. This phenomenon was found to be crucial for cell survival. However, mitochondrial transfer in the vascular system has not been described. We investigated whether mitochondrial transfer from MSCs could mediate EC survival during engraftment. MSCs were transfected with a piggyBac transposon containing a mito-tag, rendering all mitochondria DsRed+ (referred to as mitoRed-MSCs; Fig.la and Figs.7a-7d). Histological examination of 24-hour grafts revealed ultrafine and long protrusions extending from MSCs containing DsRed+ mitochondria (Fig.la). These structures were also observed in vitro upon co-culture of mitoRed-MSCs with ECs (Fig.lc) and resembled tunneling nanotubes (TNTs), previously implicated in mitochondrial transfer. These protrusions had aligned DsRed+ mitochondria, F-actin, and microtubules (Figs.8a-8c), consistent with TNTs. Flow cytometric and immunofluorescence analyses of grafts seeded with ECs and mitoRed-MSCs revealed that, after 24 hours in vivo, -25% of ECs (hCD90- / hCD31+ cells) contained DsRed+ mitochondria, suggesting mitochondrial transfer from mitoRed-MSCs (Fig.lb and Figs.9a- 10c). Notably, mitochondrial transfer required direct MSC-EC communication, was affected by cell proportions, and increased significantly under hypoxic (1% oxygen) stress (Figs.9a- 9d). TNT formation was largely unidirectional, with minimal mitochondrial transfer from ECs to MSCs (Figs.lla-lle)

[0197] We examined the role of TNFa, a potent inducer of TNT formation that is upregulated in activated ECs (Figs.l2a-12b). Blocking TNFa with a monoclonal antibody significantly reduced the presence of mitochondria-containing TNTs in our co-culture model (Fig.lc). Furthermore, transcriptional abrogation of TNFAIP2, a regulator of TNT formation, in mitoRed-MSCs (via shRNA) diminished TNT appearance (Fig.lc and Figs.l3a-13c). Importantly, inhibiting TNT formation through TNFAIP2 silencing significantly blocked mitochondrial transfer (Fig.ld and Figs.l3a-13c), reinforcing the idea that mitochondrial transfer from MSCs to ECs occurs via TNTs.

[0198] In vivo, mitochondrial transfer was transient. Transfer initiated immediately upon cell transplantation (24 hours; Fig.lb) and persisted during blood vessel formation, with DsRed+ mitochondria observed in ECs lining newly formed blood vessels on day 7 (Fig.le and Figs.l4a-14d). By day 14, once the vasculature stabilized, DsRed+ mitochondria were restricted to perivascular MSCs and largely absent in ECs (Fig.le), indicating that mitochondrial transfer had ceased. Interestingly, local administration of TNFa on day 14 reactivated mitochondrial transfer from MSCs, resulting in DsRed+ mitochondria presence in ECs (Fig.le and Figs.l4a-14d). These data suggest a dynamic relationship between mitochondrial transfer and the vasculature's physiological state (stressed and activated vs. stable).

[0199] Example 3: Mitochondrial transfer facilitates endothelial cell engraftment

[0200] The impact of mitochondrial transfer on EC engraftment was substantial. Transcriptional abrogation of TNT formation (via shRNA silencing of TNFAIP2; Fig.13) or mitochondrial mobilization within TNTs (silencing Mitochondrial Rho GTPase 1 (MIR01) expression; Figs.l5a-15e) in MSCs significantly impaired EC engraftment (Fig.lf,lg). ECs demonstrated a reduced ability to form perfused microvessels by day 7 when implanted with either shTNFAIP2-MSCs or shMIROl-MSCs compared to control MSCs (Fig.lh,li and Figs.l5a-15e). Thus, the formation of TNTs and subsequent mitochondrial transfer from MSCs were essential for robust EC engraftment.

