Methods of vascular cell therapy and endothelial cell engraftment via mitophagy inducers

Abstract

Described herein, inter alia, are compositions comprising and methods of making and using endothelial cells (ECs) comprising one or more mitophagy-inducing agents (e.g., rapamycin, SKF-96365, lithium, carbamazepine, latrepirdine, spermidine, resveratrol, sodium valproate, trehalose, kaempferol, metformin, AICAR, nilotinib, ambroxol, curcumin, and VL-004). 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 ECs comprising a mitophagy-inducing agent (MIA-ECs).

Description

[0001] Methods of Vascular Cell Therapy and Endothelial Cell Engraftment Via Mitophagy Inducers

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial Nos. 63 / 548,467, filed on November 14, 2023, and 63 / 707,329, filed on October 15, 2024. 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. However, inadequate revascularization due to challenges in regenerating microvascular beds in ischemic areas persists. While angiogenic growth factor delivery has been explored to stimulate local angiogenesis, this approach is limited by the disrupted tissue microenvironment in ischemic regions. Consequently, endothelial cell (EC) transplantation remains a priority in vascular medicine.

[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, co-transplantation with perivascular cells is essential for robust engraftment and functional blood vessel formation in vivo. Perivascular cell sources include smooth muscle cells, pericytes, fibroblasts, and mesenchymal stromal / stem cells (MSCs). 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] The mechanisms by which perivascular cells facilitate EC engraftment are not fully understood. 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 the enhanced engraftment observed for endothelial cells treated with an agent that induced mitophagy without the use of a support cell. Ischemic diseases affect millions worldwide, including critical limb ischemia and myocardial infarction. Transplanting endothelial cells (EC or 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] Described herein, inter alia, are methods and compositions based on strategy of preemptively inducing mitophagy in ECs to enhance engraftment in vivo. Without being bound by theory, this approach induced a transient cytoprotective effect (via mitophagy), enabling EC engraftment without MSC support. The findings described herein suggest potential for a new single-cell therapy strategy based on, inter alia, autologous EC engraftment, which may facilitate clinical translation.

[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 mitophagy-inducing agent; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

[0013] In some embodiments of any of the methods described herein, a population of support cells is also administered to the subject. In some embodiments, the population of support cells is administered to the subject with the population of endothelial cells (e.g., simultaneously). In some embodiments, the population of support cells is administered to the subject before the population of endothelial cells (e.g., at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 24, 36, or 48 hours; or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days; or at least about 1, 2, 3, 4, or 5 weeks). In some embodiments, the population of support cells is administered to the subject after the population of endothelial cells (e.g., at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 24, 36, or 48 hours; or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days; or at least about 1, 2, 3, 4, or 5 weeks).

[0014] In some embodiments of any of the methods described herein, the subject is not administered a population of support cells with the population of endothelial cells. Also described herein, inter alia, are methods of transplanting a population of endothelial cells (ECs) in a subject comprising: means for including mitophagy in a population of endothelial cells; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

[0015] In some embodiments of any of the methods described herein, the subject is not administered a population of support cells with the population of endothelial cells.

[0016] 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 mitophagy-inducing agent; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

[0017] 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 mitophagy-inducing agent; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

[0018] 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 mitophagy-inducing agent; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

[0019] In some embodiments, the mitophagy-inducing agent is any one or more of: rapamycin, SKF-96365, lithium, carbamazepine, latrepirdine, spermidine, resveratrol, sodium valproate, trehalose, kaempferol, metformin, AICAR, nilotinib, ambroxol, curcumin, VL-004, and combinations thereof. A useful mitophagy-inducing agent can be any agent (e.g., small molecule, pharmaceutical drug) known in the art to induce mitophagy (e.g., G.W. Dorn, II (2022) J Cardiovasc Aging 2(4) 45; Y. Lu, et al. (2023) Theranostics 13(2): 736-766; T. Eisenberg, et al (2016) NatMed22(V2): 1428-1438; P. Spilman, et al, (2010) PLoS One 5(4): e9979; Q. Li, et al. (2014) Biochem Biophys Res Commun 444(2) 182-8; D. Ebrahimi-Fakhari, et al. (2016) Cell Rep 17(4): 1053-1070; J.L. Gollihue and A.G. Rabchevsky, (2017) Mitochondrion 35: 70-79).

