Stable three-dimensional blood vessels and a method for forming the same

Abstract

A method for forming blood vessels having a stable three-dimensional vasculature is provided. [Solution] A method for producing stable three-dimensional blood vessels, comprising: a) culturing endothelial cells containing exogenous nucleic acid encoding the ETV2 transcription factor on a matrix under conditions in which the endothelial cells express the ETV2 transcription factor for at least 3 to 4 weeks to induce angiogenesis; and b) isolating the stable three-dimensional blood vessels.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 454,161, filed on February 3, 2017, the entire content of which is incorporated herein by reference.

[0002] Incorporation by Reference of Sequence Listing The sequence listing in the ASCII text file named 34633_7600_02_PC_PCTSequenceListing.txt, 8KB, created on February 1, 2018 and submitted to the United States Patent and Trademark Office via EFS - Web, is incorporated herein by reference.

[0003] The present disclosure generally relates to methods for forming stable three - dimensional vascular structures such as blood vessels. More specifically, the present disclosure is directed to stable three - dimensional artificial blood vessels, methods that do not use scaffolds for forming such vessels, and their use.

Background Art

[0004] Embryonic organogenesis and tissue regeneration rely on vasculogenesis by functional long - lasting blood vessels. Non - Patent Document 1. Blood vessels are important for maintaining tissue - specific homeostasis and for supplying paracrine angiocrine factors to guide proper patterning and morphogenesis of developing tissues. See, for example, Non - Patent Document 2; and Non - Patent Document 3.

[0005] Attempts to induce the formation of new blood vessels to repair damaged organs, a process known as angiogenesis, have faced difficulties. See Non-Patent Document 4. To date, injection of angiogenic factors into damaged organs to induce vasculogenesis has not been effective in establishing functionally durable capillaries for long-term use. Furthermore, approaches to fabricating stable blood vessels in vitro for translation to the clinical setting have been cumbersome and time-consuming. For example, current approaches require the fabrication of scaffolds to form three-dimensional vasculature, the use of pericytes surrounding the blood vessels, and forced perfusion to reproduce the hemodynamic microenvironment. See Non-Patent Document 5.

[0006] In addition, existing models use clinically incompatible artificial extracellular matrices such as Matrigel™ (Corning), which limits the remodeling and patterning of the putative capillary network in culture. Mature adult human endothelial cells form vascular networks in Matrigel™ (Corning) matrices in vitro or in vivo, but these blood vessels are not stable, have limited remodeling potential, and regress within a few weeks.

[0007] Technologies for fabricating organ-on-chip models (see Non-Patent Document 5) and three-dimensional bioprinting (see Non-Patent Document 6) have advanced disease modeling and drug screening remarkably. However, the ability to incorporate physiologically relevant vascular cells into these approaches has lagged. Specifically, in this approach, direct endothelial cell interactions with tissue-specific epithelial cells and tumor cells are limited due to physical constraints imposed by artificial biomaterials. Furthermore, this organ-on-chip or vascular scaffold approach requires separation by a layer of semipermeable artificial biomaterials, which impairs essential physical cell-cell interactions between endothelial cells and non-vascular cells, thereby limiting adaptive or maladaptive endothelial cell remodeling See Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; and Non-Patent Document 10.

[0008] The need for scaffolds, physical barriers, and artificial matrices limits in vitro studies of endothelial cell crosstalk with organoid cultures and the transition to tissue-specific artificial vasculature. Thus, a new approach using endothelial cells that can mix with and remodel organotypic stem cells to vascularize tumor organoids or tissue-specific organoids or decellularized organs would improve translation to clinical settings for regeneration, drug screening, and tumor targeting.

[0009] The stable three-dimensional blood vessels and methods of the present disclosure utilize unrecognized reprogramming elements that define the formation of stable three-dimensional artificial blood vessels that are fully functional in vivo for a much longer duration than current artificial vasculature. Further, the compositions and methods disclosed herein generate organ-specific blood vessels capable of vascularizing injured tissues, organoids, and decellularized organs.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non - Patent Document 7

Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0011] The present disclosure reveals that the ETS transcription factor variant 2, ETV2 (ER71, ETSRP71), directs endothelial cell development and plays an essential role in reprogramming differentiated endothelial cells into primordial - like endothelial cells that are more plastic, malleable, and responsive to environmental stimuli and that can accommodate other non - vascular cell types, including normal epithelial cells and tumor cells. More specifically, the methods and compositions of the present disclosure identify that exogenous expression of the ETV2 transcription factor in endothelial cells cultured on a matrix containing extracellular matrix components results in the formation of stable and functional three - dimensional artificial blood vessels in vitro and in vivo without the use of pericytes, perfusion, and cumbersome scaffolds. Thus, the present disclosure is directed to the discovery that reprogrammed endothelial cells cultured on a matrix, such as a matrix containing laminin, entactin, and / or collagen IV, form long - lasting functional three - dimensional artificial blood vessels in vitro and, in addition, in vivo.

Means for Solving the Problems

[0012] Accordingly, a first aspect of the present disclosure is directed to a method for forming a stable three-dimensional vascular structure, such as a vascular tubule having a lumen. A significant advantage of the methods described herein is that they do not utilize a three-dimensional scaffold, thereby advantageously eliminating the costly and time-consuming elements currently required to form artificial three-dimensional blood vessels. A further advantage of the method is the identification and use of a biocompatible matrix, which results in the remodeling and patterning of a stable vascular network. Thus, the method eliminates many of the current drawbacks that undermine existing methods for forming blood vessels.

[0013] In one embodiment of the present disclosure, a method for forming a stable three-dimensional blood vessel is provided that includes culturing endothelial cells containing an exogenous nucleic acid encoding an ETV2 transcription factor on a matrix such that the endothelial cells express the ETV2 transcription factor protein for at least three weeks.

[0014] Exogenous expression of the ETV2 transcription factor protein in differentiated endothelial cells reprograms the differentiated endothelial cells and provides them with the ability to self-assemble into stable functional three-dimensional vascular structures such as lumen-bearing blood vessels, which can then be isolated and used for a number of purposes such as vascular introduction into injured tissue, vascular introduction into organoids, or vascular reintroduction into decellularized organs. Thus, in some embodiments, the endothelial cells used in the method are differentiated endothelial cells. In certain embodiments, the differentiated endothelial cells are human endothelial cells. In specific embodiments, the differentiated human endothelial cells are human umbilical vein-derived endothelial cells (HUVECs), human adipose-derived endothelial cells, or tissue / organ-specific human endothelial cells. In some embodiments of the method, the differentiated endothelial cells are organ-specific endothelial cells, including but not limited to endothelial cells of the heart, kidney, testis, ovary, retina, liver, pancreas, brain, lung, spleen, large intestine, or small intestine. In other embodiments, the differentiated endothelial cells are tissue-specific endothelial cells derived from muscle, lymphoid tissue, olfactory tissue, bone-forming tissue, oral (tooth) tissue, or glandular tissue (e.g., endocrine gland, thymus).

[0015] In certain embodiments of the method, the exogenous ETV2-encoding nucleic acid is present within the endothelial cells. In one embodiment, the exogenous ETV2-encoding nucleic acid corresponds to the human ETV2 gene nucleotide sequence shown in SEQ ID NO: 1, which encodes a human ETV2 transcription factor protein having an amino acid sequence as shown in SEQ ID NO: 2. In other embodiments, the exogenous ETV2-encoding nucleic acid is an ETV2 ribosomal nucleic acid (RNA) transcript encoding the ETV2 transcription factor protein.

[0016] In certain embodiments, the exogenous ETV2 nucleic acid is provided to endothelial cells by transfection or transduction. In certain embodiments, the nucleic acid encoding ETV2 is transduced into endothelial cells using a viral vector such as a lentiviral vector. In one embodiment, the exogenous ETV2 transcription factor-encoding nucleic acid is provided to differentiated endothelial cells using an inducible expression system such as, for example, the reverse tet transactivator (rtTA)-doxycycline inducible expression system.

[0017] In some embodiments, the method includes culturing endothelial cells comprising the exogenous ETV2-encoding nucleic acid under conditions that express the ETV2 transcription factor protein. The ETV2 protein can be expressed constitutively or transiently. In some cases, the exogenous ETV2 transcription factor is expressed in differentiated endothelial cells for at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or longer to reprogram differentiated endothelial cells and induce angiogenesis with lumens. In certain embodiments, the exogenous ETV2 protein is expressed for at least 3 - 4 weeks to induce angiogenesis.

[0018] In other embodiments, the method comprises culturing endothelial cells having exogenous ETV2-encoding nucleic acid under conditions that express the ETV2 transcription factor protein, followed by a further culture period under conditions where the endothelial cells do not express exogenous ETV2. In this case, the endothelial cells are first cultured for a first period under conditions that express the exogenous ETV2 transcription factor, and then a second culture can be performed under conditions where exogenous ETV2 is not expressed, such as in the absence of a substance capable of activating an inducible promoter (e.g., doxycycline, tetracycline). Optionally, the method comprises culturing endothelial cells having exogenous ETV2-encoding nucleic acid for at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or longer under conditions that transiently express the ETV2 transcription factor protein (e.g., in the presence of doxycycline), followed by a second culture for several days, several weeks, or several months under conditions where exogenous ETV2 is not expressed, such as in the absence of doxycycline.

[0019] In some embodiments of the method, endothelial cells containing exogenous ETV2-encoding nucleic acid are cultured on a matrix. In certain embodiments, the matrix is composed of defined extracellular matrix components such as laminin, entactin, and collagen. In a particular embodiment, the matrix used in the method is composed of a combination of laminin, entactin, and collagen IV (L.E.C.). In one embodiment, the matrix may contain laminin and entactin at a combined concentration of at least 5 mg / mL. In an exemplary embodiment, ETV2-expressing endothelial cells are cultured on a matrix containing laminin and entactin at a combined concentration of 5 mg / mL. Since laminin and entactin can bind to each other to form a complex, the matrix may contain a complex of laminin and entactin. For example, the matrix may contain at least 5 mg / mL of a complex of laminin and entactin. For example, endothelial cells can be cultured on a matrix containing at least 5 mg / mL of a complex of laminin and entactin and at least 0.2 mg / mL of collagen IV to form a long-lasting functional three-dimensional artificial blood vessel. In a particular embodiment, endothelial cells are cultured on a matrix composed of laminin and entactin at a combined concentration of 5.25 mg / mL and 0.2 mg / mL of collagen IV. In other embodiments, the matrix is Matrigel™ (Corning).

[0020] In one embodiment of the method, endothelial cells containing exogenous ETV2-encoding nucleic acid are cultured in serum-free medium. In some embodiments, the method includes culturing the endothelial cells in serum-free medium for at least 7 days, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or longer. In other embodiments, the method includes culturing the endothelial cells containing exogenous ETV2-encoding nucleic acid in serum-free medium at a low oxygen pressure, i.e., less than the atmospheric oxygen pressure (20% oxygen pressure). In certain embodiments, the cells are cultured in serum-free medium at an oxygen pressure between 4% and 15%, between 5% and 10%, between 4% and 8%, or between 4% and 6%. In one embodiment, the cells are cultured in serum-free medium at an oxygen pressure of 5% for at least 7 days.

[0021] In some embodiments, the method includes culturing endothelial cells containing exogenous ETV2-encoding nucleic acid on a matrix under conditions that express the ETV2 transcription factor to induce the formation of stable three-dimensional blood vessels having lumens, and isolating the stable three-dimensional blood vessels.

[0022] The present disclosure also reveals that exogenous expression of the ETV2 protein in differentiated endothelial cells results in the autonomous self-assembly of stable functional three-dimensional blood vessels having the ability to form functional vascular networks in vitro and in vivo in the absence of pericytes, perfusion, and scaffolds.

[0023] Accordingly, in another aspect of the present disclosure, there is provided a stable three-dimensional blood vessel capable of autonomously forming a three-dimensional vascular network.

