Endothelial and smooth muscle-like tissues produced from urine cells and related uses thereof

Abstract

The present invention relates to endothelial and smooth muscle-like vascular tissues produced from urine cells. In certain embodiments, the present invention relates to a method for producing endothelial and smooth muscle-like vascular tissues, wherein the urine-derived cells containing ETV2 are exposed to a first growth medium under conditions such that the cells are modified to form a cell pool with increased expression levels of endothelial surface markers, and then, under conditions such that the cells are modified to form tissues containing cells (the cells have increased expression levels of smooth muscle surface markers in addition to endothelial cell surface markers), the cell pool is exposed to a second growth medium. In certain embodiments, the present invention relates to the use of cells and tissues reported herein to treat vascular, cardiac and wound healing-related symptoms.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 903,154, filed on September 20, 2019. The entire contents of that application are incorporated herein by reference for all purposes.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Grants DK108245, HL127759, and HL129511 from the National Institutes of Health. The government has certain rights in this invention.

[0005] Incorporation by reference of materials submitted as text files through the Office Electronic Filing System (EFS-WEB)

[0006] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated into this specification by reference. The text file containing the sequence listing is named 19161PCT_ST25.txt. The text file is 11KB, created on July 9, 2020, and submitted electronically via EFS-Web. Background Art

[0007] Ischemic cardiovascular disease is a leading cause of morbidity and mortality in the population of industrialized countries. Risk factor management, medical therapy, and surgical revascularization are current treatment options but are not always effective when permanent vascular loss occurs. Despite significant efforts over the past decades, the treatment of patients with ischemic heart and vascular disease remains a challenge. Therefore, there is an urgent need to develop therapies that restore blood supply to host organs through neovascularization.

[0008] Endothelial cells (ECs) are an important component of the vascular system and are indispensable in repairing damaged or ischemic tissues. For many years, attempts may have been made to generate ECs for cell therapy. Despite early enthusiasm, adult stem cells or progenitor cells were found to have minimal endothelial transdifferentiation potential. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have emerged as promising alternatives; however, issues such as tumorigenic potential or low efficiency of cell production have limited their clinical application. Therefore, there is an urgent need to identify improvements.

[0009] Lee et al. reported the reprogramming of human dermal fibroblasts into endothelial cells using ER71 / ETV2.

[0010] Veldman et al. report rapid skeletal muscle transdifferentiation into functional endothelial cells in vivo via the transcription factor Etv2. PLoS Biol, 2013, 11(6): e1001590.

[0011] Bharadwaj et al. reported multipotent differentiation of human urine-derived stem cells. Stem Cells 2013, 31:1840–1856.

[0012] No admission is made that the references cited herein are prior art. Summary of the invention

[0013] The present invention relates to endothelial and smooth muscle-like vascular tissues produced from urine cells. In certain embodiments, the present invention relates to a method for producing endothelial and smooth muscle-like vascular tissues, wherein the method is performed by exposing urine-derived cells containing ETV2 to a first growth medium under conditions such that the cells are modified to form a cell pool with increased expression levels of endothelial surface markers, and then exposing the cell pool to a second growth medium under conditions such that the cells are modified to form tissues containing cells (the cells, in addition to expressing endothelial cell surface markers, have increased expression levels of smooth muscle surface markers). In certain embodiments, the present invention relates to treating vascular, cardiac and wound healing-related symptoms using cells and tissues reported herein.

[0014] In certain embodiments, the present invention relates to a method for producing endothelial and smooth muscle-like vascular tissue, comprising: i) concentrating urine cells from a subject; ii) replicating the concentrated urine cells in a first growth medium comprising a) EGF, b) hydrocortisone, c) epinephrine, and d) human serum or animal serum; providing purified concentrated urine-derived cells; iii) exposing the purified concentrated urine-derived cells to ETV2; iv) culturing the purified concentrated urine-derived cells in a first growth medium providing endothelial-like urine-derived cells; v) culturing endothelial-like urine-derived cells in a second growth medium comprising a) EGF, b) VEGFA, c) bFGF, d) heparin, e) L-ascorbic acid, and d) human serum or animal serum; providing endothelial and smooth muscle-like vascular tissue. In certain embodiments, the human serum is from a subject to be treated or implanted with vascular tissue.

[0015] In certain embodiments, the purified concentrated urine-derived cells are exposed to ETV2 by mixing the purified concentrated urine-derived cells with a recombinant virus that infects the purified concentrated urine-derived cells, the concentrated urine-derived cells comprising a gene encoding ETV2 and expressing ETV2 after infection. In certain embodiments, the recombinant virus is an adenovirus or a lentivirus.

[0016] In certain embodiments, the first or second growth medium comprises glucose, amino acids, vitamins, glutamine, and sodium pyruvate.

