Method for Preparation and Use of IPSC-Derived Wall Cell Prototypes via NKX3.1 Activation

Abstract

In particular, compositions comprising iPSC-derived parietal cell precursors (iMPCs) generated using NK3 homeobox 1 (NKX3.1; parietal cell fate-determining transcription factor) and methods for their preparation and use are described herein. In addition, in particular, methods for maturing iMPCs into functional parietal cell subtypes, including smooth muscle cells, perivascular cells, and fibroblasts, as well as methods for increasing angiogenesis, angiogenesis, and cell junctions, are described herein. iMPCs mediate the formation of functional blood vessels when transplanted together with endothelial cells (ECs); thus, methods for modeling vascular diseases (e.g., 3D vascular organoids (VOs)) and therapeutic vascularization, comprising a method of administering iMPCs and ECs and a step of administering iMPCs and ECs, are also described herein.

Description

Technology Field

[0001] Claim of priority

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 544,104, filed October 13, 2023; U.S. Provisional Application Serial No. 63 / 653,051, filed May 29, 2024; and U.S. Provisional Application Serial No. 63 / 695,667, filed September 17, 2024. The full text of the above is incorporated herein by reference.

[0003] Government Support Statement

[0004] The present invention was made with government support under approval number HL128452, granted by the National Institutes of Health. The government holds specific rights to the present invention. Background Technology

[0005] Parietal cells, including perivascular cells and smooth muscle cells (SMCs), are critical to vascular development, function, and stability. Dysregulation of parietal cells can lead to vascular abnormalities, highlighting the need to generate functional parietal cells to explore novel therapeutic approaches in vascular disorders, tissue repair, and regenerative medicine. Human induced pluripotent stem cells (iPSCs) offer a promising means to obtain patient-specific parietal cells; however, conventional chemo-based differentiation methods are limited in scope and precision. Inducible transcription factors (TFs) have garnered attention as differentiation strategies, offering the potential for precise temporal control and the simultaneous differentiation of multiple cell types. However, identifying TFs for parietal cell differentiation remains challenging. Furthermore, there is an urgent need to address vascular disorders in blood vessels, which can lead to various health problems that are severe and even potentially fatal.

[0006] The present application is based, in particular, on the discovery that NK3 homeobox 1 (NKX3.1) determines the fate of a parietal cell lineage and that NKX3.1 activation in iPSC-derived mesenchymal precursors (MePCs) effectively produced iPSC-derived parietal cell precursors (iMPCs), which provides a novel method for generating unrestricted functional parietal cells for regenerative medicine.

[0007] The group of inventors previously demonstrated the successful generation of vascular endothelial cells (iECs) from iPSCs using ETV2, a pioneering TF (Wang et al., Sci Adv 2020). Recently, the inventors discovered that another TF, NK3 Homeobox 1 (NKX3.1), can be used to generate parietal cell precursors (iMPCs) from iPSCs. The data presented herein reveal efficient iMPC production upon transient activation of NKX3.1 in iPSC-derived mesenchymal precursors (MePCs). These iMPCs display definitive parietal cell characteristics (e.g., calcium influx, contractile properties, and extracellular matrix synthesis), which match them to control parietal cells such as primary SMCs. Furthermore, the inventors discovered that iMPCs mature into fully differentiated parietal cells upon interaction with ECs. This interaction enhances the ability of iMPCs to modulate EC functions, including the formation of in vivo vascular networks. In addition, the inventors' single-cell RNA sequencing analysis demonstrates the maturation of iMPCs and the resulting parietal cell heterogeneity.

[0008] The group of inventors has also developed a novel vascular organoid (VO) model that allows for the simultaneous co-differentiation of iPSCs into iECs and iMPCs. This VO model is expected to facilitate the maturation of iMPCs and aid in studying the mechanisms involved in wall cell reprogramming and maturation.

[0009] In particular, a method for preparing an iPSC-derived wall cell precursor (iMPC), comprising the following, is described herein:

[0010] A step of contacting a population of induced pluripotent stem cells (iPSCs) with a nucleic acid encoding NK3 homeobox 1 (NKX3.1) or a functional variant thereof;

[0011] A step of converting iPSCs into mesodermal precursors (MePCs); and

[0012] A step of inducing MePCs to express NKX3.1 for a period sufficient to generate iMPCs. In some embodiments, the nucleic acid is a vector (e.g., a PiggyBac transposon vector or a viral vector). In some embodiments, the vector is a viral vector (e.g., a retrovirus, a lentivirus). In some embodiments, the nucleic acid comprises an inducible promoter that controls the expression of NKX3.1 (e.g., a doxycycline-inducible promoter or other known in the art). In some embodiments, the step of converting an iPSC into a MePC includes activating the Wnt pathway and / or the Nodal pathway for a period sufficient to generate a MePC (e.g., about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours). In some embodiments, the period sufficient to generate an iMPC is about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours.

[0013] Also described herein is a method for generating a population of wall cells, including perivascular cells, smooth muscle cells, and fibroblasts, comprising the following:

[0014] A step of generating an iMPC using the method of any one of claims 1 to 6;

[0015] A step of co-cultured iMPCs with a population of endothelial cells (EC or iEC) for a period sufficient to produce a population of wall cells including perivascular cells, smooth muscle cells, and fibroblasts. In some embodiments, the period sufficient to produce a population of wall cells including perivascular cells, smooth muscle cells, and fibroblasts is about 1, 2, 3, 4, 5, 6, or 7 days.

[0016] Also described herein are a method for increasing angiogenesis in a subject, a method for vascular cell therapy, and / or a method for angiogenesis or regeneration, comprising the step of administering a therapeutically effective amount of a group of ECs and iMPCs to a subject requiring increased angiogenesis, vascular cell therapy, and / or angiogenesis or regeneration. In some embodiments, the method further comprises the step of identifying a subject requiring increased angiogenesis, vascular cell therapy, angiogenesis, and / or angiogenesis.

[0017] In particular, methods for maturing iMPCs into functional wall cell subtypes, including smooth muscle cells, perivascular cells, and fibroblasts, through the step of administering iMPCs to a subject, as well as methods for increasing vascular development, angiogenesis, and cell junctions, are described herein. As presented herein, iMPCs mediate the formation of functional blood vessels when transplanted together with endothelial cells (ECs); thus, methods for modeling vascular diseases (e.g., 3D vascular organoids (VOs)) and therapeutic vascularization, comprising a method of administering iMPCs and ECs and the step of administering iMPCs and ECs, are also described herein.

[0018] In some embodiments, the method described herein involves the use of a nucleic acid encoding NKX3.1 (e.g., UnitProtKB Q99801-1, UnitProtKB Q99801-2, UnitProtKB Q99801-3, UnitProtKB Q99801-4, UnitProtKB Q99801-5, PDB NP_006158.2, and PDB NP_001243268.1). In some embodiments, the nucleic acid is an exogenous DNA molecule or an exogenous RNA molecule. Non-limiting examples of useful RNA are modified mRNA (modRNA; e.g., US20230364267A1), mRNA, or functional variants thereof. Non-limiting examples of useful DNA may be a vector, plasmid, transposon, or functional variant thereof.

[0019] In some embodiments, the method described herein is E26 transform-specific variant 2 ( ETV2 It includes the use of nucleic acids encoding ). In some embodiments, the nucleic acid is an exogenous DNA molecule or an exogenous RNA molecule. Non-limiting examples of useful RNA are modified mRNA (modRNA; e.g., US20230364267A1), mRNA, or functional variants thereof. Non-limiting examples of useful DNA may be a vector, plasmid, transposon, or functional variant thereof.

[0020] In addition, a population of cells comprising at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% iPSC-derived wall cell precursors (iMPCs) is described herein. In some embodiments, the iMPCs comprise a nucleic acid encoding NKX3.1 (optional, exogenous nucleic acid) or transiently express NKX3.1 (optional, from a degradable nucleic acid (e.g., mRNA or modRNA)). In some embodiments, the iMPCs express PDGFRβ (CD140b) and aminopeptidase N (CD13). In some embodiments, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1% of the iMPCs express the TRA1-81 antigen. In some embodiments, the iMPCs ACTA2 , CNN1 , TAGLN , MYOCD , MYH11 , CSPG4 , DES , PDE5A , and THY1 It expresses.