[0024] In certain embodiments, the stable three-dimensional blood vessels of the present disclosure include a tubular structure such as a blood vessel having a lumen, which is composed of at least one continuous layer of reprogrammed endothelial cells. In some embodiments, the reprogrammed endothelial cells contain exogenous ETV2 transcription factor-encoding nucleic acid.

[0025] In certain embodiments, the endothelial cells of the stable three-dimensional blood vessels express an exogenous ETV2 transcription factor-encoding nucleic acid. In one embodiment, the expression of the ETV2 transcription factor is transient, i.e., for a limited period such as for several days, weeks, or months. In certain embodiments, the transient expression of the ETV2 transcription factor is regulated by an inducible expression system, such as the reverse tet transactivator (rtTA)-doxycycline inducible expression system. In other embodiments, the stable three-dimensional blood vessels of the present disclosure contain endothelial cells that constitutively (i.e., permanently) express an exogenous ETV2 transcription factor. In other embodiments, the stable three-dimensional blood vessels are composed of a combination of endothelial cells that express an exogenous ETV2 transcription factor and endothelial cells that do not express exogenous ETV2. In yet another embodiment, the stable three-dimensional blood vessels are composed of reprogrammed endothelial cells that do not express an exogenous ETV2 transcription factor.

[0026] In some embodiments, the stable three-dimensional blood vessels of the present disclosure are isolated. In this case, the blood vessels are removed, in whole or in part, from the environment in which they were formed. In certain embodiments, the isolated stable three-dimensional blood vessels do not contain a medium, medium components and additives, and any matrix in which they were formed, such as Matrigel™ or L.E.C. matrix. In other embodiments, the isolated stable three-dimensional blood vessels of the present disclosure have been removed from a medium, medium components and additives, but contain at least a portion of a matrix such as Matrigel™ or L.E.C. matrix.

[0027] In one embodiment of the present disclosure, a stable three-dimensional blood vessel is functional. In certain embodiments, a functional stable three-dimensional blood vessel has the ability to allow a fluid to pass through (perfuse) within the blood vessel. In other embodiments, a functional and stable three-dimensional blood vessel has the ability to allow blood to pass through within the blood vessel. In yet another embodiment, a functional and stable three-dimensional blood vessel has the ability to form a three-dimensional blood vessel network. In one embodiment, a stable three-dimensional blood vessel forms (vascularizes) a functional three-dimensional blood vessel network that includes at least one capillary, at least one arteriole, at least one venule, at least one lymphatic vessel, or a combination thereof, which has the ability to transport blood to tissue.

[0028] In some embodiments, the functional and stable three-dimensional blood vessels of the present disclosure are functional for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or longer. In certain embodiments, the stable three-dimensional blood vessels of the present disclosure are stable for 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or longer after angiogenesis.

[0029] Since the present disclosure shows that endothelial cells containing exogenous ETV2 can autonomously organize into a patterned stable artificial blood vessel capable of vascularizing in vivo, the present disclosure also provides a method for promoting vascularization. Therefore, in another aspect, a method for promoting vascularization in a subject is provided, which includes administering a stable three-dimensional blood vessel to the subject. In some embodiments, the subject has injured tissue such as an organ in need of vascularization. In certain embodiments, the subject has injured heart tissue, injured liver tissue, injured lymphatic tissue, injured kidney tissue, injured testicular tissue, injured ovarian tissue, injured retina, injured pancreatic tissue, injured brain tissue, injured lung tissue, injured intestinal tissue, injured glandular tissue, injured muscle tissue, or a combination thereof.

[0030] In one embodiment, the method includes administering stable three-dimensional blood vessels to a subject by surgical implantation into the damaged tissue of the subject that requires vascularization. In certain embodiments, the stable three-dimensional blood vessels are implanted directly into the damaged organ or tissue thereof. In other embodiments, the method includes administering stable three-dimensional blood vessels to a subject by injection, such as direct injection into the damaged tissue. In certain embodiments, the stable three-dimensional blood vessels are administered to the subject by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or combinations thereof.

[0031] In some embodiments, a method for promoting vascularization in a subject includes administering at least one stable three-dimensional blood vessel formed in vitro to the subject. In other embodiments, the method includes administering two or more stable three-dimensional blood vessels of the present disclosure formed in vitro. In certain embodiments, the method includes directly administering a plurality of stable three-dimensional blood vessels on a matrix composed of laminin, entactin, and collagen IV to a tissue that requires vascularization, such as damaged tissue.

[0032] Once the stable three-dimensional blood vessels are administered to the subject, the functional and stable three-dimensional blood vessels establish a three-dimensional blood vessel network capable of vascularizing the endogenous tissue. In some embodiments, the stable three-dimensional blood vessels form a three-dimensional blood vessel network that includes at least one capillary, at least one arteriole, at least one venule, at least one lymphatic vessel, or combinations thereof, capable of transporting blood to the tissue.

[0033] In another aspect of the present disclosure, a method for vascularizing an organoid is provided. In this case, the method for vascularizing an organoid includes culturing the organoid with endothelial cells containing an exogenous nucleic acid encoding the ETV2 transcription factor on a matrix under conditions that express exogenous ETV2 protein in the endothelial cells to form a stable three-dimensional blood vessel network on the organoid.

[0034] In some embodiments, a method for vascularizing an organoid includes culturing the organoid with endothelial cells on a matrix. In certain embodiments, the matrix is composed of defined extracellular matrix components such as laminin, entactin, and collagen. In a particular embodiment, the matrix used in the method is composed of a combination of laminin, entactin, and collagen IV (L.E.C.). In one embodiment, the matrix may contain laminin and entactin at a combined concentration of at least 5 mg / mL. In an exemplary embodiment, the organoid is cultured with endothelial cells on a matrix containing laminin and entactin at a combined concentration of 5 mg / mL. Since laminin and entactin can bind to each other to form a complex, the matrix may contain a complex of laminin and entactin. For example, the matrix may contain at least 5 mg / mL of a complex of laminin and entactin. For example, to vascularize an organoid, endothelial cells can be cultured on a matrix containing at least also a complex of laminin and entactin at a concentration of 5 mg / mL, and at least 0.2 mg / mL of collagen IV. In a particular embodiment, the matrix is composed of laminin and entactin at a combined concentration of 5.25 mg / mL, and 0.2 mg / mL of collagen IV. In other embodiments, the matrix is Matrigel™ (Corning).

[0035] In one embodiment, a method for vascularizing an organoid includes culturing the organoid with endothelial cells on a matrix in a medium containing fibroblast growth factor (FGF). In certain embodiments, the medium contains basic fibroblast growth factor (FGF2). In some embodiments, the medium contains heparin. In a particular embodiment, a method for vascularizing an organoid includes culturing the organoid with endothelial cells on a matrix in a medium containing FGF2 and heparin.

[0036] Organoids and endothelial cells containing exogenous nucleic acids encoding the ETV2 transcription factor, on a matrix, under conditions that express exogenous ETV2 protein in the endothelial cells, induce the formation of a functional three-dimensional vascular network in the organoids, i.e., they can be cultured for any period necessary to introduce vasculature into the organoids. For example, in certain embodiments, the organoids and differentiated endothelial cells are cultured for at least 1 week (7 days), at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or longer, such that the endothelial cells are reprogrammed and the organoids are induced to form patterned, long-lasting artificial vasculature. In some embodiments, the vasculature-introduced organoids have a three-dimensional vascular network that includes at least one capillary, at least one arteriole, at least one venule, at least one lymphatic vessel, or a combination thereof, that is capable of transporting blood across the organoid.

[0037] The method for introducing vasculature into organoids can be applied to any type of organoid. For example, the organoids used in the method can be small intestine organoids, colon organoids, kidney organoids, heart organoids, or tumor organoids. In some embodiments, the method can be used to introduce vasculature into tumor organoids, such that the tumor organoids can be tissue-specific, such as breast cancer organoids, colon cancer organoids, kidney cancer organoids, or intestinal cancer organoids.

[0038] The vasculature-introduced organoids of the present disclosure can be used by those skilled in the art for a number of purposes. Thus, in some embodiments, the method for introducing vasculature into organoids includes isolating the vasculature-introduced organoids.

[0039] Once a vasculature-introduced organoid is obtained, the vasculature-introduced organoid can be administered to a subject such as a human or an animal (e.g., mouse, rat, dog, monkey). In certain embodiments, the vasculature-introduced organoid can be administered to a mouse by surgical implantation.

[0040] In other embodiments, the isolated vasculature-introduced organoid can be used to determine the efficacy of a therapeutic agent. In this case, the vasculature-introduced organoid is contacted with the therapeutic agent and cultured for a period of time in the presence of the therapeutic agent. Thereafter, during the culture, the organoid function, cell proliferation and health status can be analyzed to determine the efficacy of the therapeutic agent.

[0041] The present disclosure also reveals that culturing a decellularized organ together with endothelial cells containing an exogenous nucleic acid encoding the ETV2 transcription factor under conditions that result in the expression of exogenous ETV2 protein in the endothelial cells results in the generation of functional vascular structures in the decellularized organ. Therefore, one aspect of the present disclosure provides a method for reintroducing vasculature into a decellularized organ.

[0042] In some embodiments, a method for vascular reintroduction into a decellularized organ comprises culturing the decellularized organ with reprogrammed endothelial cells on a matrix. In certain embodiments, the matrix used in the method is composed of a combination of laminin, entactin, and collagen IV (L.E.C.). In certain embodiments, the matrix is composed of defined extracellular matrix components such as laminin, entactin, and collagen. In one embodiment, the matrix can contain laminin and entactin at a combined concentration of at least 5 mg / mL. In an exemplary embodiment, the organ is cultured with endothelial cells on a matrix containing laminin and entactin at a combined concentration of 5 mg / mL. The matrix can contain a complex of laminin and entactin at a concentration of at least 5 mg / mL. For example, to vascularize an organ, endothelial cells can be cultured on a matrix containing a complex of laminin and entactin at a concentration of at least 5 mg / mL and at least 0.2 mg / mL collagen IV. In certain embodiments, the matrix is composed of laminin and entactin at a combined concentration of 5.25 mg / mL and 0.2 mg / mL collagen IV. In other embodiments, the matrix is Matrigel™ (Corning).

[0043] In some embodiments, a method for vascular reintroduction into a decellularized organ comprises culturing the decellularized organ in a bioreactor with endothelial cells on a matrix under conditions that express an exogenous ETV2 transcription factor protein.

[0044] In one embodiment, a method for vascular reintroduction into a decellularized organ comprises culturing the decellularized organ with endothelial cells on a matrix under conditions that induce the formation of a functional three-dimensional vascular network in the endothelial cells, i.e., culturing for any period necessary to effect vascular reintroduction into the organ. For example, in certain embodiments, the decellularized organ and reprogrammed endothelial cells are cultured for at least 1 week (7 days), at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or longer, such that the endothelial cells are induced to organize into long-lived vessels with lumens on the decellularized organ. In other embodiments, the endothelial cells form a functional three-dimensional vascular network in the decellularized organ that includes at least one capillary, at least one arteriole, at least one venule, at least one lymphatic vessel, or a combination thereof, capable of transporting blood across the organ.

[0045] The method can be applied to any type of decellularized organ. For example, the endothelial cells can be cultured on a decellularized organ such as a decellularized heart, kidney, testis, ovary, retina, bone, endocrine gland, thyroid, trachea, lymph node, liver, pancreas, brain, lung, spleen, large intestine, or small intestine under conditions that express exogenous ETV2 protein in the endothelial cells.

[0046] The vascular reintroduced organs of the present disclosure can be used by those skilled in the art for a number of purposes. Thus, in some embodiments, the method for vascular reintroduction into a decellularized organ comprises isolating the vascular reintroduced organ. In an exemplary embodiment, the vascular reintroduced organ can be isolated and then administered to a subject such as a human or an animal (e.g., mouse, rat, dog, monkey). In certain embodiments, the vascular reintroduced decellularized organ is isolated and administered to a human subject by surgical implantation.

[0047] In other embodiments, the isolated vascular reintroduction decellularized organ can be used to determine the efficacy of a therapeutic agent. In this case, the vascular reintroduction decellularized organ is contacted with the therapeutic agent and cultured for a period of time in the presence of the therapeutic agent. Thereafter, during the culture, organ function, cell proliferation and health status can be analyzed to determine the efficacy of the therapeutic agent.