[0017] In certain embodiments, the methods disclosed herein further comprise the step of folding the endothelial and smooth muscle-like vascular tissue into a three-dimensional structure. In certain embodiments, the methods disclosed herein further comprise implanting the endothelial and smooth muscle-like vascular tissue into a subject.

[0018] In certain embodiments, the endothelial-like vascular tissue and smooth muscle-like vascular tissue are implanted by contacting the endothelial-like vascular tissue and smooth muscle-like vascular tissue with a vein, an artery, a capillary, or the myocardium.

[0019] In certain embodiments, the present invention relates to a method for producing endothelial cells or endothelial-like cells, the method comprising exposing expanded urine cells comprising a recombinant vector encoding ETV2 (the vector being operably combined with a promoter) to a promoter stimulus under conditions where ETV2 is formed in the cells and the expanded urine cells are modified to form a cell pool with increased expression levels of endothelial surface markers, thereby providing endothelial-like cells, wherein the surface markers are KDR and CDH5.

[0020] In certain embodiments, the cell pool has increased expression levels of surface markers KDR and CDH5. In certain embodiments, the cell pool has increased expression levels of surface markers PECAM1 and TEK.

[0021] In certain embodiments, the urine cells or expanded cells contain or do not contain a recombinant vector encoding ERG or FLI1, or contain or do not contain a recombinant vector encoding FOXC2, MEF2C, SOX17, NANOG, or HEY1.

[0022] In certain embodiments, the urine cells or expanded urine-derived cells are contacted or not contacted with a culture medium comprising a TGFβ inhibitor.

[0023] In certain embodiments, the methods disclosed herein further include a step of purifying the purified urine-derived cell pool by selecting cells expressing KDR, thereby providing a purified KDR urine-derived cell pool. In certain embodiments, the methods disclosed herein further include a step of purifying the purified urine-derived cell pool by selecting cells that do not express KDR, thereby providing a purified KDR-negative urine-derived cell pool.

[0024] In certain embodiments, the invention relates to compositions comprising cells prepared by the methods described herein.

[0025] In certain embodiments, the methods disclosed herein further comprise the step of producing endothelial-like cells, the step comprising contacting the cells produced herein with valproic acid.

[0026] In certain embodiments, the methods disclosed herein further comprise the step of producing a pool of modified cells comprising contacting the cells produced herein with a promoter stimulator.

[0027] In certain embodiments, the methods disclosed herein further comprise the step of producing a pool of modified cells comprising contacting the cells produced herein with collagen.

[0028] In certain embodiments, the invention relates to a method of treating or preventing a skin disorder, disease, injury, contusion, open wound, laceration, vascular disorder, disease, cardiac disorder, disease, atherosclerosis, coronary artery disease, or ischemia, the method comprising administering to a subject in need thereof an effective amount of a cell or tissue produced herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A method for generating vascular mimic tissue is presented. Human urine cells were collected by urine centrifugation. The cell pellet was suspended in growth medium (urine cell growth medium: 10% fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM), DMEM / nutrient mixture F-12 (DMEM / F-12 does not contain protein, lipid or growth factors). Growth factors EGF, hydrocortisone and epinephrine were added. The cell suspension was incubated at 37°C, CO 2 (5%) incubator for 14 days. The selected cells were expanded to form colonies. Reprogramming of replicating urine cells was caused by infection with adenovirus encoding ETV2. Urine-derived cells were grown for 24 hours. Thereafter, the culture medium was changed to a medium including VEGFA, EGF, bFGF, heparin and vitamin C.

[0030] Figure 2 The data presented demonstrate that endothelial genes are significantly induced in ETV2-treated replicating urine cells.

[0031] Figure 3 The tubular network structure on the vascular mimic tissue is shown.

[0032] Figure 4 The data presented demonstrate that the non-EC population (KDR negative) is significantly enriched for smooth muscle cell specific genes.

[0033] Figure 5 The preparation of vascular mimic tissue is shown.

[0034] Figure 6Shown is a sequence comparison of human (H. sapiens, query sequence, NCBI accession number NP_055024.2) and mouse (M. musculus, target sequence, NCBI accession number NP_031985.2) ETV2 subtype 1. 232 / 344 (67% identical), 250 / 344 (73% positive) and 11 / 344 (3% gaps). DETAILED DESCRIPTION

[0035] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, and therefore can certainly be varied. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0037] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and is incorporated herein by reference to disclose and describe the methods and / or materials in connection with the cited publication. Citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided herein may be different from the actual publication dates, which may need to be independently confirmed.

[0038] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order that is logically possible.

[0039] Unless otherwise indicated, embodiments of the present invention will employ techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, etc., which are within the skill of the art and are fully explained in the literature.

[0040] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and the claims that follow, reference will be made to certain terms, which are defined to have the following meanings unless a contrary intention is evident.