[0021] Also described herein are populations of cells comprising endothelial cells (EC):iPSC-derived wall cell precursors (iMPCs) in a ratio of approximately 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:1. In some embodiments, iMPCs contain a nucleic acid encoding NKX3.1 (optional, exogenous nucleic acid) or transiently express NKX3.1 (optional, from a degradable nucleic acid (e.g., mRNA or modRNA)). In some embodiments, iMPCs express PDGFRβ (CD140b) and aminopeptidase N (CD13). In some embodiments, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1% of iMPCs express the TRA1-81 antigen. In some embodiments, iMPCs ACTA2 , CNN1 , TAGLN , MYOCD , MYH11 , CSPG4 , DES , PDE5A , and THY1 It expresses. In some embodiments, the EC comprises any one or more of iPSC-derived ECs (iECs), human vein endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs, or organ-specific endothelial cells. In some embodiments, the organ-specific endothelial cells are from an organ selected from the following: heart, muscle, kidney, testis, ovary, lymphatic system, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine. In some embodiments, the iEC contains a nucleic acid encoding ETV2 (optional, exogenous nucleic acid) or transiently expresses ETV2 (e.g., from a degradable nucleic acid (e.g., mRNA or modRNA)). In some embodiments, NKX3.1 expression is controlled by an inducible promoter and ETV2 expression is controlled by an inducible promoter. In some embodiments, the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are the same (optional, doxycycline). In some embodiments, the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are not the same.

[0022] In addition, a population of vascular organoids (VO) or a three-dimensional (3D) cell culture comprising a population of cells described herein is described herein.

[0023] Also described herein is a method for preparing vascular organoids (VO) or three-dimensional (3D) cell cultures, comprising:

[0024] (i) a step of incubating a first population of iPSC-derived mesenchymal cell precursors ("ETV2 / MePC") containing a nucleic acid encoding ETV2 or its functional variant (optional, exogenous nucleic acid) with a second population of MePCs ("NKX3.1 / MePC") containing a nucleic acid encoding NKX3.1 or its functional variant (optional, exogenous nucleic acid), and

[0025] Here, a step in which the expression of NKX3.1 is controlled by an inducible promoter and the expression of ETV2 is controlled by an inducible promoter;

[0026] (ii) inducing the expression of NKX3.1 in NKX3.1 / MePCs to thereby generate iPSC-derived wall cell precursors (iMPCs) and inducing the expression of ETV2 in ETV2 / MePCs to thereby generate iPSC-derived endothelial cells (iECs); and

[0027] (iii) a step of culturing cells for a sufficient period to produce a VO or 3D cell culture, wherein the VO or 3D cell culture comprises iECs that are h-CD31+ and iMPCs that are PDGFRβ+ and h-CD31-.

[0028] In some embodiments, the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are the same (optional, doxycycline). In some embodiments, the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are not the same.

[0029] In some embodiments, the period sufficient to produce VO or 3D cell culture is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0030] In some embodiments, (i) the culture step occurs for about 1 day or 2 days and / or; wherein (i) the culture step occurs for about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours and / or; wherein the culture occurs for a period sufficient to produce aggregates containing both NKX3.1 / MePC and ETV2 / MePC.

[0031] In some embodiments, (i) the culture step includes culturing cells using a non-adhesive culture plate and a rotary shaker.

[0032] In some embodiments, a group of NKX3.1 / MePC and a group of ETV2 / MePC are mixed in a ratio of about 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:1 to NKX3.1 / MePC:ETV2 / MePC.

[0033] In some embodiments, VO is of uniform size and / or about 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, or 550 μm diameter and / or; Here, the average diameter size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or; Here, the median size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or;VO or 3D cell cultures contain approximately 1,000, 1,500, 2,000, 2,500, 2,550, 2,600, 2,650, 2,700, 2,750, 2,800, 2,850, 2,900, 2,950, 3,000, 3,050, 3,100, 3,150, 3,200, 3,250, 3,300, 3,350, 3,400, 3,450, 3,500, 4,000, 4,500, or 5,000 cells.;

[0034] In some embodiments, the cells were self-assembled into a network of CD31+ vascular structures containing mature α-SMA+ perivascular wall cells. In some embodiments, VO comprises a network of strengthened blood vessels having apical-basal polarization and / or, wherein VO comprises arteries, veins, and / or capillary ECs. In some embodiments, VO CDH5+ and VWF + EC and ACTA2 +, MYH11 +, TAGLN +, and CNN1 + It includes wall cells. In some embodiments, the method includes the formation of arteries, veins, capillaries, arterioles, venules, or any combination thereof.

[0035] In addition, vascular organoids (VO) or 3D cell cultures prepared by any one of the methods described herein are described herein.

[0036] Also described herein is a method for preparing vascular organoids (VO) or three-dimensional (3D) cell cultures, comprising the following:

[0037] (i) transfecting a population of iPSC-derived mesenchymal precursors (MePCs) with a nucleic acid (optional, DNA, RNA, mRNA, modRNA) encoding NKX3.1 or a functional variant thereof, thereby generating a population of iPSC-derived wall cell precursors (iMPCs);

[0038] (ii) a step of mixing a population of iMPC and a population of EC (arbitrarily, in a ratio of approximately 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:1 iMPC:EC; and

[0039] (iii) a step of culturing cells for a sufficient period to produce a VO or 3D cell culture, wherein the VO or 3D cell culture comprises iECs that are h-CD31+ and iMPCs that are PDGFRβ+ and h-CD31-.

[0040] In some embodiments, the EC comprises any one or more of iPSC-derived ECs (iECs), human venous endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs, or organ-specific endothelial cells.

[0041] In some embodiments, organ-specific endothelial cells are from organs selected from the following: heart, muscle, kidney, testis, ovary, lymphatic system, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine.

[0042] In some embodiments, the method further comprises the step of transfecting a population of iPSCs with a nucleic acid encoding ETV2 or a functional variant thereof before mixing with a population of iMPCs to thereby generate a population of ECs.

[0043] In some embodiments, VO is of uniform size and / or about 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, or 550 μm diameter and / or; Here, the average diameter size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or; Here, the median size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or;VO or 3D cell cultures contain approximately 1,000, 1,500, 2,000, 2,500, 2,550, 2,600, 2,650, 2,700, 2,750, 2,800, 2,850, 2,900, 2,950, 3,000, 3,050, 3,100, 3,150, 3,200, 3,250, 3,300, 3,350, 3,400, 3,450, 3,500, 4,000, 4,500, or 5,000 cells.;

[0044] In some embodiments, the cells were self-assembled into a network of CD31+ vascular structures containing mature α-SMA+ perivascular wall cells. In some embodiments, VO comprises a network of strengthened blood vessels having apical-basal polarization and / or, wherein VO comprises arteries, veins, and / or capillary ECs. In some embodiments, VO CDH5+ and VWF + EC and ACTA2 +, MYH11 +, TAGLN +, and CNN1 + It includes wall cells. In some embodiments, the method includes the formation of arteries, veins, capillaries, arterioles, venules, or any combination thereof.

[0045] In addition, vascular organoids (VO) or 3D cell cultures prepared by any one of the methods described herein are described herein.

[0046] In some embodiments, the VO or 3D cell culture is of uniform size and / or; Here, VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, or 550 μm diameter and / or; Here, the average diameter size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or; Here, the median size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or;VO or 3D cell cultures contain approximately 1,000, 1,500, 2,000, 2,500, 2,550, 2,600, 2,650, 2,700, 2,750, 2,800, 2,850, 2,900, 2,950, 3,000, 3,050, 3,100, 3,150, 3,200, 3,250, 3,300, 3,350, 3,400, 3,450, 3,500, 4,000, 4,500, or 5,000 cells.;

[0047] In some embodiments, the VO or 3D cell culture comprises CD31+ vascular structures containing mature α-SMA+ perivascular wall cells. In some embodiments, the VO or 3D cell culture comprises a network of strengthened blood vessels having apical-basal polarization and / or arteries, veins, and / or capillary ECs.