[0048] This patent file contains at least one drawing created in color. Copies of this patent including the color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fees.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0050] It is recognized herein that exogenous expression of the ETS transcription factor variant 2, ETV2 (ER71, ETSRP71), in endothelial cells (ECs) plays an essential role in reprogramming differentiated endothelial cells into more primitive endothelial cells capable of autonomously forming functional and stable three-dimensional blood vessels and vascular networks. Accordingly, this disclosure provides methods for forming stable three-dimensional blood vessels by exogenously expressing ETV2 in endothelial cells in the absence of pericytes, forced perfusion, and artificial scaffolds, stable and functional three-dimensional artificial blood vessels produced by these methods, and methods for using the same.

[0051] Method for forming a stable three-dimensional blood vessel A first aspect of the present disclosure is directed to methods for forming stable three-dimensional vascular structures such as artificial blood vessels. Without wishing to be bound by any particular theory, it is believed that ETV2 expression in endothelial cells for an appropriate period of time, in vitro as well as in vivo, can reprogram the endothelial cells in culture to form long-lasting functional three-dimensional blood vessels. When cultured on a defined matrix, exogenous expression of the ETV2 transcription factor in differentiated endothelial cells results in the autonomous formation of functional and stable three-dimensional blood vessels. Since the methods provided herein do not utilize three-dimensional scaffolds, the methods provided herein advantageously eliminate the costly and time-consuming elements currently required to form three-dimensional blood vessels.

[0052] Generally, the method for forming a stable three-dimensional blood vessel involves culturing endothelial cells containing an exogenous nucleic acid encoding the ETV2 transcription factor on a biocompatible matrix for at least three weeks under conditions that result in the expression of the exogenous ETV2 transcription factor. Exogenous expression of the ETV2 transcription factor protein in differentiated endothelial cells reprograms the differentiated endothelial cells and confers upon the cells the ability to self-assemble into stable functional three-dimensional vascular structures such as blood vessels having lumens, which can then be isolated and used for a number of purposes such as vascular introduction into injured tissue, vascular introduction into organoids, or vascular reintroduction into decellularized organs.

[0053] In one embodiment, the endothelial cells used in the method are differentiated endothelial cells (ECs). As used herein, the term "differentiated" or "differentiated endothelial cells" refers to the process of development in which endothelial cells become specialized for a particular function, e.g., the cells acquire one or more morphological features and / or functions that are different from the original cell type. The term "differentiation" includes both lineage commitment and the development of the cell to its fully differentiated adult endothelial cell state of maturity. Differentiation can be evaluated, for example, by monitoring the presence or absence of cell lineage markers using immunohistochemistry or other procedures known to those skilled in the art.

[0054] Endothelial cells can be obtained by methods known in the art. For example, endothelial cells can be isolated from tissues using a collagenase-based digestion approach as described in Ginsberg, M. et al. Cell (2012) 151, pp. 559-575 and U.S. Patent No. 6,899,822 to Ferrara et al. The endothelial cell lineage can be verified, for example, by staining with anti-CD31 antibody, VE-cadherin, or anti-factor von Willebrand antibody. Isolation of ECs can be achieved using antibodies specific for EC surface markers such as VE-cadherin, CD31, or VEGFR2 that are attached to magnetic beads or fluorophores used in magnetic or fluorescence-activated cell sorting (MACS or FACS).

[0055] Alternatively, endothelial cells can be obtained from commercial sources. Endothelial cells can be cultured and maintained (propagated) under conditions that maintain their differentiated cell lineage and ability to replicate. Such conditions are well established in the art. For example, isolated endothelial cells can be cultured in a complete medium containing an endothelial cell growth supplement in a coated tissue culture dish. Thereafter, the endothelial cells can be split and passaged until use.

[0056] In some embodiments, the differentiated endothelial cells are human endothelial cells. In certain embodiments, the differentiated human endothelial cells are human umbilical vein endothelial cells (HUVECs), human adipose-derived endothelial cells, or tissue / organ-specific human endothelial cells. In some embodiments of the method, the differentiated endothelial cells are organ-specific endothelial cells, including, but not limited to, endothelial cells of the heart, kidney, testis, ovary, retina, liver, pancreas, brain, lung, spleen, large intestine, or small intestine. In other embodiments, the differentiated endothelial cells are tissue-specific endothelial cells derived from muscle, lymphoid tissue, olfactory tissue, bone-forming tissue, oral (tooth) tissue, or glandular tissue (e.g., endocrine gland, thymus).

[0057] Differentiated endothelial cells can be cultured to induce the formation of stable three-dimensional blood vessels. The cells can be cultured in any medium capable of sustaining the proliferation of endothelial cells, including, but not limited to, DMEM (high glucose or low glucose), advanced DMEM, DMEM / MCDB 201, Eagle's basal medium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Hayflick's medium, Iscove's modified Dulbecco's medium, mesenchymal stem cell growth medium (MSCGM), DMEM / F12, RPMI 1640, and CELL-GRO-FREE (Corning cellgro, Corning, NY). The medium can have one or more components added thereto, including, for example, fetal bovine serum, preferably about 2-15% (v / v), either alone or in combination; equine serum; human serum; fetal calf serum; β-mercaptoethanol, preferably about 0.001% (v / v); one or more growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF), and erythropoietin; amino acids, such as L-valine; and one or more antibiotics and / or antifungal agents, such as penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, for controlling microbial contamination.

[0058] Endothelial cells can be cultured to increase the cell number prior to reprogramming. Although a sufficient number of endothelial cells may be isolated from the initial sample, even if an acceptable number of differentiated endothelial cells are present in the initial sample, the proliferation of the cells in culture can provide a greater supply of endothelial cells for reprogramming. Methods for culturing and growing cells are known in the art. See, for example, Helgason et al., Basic Cell Culture Protocols, 4th Edition, Human Press Publishing, 2013; and Mitry et al., Human Cell Culture Protocols, 3rd Edition, Human Press Publishing, 2012.

[0059] Differentiated endothelial cells can be “reprogrammed” by the incorporation and expression of exogenous ETV2-encoding nucleic acid by the differentiated endothelial cells.

[0060] Endothelial cells are “reprogrammed” (directed to dedifferentiate into a more plastic state) by expressing an exogenous ETV2 transcription factor protein that alters the ability of the endothelial cells to form tubular (lumen-containing) blood vessels and other vascular structures according to the methods disclosed herein.

[0061] The term “ETV2” or “ETV2 transcription factor” refers to a RefSeq Gene that encodes a DNA-binding transcription factor protein having the amino acid sequence set forth in NP 055024 As used interchangeably herein, ID 2116 refers to human ETS-transcription factor variant 2, ETV2 (ER71, ETSRP71), as set forth in NCBI reference sequence No. NC_000019.10. The ETV2 nucleic acids of the disclosure can include an ETV2 DNA sequence or a portion thereof, and its RNA transcript, such as those set forth in accession numbers: NM_001300974.1, NM_014209.3, and NM_001304549.1. Functional derivatives and homologs of ETV2 are further contemplated for use in the disclosed methods. As used herein, a "functional derivative" is a molecule having the ability to perform the biological function of ETV2. For example, a functional derivative of ETV2 as disclosed herein is a molecule that can bind DNA and reprogram differentiated endothelial cells, as the ETV2 transcription factor does. Functional derivatives include fragments, variants, parts, portions, equivalents, analogs, mutants, mimetics, including fusion proteins, from natural, synthetic, or recombinant sources. A "homolog" is a protein related to the ETV2 transcription factor by derivation from a common ancestral nucleic acid sequence. Homologs contemplated herein include, but are not limited to, ETV2 proteins derived from different species such as mouse, rat, and monkey.

[0062] In certain embodiments of the method, the exogenous ETV2-encoding nucleic acid present in endothelial cells is human ETV2 as set forth in SEQ ID NO: 1, which encodes the human ETV2 transcription factor protein set forth in SEQ ID NO: 2. In another embodiment, the exogenous ETV2-encoding nucleic acid present in endothelial cells is a human ETV2 ribosomal nucleic acid (RNA) transcript that encodes the human ETV2 transcription factor protein. In other embodiments, the exogenous ETV2-encoding nucleic acid provided to differentiated endothelial cells is a modified synthetic RNA. Modified synthetic RNA molecules can be prepared by methods known to those of skill in the art, such as the methods set forth in Machnicka, M. A. et al. Nucleic Acids Res. (2013) 41: D262-D267. Exemplary modified synthetic molecules used in the present invention include chemical modifications to the RNA polynucleotide that modulate stability (altering nuclease resistance) or cellular uptake (e.g., conjugation of the RNA polynucleotide to cholesterol, linker, lipid, polymer, peptide, or aptamer).

[0063] The nucleic acid encoding the ETV2 transcription factor can be provided to cells by methods well known to those of skill in the art. For example, the ETV2-encoding nucleic acid can integrate the ETV2 nucleic acid sequence into the endothelial cell genome or be non-integrating, i.e., the ETV2 gene may be expressed from an extrachromosomal location. In some embodiments, the ETV2-encoding nucleic acid sequence is provided by a vector in which the nucleic acid sequence has been cloned by techniques known in the art. The vector can be introduced by any suitable method, such as transfection or virus-mediated transduction.

[0064] Vectors used to express the ETV2 transcription factor include, for example, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and other vectors, which, once introduced into cells, integrate into chromosomal positions within the genome of interest to provide stable long-term expression of ETV2. Other vectors include episomal vectors, in addition to engineered lentiviral vectors that are non-integrating. In this case, the ETV2 nucleotide sequence is cloned into the vector sequence; the vector is used to proliferate in differentiated endothelial cells and reprogram endothelial cells using the methods described herein.

[0065] In one embodiment, the ETV2 nucleic acid is contained in a lentiviral vector and provided to endothelial cells by lentivirus-mediated transduction. In certain embodiments, the nucleic acid encoding ETV2 of SEQ ID NO: 1 is transduced into differentiated endothelial cells using a lentiviral vector. In one embodiment, the lentiviral vector is a lenti pgk-vector. In certain embodiments, the exogenous ETV2-encoding nucleic acid of SEQ ID NO: 1 is provided to endothelial cells by transduction in an inducible expression system, such as, for example, the reverse tet transactivator (rtTA)-doxycycline inducible expression system.

[0066] In other embodiments, the ETV2 nucleic acid is an RNA transcript delivered to endothelial cells. In certain specific embodiments, the ETV2 RNA delivered to cells is a modified synthetic RNA molecule. Methods for introducing RNA molecules into cells are well known to those skilled in the art and such methods can be used here. For example, ETV2 RNA transcripts, such as mRNA transcripts, can be delivered to endothelial cells by transfection. In another non-limiting example, the ETV2 RNA transcript is delivered to cells by electroporation.

[0067] This method includes culturing differentiated endothelial cells containing exogenous ETV2-encoding nucleic acid under conditions that express the ETV2 transcription factor protein. In certain embodiments, the ETV2 protein is constitutively expressed. In other embodiments, the ETV2 protein is transiently expressed, such as under the control of an inducible promoter. In certain embodiments, the exogenous ETV2 transcription factor is expressed in endothelial cells for at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer, to induce endothelial cells to form blood vessels having lumens. In certain embodiments, the exogenous ETV2 protein is expressed for at least 4 weeks to induce angiogenesis. In another embodiment, the exogenous ETV2 protein is expressed for at least 3 to 4 weeks to induce lumen formation.

[0068] In other embodiments, the method includes culturing endothelial cells having exogenous ETV2-encoding nucleic acid under conditions that express the ETV2 transcription factor protein, followed by a further culture period under conditions where the endothelial cells do not express exogenous ETV2. In this case, the endothelial cells are first cultured under conditions that express the exogenous ETV2 transcription factor for a first period, and then a second culture can be performed under conditions where exogenous ETV2 is not expressed, such as in the absence of a substance capable of activating the inducible promoter (e.g., doxycycline, tetracycline). In one embodiment, the exogenous ETV2-encoding nucleic acid is a modified synthetic RNA molecule that results in transient ETV2 transcription factor expression during culture.