[0041] As used herein and in the claims, the words "comprising" (and any form of including, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes"), or "containing" (and any form of containing, such as "contain" and "contains") have the meanings ascribed to them in U.S. patent law, i.e., they are inclusive or open-ended and do not exclude other unrecited elements or method steps. For example, when the term "comprising" refers to an oligonucleotide having a nucleic acid sequence, it may include an oligonucleotide having additional 5' (5' end) or 3' (3' end) nucleotides, i.e., the term is intended to include an oligonucleotide sequence within a larger nucleic acid. "Consisting essentially of" or "consisting of", etc., when applied to the methods and compositions covered by the present invention, refers to compositions similar to those disclosed herein, which exclude certain prior art elements to provide the inventive features of the claims, but may contain additional composition ingredients or method steps, etc., which do not materially affect the nature and novelty of the composition or method compared to the corresponding composition or method disclosed herein.

[0042] The term "serum" refers to the blood product obtained when animal blood is allowed to coagulate and separated from the blood. Fetal bovine serum is serum extracted from the blood of a cow fetus after the fetus is removed from a slaughtered cow.

[0043] As used herein, "growth medium" or "culture medium" refers to a composition containing ingredients that promote cell maintenance and growth through protein biosynthesis, such as vitamins, amino acids, inorganic salts, buffers and nutrients, such as acetate, succinate, sugars and / or optional nucleotides. In addition, the growth medium may contain phenol red as a pH indicator. The ingredients in the growth medium can be from serum, or the growth medium can be serum-free. The growth medium can be selectively supplemented with albumin, lipids, insulin and / or zinc, transferrin or iron, selenium, ascorbic acid and antioxidants, such as glutathione, 2-mercaptoethanol or 1-thioglycerol. Other expected components expected in the growth medium include ammonium metavanadate, copper sulfate, manganese chloride, ethanolamine and sodium pyruvate. Minimum Essential Medium (MEM) is a technical term for a growth medium containing calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium phosphate and sodium bicarbonate), essential amino acids and vitamins: thiamine (vitamin B1), riboflavin (vitamin B2), niacinamide (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), folic acid (vitamin B9), choline and inositol (originally known as vitamin B8). Various growth media are known in the art. Dulbecco's Modified Eagle's Medium (DMEM) is a growth medium with additional ingredients such as glycine, serine and ferric nitrate, and increased levels of vitamins, amino acids and glucose, as shown in Table 1 below.

[0044] Table 1—Composition of Dulbecco's Modified Eagle's Medium

[0045]

[0046] Ham's F-12 medium contains high levels of amino acids, vitamins and other trace elements. The formula contains putrescine and linoleic acid. See Table 2 below.

[0047] Table 2 - Composition of Ham's F-12 Medium

[0048]

[0049]

[0050] In certain embodiments, the present invention contemplates the use of a mixture of DMEM and F-12 medium (a 1:1 mixture of DMEM and Ham's F-12) as the growth medium disclosed herein. The optimal carbon dioxide content of DMEM and F-12 is 10% and 5%, respectively. Since the medium is a mixture, the optimal carbon dioxide concentration is generally 5% to 8%.

[0051] As used herein, "heparin" refers to an anticoagulant polymer with variably sulfated repeating disaccharide units. The common disaccharide unit consists of 2-O-sulfo-α-L-iduronic acid and 2-deoxy-2-sulfonamido-α-D-glucopyranosyl-6-O-sulfate.

[0052] The terms "ETS translocation variant 2" and "ETV2" refer to a transcription factor involved in hematopoiesis and vascular development. ETV2 deficiency in mice results in a complete block in hematopoiesis and angiogenesis and leads to embryonic lethality. Human recombinant ETV2 is a commercially available protein with the NCBI reference sequence: NP_055024.2 (SEQ ID NO: 1). Adenovirus encoding ETV2 can also be used to express ETV2 mRNA, see NCBI reference sequence: NM_014209.4.

[0053] In certain embodiments, the present invention contemplates exposure of urine-derived cells using ETV2 under conditions such that ETV2 is optionally produced as a C-terminal or N-terminal fusion with a cell-penetrating peptide (CPP, such as polyarginine) and the ETV2 fusion is contacted with urine-derived cells. Warren et al. reported pluripotency reprogramming and directed differentiation of human cells using synthetic modified mRNA. Cell stem cell, 2010, 7: 618-630. In certain embodiments, the present invention contemplates exposure of urine-derived cells with mRNA of ETV2, for example, by using electroporation or by complexing RNA with a cationic carrier to promote endocytic uptake, delivering mRNA to cells.