[0201] We investigated whether MSCs could preemptively transfer mitochondria to ECs before transplantation. ECs sorted as CD3 H7DsRed+ cells from a 24-hour co-culture with mitoRed-MSCs (referred to as mitoT-ECs) exhibited significantly enhanced engraftment ability. Grafts containing only mitoT-ECs displayed extensive networks of perfused human microvessels surrounded by murine U-SMA+ perivascular cells (Fig. lj-11). Conversely, grafts with ECs that did not receive transferred DsRed+ mitochondria from MSCs (unprimed- ECs) failed to vascularize (Fig. lj-11). Our findings suggest that co-culturing with MSCs allowed a subset of ECs to receive mitochondria via TNTs, conferring enhanced biological properties. Specifically, these primed mitoT-ECs demonstrated an improved capacity to engraft and form functional blood vessels in vivo without direct MSC support.

[0202] Example 4: Exogenous mitochondrial transplantation boosts endothelial cell engraftment

[0203] Inspired by the natural mitochondrial transfer from MSCs (Figs.la-11), we investigated whether artificially transplanting exogenous mitochondria into ECs could enhance engraftment. Mitochondria were isolated from MSCs and incubated with EC cultures, where uptake occurred in -4 hours (Fig.2a and Figs.l6a-16d; ECs that artificially received mitochondria are referred to as mitoAT-ECs). The number of mitochondria used for artificial transplantation corresponded to a 3: 1 ratio (i.e., mitochondria from 3 donor MSCs for each recipient EC). Flow cytometry analysis revealed that the percentage of mitoAT-ECs that effectively received exogenous mitochondria was exceptionally high (-90%; Fig.l6c). This process of mitochondrial incorporation into cells by simple co-incubation is regulated via micropinocytosis (a clathrin-independent form of endocytosis)30. Internalization of exogenous mitochondria was confirmed with confocal microscopy using mitoRed-MSCs as donor cells and tracing the DsRed+ signal at appropriate focal planes (Fig.l6d).

[0204] Transmission electron microscopy (TEM) further confirmed the internalization of mitochondria isolated from mitoAPEX2-MSCs and the presence of APEX2 -labeled mitochondria in ECs (Figs.l7a-17e).

[0205] Artificial mitochondrial transplantation into ECs mimicked the effects of natural mitochondrial transfer from MSCs through TNTs (Figs.2b-2f), resulting in cytoprotective attributes, such as enhanced metabolic activities (Figs.2b-2d and Figs.l8a-18d), resistance to apoptosis (Fig.2f and Figs.l9a-19b), and increased cellular mobility (Fig.2e and Figs.20a- 20b). Of note, transplantation of other organelles (namely, lysosomes) did not produce these functional enhancements in ECs (ED Figs.20c-20e). Transplanting mitochondria from eight different types of donor cells, including primary cells from various species and immortalized cell lines, produced similar effects on mitoAT-ECs (Figs.20a-20b), suggesting the origin of the exogenous mitochondria might not be critical.

[0206] MitoAT-EC engraftment significantly improved upon mitochondrial transplantation without the need for MSCs (Figs.2g,2h). Subcutaneous grafts seeded with only mitoAT-ECs that had received exogenous mitochondria 4 hours before implantation contained extensive networks of perfused human microvessels by day 7 (Figs.2i,2k), while control EC grafts failed to vascularize. The microvessel density in grafts with mitoAT-ECs alone was comparable to that of grafts containing ECs with MSCs (Fig.2j), indicating that exogenous mitochondrial transplantation could compensate for the absence of supporting MSCs during engraftment.

[0207] MitoAT-EC engraftment was also effective in ischemic tissues. MitoAT-EC engraftment into ischemic hind limbs (femoral ligation) in diabetic nude mice was significantly higher than control EC engraftment (Fig.21 and Fig.21), resulting in a robust presence of human vessels in the ischemic muscle by day 7 (Fig.2m). This enhanced engraftment translated into improved blood flow in the affected limbs for over 3 weeks (Figs.2n-2o and 22a-22c) and prevented necrotic tissue development in mice receiving mitoAT-ECs (Fig.2p). Our findings demonstrate that artificial transplantation of exogenous mitochondria can enhance the intrinsic ability of human ECs to engraft and revascularize ischemic tissues without additional supporting cells. Example 5: Exogenous mitochondria become compromised due to calcium overload