[0020] In some embodiments, the mitophagy-inducing agent does not include mitochondria or a composition comprising mitochondria. In some embodiments, the mitophagy-inducing agent does not include a protein or an organelle. In some embodiments, the mitophagy-inducing agent can be a small molecule, a drug, a protein, an organelle, and combinations thereof. In some embodiments, the mitophagy-inducing agent is not isolated mitochondria or a composition comprising isolated mitochondria.

[0021] 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). 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.

[0022] 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: smooth muscle cells (SMCs), pericytes, fibroblasts, mesenchymal stromal cells (MSCs), and / or perivascular cells derived from human induced pluripotent stem cells (iPSCs).

[0023] In some embodiments, a population comprising support cells are administered in addition to administering the population of ECs. In some embodiments, the population of support cells comprise perivascular cells. In some embodiments, the perivascular cells are any one or more of: SMCs, pericytes, fibroblasts, mesenchymal stromal cells (MSCs), and / or perivascular cells derived from human induced pluripotent stem cells (iPSCs).

[0024] In some embodiments, the population of support cells is administered to the subject with the population of endothelial cells (e.g., simultaneously). In some embodiments, the population of support cells is administered to the subject before the population of endothelial cells (e.g., at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 24, 36, or 48 hours; or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days; or at least about 1, 2, 3, 4, or 5 weeks). In some embodiments, the population of support cells is administered to the subject after the population of endothelial cells (e g., at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 24, 36, or 48 hours; or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days; or at least about 1, 2, 3, 4, or 5 weeks).

[0025] 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. In some embodiments, a population of support cells can be administered to the subject using the same administration method or a different administration method and the same frequency or a different frequency as the population of endothelial cells. In some embodiments, the population of endothelial cells are autogeneic or allogeneic. In some embodiments, the support cells are autogeneic or allogeneic.

[0026] In some embodiments, the subject has or is at risk of a metabolic disorder, a cancer, an immunological disease, or a mitochondrial dysfunction disorder. 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.

[0027] 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.

[0028] Also disclosed herein, inter alia, are populations of endothelial cells comprising one or more mitophagy inducing agent(s), MIA-ECs (Mitophagy Inducing Agent Endothelial Cells). In some embodiments, the populations of endothelial cells include a single cell type: ECs (e.g., MIA-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 organspecific 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.

[0029] In some embodiments, the populations of endothelial cells described herein (e.g., a population of MIA-ECs) do not include multiple cell types. In some embodiments, the populations of endothelial cells (e.g., a population of MIA-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 MIA-ECs) do not include any perivascular cells.

[0030] 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).

[0031] In some embodiments, the populations of endothelial cells (e.g., a population of MIA- ECs) do 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 (SMCs), pericytes, fibroblasts, 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 MIA-ECs) do not include any perivascular cells.

[0032] 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). In some embodiments, the populations of endothelial cells include multiple cell types, including 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, a population of cells from the organ, tissue, or cell transplant, and a population of support cells, (e.g., perivascular cells, mural cells, SMCs, and / or MSCs).

[0033] Also described herein are methods of making populations of ECs comprising a mitophagy-inducing agents (MIA-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 a mitophagy-inducing agent, thereby creating the population of ECs comprising the mitophagy-inducing agent (MIA- ECs).

[0034] 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 organspecific 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.

[0035] 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.

[0036] In some embodiments, the sample is obtained from an autogenous source, an allogeneic source, and / or a xenogeneic source.