[0054] The terms "epidermal growth factor" and "EGF" refer to a protein of approximately 6-kDa. The human EGF gene encodes a preproprotein that, after proteolytic processing, produces a peptide whose function is to stimulate the division of epidermal and other cells. Human recombinant VEGFA is commercially available as a 54-amino acid protein with the following sequence:

[0055] MNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQ YRDLKWWELR(SEQ ID NO:3)

[0056] The term "vascular endothelial growth factor A" or "VEGFA" refers to a heparin-binding protein that exists as a disulfide-linked homodimer and can induce proliferation and migration of vascular endothelial cells. Human recombinant VEGFA is commercially available as a 165 amino acid protein with the following sequence:

[0057] APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR (SEQ ID NO: 4).

[0058] The term "basic fibroblast growth factor" or "bFGF" refers to a protein with a beta-trefoil structure that binds to members of the FGF receptor (FGFR) family. Human recombinant bFGF is commercially available as a 154 amino acid protein with the following sequence:

[0059] AAGSITTTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS (SEQ ID NO: 5).

[0060] The protein variants disclosed herein can be readily prepared by those skilled in the art. One can predict functional variants with structural similarities through computer modeling. Intrinsic activity can be confirmed by testing using procedures outlined in the literature or in this specification. One skilled in the art will appreciate that a large number of operable variants can be produced that are expected to have the desired properties. Genes are known and members have significant homology between species. Figure 6As shown by the differences between the human and mouse sequences SEQ ID NO:1 and SEQ ID NO:2, these sequences are not identical. As shown by the differences between the human and mouse sequences disclosed in the specification, these sequences are not identical. Only 232 of the 344 amino acids (67%) are identical. Some are conservative substitutions (plus signs). Some are non-conservative substitutions. In order to construct functional variants, a person skilled in the art would not blindly try random combinations, but would use a computer program to make stable substitutions. A person skilled in the art would know that certain conservative substitutions are desirable. In addition, a person skilled in the art would generally not change evolutionarily conserved positions. See Saldano et al., Evolutionary Conserved Positions Define Protein Conformational Diversity, PLoS Comput Biol. 2016, 12(3): e1004775.

[0061] Guidance in determining which and how many amino acid residues can be substituted, inserted or deleted without abolishing biological activity can be obtained using computer programs in conjunction with public databases well known in the art, such as RAPTROX, ESyPred3D, HHpred, Homology Modeling Professional for HyperChem, DNAStar, SPARKS-X, EVfold, Phyre and Phyre2 software. See the Phyre2 protein modeling, prediction and analysis portal reported by Kelley et al. Nat Protoc. 2015, 10(6): 845-58. See also Marks et al., Protein structure from sequence variation, Nat Biotechnol, 2012, 30(11): 1072-80; Mackenzie et al., Curr Opin Struct Biol, 2017, 44: 161-167; Mackenzie et al., Proc Natl Acad Sci US A. 113(47): E7438-E7447 (2016) and Wei et al., Int. J. Mol. Sci. 2016, 17(12), 2118.

[0062] In certain embodiments, the present invention contemplates the use of polypeptide sequence variants disclosed herein having greater than 50%, 60%, 70%, 80%, 90%, 95% or more identity. "Sequence identity" refers to a measure of the correlation between two or more nucleic acids or proteins, usually expressed as a percentage relative to the total length of the control. The identity calculation takes into account the amino acid residues that are identical and in the same relative position in each larger sequence. The calculation of identity can be performed using default parameters by the algorithm contained in the computer program, such as "GAP" (Genetics Computer Group, Madison, Wisconsin) and "ALIGN" (DNAstar, Madison, Wisconsin). In certain embodiments, sequence "identity" refers to the number of residues that are fully matched in the sequence alignment between the two sequences being compared (expressed as a percentage). In certain embodiments, the percentage consistency of the comparison can be calculated using the number of identical positions divided by the larger of the shortest sequence or the number of equivalent positions that do not include internal spaces and are counted as overhangs of equivalent positions. For example, the sequence identity between polypeptides GGGGGG (SEQ ID NO: 6) and GGGGT (SEQ ID NO: 7) is 4 / 5 or 80%.For example, the sequence identity between polypeptides GGGPPP (SEQ ID NO: 8) and GGGAPPP (SEQ ID NO: 9) is 6 / 7 or 85%.

[0063] In certain embodiments, for any imagined percentage sequence identity, it is also envisioned that the sequence can have the same or higher percentage sequence similarity. "Similarity" percentage is used to quantify the similarity of amino acids between two sequences, such as hydrophobicity, hydrogen bond potential, electrostatic charge. This method is similar to determining identity, except that some amino acids do not necessarily have to be exactly the same to match. In certain embodiments, sequence similarity can be calculated with a known computer program using default parameters. Usually, if amino acid belongs to a group with similar properties, it is classified as matching amino acids, for example, according to the following amino acid groups: aromatic-F YW; hydrophobic-AV IL; positive charge: RKH; negative charge-DE; polarity-STNQ.

[0064] "Subject" refers to any animal, but preferably a mammal, such as a human, monkey, mouse, or rabbit.