[0208] Receiving exogenous mitochondria by natural cell-to-cell transfer (Figs. la-11) or artificial transplantation (Figs.2a-2p) improved EC fitness, suggesting that transplanted mitochondria must have remained functional and incorporated into the recipient cells' endogenous mitochondrial pool. However, exogenous mitochondria did not maintain their functional competence during isolation from MSCs and incubation with ECs. Upon exposure to EGM2 culture medium containing high Ca2+concentration (~1.6 mM), mitochondria were immediately and irreversibly compromised, with depolarization of their membrane potential due to rapid mitochondrial Ca2+uniporter (MCU)-mediated Ca2+overload (Fig.3a,3b and Figs.23a-23e). Despite this, the artificial transplantation of depolarized exogenous mitochondria consistently enhanced EC fitness (Figs.2a-2p), which was puzzling.

[0209] Example 6: Transplanted mitochondria do not incorporate into the recipient mitochondrial pool

[0210] We investigated whether transplanted mitochondria integrate and persist in recipient ECs using a mitochondrial DNA (mtDNA) genotyping approach that detects heteroplasmy. After sequencing mtDNA in ECs and MSCs from two separate subjects, we identified four loci at the hypervariable regions (HVR1 & HVR2) with single-nucleotide differences (Figs.24a-24c). Following artificial mitochondrial transplantation, we estimated the percentage of donor (MSCs) vs. endogenous (ECs) mtDNA in recipient mitoAT-ECs by measuring heteroplasmy in the selected loci. This analysis revealed that 24 hours after transplantation, -12% of total mtDNA in mitoAT-ECs corresponded to exogenous mitochondria, but by day 7, donor mtDNA was undetectable, indicating the transplanted mitochondria persisted only for a limited time in recipient mitoAT-ECs (Fig.3c and Figs.24a- 24c).

[0211] To determine if transplanted mitochondria fuse with the recipient mitochondrial pool, we used a split-GFP system. We genetically split GFP into two components (GFP-1-10 and GFP-11; only when these two components are together can GFP emit its fluorescent signal) and added a mito-tag (mt) to send them to the mitochondrial matrix (Figs.25a-25e). We then transduced donor mitoRed-MSCs with mtGFP-11 and recipient ECs with mtGFP-1-10 and investigated mitochondrial fusion following both natural transfer (co-culture) and artificial transplantation. Direct co-culture with mitoRed-MSCs(mtGFP-l 1) resulted in minimal fusion of transferred mitochondria with the endogenous mitochondrial pool in ECs. Transferred DsRed+ mitochondria were -13.3% of the total mitochondrial content in recipient ECs by 24 hours, but only -1.7% effectively fused (i.e., DsRed+ / GFP+) (Figs.25a-25e). However, with artificially transplanted mitochondria (-8.2% of all mitochondria at 24 hours), the GFP signal was virtually undetectable in mitoAT-ECs (Fig.3d and Figs.25a-25e), suggesting negligible mitochondrial fusion.

[0212] Example 7: mtDNA-free mitochondria enhances EC function

[0213] To further confirm that donor mitochondria do not need to be functional, we generated mitoRed-MSCs without mtDNA (called mitoRed-pO-MSCs). These mitoRed-pO- MSCs contained a doxycycline (Dox)-inducible gene encoding an EcoRI restriction endonuclease fused to a mitochondrial targeting sequence (MTS) and a GFP reporter (Fig.26a). After administering Dox for 48 hours, the MTS-EcoRI-GFP complex co-localized with DsRed+ mitochondria, completely degrading the endogenous mtDNA in mitoRed-pO- MSCs (Fig.3e and Fig.26b). Although dysfunctional, mitoRed-pO-MSCs were viable. Transferring mtDNA-free mitochondria from mitoRed-pO-MSCs (Fig.3f) produced similar effects on ECs as those observed with normal functional mitochondria, including increased ATP (Fig.3g), migratory activity (Fig.3h), and in vivo engraftment ability (Fig.3i-3k). However, transplanting structurally damaged mitochondria from a freeze-thaw process did not improve mitoAT-ECs performance (Fig.26d), suggesting the need for physical integrity preservation. These results indicate that donor mitochondria do not need to be functional to enhance EC fitness, implying an alternative role as a trigger.