[0037] In some embodiments of any of the populations of cells, compositions, and methods described herein, the mitophagy-inducing agent is selected from rapamycin, SKF-96365, lithium, carbamazepine, latrepirdine, spermidine, resveratrol, sodium valproate, trehalose, kaempferol, metformin, AICAR, nilotinib, ambroxol, curcumin, VL-004, and combinations thereof.

[0038] In some embodiments, the mitophagy inducing agent(s) are incubated with the ECs in the contacting step for a time sufficient for at least a portion of the ECs to internalize at least a portion of the mitophagy inducing agent(s). In some embodiments, the mitophagy inducing agent(s) 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 mitophagy inducing agent(s). In some embodiments, the population of 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% MIA-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 ECs comprise mitophagy inducing agent(s).

[0039] Also disclosed herein are populations of endothelial cells (e.g., MIA-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.). In some embodiments, any of the populations of cells and compositions described herein are for use in treating a subject having (or is at risk of having) a disease or disorder of the blood vessels, an organ transplant, a tissue transplant, a cell transplant, 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 tel angiectasias, 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.

[0040] 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 section headings, subheadings, titles, and subtitles herein are solely present for reader comfort, are not intended to be limiting, and are intended to be considered together. 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.

[0041] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0042] DESCRIPTION OF DRAWINGS

[0043] FIG. 1. Effect of rapamycin and Metformin on NRF1 and TFAM expression. Gene expression evaluated on human ECFCs at the mRNA level by qPCR. Genes analyzed are Nuclear Respiratory Factor 1 (NRF ) and Transcription Factor A, Mitochondrial TFAM). Both, NRF1 and TFAM, are genes associated with mitochondrial biogenesis, which is one of the downstream effects of mitochondrial autophagy - i.e., mitophagy. Our data revealed an increase in NRFI and TFAM expression that was similar to that observed in ECFCs after artificial mitochondrial transplantation (mitoT) and with starvation.

[0044] FIGS. 2A-2C show enhanced engraftment of ECFCs after treatment with Metformin. Human ECFCs were treated with 10 pM Metformin for 24 h and then were transplanted into immunodeficient nude mice for 7 days using our hydrogel graft model. FIG. 2A, H&E images show that grafts containing ECFCs that were treated with Metformin contained perfused vascular networks (yellow arrowheads). In contrast, grafts that contained untreated ECFCs failed to form blood vessels. Insets are macroscopic views of the explanted grafts at day 7. Yellow arrowheads point the perfused blood vessels containing red blood cells. Scale bar = 50 pm. FIG. 2B, Microvessel density (MVD) evaluation of the grafts indicated the enhanced engraftment and vascularization of ECFCs after treatment with Metformin. FIG. 2C, Humanspecific vimentin and UEA1 staining confirmed that some of the blood vessels were formed by human ECFCs treated with Metformin. Green arrowheads point at the vimentin and UEA1 double-positive human vessels. Scale bar = 50 pm.

[0045] FIGS. 3A-3F show engraftment of human ECs requires support from stromal cells. FIG. 3A, 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. 3B, 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. 3C, Perfused microvessel density at day 7 in grafts seeded with different human EC types with or without MSCs. **P 0.01, ***P 0.001 (n=3; unpaired two-tailed t- test) FIG. 3D, Immunofluorescent staining revealed the presence of blood vessels lined by human-specific ECs (UEA-1+) and surrounded by a-SMA+ perivascular cells at day 7 in grafts containing ECs and MSCs. Scale bars, 100 pm. FIG. 3E, Bioluminescence imaging of grafts containing lucif-ECs with or without MSCs. FIG. 3F, Quantification of bioluminescence signal at different time points. *P 0.05, **P 0.01, ***P < 0.001. (n=3; unpaired two-tailed t-test). All data are mean ± s.e.m.