[0048] In some embodiments, VO or 3D cell cultures CDH5+ and VWF + EC and ACTA2 +, MYH11 +, TAGLN +, and CNN1 + includes wall cells.

[0049] In addition, compositions comprising any of the cell populations described herein and / or any of the VO or 3D cell cultures described herein are described herein. In some embodiments, any composition may further comprise one or more of an agent, an excipient, a matrix, or a gel. In some embodiments, any composition may further comprise a gel or matrix comprising a hydrogel. In some embodiments, any composition may further comprise a gel or matrix comprising gelatin, collagen, fibrinogen, thrombin, fibrin, or any combination thereof. In some embodiments, any composition may further comprise a gel or matrix comprising about 1.5 mg / mL collagen, about 30 μg / mL fibrinogen, and about 1 mg / mL human fibronectin. In some embodiments, the matrix may contain collagen and / or fibrin. In some embodiments, fibrin is formed from fibrinogen and thrombin (optional, about 50 μg / mL thrombin). In some embodiments, any composition may further comprise a gel or matrix comprising any one or more of gelatin, collagen, fibrinogen, laminin, entactin, or combinations thereof. In some embodiments, any composition may further comprise a gel or matrix comprising laminin, entactin, and collagen. In some embodiments, any composition may further comprise a gel or matrix comprising about 5.25 mg / mL laminin, about 5.25 mg / mL entactin, and about 0.2 mg / mL collagen IV. In some embodiments, any composition may further comprise a gel, or the matrix is ​​Matrigel™. Matrigel is known in the art (U.S. Patent No. 4,829,000).

[0050] In addition, particularly, a method of transplanting any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein is described herein. In some embodiments, the method comprises the step of administering an effective amount of a cell population, a vascular organoid, or a composition to a subject.

[0051] Additionally, a method for increasing angiogenesis is described herein, comprising the step of administering, in particular, an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein to a subject requiring increased angiogenesis. In some embodiments, the method comprises the following:

[0052] A step of identifying a subject requiring increased angiogenesis; and

[0053] A step of administering to a subject an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein.

[0054] Additionally, a method for increasing angiogenesis or vascular regeneration is described herein, comprising the step of administering an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein to a subject who requires increased angiogenesis or vascular regeneration. In some embodiments, the method comprises the following:

[0055] A step of identifying a subject requiring angiogenesis or angiogenesis;

[0056] A step of administering to a subject an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein.

[0057] In addition, a method for vascular cell therapy is described herein, comprising the step of administering, in particular, to a subject requiring vascular cell therapy an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein. In some embodiments, the method comprises the following:

[0058] Step of identifying subjects requiring vascular cell therapy;

[0059] A step of administering to a subject an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein.

[0060] In some embodiments of any of the methods described herein, the subject or patient may be receiving an organ transplant, be selected to receive an organ transplant, or require vascularization of the organ. In some embodiments, the organ is selected from the group consisting of skin, heart, kidney, testis, ovary, bone, lymph, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine.

[0061] In some embodiments of any of the methods described herein, vascularization includes the formation of arteries, veins, capillaries, arterioles, venules, or any combination thereof.

[0062] In some embodiments of any of the methods described herein, the subject has (or has or is at risk of having) any one or more of the following: diabetes mellitus, diabetic retinopathy, ischemic injury, vascular disease or disorder, atherosclerosis, age-related macular degeneration (AMD), pulmonary hypertension (PAH), hereditary hemorrhagic telangiectasia (HHT), peripheral arterial disease (PAD), arteriovenous fistula (e.g., in dialysis patients), tumor angiogenesis, tumor metastasis, stroke, and / or wound (optional, chronic wound; e.g., diabetic ulcer).

[0063] In some embodiments of any of the methods described herein, the method normalizes and / or corrects an abnormal vascular structure; for example, wherein the vascular structure lacks or has deficient parietal cells. In some embodiments of any of the methods described herein, the subject has a disorder characterized by a vascular structure lacking or having deficient parietal cells (optional, diabetic retinopathy, tumor angiogenesis, tumor metastasis, stroke, ischemic injury, atherosclerosis, age-related macular degeneration (AMD), pulmonary hypertension (PAH), hereditary hemorrhagic telangiectasia (HHT), peripheral artery disease (PAD), arteriovenous fistula (e.g., dialysis patients), and / or a wound (optional, chronic wound; e.g., diabetic ulcer)).

[0064] Without being bound by theory, tumors typically exhibit abnormal vascular structures characterized by a lack of adequate wall cell coverage, which leads to leakage and dysfunction of blood vessels. Introducing any of the iMPC, cell population, VO, and compositions of the present invention may help stabilize these blood vessels, improve the delivery of therapeutic agents, and / or reduce metastasis.

[0065] Without being bound by theory, in diabetic retinopathy, loss of perivascular cells leads to a weakened blood-retinal barrier, which results in retinal ischemia and neovascularization. The iMPCs, cell populations, VO, and / or compositions described herein may potentially restore the integrity of retinal vascular structures and / or reduce the progression of the disease.

[0066] Without being bound by theory, after a stroke or ischemic injury, there is usually a loss of vascular integrity and a need for vascular repair. The iMPCs, cell populations, VO, and / or compositions described herein can help re-establish stable blood vessels and / or promote the recovery of diseased tissues.

[0067] Without being bound by theory, in atherosclerosis, vascular stability is impaired due to inflammatory processes and endothelial dysfunction. Parietal cells derived from iMPCs, iMPCs, cell populations, VO, and / or compositions described herein may help strengthen blood vessel walls and / or alleviate the progression of atherosclerotic plaques.

[0068] Without being bound by theory, in wound healing, chronic wounds, such as diabetic ulcers, usually suffer from poor vascularization and lack of wall cell coverage. The iMPCs, cell populations, VO, and / or compositions described herein can promote angiogenesis and vascular stability, thereby facilitating better wound healing outcomes.

[0069] Without being bound by theory, in age-related macular degeneration (AMD), particularly in the wet form, choroidal neovascularization occurs with a lack of perivascular cell support, which leads to fragile and leaking vessels. The iMPCs, cell populations, VO, and / or compositions described herein may help provide the necessary support to these new vessels, reduce leakage, and / or reduce vision loss.

[0070] Without being bound by theory, pulmonary arterial hypertension (PAH) is characterized by abnormal proliferation of pulmonary vascular cells and deficient perivascular cell coverage, which leads to vascular remodeling and hypertension. The iMPCs, cell populations, VO, and / or compositions described herein may stabilize these blood vessels and / or alleviate one or more symptoms (e.g., hypertension).

[0071] Without being bound by theory, hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder that causes abnormal blood vessel formation with deficient wall cell coverage, resulting in hemorrhage and arteriovenous malformations. The iMPCs, cell populations, VO, and / or compositions described herein may potentially normalize these vessels and / or reduce hemorrhagic episodes.

[0072] In some embodiments of any of the methods described herein, a population of cells, vascular organoids, or a composition is administered to a subject before, during, or after cell transplantation, tissue transplantation, or organ transplantation.

[0073] In addition, methods for tissue manipulation (e.g., small-diameter vascular grafts) are described herein. Tissue-manipulated small-diameter vascular grafts are biomanipulated structures designed to replace damaged or diseased blood vessels. These grafts are typically generated from scaffolds seeded with cells, such as smooth muscle cells (SMCs), that provide structural support and functionality. The cell populations described herein (e.g., NKX3.1-derived iMPCs and their mature wall cell derivatives) may be incorporated into these scaffolds as SMCs, which facilitates the generation of grafts that can be surgically implanted into a patient as vascular replacements. Small-diameter vascular grafts are particularly relevant for clinical applications, such as coronary artery bypass grafts (CABG), peripheral artery disease (PAD), and arteriovenous fistulas for dialysis patients.