[0214] Example 8: Transplanting mitochondria transiently triggers mitophagy in ECs

[0215] To gain insights into the transcriptional changes produced in ECs by incorporating exogenous mitochondria through either co-culture with MSCs (mitoT-ECs) or artificial transplantation (mitoAT-ECs), we performed RNA sequencing (RNA-seq) analysis (Figs.28a-28f). ECs that were co-cultured with MSCs but did not receive exogenous mitochondria (i.e., unprimed-ECs) served as controls. Globally, there were hundreds of differentially expressed genes across all EC groups (Fig.28b). Hierarchical clustering analysis of differentially expressed genes revealed transcriptional proximity between samples from each EC group, associations confirmed by principal components analyses (Fig.28c). To gain more insight into the transcriptional differences, we carried out gene ontology (GO) enrichment analysis. Notably, both mitoT-ECs and mitoAT-ECs exhibited significant enrichment in genes associated with mitochondrial respiration, ATP synthesis, electron transport chain, and oxidative phosphorylation compared to unprimed-ECs (Figs.28e-28f), consistent with their enhanced metabolic activities (Figs.2b-2d and 18a-20e). Furthermore, qPCR analysis of gene expression in mitoAT-ECs revealed significant upregulation of several genes associated with mitochondrial biogenesis (Fig.28g,29a-29b). Indeed, 24 hours post mitochondrial transplantation, mitoAT-ECs had significantly more mitochondria (~2- fold; normalized to nuclear protein content) compared to control ECs (Fig.28h). Additionally, Western blot analyses confirmed significantly more mitochondrial content (TOM20 normalized to P-Actin) in mitoAT-ECs than control ECs, and this increase was higher than the mere presence of exogenous mitochondria (Fig.28i).

[0216] Our results did not support the idea that transplanted mitochondria functionally incorporate into recipient ECs. Instead, 6-12 hours after artificial transplantation, exogenous mitochondria co-localized with autophagosomes (Fig.4a), and ULK1 -mediated autophagic activity significantly increased in mitoAT-ECs (Fig.4b and Figs.27a-27c). Most exogenous DsRed+ mitochondria (including those using mtDNA-free mitochondria from mitoRed-pO- MSCs) localized in LC3B+ autophagosomes (Fig.4c), suggesting rapid degradation.

[0217] Mitophagy refers to the selective autophagic degradation of mitochondria. To further investigate whether transplanting mitochondria triggered mitophagy in mitoAT-ECs, we targeted PTEN-induced putative protein kinase 1 (PINK1) and the E3 ubiquitin ligase PARKIN, both well-established mediators of mitophagy. During mitophagy, PINK1 accumulates on the outer membrane (OMM) of depolarized mitochondria and recruits Parkin from the cytosol. Parkin initiates the ubiquitination of numerous OMM proteins, leading to phagophore recruitment and autophagosome assembly. We have several lines of evidence suggesting the involvement of the PINK 1 -Parkin pathway upon mitochondrial transplantation. First, we detected the presence of PINK1 in the isolated mitochondria by both flow cytometer and western blot analyses (Figs.31c-31d). Also, immunofluorescence analysis of mitoAT-ECs revealed that approximately 23% of the DsRed-labeled exogenous mitochondria co-localized with cytoplasmic Parkin 24 hours after transplantation (Fig.31d). Moreover, genetic ablation of PINK1 (shRNA) in donor mitochondria prevented autophagosome formation in mitoAT-ECs (Fig.4d and Figs.27a-27c, 31a-31i). However, silencing PINK1 in recipient ECs did not affect LC3B assembly, indicating that only internalized exogenous mitochondria triggered mitophagy. Similarly, silencing PARKIN in recipient ECs abrogated LC3B+ autophagosome formation in mitoAT-ECs, whereas silencing PARKIN in mitochondrial donor MSCs had no effect (Fig.4d and Fig.27a-27d, 31a-31i)

[0218] Collectively, these findings were consistent with the notion of activation of mitophagy upon the internalization of exogenous mitochondria, which, in turn, enhanced mitochondrial biogenesis and resulted in mitoAT-ECs exhibiting improved respiration and cellular functions.