[0046] FIGS. 4A-4D show MSC paracrine contribution is insufficient to enable robust EC engraftment. FIG. 4A, 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). EC apoptosis assessed by flow cytometry at 24 h post-implantation with / without MSCs, MSC-CM, VEGF, bFGF, and AG1296; *P < 0.05, ***P < 0.001 (n=3; unpaired t-test). FIG. 4B, 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 ImageJ. FIG. 4C, 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. 4D, Perfused microvessel density at day 7. 0.001 (n=3; unpaired two-tailed t-test). All data are mean ± s.e.m.

[0047] DETAILED DESCRIPTION

[0048] Without being bound by theory, the data presented herein demonstrates 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 inducing mitophagy in ECs enhances engraftment in vivo. The mitophagy is a process that removes damaged or excess mitochondria from a cell. This disclosure demonstrates that mitophagy can induce a transient cytoprotective effect, enabling EC engraftment without the use of any supporting cell (e.g., without perivascular cells or MSCs). The findings herein suggest potential for a new single-cell therapy strategy based on EC engraftment, which may facilitate clinical translation.

[0049] The studies herein have direct translational implications. The data showed that inducing mitophagy could effectively improve the engraftment capacity of ECs in ischemic tissues, where they form new blood vessels and provide robust therapeutic effects without the need for additional (or support) cell types, e.g., perivascular cells or perivascular cell sources, e.g., smooth muscle cells (SMCs), pericytes, fibroblasts, and mesenchymal stromal / stem cells (MSCs). The reliance on a single cell type for the methods presented herein simplifies clinical translation and applications enormously. This mitophagy inducement approach could become the basis for a new, more streamlined, and more efficient strategy in vascular cell therapies. Whether this strategy could benefit other forms of cell therapies remains to be determined. Populations of Endothelial Cells (ECs) and Methods of Making EC Populations

[0050] Compositions described herein and useful in any of the methods herein include populations of endothelial cells (ECs) that have been contacted with a composition comprising one or more mitophagy -inducing agent(s). In some embodiments, 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 a mitophagy-inducing agent, MIA-ECs).

[0051] Populations of MIA-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.

[0052] In some embodiments, the populations of endothelial cells described herein (e.g., a population of MIA-ECs) do not include multiple cell types. In some embodiments, the populations of endothelial cells (e.g., a population of MIA-ECs) do not include support cells (e.g., perivascular cells). In some embodiments, the populations of endothelial cells (e.g., a population of MIA-ECs) do 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 (SMCs), pericytes, fibroblasts, 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 MIA-ECs) do not include any perivascular cells.

[0053] 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). In some embodiments, the populations of endothelial cells include multiple cell types, including 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, a population of cells from the organ, tissue, or cell transplant, and a population of support cells, (e.g., perivascular cells, mural cells, SMCs, and / or MSCs).

[0054] Also described herein are methods of making populations of ECs comprising a mitophagy-inducing agents (MIA-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 a mitophagy-inducing agent, thereby creating the population of ECs comprising the mitophagy-inducing agent (MIA- ECs).

[0055] 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 is obtained from an autogenous source, an allogeneic source, and / or a xenogeneic source.

[0056] 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 iECs, 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 1 1,898,169; 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).

[0057] 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.

[0058] In some embodiments, a population of MIA-ECs described herein can be made contacting the population of ECs with one or more mitophagy -inducing agent(s). In some embodiments, the mitophagy-inducing agent is any one or more of: rapamycin, SKF-96365, lithium, carbamazepine, latrepirdine, spermidine, resveratrol, sodium valproate, trehalose, kaempferol, metformin, AICAR, nilotinib, ambroxol, curcumin, VL-004, and combinations thereof. A useful mitophagy-inducing agent can be any agent (e.g., small molecule, pharmaceutical drug, a protein, an organelle, and combinations thereof) known in the art to induce mitophagy. See, e.g., G.W. Dorn, II (2022) J Cardio ’asc Aging 2(4): 45; Y. Lu, et al. (2023) Theranostics 13(2): 736-766; T. Eisenberg, et al (2016) Nat Med 22(12): 1428-1438; P. Spilman, et al, (2010) PLoS One 5(4): e9979; Q. Li, et al. (2014) Biochem Biophys Res Commun 444(2) 182-8; D. Ebrahimi-Fakhari, et al. (2016) Cell Rep 17(4): 1053-1070; J.L. Gollihue and A.G. Rabchevsky (2017) Mitochondrion 35: 70-79).