[0074] In some embodiments, any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein (e.g., compositions comprising NKX3.1-derived iMPCs and / or their mature wall cell derivatives) are used as cell sources for tissue manipulation applications, particularly in the development of small-diameter vascular grafts. For example, the iMPC-derived cell population described herein may function as smooth muscle cells (SMCs) within the graft scaffold of a small-diameter vascular graft and may be surgically implanted into subjects requiring this (e.g., subjects requiring vascular replacement, subjects receiving or at risk of receiving coronary artery bypass graft (CABG), subjects having or at risk of having peripheral artery disease (PAD), and subjects having or at risk of having an arteriovenous fistula (e.g., dialysis patients)). Unbound by theory, the incorporation of iMPC cells into structural scaffolds enables the creation of functional, living vascular grafts that can be transplanted to repair or replace damaged blood vessels, offering significant potential for clinical scenarios with high demand.

[0075] In some embodiments of any of the methods described herein, a population of cells, vascular organoids, or compositions are administered to a subject by direct injection into a blood vessel or by subcutaneous, intradermal, intramuscular, intralymphatic, intravenous, intraprostatic, intratumoral, intralymphatic, and intraperitoneal injection.

[0076] "Effective dose" is an amount sufficient to produce a beneficial or desired result (e.g., an amount sufficient for the formation of arteries, veins, capillaries, arterioles, venules, or any combination thereof in a patient or subject). For example, a therapeutic dose is one that achieves a desired therapeutic effect (e.g., an amount sufficient for the formation of arteries, veins, capillaries, arterioles, venules, or any combination thereof; an amount sufficient to increase blood flow in a patient or subject; an amount sufficient to improve at least one symptom of a disease or condition in a patient or subject).

[0077] As used herein, the term “about” means within a statistically meaningful range of a value, e.g., a specified concentration range, period, molecular weight, particle size, temperature, or pH. Such ranges may be within one digit of the indicated value or range, typically within 20%, more typically within 10%, and even more typically within 5%. Occasionally, such ranges may be within the experimental error typical of the standard method used to measure and / or determine the provided value or range. The acceptable variation included by the term “about” will depend on the specific system under study and will be readily recognizable by a person skilled in the art. Whenever a range is referred to within this application, all integers within the range are also considered as embodiments of this disclosure.

[0078] An effective dose may be administered in one or more doses, applications, or dosages. A person skilled in the art will recognize that certain factors, including but not limited to the severity of the subject's disease or disorder, prior treatment, overall health and / or age, and other existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of the subject with any of the cell therapeutic populations, VO, 3D cell cultures, or compositions described herein with a therapeutic effective dose may include a single treatment or a series of treatments.

[0079] A person skilled in the art will be able to determine and identify a subject or patient suitable for any of the methods described herein (e.g., a subject who has or is at risk of having a stroke, a subject who has or is at risk of having diabetes, a subject who has or is at risk of having diabetic retinopathy, a subject who has or is at risk of having ischemic injury, a subject who has or is at risk of having vascular disease or disorder, and / or a subject who is or is selected to receive a transplant).

[0080] Parietal cells are pivotal to vascular integrity and function. An innovative use of the transcription factor NKX3.1 to guide the differentiation of human induced pluripotent stem cells into parietal cell precursors (iMPCs) is described herein. By transiently activating NKX3.1 at mesodermal intermediates, the method described herein departs from traditional growth factor-based differentiation techniques. This approach efficiently generates a potent iMPC population capable of maturing into various functional parietal cell subtypes, including smooth muscle cells and perivascular cells. These iMPCs exhibit key parietal cell functionalities, such as contractility, extracellular matrix deposition, and the ability to support endothelial cell-mediated vascular network formation in vivo. These findings highlight not only the fate-determining importance of NKX3.1 in parietal cell differentiation but also the therapeutic potential of these iMPCs. The inventors envision these insights paving the way for broader applications of iMPCs in vascular biology and regenerative medicine.

[0081] Further methods for the preparation, formulation, and administration of VO and 3D cell cultures are known in the art (see, for example, US 2019 / 0376044 A1; US ​​2020 / 0199541 A1; US ​​2023 / 0287357 A1; US ​​2023 / 0174949 A1; US ​​2023 / 0364267 A1; US ​​Patent No. 11,214,768; WO 2022 / 226337; WO 2023 / 196683).

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are not intended to be merely illustrative and limiting. All published documents, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0083] Other features and advantages of the present invention will be apparent from the following detailed description, drawings, and claims. Brief explanation of the drawing