[0219] Example 9: Activation of mitophagy mediates the enhanced engraftment ability of ECs

[0220] We next examined if mitophagy regulated the engraftment of mitoAT-ECs. To do this, we first inhibited mitophagy in mitoAT-ECs by either silencing PARKIN in the recipient ECs (shPARKIN-ECs) or PINK1 in the mitochondrial donor MSCs (shPINKl-MSCs). Silencing ULK1, a central enzyme in autophagy, in ECs served as a control. When the PINKl-PARKIN-dependent mitophagy pathway was impaired, mitochondrial transplantation failed to enable the engraftment of mitoAT-ECs into immunodeficient mice (Figs.4e-4g). Grafts with abrogated mitophagy showed a significantly reduced presence of perfused vessels at day 7 (Fig.4g). In contrast, control mitoAT-ECs exhibited robust engraftment in the form of perfused blood vessels. Mechanistically, these results confirmed that the PINK1 -PARKIN pathway mediated mitophagy, which in turn regulated the enhanced engraftment ability displayed by mitoAT-ECs after mitochondrial transplantation (Fig.4h).

[0221] Mitophagy also played a role in regulating the engraftment ability of ECs when supported by MSCs. Vascularization at day 7 in grafts containing ECs and MSCs was significantly reduced when using either shPARKIN-ECs or shPINKl-MSCs (Figs.4i-4k and Figs.30a-30b), confirming that mitophagy was implicated in regulating the engraftment of ECs in the co-implantation model.

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[0278] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of transplanting a population of endothelial cells (ECs) in a subject comprising: contacting a population of endothelial cells with isolated mitochondria; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

2. A method of promoting blood vessel formation in a subject, comprising: identifying a subject at in need of increased blood vessel formation; contacting a population of endothelial cells with isolated mitochondria; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

3. A method of vascular cell therapy comprising: identifying a subject at in need of vascular cell therapy; contacting a population of endothelial cells with isolated mitochondria; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

4. A method of increasing vascular generation or vascular regeneration in a subject comprising: identifying a subject at in need of vascular generation or vascular regeneration; contacting a population of endothelial cells with isolated mitochondria; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

5. The method of any one of the preceding claims, wherein the mitochondria are not functionally active, are not respiration competent, and / or do not have mitochondrial DNA (mtDNA).

6. The method of any one of the preceding claims, wherein the population of ECs comprises human ECs.

7. The method of any one of the preceding claims, wherein the ECs comprise any one or more of human umbilical vein endothelial cells (HUVECs), endothelial colonyforming cells (ECFCs), adipose tissue-derived ECs (e.g., white adipose tissue-derived ECs, watECs), organ- specific endothelial cells, and / or ECs derived from human induced pluripotent stem cells (iPSCs).

8. The method of claim 7, wherein the organ-specific endothelial cells are from an organ selected from: heart, muscle, kidney, testis, ovary, lymphoid, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine, and small intestine.

9. The method of any one of claims 1-8, wherein no support cells are administered with the population of ECs.

10. The any one of the preceding claims, wherein the support cells are perivascular cells.

11. The method of claim 10, wherein the perivascular cells are any one or more of: smooth muscle cell, pericytes, fibroblast, mesenchymal stromal cells (MSCs), and / or perivascular cells derived from human induced pluripotent stem cells (iPSCs).

12. The method of any one of the preceding claims, wherein the population of endothelial cells is administered to the subject by injecting the composition into a blood vessel of the subject.

13. The method of claim 12, wherein the blood vessel is the hepatic portal vein of the subject, the coronary artery of the subject, the renal artery of the subject, the pulmonary artery of the subject, or the prostate artery of the subject.

14. The method of any one of claims 1-11, wherein administering is performed by intravenous, intra-articular, subcutaneous, intraperitoneal, intramuscular, intradermal, or intracardiac injection.

15. The method of any one of the preceding claims, wherein the subject is administered a single dose of the population of ECs.