[0069] In some embodiments, the method comprises culturing endothelial cells having exogenous ETV2-encoding nucleic acid for at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer, under conditions that express the ETV2 transcription factor protein, and then performing a second culture for several days, several weeks, or several months, under conditions that do not express exogenous ETV2, such as in the absence of doxycycline. In certain embodiments, the endothelial cells are cultured for at least 3-4 weeks under conditions that express the exogenous ETV2 transcription factor protein, and then the endothelial cells are cultured for a period ranging from 7 days to 6 months, from 7 days to 5 months, from 7 days to 4 months, from 7 days to 3 months, from 7 days to 2 months, or from 7 days to 1 month, under conditions that do not express the ETV2 transcription factor. Regardless of the amount of time the reprogrammed endothelial cells are cultured under conditions that express the ETV2 protein, the endothelial cells will continue to generate stable, functional three-dimensional vascular-like structures, such as those shown in FIGS. 4A-4H.

[0070] The method comprises culturing endothelial cells containing exogenous ETV2-encoding nucleic acid on a matrix. Indeed, the present disclosure has identified essential extracellular matrix components, namely laminin, entactin, and collagen IV, which, when used to culture reprogrammed endothelial cells, result in the formation of stable and functional three-dimensional artificial blood vessels in vitro and in vivo, without the use of pericytes, perfusion, and cumbersome scaffolds. Thus, in certain embodiments, the matrix is composed of extracellular matrix components such as laminin, entactin, and / or collagen.

[0071] In one embodiment, the matrix used in the method may contain laminin and entactin at a combined concentration of at least 5 mg / mL. Since laminin and entactin can bind to each other to form a complex, the matrix used in the method may contain a complex of laminin and entactin. For example, the matrix may contain at least 5 mg / mL of a complex of laminin and entactin. In an exemplary embodiment, ETV2-expressing endothelial cells are cultured on a matrix containing laminin and entactin at a combined concentration of 5 mg / mL. In certain embodiments, the matrix used in the method is composed of a combination of laminin, entactin, and collagen IV (L.E.C.). For example, endothelial cells can be cultured on a matrix containing at least 5 mg / mL of laminin and entactin and at least 0.2 mg / mL of collagen IV to form a long-lasting functional three-dimensional artificial blood vessel. In certain embodiments, endothelial cells are cultured on a matrix composed of laminin and entactin at a combined concentration of 5.25 mg / mL and 0.2 mg / mL of collagen IV. In other embodiments, the matrix is Matrigel™ (Corning).

[0072] Methods for determining the concentration of a substance (e.g., a molecule and a protein, or a complex thereof) in a solution or a material are known to those skilled in the art. For example, spectrophotometry can be used to determine the concentration of a substance in a sample.

[0073] In one embodiment of the method, endothelial cells containing exogenous ETV2-encoding nucleic acid are cultured in a serum-free medium for a period of time. In some embodiments, the method includes culturing endothelial cells in a serum-free medium for at least 7 days, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or longer.

[0074] In other embodiments, the method comprises culturing endothelial cells comprising exogenous ETV2-encoding nucleic acid at low oxygen pressure in serum-free medium. In some embodiments, the endothelial cells are cultured in serum-free medium at less than atmospheric oxygen pressure, i.e., less than 20% oxygen pressure. In certain embodiments, the cells are cultured in serum-free medium at an oxygen pressure between 4% and 15%, between 5% and 10%, between 4% and 8%, or between 4% and 6%. In one embodiment, the cells are cultured in serum-free medium at 5% oxygen pressure. In certain embodiments, the method comprises culturing endothelial cells comprising an exogenous ETV2-encoding vector in serum-free medium at 5% oxygen pressure for 7 days.

[0075] Regardless of the duration or specific culture conditions, the method comprises culturing endothelial cells comprising exogenous ETV2-encoding nucleic acid on a matrix under conditions that express the ETV2 transcription factor to induce the formation of stable and functional three-dimensional blood vessels having lumens, and isolating the same.

[0076] "Stable" means that the three-dimensional blood vessels maintain their structure and function for a long period of time, e.g., at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or longer. Stable three-dimensional blood vessels have an intact lumen, exhibit proper apical membrane side-basolateral polarity, and maintain a tubular (lumen-containing) structure without collapsing, puncturing, or dissociating for at least 1 month without the use of scaffolds, forced perfusion, or perivascular support, meaning a tubular structure of endothelial cells.

[0077] As used herein, the term "functional" means that a stable three-dimensional blood vessel can perform processes typically carried out by natural (endogenous) blood vessels. For example, the functional blood vessels of the present disclosure can perfuse a fluid such as blood, anastomose to other vasculature (e.g., capillaries, veins, arteries, arterioles, venules, or lymphatic vessels) or tissue, and autonomously form an integrated, patterned (branched) network of blood vessels on the tissue, such that blood and / or fluid is supplied ( "vasculature introduced") to the tissue without the use of scaffolds, forced perfusion, or perivascular support.

[0078] As shown in FIGS. 2A-2B and 3A-3M, the stable three-dimensional blood vessels produced by the present method can form functional blood vessels that perfuse a fluid (FIG. 2A), and when isolated and implanted into a subject, can form a three-dimensional blood vessel network including capillary and arteriole structures (FIGS. 3L-3M). Further, as shown in FIG. 3M, the implanted stable three-dimensional blood vessels anastomosed to the surrounding tissue by integrating endogenous pericytes into the vasculature having its lumen. In addition, as shown in FIG. 1E, the stable three-dimensional blood vessels of the present disclosure remain stable and functional for over four months (16 weeks).

[0079] Thus, in some embodiments, the stable three-dimensional blood vessels of the present disclosure are stable and functional for at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, at least thirteen weeks, at least fourteen weeks, at least sixteen weeks, at least seventeen weeks, at least eighteen weeks, at least nineteen weeks, at least twenty weeks, or longer. In certain embodiments, the three-dimensional blood vessels formed by the present method are stable and functional for at least one month or at least two months. In other embodiments, the three-dimensional blood vessels formed by the present method are stable and functional for at least three months, at least four months, at least five months, at least six months, or longer. In one embodiment, the three-dimensional blood vessels formed by the present method are stable and functional for four months.

[0080] Stable three-dimensional blood vessel composition and method of therapeutic use The culture method disclosed herein enables the reproducible production of stable three-dimensional blood vessels, which are useful for several purposes such as organoid vasculature introduction and therapeutic vasculature introduction (tissue regeneration) of injured tissues. The present disclosure also reveals that exogenous expression of the ETV2 protein in differentiated endothelial cells results in the autonomous self-assembly of stable functional three-dimensional blood vessels that have the ability to form functional vascular networks in vitro and in vivo in the absence of pericytes, perfusion, and scaffolds. Accordingly, in a further aspect, the present disclosure provides a composition comprising stable three-dimensional blood vessels capable of autonomously forming a three-dimensional vascular network.

[0081]

[0082] ​In certain embodiments, the stable three-dimensional blood vessels of the present disclosure comprise a vascular-like tubular structure (lumen) composed of at least one continuous layer of reprogrammed endothelial cells. In other embodiments, the stable three-dimensional blood vessels comprise a lumen composed of at least two layers of reprogrammed endothelial cells. In yet another embodiment, the stable three-dimensional blood vessels have a lumen composed of at least one layer of reprogrammed endothelial cells and at least one other layer of cells, such as differentiated vascular endothelial cells or non-vascular cells. In other embodiments, the stable three-dimensional blood vessels of the present disclosure are composed of a combination of reprogrammed endothelial cells that express an exogenous ETV2 transcription factor and endothelial cells that do not express exogenous ETV2.

[0083] In certain embodiments, the endothelial cells of the stable three-dimensional blood vessels of the present disclosure express an exogenous ETV2 transcription factor-encoding nucleic acid. In one embodiment, the expression of the ETV2 transcription factor is transient, i.e., for a limited period such as a few days, weeks, or months. In certain embodiments, the transient expression of the ETV2 transcription factor is regulated by an inducible expression system, such as a reverse tet transactivator (rtTA)-doxycycline inducible expression system. In other embodiments, the transient expression of the ETV2 transcription factor is regulated by the introduction of a modified synthetic RNA molecule encoding the ETV2 protein. In other embodiments, the stable three-dimensional blood vessels of the present disclosure contain endothelial cells that constitutively (i.e., permanently) express an exogenous ETV2 transcription factor. In other embodiments, the stable three-dimensional blood vessels are composed of a combination of endothelial cells that express an exogenous ETV2 transcription factor and endothelial cells that do not express exogenous ETV2. In yet another embodiment, the stable three-dimensional blood vessels are composed of reprogrammed endothelial cells that do not express an exogenous ETV2 transcription factor.

[0084] In some embodiments, the stable three-dimensional blood vessels of the present disclosure are isolated blood vessels.

[0085] The term "isolated", when used with respect to a stable three-dimensional blood vessel or reprogrammed endothelial cell, means that the blood vessel or cell has been removed from the environment in which it naturally occurs, or in which it was formed, and is substantially free of other molecules or media. "Substantially free of" means that the isolated three-dimensional blood vessel or isolated cell comprises at least 60%, 70%, 80%, 90%, or 95% (by dry weight or volume) of the composition or preparation. For example, an isolated stable three-dimensional blood vessel can be substantially free of media, i.e., the media corresponds to less than about 20% of the volume of the preparation, less than about 10% of the volume of the preparation, or less than about 5% of the volume of the preparation. The level of purification can be based on the intended use. In certain non-limiting examples, the isolated stable three-dimensional blood vessels of the present disclosure can be removed from the cell culture media used to form the stable three-dimensional blood vessels, and any molecules added to the cell culture media, such as cytokines, growth factors, or amino acids. In another example, reprogrammed endothelial cells can be isolated by removing the cells from the cell culture media used to form the reprogrammed endothelial cells, and any cell culture additives, although at least a portion of the matrix remains with the isolated cells.

[0086] In some embodiments, the isolated stable three-dimensional blood vessels are the environment in which they were formed , for example, is taken in whole or in part from a cell culture, a bioreactor, or a subject. In certain embodiments, the isolated stable three-dimensional blood vessel is free of media, media components and additives, and any matrix in which it was formed, such as Matrigel™ or L.E.C matrix. In other embodiments, the isolated stable three-dimensional blood vessel of the present disclosure has been removed from media, media components and additives, but includes at least a portion of a matrix such as Matrigel™ or L.E.C. matrix. In one embodiment of the present disclosure, the stable three-dimensional blood vessel is functional. In certain embodiments, the functional stable three-dimensional blood vessel is capable of passing (perfusing) fluid through the blood vessel having its lumen. In one embodiment, the functional and stable three-dimensional blood vessel is capable of passing blood through the blood vessel. In yet another embodiment, the functional and stable three-dimensional blood vessel is capable of forming a three-dimensional blood vessel network. In some embodiments, the three-dimensional blood vessel network includes an integrated patterned network of blood vessels on tissue without the use of a scaffold, forced perfusion, or perivascular support, such that blood and / or fluid is supplied ( "vascularized") to the tissue. In certain embodiments, the three-dimensional blood vessel network is formed autonomously by stable three-dimensional blood vessels, and at least one capillary, at least one arteriole, at least one venule, at least one lymphatic vessel, or combinations thereof.

[0087] In some embodiments, the functional and stable three-dimensional blood vessels of the present disclosure are functional for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or longer. In certain embodiments, the stable three-dimensional blood vessels of the present disclosure are functional for 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or longer. In one embodiment, the three-dimensional blood vessels of the present disclosure are stable for at least 4 months (16 weeks), as shown in FIG. 1E.

[0088] Method for promoting vasculogenesis Figures 3L and 3M of the present disclosure show that the stable three-dimensional blood vessels formed by culturing endothelial cells containing exogenous ETV2 can autonomously organize into a patterned and long-lasting vascular network capable of vasculogenesis into the endogenous tissue after implantation. Therefore, the stable three-dimensional blood vessels of the present disclosure are also used to promote vasculogenesis of damaged tissues in a subject.