[0084] The patent or application file contains at least one drawing in color. A copy of the published document of the patent or patent application containing the color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fees. Figs. 1a-1o. Efficient differentiation of iPSCs into iMPCs using NKX3.1 activation. (1a) Schematic of the 2-step, feeder-free, chemically defined protocol used for wall cell differentiation. (1b) Flow cytometry analysis of CD13 and CD140b (wall cell markers) across differentiation stages demonstrating extremely high-efficiency conversion to CD140b+ / CD13+ iMPCs. (1c) Flow cytometry analysis of TRA1-81 (pluripotency marker) across differentiation stages. The presence of undifferentiated iPSCs expressing TRA1-81 antigen is negligible. (1d) Immunofluorescence staining demonstrating the expression of wall-specific contractile proteins and cytoskeletal markers: α-SMA, SM22, vimentin, and calponin, using DAPI nuclear staining in iMPCs after one passage in culture (scale bar: 50 μm). mRNA expression (qPCR) of (1e) SMC markers and (1f) perivascular cell markers in iMPCs, SMCs, and MSCs. Smooth muscle markers of SMC gene expression in 1e (n=4-7) and perivascular cell markers presenting perivascular cell-specific gene expression in 1f (n=4-7) are qRT-PCR quantification analyses shown in the bar graph presenting mean ± SEM. (1g) NKX3.1 expression: Time-course qRT-PCR analysis demonstrating NKX3.1 upregulation during differentiation (n=4-8; ***P<0.001; mean ± SEM). (1h) Morphological progression: Phase-contrast microscopy revealing morphological evolution at various stages (scale bar: 100 μm; inset 50 μm). (1i) Differentiation schematic illustrating stepwise differentiation from iPSC to iMPC, detailing mesoderm induction, parietal cell specification, and extension. (1j-1m) Doxycycline-induced expression of NKX3.1 and retention of pluripotency markers in genetically engineered iPSCs. (1j) Schematic illustrating the induction of NKX3.1 expression in human iPSCs upon treatment with doxycycline (Dox). Phase contrast image of an iPSC-Dox-NKX3.1 clone in culture.Scale bar: 200 μm. (1k) Immunofluorescence imaging of iPSC-Dox-NKX3.1 clones without Dox (-Dox) and 24 hours after Dox treatment (+Dox 24h). Cells are stained for NKX3.1 (green) and nuclei are counterstained with DAPI (blue). Scale bar: 50 μm. (1l) Representative immunofluorescence image demonstrating retention of pluripotency markers OCT4, SOX2, and NANOG in iPSC-Dox-NKX3.1 clones. Nuclei are counterstained with DAPI (blue). Scale bar: 50 μm. (1m) Pluripotency markers in iPSC-Dox-NKX3.1 compared to parental iPSC. OCT4 , SOX2 , and NANOG qRT-PCR-based expression analysis (n=4; independent two-sided t-test; ns indicates insignificant differences; mean + / - SEM). (1n-1o) Reproducibility of NKX3.1-induced differentiation in three independent iPSC cell lines. (1n) Flow cytometry analysis of CD13 and CD140b (wall cell markers) in iMPCs generated from three independent iPSC-Dox-NKX3.1 clones (BJ273, 3C18, and 11C23). (1o) qRT-PCR-based expression analysis of wall cell markers in iMPCs generated from iPSC-Dox-NKX3.1 clones 3C18 and 11C23 (n=3; mean ± SEM). For all experiments in Fig. 1a-1o: *P<0.05, **P<0.01, ***P<0.001. All PCR data are normalized to GAPDH. Figs. 2a-2e. Comparative analysis of NKX3.1-induced iMPCs and chemically-induced iSMCs. (2a) Expression (qPCR) of NKX3.1 (top bar graph) and TBXT (bottom bar graph) in both NKX3.1-induced (iMPC; right column of the graph) and chemically-induced (iSMC; left column of the graph) differentiation protocols. Both protocols show transient expression of NKX3.1 starting at 48 h (n=4-6; Bonferroni post-hoc analysis followed by ANOVA; mean ± SEM). (2b) mRNA expression of SMC markers and (2c) perivascular cell markers (qPCR) in iMPCs and iSMCs at 96 h. Analysis of qRT-PCR-based expression analysis of SMC markers in iMPCs and iSMCs in 2b and perivascular cell markers in iMPCs and iSMCs in 2c is plotted in a bar graph presenting mean+SEM (n=4-7; independent two-sided t-test). (2d) Schematic of a chemically-induced protocol for generating iSMCs from iPSCs via MePC intermediates through exposure to PDGF-BB (10 ng / mL) and Activin A (2 ng / mL). (2e) Schematic illustrating a dox-induced protocol for differentiating iPSCs into iMPCs through transient expression of NKX3.1. *P<0.05, **P<0.01, ***P<0.001; ns indicates insignificant differences. Figs. 3a-3g. Generation of iMPCs using a non-genome footprint approach with modified mRNA. (3a) Schematic representation of the modRNA-induced protocol. (3b) Transfection of iPSCs with modRNA and expression of NKX3.1 by qPCR after 48 h. (3c) FACS analysis showing ~95% conversion efficiency to iMPCs after modRNA-induced NKX3.1 activation in MePCs. (3d) mRNA expression of wall cell markers at 96 h (qPCR). qRT-PCR-based expression analysis of SMC and perivascular cell markers in Dox versus modRNA-generated iMPCs is plotted in a bar graph presenting mean+SEM (n=5; independent two-sided t-test; *P<0.05, ns indicates insignificant difference). (3e) Flow cytometric analysis of CD13 and CD140b (wall cell markers) expression in iMPCs generated by modRNA versus Dox at 96 hours. (3f) Schematic of a timeline illustrating the differentiation protocol from iPSCs to induced wall cell precursors (iMPCs) via mesodermal intermediates (MePCs) using modRNA encoding NKX3.1. (3g) Time course after modRNA transfection (24 h, 48 h, and passages P1 and P2) NKX3.1 Quantitative PCR analysis of expression. Untransfected MePCs served as controls (n=4; Bonferroni post-hoc analysis following ANOVA; ***P<0.001, ns indicates insignificant difference). For all PCR analyses, the expression values ​​were for the housekeeping gene GAPDH Normalized with respect to . **P<0.01. Figs. 4a-4j. Functional characterization and secretion profiles of iMPCs. (4a) Calcium imaging shows increased intracellular calcium in iMPCs in response to endothelin-1 and carvacol. (4b) Collagen contractility assay shows the comparable responses of iMPCs, MSCs, and iSMCs to U46619 (TXA2 analog; vasoconstrictive stimulus). Collagen gel contraction imaging is presented under gel contractility analysis (n=3; ***P< 0.001; mean ± SEM). (4c) Increased fibronectin production in iMPCs upon TGF-β treatment, and its inhibition by the TGF-β signaling inhibitor, SB31542. (4d) Array of angiogenic proteins in conditioned medium (CM) from iMPCs, SMCs, and MSCs. (4e) Secreted angiogenesis-inducing factors by Luminex protein assay: Multiplex assay of angiogenesis factors in conditioned medium including FGF-2, HB-EGF, HGF, PLGF, VEGF-A, and VEGF-C (n=2; *P<0.05, **P<0.01, ***P<0.001; mean ± SEM). (4f) Calcium imaging: Intracellular calcium flow in iMPCs visualized using green fluorescent indicator after stimulation with endothelin-1, carvacol, or PBS. Pseudo-colors indicate intensity, and blue and red indicate lower and higher calcium levels, respectively (scale bar: 40 μm). Quantitative analysis of peak calcium uptake is presented on the right. (4g) Peak calcium response: Comparative uptake in MSCs, SMCs, and iMPCs upon endothelin-1 and carvacol stimulation, presented by delta fluorescence (n=5; ***P< 0.001; mean ± SEM). (4h-4i) Fibronectin deposition: Immunofluorescence staining of iMPCs treated with TGFβ and the TGFβ inhibitor SB431542 (scale bar: 100 μm) and quantification of fibronectin intensity per cell presented in 4i (n=6; **P<0.01, ***P< 0.001; mean ± SEM).(4j) FN1 expression: RT-qPCR analysis of FN1 normalized to GAPDH (n=4; ***P< 0.001; mean ± SEM). *P<0.05, **P<0.01, ***P<0.001. Figs. 5a-5m. Modulation of EC function by iMPCs. (5a) EC proliferation under indirect co-culture with iMPCs. Schematic diagram illustrating ECs cultured with wall cells and proliferation evaluation when co-cultured with SMCs, MSCs, and iMPCs (Right; n=7; *P< 0.05; mean ± SEM). (5b) EC proliferation upon exposure to iMPC-conditioned medium (CM-(iMPC)). Growth quantification in ECs exposed to 2-fold enriched conditioned medium from SMCs, MSCs, and iMPCs (***P< 0.001; mean ± SEM). (5c) Enhancement of EC migration and re-endothelialization by CM-(iMPCs). (5d) Scratch assay comparing EC migration from iMPCs in conditioning medium and basal medium after 24 hours (left; scale bar: 200 μm) and quantified percentage migration of gap closure normalized relative to the basal medium control (right; n=4; ***P< 0.001; mean ± SEM). (5d) Formation of capillary-like structures in 3-dimensional cultures exposed to CM-(iMPC). Tube formation assay on Matrigel using conditioning medium, representative image (scale bar: 200 μm), and quantification of total tube length (right; n=4; **P< 0.01; mean ± SEM). (5e) Hemoperfusion in subcutaneous grafts containing EC+ wall cells (SMC, MSC, or iMPC) 1 week after transplantation into nude mice, and visual evaluation of the explanted graft on day 7 (scale bar: 4 mm). (5f) H&E staining confirming perfused vessels in the graft on day 7 (yellow arrow) (scale bar: 50 μm). H&E staining suggests the formation of perfused vessels containing murine erythrocytes in grafts seeded with EC + iMPC, but not in grafts with EC alone. (5g) mm2 Analysis of microvascular density per area (n=7; *P< 0.05; mean ± SEM). Mean microvascular density at day 7 across grafts with different wall cell populations. (5h) Staining of newly formed human blood vessels for human-specific CD31 and surrounding α-SMA-positive perivascular cells. (5i) Human blood vessel identification: IHC presenting human-specific ECs (h-CD31+) and human perivascular cells (h-vimentin+) (scale bar: 50 μm; inset 10 μm). (5j) iMPC tracking: GFP and α-SMA staining to track GFP-labeled iMPCs within in vivo perivascular niches (scale bar: 50 μm; inset 10 μm). *P<0.05, **P<0.01, ***P<0.001. (5k) Schematic illustration and fluorescence image presenting the co-culture of ECs and iMPCs (P1) in a microfluidic on-a-chip model. GFP-labeled iMPCs and DsRed-labeled ECs were embedded in fibrin gel, and the formation of vascular structures was observed after 2 days. (Scale bar: 500 μm). (5l) Immunofluorescence staining of the vascular network formed within the microfluidic chip. ECs are labeled by CD31 and VE-cadherin (red), and iMPCs are identified by α-SMA and SM22 (green). Nuclei are counterstained with DAPI (blue). The inset presents an enlarged view of the endothelial lumen surrounded by parietal cells (yellow arrowheads). (Scale bar: 100 μm). (5m) Quantification of the percentage of human blood vessels with human wall cell coverage, comparing ECs transplanted with SMC, MSC, and iMPC (n=4; mean ± SEM). All experiments in this figure used neonatal iMPCs immediately after differentiation (96 h). Figs. 6a-6j. Maturation of iMPCs upon interaction with ECs. (6a) Schematic of iMPCs co-cultured with ECs for 7 days and subsequently isolated as CD31- cells for bulk RNA-seq analysis. The bottom panel shows up-regulated and down-regulated gene coefficients in co-cultured iMPCs (co-iMPCs) compared to single-cultured iMPCs. (6b) Principal component analysis of differentially expressed genes demonstrating that co-iMPCs exhibit closer transcriptional proximity to primary SMCs and MSCs than iMPCs. Transcriptional comparison of co-iMPCs versus primary SMCs and MSCs (n=3). (6c) Gene ontology (GO) analysis highlighting significant enrichment of genes associated with mature wall cell function in co-iMPCs. Up-regulated genes associated with mature wall cell function in co-iMPCs. (6d) qPCR analysis confirming significant upregulation of genes associated with wall cell markers in co-iMPCs. (6e) Transcriptome correlation: Pearson correlation plots describing transcriptional profiles among SMC, co-iMPCs, MSCs, iMPCs, iSMCs, and iPSCs. (6f-6g) Marker gene expression: RT-qPCR analysis of SMCs and perivascular cell markers showing enhanced expression in co-iMPCs compared to single-cultured iMPCs (n=3-9; *P<0.05, **P<0.01, ***P<0.001; mean ± SEM). All PCR data are normalized to GAPDH. (6h) Immunofluorescence characterization of co-iMPCs: Co-iMPCs were sorted from co-cultures as CD31- cells and stained for α-SMA (green) along with 3G5 (perivascular cell marker, red), and nuclei were counterstained with DAPI (blue), demonstrating the presence of both SMCs (α-SMA+- / 3G5-) and perivascular cells (α-SMA- / 3G5+) (scale bar: 100 μm). (6i) Gene expression heatmap: Differential gene expression patterns in co-iMPCs compared to iMPCs. *P<0.05, **P<0.01, ***P<0.001. (6j) Classified cells stained for α-SMA (green), MYH11 (red), and DAPI (blue). Co-localization of MYH11 and α-SMA indicates cells with a more mature SMC phenotype (yellow arrowheads). (Scale bar: 50 μm). All experiments in this figure used nascent iMPCs immediately after differentiation (96 h). FIG. 7a-7c. Single-cell RNA sequencing analysis of wall cell heterogeneity. (7a) Time points of analysis during the differentiation protocol and after co-culture with ECs. (7b) Integrated clustering analysis using Seurat (UMAP plot). The inventors identified eight distinct clusters by manual annotation. (7c) UMAP plot with the temporal appearance of clusters presenting the transition from iPSCs to MePCs and iMPCs, and the final maturation of iMPCs into distinct wall cell types (SMCs, perivascular cells, and