16. The method of any one of claims 1-14, wherein the subject is administered multiple doses of the population of ECs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses).

17. The method of claim 16, wherein the multiple does are administered at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 1 week, 2 weeks, 3 weeks, 1 month, 2, months, 3, months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months apart.

18. The method of any one of the preceding claims, wherein the mitochondria are autogeneic, allogeneic, or xenogeneic.

19. The method of any one of the preceding claims, wherein the population of endothelial cells are autogeneic or allogeneic.

20. The method of any one of the preceding claims further comprising, prior to the administering step, a step of collecting the isolated mitochondria from cells (e.g., donor cells).

21. The method of any one of the preceding claims, wherein the subject has or is at risk of a disease or disorder of the blood vessels, aberrant vasculature, leaky blood vessels, narrow blood vessels, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, a myocardial infarction, cardiotoxicity (e.g., caused by chemotherapy), cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, valvular heart disease, cerebral cavernous malformations, hemorrhagic strokes, hereditary hemorrhagic telangiectasias, arteriovenous malformations, a metabolic disorder, diabetes, diabetic retinopathy, an ischemic injury, an IRI injury, atherosclerosis, Age-related Macular Degeneration (AMD), Pulmonary Arterial Hypertension (PAH), Hereditary Hemorrhagic Telangiectasia (HHT), peripheral artery disease (PAD), arteriovenous fistulas (e.g., dialysis patients), tumor angiogenesis, tumormetastasis, a cancer, a metabolic disease, a stroke, a wound (optionally, a chronic wound; e.g., a diabetic ulcer), and / or a mitochondrial dysfunction disorder.

22. A method of making a population of endothelial cells (ECs) comprising transplanted mitochondria (mitoAT-ECs), the method comprising: i. obtaining a sample (e.g., a sample comprising ECs) from a subject (e.g., blood or a tissue sample); ii. isolating a population of EC from the sample; iii. contacting the population of ECs with isolated mitochondria (optionally, exogenous mitochondria), thereby creating the population of mitoAT-ECs.

23. The method of claim 22, wherein the sample comprises ECs, optionally wherein the sample is a human sample, optionally wherein the sample is blood (e.g., human umbilical cord blood) or tissue (e.g., subcutaneous adipose tissue).

24. The method of claim 22 or 23, wherein the mitochondria are isolated from a population of donor cells (e.g., mesenchymal stromal / stem cells (MSCs)).

25. The method of claim 24, wherein the mitochondria are isolated from a number of donor cells that is a ratio of donor cells:ECs of 5:1, 4: 1 3: 1, 2: 1, or 1 : 1.

26. The method of claim 24, wherein the mitochondria are isolated from a number of donor cells that is a ratio of donor cells:ECs of 3:1 (e.g., mitochondria from 3 donor cells (e.g., MSCs) for each recipient EC).

27. The method of any one of claims 22-26, wherein the ECs comprise any one or more of human umbilical vein endothelial cells (HUVECs), endothelial colony -forming cells (ECFCs), adipose tissue-derived ECs (e.g., white adipose tissue-derived ECs, watECs), organ-specific endothelial cells, and / or ECs derived from human induced pluripotent stem cells (iPSCs).

28. The method of claim 27, wherein the organ-specific endothelial cells are from an organ selected from: heart, muscle, kidney, testis, ovary, lymphoid, liver, pancreas, brain, lungs, bone marrow, spleen, large intestine, and small intestine.

29. The method of any one of claims 22-28, wherein the isolated mitochondria are incubated with the ECs in the contacting step for a time sufficient to internalize at least a portion the mitochondria (e.g., at least about 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 24, 26, 28, 40, 42, 44, 46, or 48 hours).

30. The method of any one of claims 22-29, wherein at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% of the ECs internalize the isolated mitochondria.

31. The method of any one of claims 22-30, wherein about 90% of the EC population are mitoAT-ECs.

32. The method of any one of claims 22-31, wherein the mitochondria are not respiratory competent.

33. The population of mitoAT-ECs made according to the method of any one of claims 22-31.

Images