[0065] As used herein, the terms "treat" and "treating" are not limited to situations where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments of the present invention also contemplate treatments that only alleviate symptoms and / or delay disease progression.

[0066] The term "nucleic acid" refers to a polymer of nucleotides or polynucleotides. The term is used to refer to a single molecule or a group of molecules. Nucleic acids can be single-stranded or double-stranded and can include regions for coding and various control elements, as described below.

[0067] The term "polynucleotide having a nucleotide sequence encoding a gene" or a nucleic acid sequence "encoding" a specific polypeptide refers to a nucleic acid sequence comprising a gene coding region, or in other words, a nucleic acid sequence encoding a gene product. The coding region may be present in the form of cDNA, genomic DNA or RNA. When present in the form of DNA, the oligonucleotide, polynucleotide or nucleic acid may be single-stranded (i.e., positive strand) or double-stranded. If necessary to allow proper initiation of transcription and / or correct processing of primary RNA transcripts, appropriate control elements, such as enhancers / promoters, splice junctions, polyadenylation signals, etc., may be placed in proximity to the gene coding region. Alternatively, the coding region used in the expression vector of the present invention may contain endogenous enhancers / promoters, splice junctions, interfering sequences, polyadenylation signals, etc., or a combination of endogenous and exogenous control elements.

[0068] The terms "operable combination", "operable sequence" and "operably linked" refer to the connection of nucleic acid sequences in such a manner that a nucleic acid molecule is produced that is capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. The term also refers to the connection of amino acid sequences in such a manner that a functional protein is produced.

[0069] The term "regulatory element" refers to a genetic element that controls some aspect of the expression of a nucleic acid sequence. For example, a promoter is a regulatory element that promotes the initiation of transcription of an operably linked coding region. Other regulatory elements include splicing signals, polyadenylation signals, termination signals, etc.

[0070] Promoters can be constitutive or regulatable. When referring to promoters, the term "constitutive" means that the promoter is able to direct the transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, a constitutive promoter is able to direct the expression of a transgene in any cell and tissue. In contrast, a "regulatable" or "inducible" promoter refers to a promoter that is able to direct the transcription level of an operably linked nucleic acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.), which is different from the transcription level of an operably linked nucleic acid sequence in the absence of the stimulus.

[0071] Enhancers and / or promoters can be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer or promoter is an enhancer or promoter that is naturally associated with a specific gene in the genome. An "exogenous" or "heterologous" enhancer or promoter refers to an enhancer or promoter that is juxtaposed to a gene by genetic manipulation (i.e., molecular biology techniques) so that transcription of the gene is directed by the connected enhancer or promoter. For example, an endogenous promoter that is operably combined with a first gene can be separated, removed, and operably combined with a second gene, thereby making it a "heterologous promoter" that is operably combined with the second gene. A variety of such combinations can be envisioned (for example, the first and second genes can be from the same species, or from different species).

[0072] Direct reprogramming of human urine cells into reprogrammed vascular tissue (rVT)

[0073] Ischemic cardiovascular disease, including coronary artery disease (e.g., myocardial infarction) and peripheral artery disease (e.g., critical limb ischemia), is a common cause of morbidity and mortality. The main cause of these clinical outcomes is the loss of blood vessels. Endothelial cells (ECs) are an important component of the vascular system and are crucial for the repair of damaged or ischemic tissues. Several methods have been developed to generate ECs for cell therapy. One approach to generate ECs is through direct lineage reprogramming using a single transcription factor (TF), ETV2 (gene or gene product, including modified mRNA, protein, exosome-containing protein), which is specific and critical for the development of ECs. This strategy has attracted much attention due to its potential advantages, including a simpler process and avoidance of the potential tumorigenicity caused by the use of pluripotent stem cells (PSCs) and induced pluripotent stem cells (IPSCs). The reprogramming methods outlined in this article can be applied to autologous cell therapy.

[0074] The present invention develops an enhanced reprogramming method that utilizes EC to mature in a short period of time to form vascular mimic tissues including smooth muscle cells. Source cells are collected from patients in a non-invasive manner and can be used for clinical applications. By using human urine cells, the pain of sampling source cells through skin dermal fibroblast biopsy can be avoided. Urine cells are replicated and converted into endothelial-like and smooth muscle-like tissues for the treatment of diseases requiring vascular reconstruction, including but not limited to coronary artery disease, myocardial infarction, heart failure, peripheral artery disease, critical limb ischemia, stroke, diabetic complications, and wound healing. The conversion of human urine cells into vascular mimic tissue is achieved by a scheme that includes special culture conditions and the introduction of the ETV2 gene into human urine cells.

[0075] Endogenous synthesis of natural bioscaffolds or biomatrices from autologous sources improves the survival rate of transplanted cells in target tissues. Natural ECM allows the deposition of growth factors released by therapeutic cells and provides a suitable microenvironment for cells to initiate angiogenesis.