fibroblasts) after co-culture with ECs. Figs. 8a-8g. Co-differentiation of iPSCs into iECs and iMPCs in 3D vascular organoids (VO). (8a) Schematic representation of the VO method using dox-ETV2-iPSC and dox-NKX3.1-iPSC cell lines. (8b) Image illustrating VO of uniform size (~200 μm) generated on day 5. (8c) Flow cytometry panel presenting iECs (h-CD31+) and iMPCs (h-CD31- / PDGFRβ+) in post-differentiated VO. (8d) Image of self-assembled CD31+ vascular structures within VO. (8e) Schematic representation of VO graft into the neocapsule of NSG mice. (8f) H&E-stained image showing an extensive network of perfused vessels within the graft. (8g) Immunofluorescence staining confirming perfused microvessels lined by human CD31+ iECs and surrounded by α-SMA+ perivascular wall cells. FIG. 9a-9c. Comparative maturation of iMPCs and iECs in the inventors' VO model. (9a) Schematic representation of the VO enzymatic digestion process and the sorting of CD31+ (VO-iEC) and CD31- (VO-iMPC) cells. (9b) Increased expression of core endothelial markers in VO-iECs compared to 2D-iECs, indicating enhanced maturation (qPCR). (9c) Significant upregulation of wall cell markers in VO-iMPCs, indicating improved maturation (qPCR). ***P<0.001. Figs. 10a-10d. Transplantation of VO into ischemic tissue improves blood flow and prevents necrosis. (10a) Schematic illustration of the procedure for inducing hindlimb ischemia in diabetic nude mice and the injection of VO. Untreated ischemic mice served as controls. (10b) Bioluminescence imaging demonstrating successful engraftment of VO in ischemic hindlimbs. (10c) Laser Doppler imaging demonstrates 50% recovery of blood flow in diseased legs 2 weeks after VO injection. (10d) Prevention of necrotic tissue development in mice receiving VO. **P<0.01. Figs. 11a-11h. Description of parietal cell heterogeneity and maturation in iMPCs via scRNA-seq. (11a) Differentiation and co-culture timeline: A schematic cartoon illustrating progression from iPSCs to iMPCs through various stages and their subsequent co-culture with ECs, with highlights of the transition points sampled for scRNA-seq. c-SMC and s-SMC refer to contracted and synthetic SMCs, respectively. (11b) Cell clustering: A UMAP projection displaying 17 identified clusters annotated with 8 cell types, including iPSCs, MePCs, iMPCs, and various parietal cells, based on gene expression markers. (11c) Differentiation trajectory: A UMAP visualization tracking differentiation from iPSCs to iMPCs and the emergence of parietal cell clusters. (11d) Marker gene expression: A dot plot summarizing the expression profiles of key markers across clusters describing cell identity. (11e) Gene expression kinetics: Volcano plots displaying up- and down-regulated genes in iMPCs after EC co-culture, with a highlight of genes and ECM components related to contractility. (11f) Pseudo-chronological analysis: UMAPs overlaid with pseudo-chronological scores indicating the progression of cell development. (11g) Pseudo-chronological trajectory: Sequential UMAP plots presenting the gradual transition from iPSCs to mature parietal cells and the effects of EC co-culture over time. (11h) Schematic summary: An example summarizing the differentiation of iMPCs into specialized parietal cells, highlighting the influence of EC interactions on iMPC maturation and the establishment of parietal cell heterogeneity. Fig. 12. Heatmap of differentially expressed genes across cell clusters. Heatmap describing the top 10 upregulated genes within each cell cluster—iPSC, MePC, iMPC, and various wall cell subtypes—identified by single-cell RNA sequencing. The color spectrum (purple for lower expression, yellow for higher expression) highlights the scale of gene expression. Rows represent individual genes, and columns represent single cells, illustrating the unique expression profiles defining each cell population. This heatmap highlights the characteristics of transcriptional diversity at the cell differentiation stage. Fig. 13. UMAP visualization of selected gene expression in cell differentiation. This feature plot presents a UMAP visualization illustrating the expression patterns of key genes across cell types derived from iPSCs. Each panel represents the expression of a specific gene from single-cell RNA sequencing data, and color intensity reflects low (gray) to high (blue) expression levels. The displayed genes are pluripotent stem cells ( SOX2 , POU5F1 ), mesoderm precursor ( MIXL1 , TBXT ), induced wall cell precursor ( NKX3.1 , DES ), wall cells ( PDGFRB , NT5E , ACTA2 , CNN1 , COL1A1 ), and endothelial cells ( PECAM1 It is a marker representing ) and provides insight into the molecular indicator characteristics of each differentiation stage. Fig. 14. Violin plot of marker gene expression in cell subtypes. Violin plots representing the distribution of expression levels for selected marker genes within distinct cell clusters identified by single-cell RNA sequencing. The clusters include induced pluripotent stem cells (iPSCs), mesenchymal cell precursors (MePCs), induced parietal cell precursors (iMPCs), fibroblasts, perivascular cells, contractile smooth muscle cells (c-SMCs), synthetic smooth muscle cells (s-SMCs), endothelial cells (ECs), and endothelial cells after co-culture with iMPCs (co--ECs). Each plot provides a visual indication of cell density at various expression levels, which offers insight into the transcriptional landscape characteristics of each cell subtype. Figs. 15a-15b. NKX3.1-induced differentiation of MePCs into iMPCs. (15a) NKX3.1 expression: Time-course qRT-PCR analysis demonstrating NKX3.1 upregulation during differentiation (n=9; ***P<0.001; mean ± SEM). (15b) Quantitative PCR analysis of wall cell marker expression with and without NKX3.1 induction. MePCs treated with doxycycline (+Dox) and without doxycycline (-Dox) for 48 hours to induce iMPCs. ACTA2 , CNN1 , TAGLN , MYOCD , TPM1 , MYH11 , PDGFRB , CSPG4 , DES , PDE5A , and THY1 Gene expression. (n=4; independent two-sided t-test; *P<0.05, **P<0.01, ***P<0.001; mean + / - SEM). All PCR data are normalized to the control group (+dox). Figs. 16a-16c. Immunofluorescence analysis of parietal cell markers in iMPCs. (16a) Immunofluorescence staining of ECFCs and iMPCs for endothelial and parietal cell markers. Control ECFCs show positive expression of EC markers (CD31, VE-cadherin, vWF) and negative expression of parietal cell markers (α-SMA, SM22, calponin), demonstrating antibody specificity. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. (16b) Immunofluorescence staining of iMPCs from three different iPSC cell lines (BJ273, 3C18, 11C23). iMPCs are positive for parietal cell markers α-SMA, SM22, calponin, and vimentin (green). Nuclei are stained with DAPI (blue). (16c) Quantification of marker expression in iMPCs. The bar graph presents the percentage of cells positive for α-SMA, SM22, calponin, and vimentin in BJ273, 3C18, and 11C23 iMPCs (n=5; mean + / - SEM). Scale bar: 50 μm. Figs. 17a-17d. In vivo verification of the ability of iMPCs to facilitate HUVEC-mediated vascular network development. Subcutaneous transplantation of HUVECs with iMPCs into nude mice. A graft containing ECFCs + iMPCs served as a control. (17a) H&E staining identifying perfused vessels on day 7 (yellow arrowheads) (scale bar: 50 μm). (17b) Microvascular density in the graft on day 7 (independent two-sided t-test; ns indicates insignificant difference; mean ± SEM). (17c) Human vessel identification: IHC shows human-specific ECs (h-CD31+) covered by human perivascular cells (h-vimentin+) in the graft on day 7 (yellow arrowheads) (scale bar: 50 μm; inset 10 μm). (17d) Quantification of the percentage of human blood vessels covered by human parietal cells (n=4; independent two-sided t-test; ns represents insignificant differences; mean ± SEM). Fig. 18. Quantitative PCR analysis of wall cell markers in iMPCs after co-culture with and without ECs. In iMPCs co-cultured with and without ECs for 7 days ACTA2 , CNN1 , TAGLN , MYOCD , TPM1 , MYH11 , and CSPG4 Gene expression. Data are normalized for conditions without EC (n=3, independent two-sided t-test; *P<0.05, **P<0.01, ***P<0.001; mean + / - SEM). Figs. 19a-19d. UMAP visualization of selected gene expression in MePCs and iMPCs. (19a) UMAP plot derived from single-cell RNA sequencing data captures the transition from mesodermal cell progenitors (MePCs) on day 2 after NKX3.1 induction to induced parietal cell progenitors (iMPCs) on day 4. (19b-19d) Feature plots capture (19b) peraxial mesodermis ( TBX6 , MSGN1 ), (19c) segment ( FOXC2 , MEOX2 , TCF15 ), and (19d) tibial segment ( PAX9 , SOX9 , NKX3.2 ) exemplifies the expression level of genes associated with. The + symbol indicates detectable gene expression, and intensity is indicated by color depth ranging from low (light gray) to high (dark purple). Fig. 20. UMAP visualization of selected gene expression in cell differentiation. This feature plot presents a UMAP visualization illustrating the expression patterns of key genes across cell types derived from iPSCs. Each panel represents the expression of a specific gene from single-cell RNA sequencing data, and color intensity reflects low (gray) to high (blue) expression levels. The displayed genes are pluripotent stem cells ( SOX2 , POU5F1 ), mesoderm precursor ( MIXL1 , TBXT ), induced wall cell precursor ( NKX3.1 , DES ), wall cells ( PDGFRB , NT5E , ACTA2 , CNN1 , COL1A1 ), and endothelial cells ( PECAM1 It is a marker representing ) and provides insight into the molecular indicator characteristics of each differentiation stage. Figs. 21a-21d. Comparative transcriptome analysis of iMPC-derived parietal cells and air-available parietal cell data. (21a, 21b) Heatmaps displaying Pearson correlation coefficients for transcriptome comparisons of iMPC-derived SMCs (clusters #6 and #7 in Fig. 5) and perivascular cells (cluster #5 in Fig. 5) with air-available primary human SMCs (accession codes GSM7073879, GSM7073881, and GSM7073883) and brain perivascular cell datasets (accession codes GSM5293256, GSM5293257, and GSM5293258), respectively. Strong correlation (correlation coefficient ~0.6, p < 0.001) indicates a high degree of similarity in gene expression profiles. (21c, 21d) Bar graphs presenting the number of iMPC-derived SMCs and perivascular cells that closely match their respective cell types in the Tabula Sapiens consortium scRNA-seq data. This analysis confirms the similarity of iMPC-derived wall cell types to both primary human cells and cells from comprehensive single-cell maps, which highlights the relevance of the inventors' differentiation model to its in vivo counterparts. Figures 22a-22e. Analysis of signaling pathways from ECs affecting parietal cell maturation. (22a, 22b) CellChat analysis illustrating predicted ligand-receptor interactions between ECs and iMPCs based on scRNA-seq data. Pathways are color-coded by communication probability, and statistical significance is indicated by symbols. (22c) Schematic of cell sorting and qPCR analysis following co-culture settings for iMPCs and ECs with treatments using inhibitors of the TGF-β (SB431542) and NOTCH (DAPT) pathways. (22d) Effect of TGF-β pathway inhibition on the expression of parietal cell markers in co-cultured iMPCs (n=5; independent two-sided t-test; **P<0.01, ***P<0.001; mean + / - SEM). (22e) Effect of NOTCH pathway inhibition on parietal cell marker expression (n=5; independent two-sided t-test; *P<0.05, **P<0.01, ***P<0.001; mean + / - SEM). FIG. 23a-23c. Analysis of DEGs between neovascular and mature perivascular cells and contracted SMCs. (23a) Gene ontology (GO) analysis comparing neovascular cells on day 4 and mature perivascular cells on day 11. Enriched pathways in day 11 perivascular cells include extracellular matrix organization, cellular response to TGF-β stimulation, and integrin-mediated signaling, which indicate a more mature wall cell phenotype. (23b) Gene ontology (GO) analysis comparing Cluster 5 (perivascular cells) and Cluster 6 (contracted SMCs), showing enrichment in extracellular matrix organization, cell-matrix adhesion, and pathways associated with TGF-β signaling in Cluster 6. (23c) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighting enriched pathways in cluster 6, including ECM-receptor interactions, vascular smooth muscle contraction, and TGF-β signaling. The size of the dots indicates the number of genes, and the color indicates the adjusted p-value. FIG. 24a-24b. Analysis of gene regulatory networks (GRNs) in iPSC-derived wall cells. (24a) Dot plot highlighting the prevalence and mean expression of various GRNs across the following different cell identities: SMC, perivascular cells, MePC, and iMPC. (24b) Detailed network diagrams for each identified GRN (GRN-1 to GRN-13) illustrate the relationships between regulatory genes within each network. Each network is color-coded to represent a distinct functional cluster, and the edges indicate regulatory interactions among the genes. Fig. 25. Pseudo-chronological analysis: Differentiation trajectories from iMPCs to perivascular cells and SMCs, and annotations indicating distinct cell identities. Specific details for implementing the invention