[0059] In some embodiments, no exogenous mitochondria are administered to the ECs. In some embodiments, the mitophagy-inducing agent does not include mitochondria or a composition comprising mitochondria. In some embodiments, the mitophagy-inducing agent does not include a protein or an organelle.

[0060] In some embodiments, the mitophagy-inducing agent is 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). In some embodiments, at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% of the ECs internalize the mitophagy-inducing agent. I some embodiments, about 90% of the EC population are MIA-ECs. In some embodiments, the one or more mitophagy-inducing agent(s) are incubated with the ECs in the contacting step for a time sufficient for at least a portion of the ECs to internalize the mitophagy-inducing agent(s). 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 mitophagy-inducing agent(s). In some embodiments, the population of MIA-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% MIA-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 MIA-ECs comprise the mitophagy-inducing agent(s). 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).

[0061] In some embodiments, disclosed herein are populations of MIA-ECs made using any of the methods disclosed herein.

[0062] Cell Compositions and Formulations

[0063] Also disclosed herein are compositions comprising populations of ECs comprising a mitophagy-inducing agent. As used herein, the term “mitophagy-inducing agent” refers to an agent that can induce mitophagy. Exemplary mitophagy-inducing agents include e.g., rapamycin, SKF-96365, lithium, carbamazepine, latrepirdine, spermidine, resveratrol, sodium valproate, trehalose, kaempferol, metformin, AICAR, nilotinib, ambroxol, curcumin, VE-004, and combinations thereof. In some embodiments, a composition comprises ECs comprising a mitophagy-inducing agent (MIA-ECs) made using any of the methods described herein (see, e.g., above section). In some embodiments, described herein, inter alia, are populations of MIA-ECs assembled into a network of CD31+ vascular structures.

[0064] In some embodiments, also described herein vascular organoids (VOs) and 3D cell cultures comprising the CD31+ vascular structures comprising a population of MIA-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.

[0065] Also described herein, inter alia, are compositions comprising any of the populations of cells described herein (e.g., MIA-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).

[0066] 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.

[0067] 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, dichlorobenzyl alcohol, amylmetacresol and antibiotics. Exemplary antibiotics include penicillins carbapenems, cephalosporins aminoglycosides, bacitracin, gramicidin, mupirocin, chloramphenicol, thiamphenicol, 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, predni carb ate, fluticasone propionate, fhirandrenolide, 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.

[0068] 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.

[0069] 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.

[0070] Methods of Using the Populations of ECs and Compositions Comprising the ECs

[0071] Described herein, inter alia, are methods of administering any of the populations of cells (e.g., MIA-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.

[0072] 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).

[0073] 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).

[0074] 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 MIA-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.

[0075] Described herein, inter alia, are methods of increasing vascular development, angiogenesis, and cell junction via administering a population of MIA-ECs to a subject. In some embodiments, described herein are methods of administering a population of MIA-ECs (e.g., therapeutic vascularization) and methods of modeling vascular diseases (e.g., a 3D vascular organoid (VO)) are also described herein.

[0076] 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 MIA-ECs) are used as a cell source for tissue engineering applications, particularly in the development of small-diameter vascular grafts.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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. 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.