[0076] Natural biomatrix that forms spontaneously from therapeutic cellular components offers an effective method to generate tissue while bypassing the complex processes required for single cell generation and artificial tissue construction. Natural biomatrix is ​​more biocompatible because it is generated from an autologous source and the production mechanism has minimal risk of pathogen transfer during the production process. Cell-mediated matrix synthesis has not been substantially studied, but studies have shown that vascular smooth muscle cells with a synthetic phenotype play a key role in natural matrix formation. Introduction of TGFB and PDGFB induced collagen synthesis. Collagen synthesis of VSMCs was also significantly increased under lactate medium, indicating that glucose metabolism may affect the synthetic phenotype of cells. Administration of ascorbic acid stimulated collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts. Ascorbic acid is a cofactor for hydroxyproline and hydroxylysine and plays a key role in α-peptide cross-linking during collagen biosynthesis.

[0077] Extraction of urine cells from human urine:

[0078] To obtain urine cells from the sample, urine was collected and centrifuged. The concentrated urine cells were collected and suspended in a growth medium containing EGF, hydrocortisone, epinephrine, and fetal bovine serum (FBS). The cell suspension was plated on a cell culture plate pre-coated with gelatin and incubated at 37°C, CO 2 (5%) atmosphere for 14 days. Urine cells formed colonies and continued to be cultured as source cells for direct reprogramming. FBS can be replaced with human serum from patients for clinical purposes in a xeno-free environment.

[0079] Reprogramming human urine cells into vascular mimic tissue / reprogrammed vascular tissue (rVT)

[0080] To generate vascular mimic tissue, human urine cells were reprogrammed into endothelial cells by overexpression of ETV2 under specific culture conditions. Reprogramming of human urine cells was initiated by infecting cells with adenovirus expressing ETV2 in urine cell growth medium for 24 h. Thereafter, the endothelial reprogramming medium was changed to include VEGFA, EGF, bFGF, heparin, and vitamin C (see Figure 1After ETV2 transduction, the morphology of urine cells was corbel stone shaped endothelial cells. The reprogramming process was monitored by changes in gene profiles and protein expression compared with non-reprogrammed control urine cells. To determine whether urine cells were reprogrammed into endothelial cells, mRNA levels of endothelial-specific genes such as KDR, CDH5, PECAM1, and VWF were measured by real-time quantitative PCR ( Figure 2 ). EC genes were significantly induced in ETV2-treated urine cells. In addition to early endothelial genes, late mature endothelial genes such as PECAM1 and VWF were also expressed ( Figure 2 ). As a result, the induction of EC markers increased rapidly, reaching higher amounts of mature endothelial surface markers such as PECAM1 (which is not present in other types of source cells, such as dermal fibroblasts). Human dermal fibroblasts did not express such high levels of PECAM1 in the short reprogramming period of 14 days by overexpression of ETV2. Urine-derived cells were also reverse-differentiated into multiple lineages, such as smooth muscle-like cells.

[0081] During endothelial cell reprogramming, urothelial cells formed specific tubular structures on top of the cell sheet. To test functional properties, fluorescent cytochemistry confirmed EC-specific uptake of acetic acid-LDL and binding of UEA1 lectin. The tissue was co-stained with EC markers and smooth muscle markers such as CDH5, PECAM1, ACTA2, and CNN1. The reprogrammed tissue was enzymatically digested and separated into EC and non-EC populations using the EC-specific marker KDR. Populations separated by EC (ETV-VMT) versus non-EC markers showed that the reprogrammed tissue was enriched in endothelial cells and smooth muscle-like cells, respectively ( Figure 4 ). Reprogrammed tissues can be mechanically acquired and folded to form vascular mimicking structures ( Figure 5 ). The tissue contained 30-50% of EC marker expressing cells such as KDR, CDH5, and PECAM1. Compared with reprogramming of human dermal fibroblasts induced by the mature EC marker ETV2, PECAM1 showed a shorter reprogramming cycle and higher induction efficiency. Non-EC marker expressing cells (KDR negative) showed high levels of smooth muscle markers such as SM22a, SMTN, ACTA2, and CNN1 ( Figure 4 ). Tissue containing recombinant endothelial cells and smooth muscle-like cells was implanted into a mouse hindlimb ischemia model. The tissue survived for more than 3 months in vivo. Therefore, this tissue is more suitable for direct transplantation or injection into ischemic tissues where high retention and cell survival rates are required during treatment.