[0085] Effective in vitro models for studying vascular diseases require the accurate representation of multiple cell types. Parietal cells are a critical component of blood vessels and play an essential role in vascular development and function. Induced pluripotent stem cells (iPSCs) possess the potential to generate parietal cells, but current differentiation protocols rely on chemical or growth factor stimulation and are limited in category and precision. Here, the inventors present a method using the transient activation of the transcription factor NKX3.1 to generate parietal cell precursors (iMPCs). These cells resemble primary parietal precursors in terms of transcriptome and functional characteristics. Furthermore, secretory analysis reveals that iMPCs produce angiogenic factors similar to those produced by mesenchymal stem cells and vascular smooth muscle cells, thereby promoting endothelial cell growth. Moreover, when co-cultured with endothelial cells, iMPCs can mature into functional parietal cell subtypes possessing upregulated genes related to vascular development, angiogenesis, and cell junctions, including smooth muscle cells, perivascular cells, and fibroblasts. Perivascular cells are primarily associated with microvessels, such as capillaries, whereas SMCs are more commonly found in larger vessels, such as arteries and veins. Parietal cells contribute to vascular stabilization, blood flow regulation, endothelial rest, and the integrity of the blood-brain barrier. However, dysregulation of these cells can lead to vascular abnormalities, including abnormal angiogenesis, vascular destabilization, and impaired vascular function. Consequently, the generation of functional parietal cells is essential for understanding vascular function in health and disease and for developing novel therapeutic approaches that target perivascular cells.