[0082] 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 a mitophagy-inducing agent. In some embodiments, the ECs are contacted with the mitophagy-inducing agent before the organ or tissue is transplanted in the subject. In some embodiments, the ECs are contacted with the mitophagy-inducing agent during the transplantation surgery. In some embodiments, the ECs are contacted with the mitophagy- inducing agent 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 mitophagy-inducing agent. In some embodiments, the ECs of an organ may be perfused with a composition comprising the mitophagy-inducing agent. The term “perfusion” is an art recognized term, and relates to the passage of a liquid, e.g., a composition comprising a mitophagy-inducing agent, 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 mitophagy-inducing agent, to remove the donor's blood from the organ. As another option, the UW solution can include the mitophagy-inducing agent. 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.

[0083] 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.

[0084] 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).

[0085] Without being bound by theory, narrowing of blood vessels result from plaque build-up 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).

[0086] 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 MIA-ECs, cell populations, VOs, and compositions herein could help stabilize these vessels, improving the delivery of therapeutics, and / or reduce metastasis. Without being bound by theory, in diabetic retinopathy, pericyte loss leads to weakened blood-retinal barriers, resulting in retinal ischemia and neovascularization. The MIA-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.

[0087] 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 MIA-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.

[0088] Without being bound by theory, in atherosclerosis, the stability of blood vessels is compromised due to inflammatory processes and endothelial dysfunction. The MIA-ECs, cell populations, VOs, and / or compositions described herein can help reinforce vascular walls and / or mitigate the progression of atherosclerotic plaques.

[0089] 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 MIA-ECs, cell populations, VOs, and / or compositions described herein can enhance angiogenesis and vascular stability, promoting better wound healing outcomes.

[0090] 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 MIA-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.

[0091] 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 MIA-ECs, cell populations, VOs, and / or compositions described herein could stabilize these blood vessels and / or alleviate one or more symptoms (e.g., hypertension).

[0092] 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 MIA-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.

[0093] In some embodiments of any of the methods described herein, the population of ECs (e.g., MIA-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 MIA-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., MIA-ECs).

[0094] 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.

[0095] 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. In some embodiments, a population of support cells can be administered to the subject using the same administration method or a different administration method and the same frequency or a different frequency as the population of endothelial cells.

[0096] In some embodiments, the population of endothelial cells are autologous to the subject. In some embodiments, the population of endothelial cells are allogeneic, optionally allogeneic iPSC-derived ECs.

[0097] 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).

[0098] 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. 11,214,768; WO 2024 / 133285; WO 2022 / 226337; and WO 2023 / 196683; each of which is incorporated herein by reference in its entirety).

[0099] EXAMPLES

[0100] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0101] Methods and Materials

[0102] Cell culture

[0103] 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 protocols . 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.

[0104] Generation of mitoDsRed-labeled cells

[0105] 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.

[0106] In vitro cell co-culture

[0107] To assess mitochondrial transfer, human ECs and mitoDsRed-labeled MSCs were cocultured on 1% (w / v) gelatin-coated plates in EC-medium. The effect of cell density on mitochondrial transfer was investigated by mixing mitoDsRed-MSCs and ECs in a 1 : 1 ratio and plating them in individual wells of a 6-well plate, with total cell numbers ranging from 0.05 to 0.8xl06per well. To evaluate the impact of donor-to-recipient ratios, mitoDsRed-MSCs and ECs were mixed in various ratios from 1 : 10 to 10: 1, maintaining a total cell count of 4xl05per well. The influence of hypoxia and 3D culture was studied by mixing mitoDsRed-MSCs and ECs in a 1 : 1 ratio with a total of 4xl05cells under either 2D (culture plates) or 3D (collagen hydrogel; 200 pL) conditions, and in normoxia (21% O2) or hypoxia (1% O2). Co-cultures were maintained for 24 hours before analysis via microscopy and flow cytometry.

[0108] Immunofluorescence staining

[0109] 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

[0110] 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 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.

[0111] Flow cytometry and cell sorting

[0112] 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).