[0089] Therefore, in another aspect, a method for promoting vasculogenesis in a subject is provided, which includes administering a stable three-dimensional blood vessel to the subject. For example, the present three-dimensional blood vessels can be used therapeutically to repair ischemic tissues, form blood vessels and heart valves, repair damaged vasculature, and induce blood vessel formation in engineered tissues (e.g., before transplantation). In some embodiments, the subject has damaged tissue, such as an organ in need of vasculogenesis.

[0090] This method includes administering a composition containing the three-dimensional blood vessels of the present disclosure to a human subject in need of such treatment to promote vasculogenesis in such tissue. Promoting vasculogenesis (angiogenesis) in tissue can be beneficial for subjects having or at risk of developing conditions including ischemic conditions such as myocardial infarction, congestive heart failure, and peripheral vascular occlusive disease, stroke, reperfusion injury, limb ischemia; neuropathy (e.g., peripheral neuropathy, or diabetic neuropathy), organ failure (e.g., liver failure, kidney failure, etc.), diabetes, rheumatoid arthritis, and osteoporosis.

[0091] The tissue in need of vasculogenesis is a tissue characterized by being damaged and having excessive cell death It can be woven, tissue at risk of injury, or artificially manipulated tissue, and in particular, can be cardiac tissue, liver tissue, pancreatic tissue, kidney tissue, muscle tissue, nerve tissue, bone tissue. In certain embodiments, the subject has injured cardiac tissue, injured liver tissue, injured lymphoid tissue, injured kidney tissue, injured testicular tissue, injured ovarian tissue, injured retina, injured pancreatic tissue, injured brain tissue, injured lung tissue, injured intestinal tissue, injured glandular tissue, injured muscle tissue, or a combination thereof.

[0092] The method includes administering a stable three-dimensional blood vessel to a tissue in need of repair or revascularization, or near such tissue, in a manner that results in delivery of the blood vessel to the tissue. Optionally, the stable three-dimensional blood vessel is administered locally, for example, directly (e.g., by injection, implantation, or any suitable means) to or near the tissue in need of revascularization. In one embodiment, the stable three-dimensional blood vessel is administered by surgical implantation into the injured tissue of a subject in need of revascularization. In certain embodiments, the stable three-dimensional blood vessel is directly implanted into the injured organ or its tissue. In other embodiments, the method includes administering a stable three-dimensional blood vessel to a subject by injection, such as direct injection into the injured tissue. In certain specific embodiments, the stable three-dimensional blood vessel is administered to the subject by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or a combination thereof.

[0093] In one embodiment, stable three-dimensional blood vessels are administered with a matrix, such as in a suspension of extracellular matrix components. In certain embodiments, stable three-dimensional blood vessels are administered on a matrix comprising a combination of laminin, entactin, and collagen IV (L.E.C.). In one embodiment, the matrix can comprise laminin and entactin at a combined concentration of at least 5 mg / mL. In an exemplary embodiment, the blood vessels are administered with a matrix comprising laminin and entactin at a combined concentration of 5 mg / mL. Since laminin and entactin can bind to each other to form a complex, the matrix can comprise a complex of laminin and entactin. For example, the matrix can comprise at least 5 mg / mL of a complex of laminin and entactin. For example, the blood vessels are administered with a matrix containing at least 5 mg / mL of a complex of laminin and entactin and at least 0.2 mg / mL collagen IV. In certain embodiments, the matrix is composed of laminin and entactin at a combined concentration of 5.25 mg / mL and 0.2 mg / mL collagen IV. In other embodiments, the matrix is Matrigel™ (Corning).

[0094] In some embodiments, a method of promoting vasculogenesis in a subject comprises administering to the subject at least one stable three-dimensional blood vessel formed in vitro. In other embodiments, the method comprises administering two or more stable three-dimensional blood vessels of the present disclosure formed in vitro.

[0095] Regardless of the route of administration, once the stable three-dimensional blood vessels are administered to the subject, the functional and stable three-dimensional blood vessels establish a three-dimensional blood vessel network capable of vasculogenicizing injured tissue. For example, the stable three-dimensional blood vessels can form a three-dimensional blood vessel network comprising at least one capillary, at least one arteriole, at least one venule, at least one lymphatic vessel, or a combination thereof, capable of transporting blood to the injured tissue.

[0096] The present disclosure has also revealed that differentiated endothelial cells expressing exogenous ETV2 transcription factor can autonomously form stable three-dimensional blood vessels when injected into a subject together with certain extracellular matrix components.

[0097] Accordingly, a further aspect of the present disclosure provides a method for promoting vasculogenesis by administering a plurality of reprogrammed endothelial cells on a matrix to a tissue or organ. For example, the method includes administering a plurality of reprogrammed endothelial cells containing an exogenous ETV2 transcription factor-encoding nucleic acid on the matrix as shown herein.

[0098] In some embodiments, the reprogrammed endothelial cells and the matrix can be directly administered to a subject in need of vasculogenesis. In this case, the reprogrammed endothelial cells and the matrix can be directly delivered to the injured tissue by intraperitoneal injection, intramuscular injection, subcutaneous injection, surgical implantation, or a combination thereof.

[0099] Method for vasculogenesis of an organoid or decellularized organ In another aspect of the present disclosure, a method for vasculogenesis of an organoid is provided. In this case, the method includes culturing the organoid on a matrix together with endothelial cells containing an exogenous nucleic acid encoding the ETV2 transcription factor under conditions that express exogenous ETV2 protein in the endothelial cells to form a stable three-dimensional vascular network on the organoid.

[0100] In some embodiments, a method for introducing vasculature into an organoid includes culturing the organoid on a matrix with reprogrammed endothelial cells. The matrix used in this method can include laminin and entactin at a combined concentration of at least 5 mg / mL. Since laminin and entactin can bind to each other to form a complex, the matrix used in this method can include a complex of laminin and entactin. For example, the matrix can include a complex of laminin and entactin at least 5 mg / mL. In an exemplary embodiment, the reprogrammed endothelial cells are cultured on a matrix containing laminin and entactin at a combined concentration of 5 mg / mL. In certain embodiments, the matrix used in this method is composed of a combination of laminin, entactin, and collagen IV (L.E.C.). For example, endothelial cells and organoids can be cultured on a matrix containing at least 5 mg / mL of laminin and entactin, and at least 0.2 mg / mL of collagen IV to form a functional three-dimensional artificial blood vessel that persists in the organoid. In certain embodiments, the matrix is composed of laminin and entactin at a combined concentration of 5.25 mg / mL, and 0.2 mg / mL of collagen IV. In other embodiments, the matrix is Matrigel™ (Corning).

[0101] Endothelial cells and organoids can be cultured in any medium capable of sustaining the growth and development of endothelial cells, including, but not limited to, DMEM (high glucose or low glucose), advanced DMEM, DMEM / MCDB 201, Eagle's basal medium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Hayflick's medium, Iscove's modified Dulbecco's medium, mesenchymal stem cell growth medium (MSCGM), DMEM / F12, RPMI 1640, and CELL-GRO-FREE (Corning cellgro, Corning, NY). The medium can have one or more components added thereto, including, for example, fetal bovine serum, preferably about 2-15% (v / v), either alone or in combination; equine serum; human serum; fetal calf serum; β-mercaptoethanol, preferably about 0.001% (v / v); one or more growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF), and erythropoietin; amino acids, such as L-valine; and one or more antibiotics and / or antifungal agents for controlling microbial contamination, such as penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin. Statins are included.

[0102] In one embodiment, a method for vasculogenic introduction into an organoid includes culturing the organoid with endothelial cells on a matrix in a medium containing fibroblast growth factor (FGF). In certain embodiments, the medium contains basic fibroblast growth factor (FGF2). In some embodiments, the medium contains heparin. In certain embodiments, a method for vasculogenic introduction into an organoid includes culturing the organoid with endothelial cells on a matrix in a medium containing FGF2 and heparin.

[0103] Organoids, and endothelial cells containing exogenous nucleic acids encoding the ETV2 transcription factor, on a matrix, under conditions that express exogenous ETV2 protein in endothelial cells, induce the formation of a three-dimensional vascular network with lumens that is functional in the organoid, i.e., it can be cultured for any period required to vascularize the organoid. During the culture of the organoid with the reprogrammed endothelial cells, a functional vascular network will arise that includes at least one capillary, at least one arteriole, at least one venule, at least one lymphatic vessel, or a combination thereof, capable of transporting blood across the organoid. For example, in certain embodiments, the organoid and endothelial cells are cultured for at least 1 week (7 days), at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or longer, such that the endothelial cells are induced to organize into long-lasting blood vessels patterned in the organoid.

[0104] The method for vascularizing an organoid can be applied to any type of organoid. For example, the organoid used in the method can be a small intestine organoid, a colon organoid, a kidney organoid, a heart organoid, or a tumor organoid. In some embodiments, the method can be used to vascularize a tumor organoid, which can be tissue-specific, such as a breast cancer organoid, a colon cancer organoid, a kidney cancer organoid, or an intestinal cancer organoid.

[0105] The vascularized organoids of the present disclosure can be used by those skilled in the art for a number of purposes. Thus, in some embodiments, the method for vascularizing an organoid includes isolating the vascularized organoid.

[0106] Once the vasculature-introduced organoid is obtained, the vasculature-introduced organoid can be administered to a subject such as a human or an animal (e.g., mouse, rat, dog, monkey). In certain embodiments, the vasculature-introduced organoid can be administered to a human subject by surgical implantation.

[0107] In other embodiments, the isolated vasculature-introduced organoid can be used to determine the efficacy of a therapeutic agent. In this case, the vasculature-introduced organoid is contacted with the therapeutic agent and cultured for a period of time in the presence of the therapeutic agent. Thereafter, the organoid function, cell proliferation and health status can be analyzed to determine the efficacy of the therapeutic agent.

[0108] Examples of therapeutic agents that can be used in the present method include, but are not limited to, anti-inflammatory drugs, anti-angiogenic molecules, antibodies, cytotoxic drugs, other toxins, radionuclides, immunomodulators, small molecules, exosomes, and gene expression products. For example, a chemotherapeutic agent is administered to a vasculature-introduced organoid (i.e., a tumor organoid) to determine the anti-tumor activity (efficacy) of the chemotherapeutic agent.

[0109] The present disclosure also demonstrates that culturing a decellularized organ together with endothelial cells containing an exogenous nucleic acid encoding the ETV2 transcription factor under conditions that result in the expression of exogenous ETV2 protein in the cells thereof results in the generation of functional vascular structures in the decellularized organ.

[0110] ​Accordingly, another aspect of the present disclosure provides a method for vascular reintroduction of a decellularized organ. A method for vascular reintroduction into a decellularized organ includes culturing the vascular-removed organ with endothelial cells on a matrix for a period sufficient to form a functional three-dimensional vascular network in the decellularized organ. During culturing with the reprogrammed endothelial cells of the decellularized organ, a functional three-dimensional vascular network will result that includes at least one capillary, at least one arteriole, at least one venule, at least one lymphatic vessel, or a combination thereof that is capable of transporting blood across the organoid. For example, in certain embodiments, the decellularized organ and endothelial cells are induced to organize the endothelial cells into patterned long-lasting blood vessels in the decellularized organ and then cultured for at least 1 week (7 days), at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or longer to form a functional vascular network for vascular reintroduction into the decellularized organ.

[0111] In some embodiments, a method for vascular reintroduction into a decellularized organ includes culturing the decellularized organ with endothelial cells on a matrix in a bioreactor under conditions that express exogenous ETV2 protein. The matrix used in the method can include laminin and entactin at a combined concentration of at least 5 mg / mL. For example, the matrix can include a complex of laminin and entactin at at least 5 mg / mL. In an exemplary embodiment, the matrix includes laminin and entactin at a combined concentration of 5 mg / mL. In certain embodiments, the matrix used in the method is composed of a combination of laminin, entactin, and collagen IV (L.E.C.). For example, the method includes culturing on a matrix containing at least 5 mg / mL of laminin and entactin, and at least 0.2 mg / mL of collagen IV to form a long-lasting functional three-dimensional artificial blood vessel. In certain embodiments, the matrix contains laminin and entactin at a combined concentration of 5.25 mg / mL, and 0.2 mg / mL of collagen IV. In other embodiments, the matrix is Matrigel™ (Corning).