[0082] Using a variety of other protocols to generate endothelial cells, such as differentiation of PSCs and direct reprogramming of somatic cells, obtaining a sufficient number of functional endothelial cells within the time required for clinical application has not been reported. A major obstacle to the use of reprogrammed endothelial cells for clinical application is the low retention efficiency of injected endothelial cells in the patient's ischemic target area. Compared with other EC production technologies, the method disclosed in this article has multiple advantages. Urine can provide unlimited source cells in a non-invasive manner. Compared with other methods, EC production is more timely. One advantage is that reprogrammed endothelial cells and smooth muscle cells are obtained simultaneously in a cell sheet structure, which has good retention in the ischemic area without the need for exogenous biomaterials. This reprogrammed vascular mimic tissue is suitable for autologous cell therapy of various ischemic diseases (such as PAD and MI). Sequence Listing <110> Emory University <120> Endothelial and smooth muscle-like tissues produced from urine cells and related uses thereof <130> 19161-PCT <150> US 62 / 903,154 <151> 2019-09-20 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 342 <212> PRT <213> Homo sapiens <400> 1 Met Asp Leu Trp Asn Trp Asp Glu Ala Ser Pro Gln Glu Val Pro Pro 1 5 10 15 Gly Asn Lys Leu Ala Gly Leu Glu Gly Ala Lys Leu Gly Phe Cys Phe 20 25 30 Pro Asp Leu Ala Leu Gln Gly Asp Thr Pro Thr Ala Thr Ala Glu Thr 35 40 45 Cys Trp Lys Gly Thr Ser Ser Ser Leu Ala Ser Phe Pro Gln Leu Asp 50 55 60 Trp Gly Ser Ala Leu Leu His Pro Glu Val Pro Trp Gly Ala Glu Pro 65 70 75 80 Asp Ser Gln Ala Leu Pro Trp Ser Gly Asp Trp Thr Asp Met Ala Cys 85 90 95 Thr Ala Trp Asp Ser Trp Ser Gly Ala Ser Gln Thr Leu Gly Pro Ala 100 105 110 Pro Leu Gly Pro Gly Pro Ile Pro Ala Ala Gly Ser Glu Gly Ala Ala 115 120 125 Gly Gln Asn Cys Val Pro Val Ala Gly Glu Ala Thr Ser Trp Ser Arg 130 135 140 Ala Gln Ala Ala Gly Ser Asn Thr Ser Trp Asp Cys Ser Val Gly Pro 145 150 155 160 Asp Gly Asp Thr Tyr Trp Gly Ser Gly Leu Gly Gly Glu Pro Arg Thr 165 170 175 Asp Cys Thr Ile Ser Trp Gly Gly Pro Ala Gly Pro Asp Cys Thr Thr 180 185 190 Ser Trp Asn Pro Gly Leu His Ala Gly Gly Thr Thr Ser Leu Lys Arg 195 200 205 Tyr Gln Ser Ser Ala Leu Thr Val Cys Ser Glu Pro Ser Pro Gln Ser 210 215 220 Asp Arg Ala Ser Leu Ala Arg Cys Pro Lys Thr Asn His Arg Gly Pro 225 230 235 240 Ile Gln Leu Trp Gln Phe Leu Leu Glu Leu Leu His Asp Gly Ala Arg 245 250 255 Ser Ser Cys Ile Arg Trp Thr Gly Asn Ser Arg Glu Phe Gln Leu Cys 260 265 270 Asp Pro Lys Glu Val Ala Arg Leu Trp Gly Glu Arg Lys Arg Lys Pro 275 280 285 Gly Met Asn Tyr Glu Lys Leu Ser Arg Gly Leu Arg Tyr Tyr Tyr Arg 290 295 300 Arg Asp Ile Val Arg Lys Ser Gly Gly Arg Lys Tyr Thr Tyr Arg Phe 305 310 315 320 Gly Gly Arg Val Pro Ser Leu Ala Tyr Pro Asp Cys Ala Gly Gly Gly 325 330 335 Arg Gly Ala Glu Thr Gln 340 <210> 2 <211> 335 <212> PRT <213> Mouse <400> 2 Met Asp Leu Trp Asn Trp Asp Glu Ala Ser Leu Gln Glu Val Pro Pro 1 5 10 15 Gly Asp Lys Leu Thr Gly Leu Gly Ala Glu Phe Gly Phe Tyr Phe Pro 20 25 30 Glu Val Ala Leu Gln Glu Asp Thr Pro Ile Thr Pro Met Asn Val Glu 35 40 45 Gly Cys Trp Lys Gly Phe Pro Glu Leu Asp Trp Asn Pro Ala Leu Pro 50 55 60 His Glu Asp Val Pro Phe Gln Ala Glu Pro Val Ala His Pro Leu Pro 65 70 75 80 Trp Ser Arg Asp Trp Thr Asp Leu Gly Cys Asn Thr Ser Asp Pro Trp 85 90 95 Ser Cys Ala Ser Gln Thr Pro Gly Pro Ala Pro Pro Gly Thr Ser Pro 100 105 110 Ser Pro Phe Val Gly Phe Glu Gly Ala Thr Gly Gln Asn Pro Ala Thr 115 120 125 Ser Ala Gly Gly Val Pro Ser Trp Ser His Pro Pro Ala Ala Trp Ser 130 135 140 Thr Thr Ser Trp Asp Cys Ser Val Gly Pro Ser Gly Ala Thr Tyr Trp 145 150 155 160 Asp Asn Gly Leu Gly Gly Glu Ala His Glu Asp Tyr Lys Met Ser Trp 165 170 175 Gly Gly Ser Ala Gly Ser Asp Tyr Thr Thr Thr Trp Asn Thr Gly Leu 180 185 190 Gln Asp Cys Ser Ile Pro Phe Glu Gly His Gln Ser Pro Ala Phe Thr 195 200 205 Thr Pro Ser Lys Ser Asn Lys Gln Ser Asp Arg Ala Thr Leu Thr Arg 210 215 220 Tyr Ser Lys Thr Asn His Arg Gly Pro Ile Gln Leu Trp Gln Phe Leu 225 230 235 240 Leu Glu Leu Leu His Asp Gly Ala Arg Ser Ser Cys Ile Arg Trp Thr 245 250 255 Gly Asn Ser Arg Glu Phe Gln Leu Cys Asp Pro Lys Glu Val Ala Arg 260 265 270 Leu Trp Gly Glu Arg Lys Arg Lys Pro Gly Met Asn Tyr Glu Lys Leu 275 280 285 Ser Arg Gly Leu Arg Tyr Tyr Tyr Arg Arg Asp Ile Val Leu Lys Ser 290 295 300 Gly Gly Arg Lys Tyr Thr Tyr Arg Phe Gly Gly Arg Val Pro Val Leu 305 310 315 320 Ala Tyr Gln Asp Asp Met Gly His Leu Pro Gly Ala Glu Gly Gln 325 330 335 <210> 3 <211> 54 <212> PRT <213> Artificial <220> <223> Synthetic constructs <400> 3 Met Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu 1 5 10 15 His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys 20 25 30 Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu 35 40 45 Lys Trp Trp Glu Leu Arg 50 <210> 4 <211> 165 <212> PRT <213> Artificial <220> <223> Synthetic constructs <400> 4 Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys 1 5 10 15 Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu 20 25 30 Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 40 45 Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu 50 55 60 Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile 65 70 75 80 Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95 Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg 100 105 110 Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe 115 120 125 Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser 130 135 140 Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys 145 150 155 160 Asp Lys Pro Arg Arg 165 <210> 5 <211> 154 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 5 Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly Gly 1 5 10 15 Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr 20 25 30 Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg Val 35 40 45 Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu Gln 50 55 60 Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg 65 70 75 80 Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val 85 90 95 Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn 100 105 110 Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg 115 120 125 Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys Ala 130 135 140 Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 <210> 6 <211> 6 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 6 Gly Gly Gly Gly Gly Gly 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic constructs <400> 7 Gly Gly Gly Gly Thr 1 5 <210> 8 <211> 6 <212> PRT <213> Artificial <220> <223> Synthetic constructs <400> 8 Gly Gly Gly Pro Pro Pro 1 5 <210> 9 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic constructs <400> 9 Gly Gly Gly Ala Pro Pro Pro 1 5