[0113] Bioluminescence imaging

[0114] 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 (8xlO5cells) 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).

[0115] Histology and immunofluorescence staining

[0116] 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 1A4). Microvessels density

[0117] 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.

[0118] RNAseq analysis

[0119] 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 * 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).

[0120] Quantitative Real-Time PCR

[0121] 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. Human cytokine protein array

[0122] Selected cytokines were evaluated in conditioned medium samples using the Proteome Profder 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 fdm (Kodak). Densitometry was carried out using image analysis (ImageJ) to estimate protein amounts in each sample.

[0123] Microscopy

[0124] 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-fluore scent images were taken with an AxioCam MRc5 camera using a 10X or 20X objective lens.

[0125] Statistical analyses

[0126] 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.

[0127] Example 1: Endothelial cell engraftment without the use of a mitophagy-inducing agent requires support from stromal cells

[0128] 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. 3A). 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 (Figs. 3B-3D). The marked difference in microvessel density due to MSC presence was evident in histological analysis (Figs. 3B-3C). Engraftment disparity was confirmed using bioluminescence imaging of luciferase-expressing ECs (luc-ECs) for up to 4 weeks (Figs. 3E-3F). 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 (Figs. 3B-3C).

[0129] 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. 4B; EC apoptosis assessed by flow cytometry at 24 h post-implantation with / without MSCs, MSC-CM, VEGF, bFGF, and AG1296). Nonetheless, supplementing MSC-secreted factors could not replace MSCs and was insufficient to support early survival (24 h post-implantation; Fig. 4A) or blood vessel-forming ability of ECs at day 7 (Fig. 4C), indicating that MSCs' supportive role is not exclusively paracrine. Moreover, inhibiting PDGFR signaling (tyrphostin AG 1295), which delays MSC-EC juxtaposition, did not affect EC engraftment (Figs. 4A, 4C, 4D), suggesting that the PDGFR signaling-mediated perivascular contact is not essential for maintaining EC viability under immediate post-implantation stress.

[0130] Example 2: Mitophagy-inducing agents Increase Expression of Genes Associated with Mitochondrial Biogenesis

[0131] Experiments were conducted to test the effect mitophagy-inducing agents have on gene expression.

[0132] The genes analyzed are Nuclear Respiratory’ Factor 1 (NRFF) and Transcription Factor A, Mitochondrial (TFAM). Both, NRF1 and TFAM, are genes associated with mitochondrial biogenesis, which is one of the downstream effects of mitochondrial autophagy - i.e., mitophagy. Briefly, human ECs were treated with two mitophagy-inducing agents, Rapamycin (mTOR pathway) and Metformin (AMPK pathway) and compared to cells without treatment (“control”), cells that were administered mitochondria to induce mitophagy (“mitoT”), and cells that were cultured under starvation conditions (2h in HBSS buffer) to induce mitophagy (Fig. 1). Specifically, human ECs were treated with either Rapamycin (10 pM) or Metformin (10 pM) for 24 h.

[0133] Gene expression was then evaluated on the human ECFCs at the mRNA level via qPCR. Our data revealed that treatment with a mitophagy-inducing agent resulted in an increase in both NRF1 and TFAM expression similar to that observed in ECFCs after artificial mitochondrial transplantation (mitoT) and with starvation.

[0134] Example 3: Enhanced Engraftment of ECFCs after Treatment with a Mitophagy-inducing agent

[0135] Experiments were then conducted to test the ability of a mitophagy-inducing agent to enhance EC engraftment and angiogenesis.

[0136] Human ECFCs were treated with 10 M Metformin for 24 h and then were transplanted into immunodeficient nude mice for 7 days using our hydrogel graft model. The H&E images show that grafts containing ECFCs that were treated with Metformin contained perfused vascular networks (arrowheads in Fig. 2A, right panel). In contrast, grafts that contained untreated ECFCs failed to form blood vessels (Fig. 2A, left panel). Insets are macroscopic views of the explanted grafts at day 7 (Fig. 2A, both panels).