[0112] The method can be applied to any type of decellularized organ. For example, endothelial cells can be cultured on a decellularized organ such as a decellularized heart, kidney, testis, ovary, retina, bone, endocrine gland, thyroid, trachea, lymph node, liver, pancreas, brain, lung, spleen, large intestine, or small intestine under conditions that express exogenous ETV2 protein.

[0113] The vascular reintroduced organs of the present disclosure can be used by those skilled in the art for a number of purposes. Thus, in some embodiments, the method for vascular reintroduction into a decellularized organ includes isolating the vascular reintroduced organ.

[0114] In an exemplary embodiment, a vascular reintroduction organ can be obtained and then administered to a subject such as a human or an animal (e.g., a mouse, a rat, a dog, a monkey). In certain embodiments, the vascular reintroduction organoid is isolated and administered to a human subject by surgical implantation.

[0115] In other embodiments, the isolated vascular reintroduction organ can be used to determine the efficacy of a therapeutic agent. In this case, the vascular reintroduction organ is contacted with the therapeutic agent and cultured for a period of time in the presence of the therapeutic agent. Thereafter, the vascular reintroduction organ function, cell proliferation, and organ health status can be analyzed to determine the efficacy of the therapeutic agent.

Examples

[0116] This description is further illustrated by the following examples, which should in no way be construed as limiting. The content of all cited references (including reference documents, issued patents, and published patent applications as cited throughout this application) is expressly incorporated herein by reference.

Examples

[0117] Materials and Methods Cell culture. Human umbilical vein endothelial cells (HUVEC) and human adipose tissue endothelial cells were isolated in the laboratory using a collagenase-based digestion approach as shown in Ginsberg, M. et al. Cell (2012) 151, pp. 559 - 575 (the entire content of which is incorporated herein by reference). Thereafter, the isolated endothelial cells were placed in a tissue culture dish coated with 0.2% gelatin in complete medium (400 ml of M199, 100 ml of heat-inactivated FBS, 7.5 ml of Hepes, 5 ml of antibiotics, 5 ml of glutamax, 5 ml of lipid mixture, and endothelial cell growth supplement In a 1 / 2 bottle (Alpha Aesar), the cells were grown. After subculture, the cells were transduced with lenti pgk-ETV2 or an empty lentiviral vector. If necessary, the cells were also labeled by using pgk-mCherry or pgk-GFP lentivirus. The cells were split 1:2 using accutase and subcultured on gelatinized plates. If necessary, the cells were frozen for future experiments. Overall, more than 10 batches of different HUVECs were used in the experiments. The endothelial cells used for the tube formation assay were at passages 5 - 10.

[0118] Human adipose-derived endothelial cells were isolated by mechanical fragmentation followed by 30 minutes of collagenase digestion. The crude cell population was seeded on plastic dishes and grown for 5 - 7 days. Then, the cells were sorted to purify endothelial cells and grown as described above. + CD31 + The endothelial cells were sorted to purify endothelial cells and grown as described above.

[0119] Tube formation assay. 24-well plates were coated with 250 - 300 μl of Matrigel™ (Corning) in an incubator for 30 minutes. At the same time, cells with or without ETV2 were treated with accutase and counted. Then, the cells were resuspended in knockout serum and cytokines (10 ng / ml Resuspended in StemSpan (Stem Cell Technologies) supplemented with FGF-2, 10 ng / ml IGF1, 20 ng / ml EGF, 20 ng / ml SCF, 10 ng / ml IL6 (Peprotech). Then, 100,000 cells, either with or without ETV2, were seeded into each well. For the remainder of the experiment, the plates were incubated at 5% oxygen. For experiments based on laminin / entactin / collagen IV (L.E.C.), high concentrations (i.e., 15 mg / ml) of laminin and entactin (Corning) were mixed with different amounts of collagen IV (Corning) in phosphate buffered saline (PBS). The most effective concentration of L.E.C. for lumen formation consisted of a final matrix of 5.25 mg / mL laminin / entactin complex with 2.0 mg / mL collagen IV and contained 200 μl (15 mg / ml) of laminin / entactin complex and 100 μl of collagen IV. Vessel density was measured over a period of 24 hours to 16 weeks. Images were taken at 4× magnification in four different randomized zones in each well for each condition and time point using an EVOS® inverted microscope. Subsequently, all images were analyzed for vessel density using tracing with ImageJ. The same procedure was used for cells transduced with ETS1 or myrAKT1.

[0120] In vitro tube staining. At 8 - 12 weeks, all media were removed from the wells. Tubes were washed once with phosphate buffered saline (PBS), then fixed for 30 minutes at room temperature, washed again with PBS, and placed in blocking buffer for 1 hour at room temperature. Tubes were then stained for VECAD (R&D), laminin (abcam), collagen IV (abcam), and / or podocalyxin (R&D). The next day, tubes were washed again in PBS, incubated with secondary antibody for 3 hours, and counterstained with DAPI. All images were acquired using a Zeiss 710 confocal microscope.

[0121] Electron microscopy. Tissues were washed with serum-free medium or phosphate-buffered saline (PBS), and then fixed with modified Karnovsky's fixative containing 2.5% glutaraldehyde, 4% paraformaldehyde, and 0.02% picric acid in 0.1 M sodium cacodylate buffer pH 7.2. After secondary fixation in 1% osmium tetroxide, 1.5% potassium ferricyanide samples were dehydrated in a series of ethanol and embedded in an Epon analog resin. Ultrathin sections were cut using a Diatome diamond knife (Diatome, Hatfield, PA, USA) on a Leica Ultracut S ultramicrotome (Leica, Vienna, Austria). Sections were collected on copper grids, further contrasted with lead, and observed in a JEM 1400 electron microscope (JEOL, USA, Inc., Peabody, MA) operating at 100 kV. Images were recorded with a Veleta 2K×2K digital camera (Olympus-SIS, Germany).

[0122] Bead flow for evaluating the perfusion capacity of three-dimensional blood vessels. Each device was formed from two layers of polydimethylsiloxane (PDMS; Sylgard 184; Dow-Corning) molded from a silicon wafer master. The device was plasma-treated with a plasma etching machine (PlasmaEtch) and subsequently treated with trimethoxysilane (Sigma) overnight. Before use, the device was washed with MiliQ H2O overnight. A mixture of 3 million reprogrammed endothelial cells (R-VEC) or control non-transfected endothelial cells (CTRL-EC) in 2.5 mg / ml bovine fibrinogen (Sigma) and 3 U / ml bovine thrombin (Sigma) was injected into the device with two 400-μm needle therapy needles (Hwato). After polymerizing the cell and gel mixture, the needles were withdrawn to form two hollow channels. ETV2-transduced endothelial cells (reprogrammed endothelial cells) and non-transduced endothelial cells (control) were seeded separately into the hollow channels, and the next day, two parent vasculatures were formed. The cells were cultured in endothelial cell growth medium 2 (Promocell), and the medium was refreshed daily until day 6. On day 6, the device was connected to a syringe pump (Harvard Apparatus), and 4-μm red fluorescent microspheres (Invitrogen) were injected at 50 μL / min into one of the parent vasculatures in each device. Low-speed imaging of the fluorescent beads flowing within the device was taken at 50-ms intervals with a Nikon Eclipse TiE (Nikon) equipped with an Andor Zyla sCMOS 5.5MP camera (Andor). The low-speed imaging images of the fluorescent beads were stacked together and overlaid with HUVEC vasculature using ImageJ software.

[0123] Exemplary in vivo evaluation of blood vessels. Human umbilical vein-derived endothelial cells (HUVECs) and human adipose-derived human endothelial cells (2 million cells / plug) transduced with a lentiviral empty vector or a lenti-ETV2 vector and labeled with a GFP or mCherry fluorescent marker were subcutaneously injected into male or female 8- to 12-week-old SCID-beige mice (Taconic). The cells were first resuspended in PBS (50 μl) and then mixed with Matrigel™ or L.E.C. matrix (350 μl) as described above. The cell / matrix suspension also contained FGF-2 (10 ng / ml), VEGF-A (20 ng / ml), and heparin (100 ng / ml). Each mouse received 2 plugs, one for control cells and one for cells expressing ETV2. At week 1, month 1, month 2, or month 5, mice were injected with VECAD (clone BV9 - Biolegend) conjugated to Alexa-647 that detects only human endothelial cells (25 μg in 100 μl of PBS) to evaluate the perfusion and anastomosis potential of ETV2-expressing human endothelial cells (reprogrammed, R-VEC) and non-ETV2-expressing human endothelial cells (control). Whole-mounted images were directly acquired with a Zeiss 710 confocal microscope using wells containing the coverslip bottom. The plugs were fixed overnight in 4% PFA, then dehydrated in ethanol, or placed in sucrose for further immunostaining. The dehydrated plugs were sectioned and stained with H&E. The sections were processed for immunostaining as follows. When ETS1-expressing cells and / or myrAKT1-expressing cells were implanted for 1 month, the same procedure was followed. Two plugs, one for control cells and one for cells expressing ETV2, were received by each mouse. At week 1, month 1, month 2, or month 5, mice were injected with VECAD (clone BV9 - Biolegend) conjugated to Alexa-647 that detects only human endothelial cells (25 μg in 100 μl of PBS) to evaluate the perfusion and anastomosis potential of ETV2-expressing human endothelial cells (reprogrammed, R-VEC) and non-ETV2-expressing human endothelial cells (control). Whole-mounted images were directly acquired with a Zeiss 710 confocal microscope using wells containing the coverslip bottom. The plugs were fixed overnight in 4% PFA, then dehydrated in ethanol, or placed in sucrose for further immunostaining. The dehydrated plugs were sectioned and stained with H&E. The sections were processed for immunostaining as follows. When ETS1-expressing cells and / or myrAKT1-expressing cells were implanted for 1 month, the same procedure was followed.

[0124] Immunostaining of tissue sections. Frozen sections (20 μM) that had been fixed in 4% PFA and processed in sucrose were washed once with PBS. The slides were then incubated in blocking buffer for 30 minutes at room temperature and overnight at 4°C in the primary antibody (1:500). The next day, the slides were washed three times for 10 minutes at room temperature and then incubated in the fluorescent-conjugated secondary antibody for 3 hours. Finally, the slides were washed three times for 10 minutes and counterstained with DAPI. A coverslip was placed on the sections, and images were acquired using a confocal microscope. For stromal staining, a PDGFRβ antibody (1:500, Biolegend) that detects mouse pericytes was used. Several images were acquired from sections derived from different parts of each plug. For each condition and time point, at least 12 images were obtained from different slides. The images were processed using ImageJ, and the percentage of the vascular area relative to the area of each image field was quantified by using the threshold function in the ImageJ software.

[0125] Transplantation of pre-formed stable three-dimensional artificial blood vessels. The artificial blood vessels 8 - 12 weeks after being formed as described above on Matrigel™ or L.E.C. matrix were isolated by removing the medium and overlaid with 200 μl of Matrigel™ or L.E.C. Within 30 minutes, the gelled plugs were subcutaneously administered to SCID-beige mice. The entire plug was surgically inserted into a pocket formed by a subcutaneous incision and sutured closed. Two weeks later, in order to detect the formation of anastomosed and perfused blood vessels, the mice were injected with an in vivo antibody against human VECAD.