Claims

1. A method for producing endothelial and smooth muscle-like vascular tissue, comprising: i) concentrating urine cells from a subject; ii) replicating the concentrated urine cells in a first growth medium, the first growth medium comprising: a) EGF, b) hydrocortisone, c) Adrenaline and d) human serum or animal serum; Providing purified and concentrated urine-derived cells; iii) exposing purified concentrated urine-derived cells to ETV2; iv) culturing the purified concentrated urine-derived cells in a first growth medium providing endothelial-like urine-derived cells; v) culturing the endothelial-like urine-derived cells in a second growth medium, the second growth medium comprising: a) EGF, b) VEGFA, c) bFGF, d) Heparin, e) L-ascorbic acid, and d) human serum or animal serum; Provides endothelial and smooth muscle-like vascular tissue.

2. The method of claim 1, wherein the human serum is from the subject.

3. The method according to claim 1, wherein the purified concentrated urine-derived cells are exposed to ETV2 by mixing the purified concentrated urine-derived cells with a recombinant virus, wherein the recombinant virus infects the purified concentrated urine-derived cells, wherein the concentrated urine-derived cells contain a gene encoding ETV2 and express ETV2 after infection. The method according to claim 3 , wherein the recombinant virus is an adenovirus or a lentivirus.

5. The method of claim 1, wherein the growth medium comprises glucose, amino acids and vitamins, glutamine and sodium pyruvate.

6. The method of claim 1, further comprising the step of folding the endothelial cells and smooth muscle-like vascular tissue into a three-dimensional structure.

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