[0137] Microvessel density (MVD) evaluation of the grafts showed the enhanced engraftment and vascularization of ECFCs after treatment with Metformin (Fig. 2B). Human-specific vimentin and UEA-1 (a lectin that binds to human ECs with high affinity but not to murine ECs) staining confirmed that some of the blood vessels were formed by the human ECFCs treated with Metformin (Fig. 2C).

[0138] Taken together, the results show treating ECs with mitophagy / autophagy inducers will transiently provide cytoprotection and enhance the engraftment ability of the cells once they are in vivo. This finding is significant because it signifies that treating ECs with a mitophagy- inducing agent can be used to significantly enhance the outcome of endothelial cell therapies, for example in ischemic diseases.

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[0193] 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 a mitophagy-inducing agent; 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 a mitophagy-inducing agent; 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 a mitophagy-inducing agent; 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 a mitophagy-inducing agent; and administering to the subject a therapeutically effective amount of the population of endothelial cells.

5. The method of any one of claims 1-4, wherein the mitophagy-inducing agent is any one or more of: rapamycin, SKF-96365, lithium, carbamazepine, latrepirdine, spermidine, resveratrol, sodium valproate, trehalose, kaempferol, metformin, AICAR, nilotinib, ambroxol, curcumin, VL-004, and combinations thereof.

6. The method of any one of the preceding claims, wherein the mitophagy-inducing agent does not comprise mitochondria or a composition comprising mitochondria.

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

8. 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 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).

9. The method of claim 8, 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.

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

11. The method of claim 10, wherein the support cells are perivascular cells.

12. The method of claim 10, wherein 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).

13. 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.

14. The method of claim 13, 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.

15. The method of any one of claims 1-12, wherein administering is performed by intravenous, intra-articular, subcutaneous, intraperitoneal, intramuscular, intradermal, or intracardiac injection.

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

17. The method of any one of claims 1-15, 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).

18. The method of claim 17, 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.

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, 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 tel angiectasias, arteriovenous malformations, a metabolic disorder, diabetes, diabetic retinopathy, an ischemic injury, an IRI injury, atherosclerosis, Age-related Macular Degeneration (AMD), PulmonaryArterial 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.

21. A method of making a population of endothelial cells (ECs) comprising a mitophagy-inducing agent, the method comprising: vii. obtaining a sample (e.g., a sample comprising ECs) from a subject (e g., blood or a tissue sample); viii. isolating a population of ECs from the sample; ix. contacting the population of ECs with a mitophagy-inducing agent, thereby creating the population of ECs comprising the mitophagy-inducing agent (MIA- ECs).

22. The method of claim 21, 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).

23. The method of any one of claims 21-22, 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), organspecific endothelial cells, and / or ECs derived from human induced pluripotent stem cells (iPSCs).

24. The method of claim 23, 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.

25. The method of any one of claims 21-24, wherein the mitophagy-inducing agent is selected from rapamycin, SKF-96365, lithium, carbamazepine, latrepirdine, spermidine,resveratrol, sodium valproate, trehalose, kaempferol, metformin, AICAR, nilotinib, ambroxol, curcumin, VL-004, and combinations thereof.

26. The method of any one of claims 21-25, wherein the mitophagy-inducing agent is incubated with the ECs in the contacting step for a time sufficient for at least a portion of the ECs to internalize at least a portion the mitophagy-inducing agent (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).

27. The method of any one of claims 21-26, wherein at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% of the ECs internalize the mitophagy-inducing agent.

28. The method of any one of claims 21-27, wherein about 90% of the EC population are MIA-ECs.29 The method of any one of claims 21-28, wherein no exogenous mitochondria are administered to the ECs.

30. The population of MIA-ECs made according to the method of any one of claims 21-29.

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