[0126] Harvest of intestinal tissue and decellularization of the organ. The intestine was harvested from Sprague Dawley rats with a body weight in the range of 250 g to 350 g. Briefly, midline laparotomy was performed under aseptic conditions to expose the intestine. A 5-cm length of intestinal segment was isolated while preserving the mesenteric artery and mesenteric vein that perfuse the isolated segment. Both blood vessels were cannulated with a 26G cannula, and the intestinal lumen was cannulated using a 1 / 4-inch spiked connector. The isolated intestinal segment was decellularized by perfusing the vasculature and lumen at 1 ml / min using a peristaltic pump (iPump). The decellularization process consisted of perfusing milliQ water for 24 hours, sodium deoxycholate (Sigma) for 4 hours, and Dnase I (Sigma) for 3 hours. The decellularized intestine was sterilized by gamma irradiation before use.

[0127] Bioreactor culture. The decellularized intestine was seeded with either 5 million GFP + ETV2 + human endothelial cells (reprogrammed ECs expressing exogenous ETV2) or 5 million GFP + control endothelial cells (CTRL-EC). The cells were seeded through the mesenteric artery and mesenteric vein. The seeded intestine was placed inside the bioreactor under aseptic conditions. After 24 hours, perfusion was initiated through the mesenteric artery at 1 ml / min using a peristaltic pump (iPump). The cells were cultured in a medium containing 20% heat-inactivated FBS, 1% Pen-Strep, 1.5% HEPES (Corning), 1% Glutamax™ (Gibco), 1% lipid In M199 / EBSS (HyClone) supplemented with a quality mixture (Gibco), 1% heparin (Sigma), and 15 μg / ml endothelial cell growth supplement (Merck), cells were grown for the first 5 days, and then the cells were grown for 2 days in StemSpan (Stemcell Technologies) supplemented with 1.1% knock-out serum (Thermo), 1% Pen-Strep, 1% Glutamax, 10 ng / ml FGF (Peprotech), 20 ng / ml EGF (Invitrogen), 10 ng / ml IGF2 (Peprotech), 20 ng / ml SCF (Peprotech), and 10 ng / ml IL6 (Peprotech). After 7 days, the re-endothelialized intestine was harvested under sterile conditions, and a 5×7 mm area was excised for implantation. Subsequently, the remaining intestinal tissue was fixed in 4% paraformaldehyde and mounted for imaging by fluorescence microscopy. To evaluate the viability of blood vessels, some re-endothelialized intestines were perfused in vivo with fluorescently labeled LDL or an antibody against human PECAM (CD31).

[0128] Heterotopic graft implantation. Immunodeficient NOD-SCID-gamma (NSG) mice aged 8 - 12 weeks were anesthetized with a 2 - 5% isoflurane-oxygen gas mixture. Buprenorphine 0.1 mg / Kg was administered at the induction of analgesia. Under sterile conditions, midline laparotomy was performed. The stomach was exteriorized from its incision, and the omentum was stretched from its highly curved state. Subsequently, the operated intestinal area was wrapped into the omentum using 8 / 0 sutures to ensure closure of the omental wrapping. The stomach and omentum were returned to the abdomen, and the laparotomy was closed using 6 / 0 sutures. The animals were allowed to eat and drink normally immediately after surgery and were not given further drug therapy during the postoperative period. One week or 4 weeks later, the mice were intravenously injected with fluorescently labeled anti-VECAD or fluorescently labeled lectin and euthanized. The grafts were recovered together with the omental wrapping, fixed in 4% paraformaldehyde, mounted for imaging by fluorescence microscopy, and prepared.

[0129] Analysis of the perfusion, anastomosis, size, shape, and density of functional vasculature. Quantification of in vitro endothelial revascularization was performed on a 5×5 area in a 10× field of view. Images were processed using ImageJ software by setting the threshold and quantifying the area covered by the CD31 signal relative to the intestinal area. In vivo quantification of GFP- and VE-cadherin-positive cells was performed on images acquired with a confocal microscope (Zeiss LSM710), and a 3×3 area in a 20× field of view was evaluated. Evaluation of vascular parameters was performed using Angiotool software (National Cancer Institute).

[0130] Organoid isolation and culture. Mouse small intestinal organoids were isolated and maintained as previously described by O’Rourke, K. P. et al. Bio Protoc. (2016) 6 (the entire content of which is incorporated herein by reference). Isolation of human colonic crypts and adenomas; culture and maintenance of organoids were performed as previously described by Sugimoto, S. & Sato, T. Methods Mol Biol (2017) 1612, pp. 97–105 (the entire content of which is incorporated herein by reference). Normal and adenomatous tissues were collected. Briefly, cultures were grown in serum-free medium containing Wnt3a, R-spondin-3, and Noggin together with 10 mM nicotinamide (Sigma-Aldrich) and expanded in Matrigel™ (Corning). Organoids were passaged every 7 days by digestion in TrypLE Select (Thermofisher) supplemented with 10 μM Y27632 (Tocris Bioscience).

[0131] Co-culture of organoids with reprogrammed human endothelial cells. MCherry ETV2 EC or control (CTRL) EC (final concentration of 4 million cells / ml) was co-cultured with mouse small intestinal organoids, normal human colon, or patient-derived tumor organoids in 24-well plates Mix in 75 μl of Matrigel™ bubbles in a well or 40 μl of Matrigel™ bubbles in a chamber slide (normal organoids were passaged at 1:2 and tumor organoids were passaged at 1:3), and culture in organoid medium supplemented with FGF-2 (10 ng / ml) and heparin (100 ng / ml). Images were acquired from the live cultures at 24 hours, 4 days, 5 days, and 7 days. On day 8, the co-cultures were fixed and the whole mounts were stained with keratin 20 (KRT20), EdU, and / or EpCam.

[0132] RNA library preparation and sequence data processing. At least 100 ng of total RNA was isolated for each sample (phenol-chloroform separation with TRIzol LS) and purified using Qiagen's RNeasy Mini Kit. The quality of the RNA was verified using an Agilent Technologies 2100 Bioanalyzer. RNA library preparations were prepared and multiplexed using the Illumina TruSeq RNA Library Preparation Kit v2 (non-stranded and polyA selection), and 10 nM cDNA was used as input for high-throughput sequencing with an Illumina HiSeq 2500 that generates 51 base pair end reads. Sequencing reads were demultiplexed (bcl2fastq), and high-quality reads were mapped to the transcriptome sequence references of the UCSC hg19 genome constructed for HUVEC samples and the UCSC mm10 genome constructed for mouse EC (TopHat2; Bowtie2). For gene quantification (FPKM values), the mapped reads were assembled and quantified to transcripts (Cufflinks). Genes with FPKM < 1 were filtered out and base-2 The log-transformed FPKM values were used to plot MDS, volcano, and heatmap plots. Gene ontology analysis was performed using DAVID Bioinformatics Resource Tools. The P-values shown in the Venn diagram were calculated based on the cumulative distribution function (CDF) of the hypergeometric distribution curve.

[0133] ChIP and antibodies. ChIP assays were performed using approximately 1×10 7 cells per experiment. Briefly, cells were cross-linked in 1% paraformaldehyde (PFA) at 37 °C for 10 minutes and then quenched with 0.125 M glycine. Chromatin was sheared using a Bioruptor (Diagenode) to generate 200 - 400 base pair fragments, and Dynabeads M-280 (Invitrogen) 75 μl was incubated with 2 - 5 μg of the antibody overnight at 4 °C. The magnetic beads were washed and the chromatin was eluted. ChIP DNA was reverse cross-linked and column purified. ChIP was performed using the following antibodies: Flag (Sigma F1804); H3K4me3 (Abcam ab8580); and H3K27ac (Abcam ab4729). RNA was isolated using the QIAGEN RNeasy kit. RNA-seq libraries were prepared with the Illumina TruSeq RNA Sample Preparation Kit. ChIP-seq libraries were prepared with the Illumina TruSeq DNA Sample Preparation Kit. Both RNA-seq libraries and ChIP-seq libraries were sequenced on an Illumina HiSeq 4000 system.

[0134] ChIP-seq reads were aligned to the reference human genome (hg19, Genome Reference Consortium GRCh37) using the BWA alignment software (version 0.5.9). Unique reads mapped to a single best matching position with mismatches less than 4% of the read length were retained for peak identification and profiling. Sequence data were visualized in IGV by normalizing against one million reads. Using sequencing data from input DNA as a control, the software MACS2 was applied to the ChIP-seq data to identify genomic enrichment (peaks) or specific histone modifications of ETV2. The resulting peaks were filtered by p-value < 0.05 for ETV2 and p-value < 0.01 for K4me3 or K27ac modifications. Read counts were calculated by HOMER at individual promoters. Each identified peak was annotated by HOMER as a promoter (±2 kb from the transcription start site), gene body, or intergenic region. The genomic enrichment (peaks) or specific histone modifications of ETV2 were identified. The resulting peaks were filtered by p-value < 0.05 for ETV2 and p-value < 0.01 for K4me3 or K27ac modifications. Read counts were calculated by HOMER at individual promoters. Each identified peak was annotated by HOMER as a promoter (±2 kb from the transcription start site), gene body, or intergenic region.

Example

[0135] Transient expression of exogenous ETV2 in endothelial cells forms long-lasting and stable three-dimensional artificial blood vessels in vitro This study shows that transient induction of ETV2 in differentiated endothelial cells reprograms the endothelial cells to acquire enhanced cell affinity for non-vascular cells and, in addition, gives rise to durability and patterning plasticity such that stable three-dimensional artificial vasculature is formed in vitro and in vivo. To determine whether ETV2 transcription factor expression alters vasculogenesis, endothelial cells transduced with ETV2 were single-cell FACS sorted and clonally expanded. Exogenous ETV2 expression in endothelial cells consistently resulted in the formation of a branched sprouting vascular network with an organized and durable geometric pattern of artificial blood vessels, often localized on the surface of the extracellular matrix.

[0136] Administration of ETV2 by a lentiviral vector was evaluated by immunostaining and Western blot analysis in both flattened endothelial cells that form typical flat monolayer cobblestone shapes and flattened endothelial cells that form three-dimensional (3D) tubes. See Figures 1A - 1C. Over a 7-day culture period, human umbilical vein endothelial cells or adult adipose-derived endothelial cells (R-VECs) expressing exogenous ETV2 under serum-free conditions at 5% oxygen pressure (normoxia) were significantly superior in forming functional 3D artificial vasculature compared to control endothelial cells without exogenous ETV2, as shown in Figures 1D - 1F, and showed an increase more than 50-fold in angiogenesis. Notably, the formed 3D artificial vasculature could be maintained in vitro for longer than 16 weeks, as shown in Figure 1E.

[0137] To determine whether the ability of differentiated endothelial cells to self-assemble into stable 3D artificial blood vessels with lumens is an attribute of other ETS family transcription factors of the tube, differentiated endothelial cells were transduced with another ETS transcription factor, ETS1. Furthermore, to examine whether ETV2 confers angiogenic function to endothelial cells by increasing their cell survival, endothelial cells were transduced with constitutively active myristoylated AKT1 (myrAKT1). As shown in Figure 1G, neither ETS1 expression nor myrAKT1 expression enabled differentiated endothelial cells to form stable 3D artificial blood vessels as ETV2 did. Thus, exogenous ETV2 expression in differentiated human endothelial cells has been shown to reprogram those cells and provide their ECs with the ability to self-assemble into stable 3D blood vessels without being constrained by existing artificial scaffolds, pericellular coatings, forced perfusion, or shear stress required by existing methods for forming artificial blood vessels.

[0138] Proper lumen formation is required for artificial 3D vascular function. As shown in Figure 1H, the presence of continuous and unbroken lumens in the stable 3D artificial blood vessels of the present disclosure has been revealed by confocal microscopy. The vasculature exhibited proper polarization, with podocalyxin expressed on the apical membrane side and laminin expressed on the basement membrane side (Figure 1I). Furthermore, as shown in Figure 1J, at 8 to 12 weeks after vasculogenesis, the artificial 3D blood vessels autonomously form branched-patterned vasculature with patent lumens and tight junctions. Therefore, the stable three-dimensional blood vessels of the present disclosure have lumens and exhibit proper apical membrane side-basement membrane side polarity.

[0139] Current approaches for in vitro vasculature patterning and organoid formation require the use of crude preparations of extracellular matrices such as Matrigel™. . Matrigel™ contains numerous digested matrix components, and therefore, it is difficult to determine whether specific components are required for vasculogenesis or their interaction with organoid cell components. Screening with numerous combinations of vascular extracellular matrices determined that the exact stoichiometry of matrix components, laminin, entactin, and collagen IV (L.E.C.) enables self-assembly of the stable 3D blood vessels of the present disclosure. Notably, as shown in Figure 1K, control naive human endothelial cells did not form a vascular network when using L.E.C. as the matrix. Furthermore, as shown in Figure 1L, the vasculature formed on L.E.C. and Matrigel™ showed similar vascular density and branching over 8 weeks. The presence of lumens in the vasculature formed on L.E.C. was confirmed by electron microscopy. See Figure 1M. Collectively, exogenous expression of ETV2 in differentiated endothelial cells forms stable three-dimensional artificial blood vessels on a matrix containing at least laminin, entactin, and collagen IV.

[0140] To test the ability of R-VEC vessels to sustain perfusion and laminar flow, endothelial cells containing fluorescently labeled, exogenous nucleic acid encoding either control-EC (CTRL-EC) or ETV2 were seeded in parallel in a microfluidic device. As shown in Figure 2A, after seeding, endothelial cells expressing exogenous ETV2 organized and self-assembled into vessels with lumens, while control cells were unable to form blood vessels. Once the vascular network of the artificial blood vessels was established, mCherry-labeled beads (4 μM) were circulated through one channel of the device to determine whether the stable three-dimensional artificial blood vessels of the present disclosure could sustain laminar flow. As shown in Figure 2A, the stable three-dimensional artificial blood vessels formed from endothelial cells expressing exogenous ETV2 withstood the flow of beads across the channel. In contrast, the beads did not flow across the channel containing control non-ETV2-transduced endothelial cells (CTRL-EC). Furthermore, as shown in Figure 2B, in the artificial blood vessels formed from endothelial cells expressing exogenous ETV2, the vessel density was significantly higher compared to those containing CTRL-EC. Thus, the vascular tubules with stable three-dimensional lumens formed from endothelial cells expressing exogenous ETV2 sustain perfusion without the need for perivascular support or constraints imposed by the scaffold.

Example

[0141] Endothelial cells expressing exogenous ETV2 form stable three-dimensional functional blood vessels in vivo The potential of endothelial cells expressing exogenous ETV2 to sustain functional patterned vasculature was evaluated using an in vivo mouse model. In this case, SCID-beige mice were subcutaneously implanted with plugs containing mCherry- or GFP-labeled control human endothelial cells, or reprogrammed endothelial cells expressing exogenous ETV2, mixed with laminin, entactin, and collagen IV (L.E.C.). One to five months after implantation, the degree of vascularization (vascular persistence and anastomosis to the existing mouse vascular structure) was evaluated by in vivo staining as shown in FIGS. 3A - M. FIG. 3A shows that mice administered plugs with reprogrammed endothelial cells (R-VECs) expressing exogenous ETV2 appeared to have more vascularization than mice administered control non-ETV2-transduced endothelial cells. By both whole-mount confocal microscopy and subsequent sectioning of the plugs, much higher vascular density, organization, and patterning were revealed in mice administered reprogrammed endothelial cells expressing exogenous ETV2 compared to control endothelial cell plugs that did not express ETV2.

[0142] Vascular function was further evaluated in vivo by examining the ability of the stable 3D artificial blood vessels to perfuse fluid and anastomose to the mouse vascular structure. See FIGS. 3B - C. Furthermore, as shown in FIG. 3D, vascular density was higher in animals injected with reprogrammed endothelial cells expressing exogenous ETV2 compared to control ECs at both the 1-month and 2-month time points. , it was deliberately high. Similar results were obtained when analyzing the functionality of artificial blood vessels formed using Matrigel™ as a matrix instead of L.E.C. at the 1-week, 1-month, and 2-month time points. See FIGS. 3E - F. Histological analysis showed that functional perfused vasculature was formed at a much higher rate for reprogrammed endothelial cells expressing exogenous ETV2 compared to control endothelial cells. FIG. 3G. Further, FIG. 3H shows that the stable 3D blood vessels of the present disclosure contain mouse pericytes, and the pericytes further stabilize the luminal geometric arrangement. Notably, endothelial cells transduced with myrAKT1 or ETS1 formed non-functional artificial vasculature. For example, these vasculatures were not perfused and could not be introduced into the vasculature in mice, as shown in FIGS. 3I - 3K. In view of the foregoing, reprogrammed endothelial cells expressing exogenous ETV2, when injected as a single cell population in a defined matrix, vasculature in vivo and form a functional vascular network.

[0143] Similarly, when stable three-dimensional blood vessels formed from endothelial cells expressing exogenous ETV2 were implanted instead of being administered as a single cell suspension, vascular functionality and vascular density were maintained over a long period. This indicates that the stable 3D blood vessels of the present disclosure can be transplanted together with organoids to regenerate organ-specific tissue. Endothelial cells expressing exogenous ETV2 were cultured for 8 weeks to form a stable vascular network in vitro, and then it was subcutaneously injected into SCID-beige mice as a fully formed vascular graft. Subsequently, VEcad antibody was injected into the mice to identify perfused vasculature. As shown in FIGS. 3L and 3M, mouse PFGFRβ +Pericytes were recruited around the implanted vasculature, stabilizing and forming a capillary-like vasculature and a network of small arterioles, so the implanted vasculature functionally anastomosed with the endogenous mouse blood vessels. Thus, the 3D blood vessels of the present disclosure are stable and functional and sustain the stress of implantation without the need for a scaffold or perivascular support. This indicates that the stable 3D blood vessels of the present disclosure are a viable vehicle for promoting vasculogenesis in a subject.

Example

[0144] Transient ETV2 expression forms functional artificial blood vessels To determine whether transient expression of ETV2 is sufficient to sustain stable 3D blood vessel formation, a reverse tet transactivator (rtTA)-doxycycline inducible expression system was used. Quantitative PCR analysis confirmed rapid downregulation of ETV2 upon doxycycline removal. Fig. 4H. The in vitro vasculogenesis assay and in vivo plug assay described above have shown that ETV2 expression is only required until proper blood vessels form at 4 weeks (i.e., 1 month), after which it is not essential. Figs. 4A - B. Indeed, as shown in Figs. 4C - D, the artificial blood vessels formed by endothelial cells expressing ETV2 during the first 4 weeks of culture sustained the vasculogenic fitness conferred by their ETV2.

[0145] Furthermore, as shown in Figs. 4E and 4F, the artificial blood vessels formed by endothelial cells expressing ETV2 acquired transcriptional similarity with freshly isolated endothelial cells when compared to endothelial cells cultured in vitro. Notably, for the 3D artificial blood vessels to become stable at 4 weeks, the expression of upregulated genes is downregulated and the cells adapt to the overall microenvironmental conditions. See Fig. 4G.

Example

[0146] Reprogrammed endothelial cells expressing ETV2 vasculogenicize organoids The potential of endothelial cells expressing ETV2 to branch, i.e., form a branched three-dimensional vascular network, was elucidated in mouse, human, or malignant epithelial organoids in culture. GFP-labeled human or mouse intestinal organoids were mixed with either reprogrammed endothelial cells expressing ETV2 (R-VEC) or naive endothelial cells (CTRL-EC). As shown in Fig. 5A, R-VEC was able to form highly organized 3D vasculature within 24 hours and associate with both stem cells containing crypt buds and differentiated villus-like central domains. Vascularization of intestinal organoids by reprogrammed endothelial cells expressing ETV2 formed patterned vasculature that was more organized and branched to spread over virtually every organoid unit by day 4 and day 7. In contrast, naive non-ETV2-transduced endothelial cells (CTRL-EC) were unable to form vasculature in the presence of organoids and thus lacked the ability to vascularize the organoids. Fig. 5A.

[0147] As shown in Figs. 5B and 5C, the vascular density per organoid and the number of capillaries formed were found to be significantly higher in organoids cultured with reprogrammed endothelial cells.

[0148] As shown in Figs. 5D-5F, for human-derived normal colon organoids, reprogrammed endothelial cells expressing ETV2 branched and formed a vascular network. Furthermore, the vascular density, number, and in addition, the organoid size of colon organoids were found to be higher when co-cultured with reprogrammed endothelial cells expressing ETV2. Thus, reprogrammed endothelial cells enable differentiated endothelial cells to bind to endogenous epithelial cells and form a functional vascular network.

[0149] ​As shown in FIGS. 5G-5J, within 24 hours, reprogrammed endothelial cells (R-VECs) expressing ETV2 migrated towards tumor organoids and actively and abundantly vasculogenic introduced into the tumor organoids, while naive endothelial cells (CTRL-ECs) were unable to do so. For example, FIG. 5G shows that after 7 days, all tumor organoids had a very high-density vasculature population in a branched and unorganized state. As shown in FIG. 5H, the vascular density was also found to be much higher in R-VEC co-cultures compared to control non-ETV2-transduced endothelial cells. Staining for the epithelial marker EpCam revealed close cell-cell interactions between tumor colon organoid cells and reprogrammed endothelial cells. See FIG. 5I. Notably, unlike the co-culture of normal intestine and colon organoids where reprogrammed endothelial cells formed an organized vascular network, the vasculature within the tumor organoids mimicked the tumor vasculature structure, was unorganized, and had abnormal shape and size. See FIGS. 5G and 5J.

[0150] Other patient-derived tumor organoids, including triple-negative breast cancer organoids, also produced similar results as illustrated in FIGS. 5K-5L.

[0151] In view of the foregoing, the reprogrammed endothelial cells and stable 3D artificial blood vessels of the present disclosure have the ability to vasculogenic introduce into organoids of many different tissue types, adapt to the environment into which they are introduced, and show remodeling and morphological changes in the setting of normal and tumorigenic organoids. For example, see FIG. 5J.

Example

[0152] Reprogrammed endothelial cells vasculogenic introduce into decellularized organs In regenerative medicine, the generation of long-lasting functional vascular structures, including capillaries and arterioles, has not yet been achieved. Specifically, large-diameter vessels can colonize with endothelial cells but have difficulty forming smaller capillary-sized vessels that can provide a blood supply distributed over the long term to an organ or an organ model. Here, reprogrammed endothelial cells expressing exogenous E TV2 were introduced into a decellularized intestinal organ model, and in that model, they were able to establish a vascularized structure in vitro inside a bioreactor. See Figures 6A - B. The results show that reprogrammed endothelial cells (R-VECs) expressing exogenous ETV2 were abundantly located in the capillaries of the decellularized intestine and achieved an even distribution across the entire area of the intestinal wall. See Figure 6C. As shown in Figures 6D - 6E, in vivo staining with a CD31 antibody and acetylated LDL uptake revealed much higher vascular coverage when using R-VECs compared to using control non-ETV2-transduced endothelial cells (CTRL-EC). As shown in Figures 6F and 6G, quantification of the re-endothelialized area and vascular network parameters support the higher vascular introduction potential of reprogrammed endothelial cells expressing exogenous ETV2. After 1 week of in vitro culture, the vascularly reintroduced intestine was implanted into the omentum of immunodeficient NOD-SCID-γ mice. At both 1 week and 4 weeks, as shown by in vivo isolectin and human VEcad injection, reprogrammed endothelial cells expressing exogenous ETV2 vascularly introduced into the decellularized intestine, maintained the continuity of large-diameter vessels (arteries and veins) and small-diameter vessels (capillaries), and anastomosed to the mouse vascular structure. See Figures 6H - 6J.

Claims

1. A stable three-dimensional blood vessel having the ability to autonomously form a three-dimensional vascular network, maintaining its structure and function of perfusing body fluids and anastomosing with other blood vessels or tissues for at least 1 month in the absence of a scaffold or perivascular support, and including a tubular structure having at least one continuous layer of reprogrammed endothelial cells, wherein the reprogrammed endothelial cells contain an exogenous nucleic acid sequence encoding ETV2, said stable three-dimensional blood vessel.

2. The stable three-dimensional blood vessel according to claim 1, which is functional for at least 2 months.

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