[0086] Importantly, the in vivo experiments described herein demonstrate that iMPCs mediate the formation of functional blood vessels when transplanted with endothelial cells, highlighting their potential to model vascular diseases and therapeutic vascularization. The findings here establish NKX3.1 as a wall cell fate-determining transcription factor and highlight the potential of these precursors in vascular biology research and the development of novel therapeutic strategies for vascular diseases.

[0087] Blood vessels are essential for mammalian development and homeostasis. However, generating a fully functional vascular system for solid organ regeneration remains a significant challenge. Parietal cells, including smooth muscle cells (SMCs) and perivascular cells, which regulate vascular development, stability, and function, are pivotal in the formation of blood vessels. Dysregulation of parietal cells in various diseases can lead to vascular abnormalities. Furthermore, parietal cells facilitate angiogenesis in therapeutic vascularization and vascular tissue manipulation. Given their significance, there is an urgent need to develop methods to generate patient-specific parietal cells. Doing so holds the potential to significantly advance regenerative vascular medicine and address unmet needs in the treatment of vascular disorders.

[0088] Human induced pluripotent stem cells (iPSCs) represent a promising, non-invasive source of patient-specific wall cells. Conventional differentiation strategies equate iPSCs through two distinct steps regulated by Wnt, Nodal, TGFβ, and PDGF signaling pathways. However, inducible transcription factors (TFs) are emerging as a more precise and versatile tool for differentiation. By using TFs, it is possible to achieve precise temporal control and the potential for simultaneous differentiation of multiple cell types, an approach that presents possibilities in tissue manipulation and organoid systems.

[0089] The emergence of human induced pluripotent stem cells (h-iPSCs) has provided a promising and non-invasive approach to obtaining patient-specific parietal cells. Conventional parietal cell differentiation methods are largely based on angiogenesis and involve the differentiation of h-iPSCs through two distinct stages. Initially, h-iPSCs differentiate into intermediate mesenchymal cell precursors (MePCs), regulated by Wnt and Nodal signaling pathways. Subsequently, the cells undergo SMC differentiation, driven primarily by TGFβ and PDGF signaling. Despite advancements in chemically induced strategies, interest in utilizing inducible transcription factors (TFs) for differentiation has grown. Using TF-based approaches offers several benefits, such as precise temporal control and the potential to develop methods for the simultaneous differentiation of multiple cell types. However, identifying TFs that can be effectively utilized for parietal cell differentiation remains challenging.

[0090] NK3 Homeobox 1 (NKX3.1) is a TF belonging to the NKX family of homeodomain-containing proteins and plays a pivotal role in the development and maintenance of various tissues, particularly in prostate and SMC differentiation. During SMC differentiation, NKX3.1 interacts with Serum Response Factor (SRF), a critical TF involved in regulating smooth muscle-specific gene expression. Furthermore, NKX3.1 cooperates with additional TFs and co-activators, such as GATA-6 and myocardin, to further modulate the transcriptional activity of SRF and promote the expression of smooth muscle-specific genes. Thus, molecular interactions mediated by NKX3.1 collectively contribute to establishing and maintaining the SMC phenotype. Nevertheless, the feasibility of utilizing NKX3.1 as a single fate-determining TF to guide iPSC differentiation into wall cells has not yet been investigated.

[0091] As described herein, transient activation of NKX3.1 in human MePCs potently drives their differentiation into cell progenitors closely resembling primary parietal cells in their gene expression profiles and functional characteristics. These iPSC-derived parietal cell progenitors (iMPCs) can further mature upon co-culture with endothelial cells (ECs) to generate heterogeneous parietal cell subpopulations, including perivascular cells and SMCs. Importantly, the iMPCs described herein exhibit potent angiogenic capabilities and support EC engraftment in the form of functional blood vessels in vivo, highlighting their therapeutic potential for vascular repair and regenerative medicine applications. By establishing NKX3.1 as a key regulator of parietal cell differentiation from iPSCs and presenting a novel strategy for generating parietal progenitors, this study opens new avenues for understanding parietal cell biology and developing innovative therapeutic approaches for vascular diseases.

[0092] The study described herein revealed the ability of NKX3.1 to rapidly drive the differentiation of human MePCs into functionally qualified iMPCs. These findings simplify the complexity associated with traditional chemically induced differentiation processes, which rely on a cascade of signaling molecules to achieve cell lineage specificity. The ability to streamline the differentiation process from iPSCs to wall cells into a 4-day window through the transient induction of a single TF has broad implications for both basic research and translational applications.

[0093] NKX3.1 has been implicated in SMC differentiation during development through cooperative interactions with other TFs and co-activators, such as serum response factors and myocardin. However, its potential to serve as a single fate-determining TF for differentiation from iPSCs into parietal cells remained an unresolved issue. The inventors previously engaged in a comprehensive, unbiased TF screen involving over 1,500 human TFs across three human PSC cell lines. This screening identified 290 TFs capable of inducing differentiation into identifiable lineages without requiring modifications in external availability or biomechanical signals. Among the identified TFs, NKX3.1 emerged as particularly noteworthy for its ability to direct human iPSCs toward fibroblast-like cells. In this study, direct activation of NKX3.1 in iPSCs, thereby bypassing the MePC intermediate stage, resulted in a predominance of fibroblast-like cells but not perivascular SMCs or perivascular cells. However, during embryonic development, the parietal cell lineage originates primarily from mesodermal precursors. Indeed, mouse models have previously demonstrated highly organized expression of Nkx3.1, which begins in the periaaxial mesoderm at E7.5 and progressively increases in concentration adjacent to the endothelium of the ventral aorta by E9.5. From E11.5 to E15.5, Nkx3.1 was co-expressed with smooth muscle myosin heavy chains (SM-MHC) in these regions. Zebrafish studies have validated this mesodermal origin, suggesting Nkx3.1 expression in mesodermal precursors specific to the trunk periavascular cell lineage.

[0094] The present disclosure suggests that, in particular, activating NKX3.1 at the MePC stage rather than directly activating it in iPSCs generates a more versatile population of wall cell precursors, which the inventors have named iMPCs. By timing the activation of NKX3.1, the inventors were able to go beyond the production of mere fibroblast-like cells, generating a homogeneous population of wall precursors capable of reproducing the complexity of wall cell heterogeneity. Importantly, these iMPCs exhibit functional qualification as perivascular cells when interacting with vascular ECs. Thus, the differentiation strategy provides remarkable efficiency and closely mimics the original developmental pathways of mesenchymal-derived wall cells. This NKX3.1 reprogramming paradigm is an ideal platform for investigating wall lineage specification processes. The simplicity and modularity of the approach provide customization, which can be further tuned to activate additional genes or pathways, thereby providing a highly adaptable means to generate arrays of wall cell types and study their diverse functional roles. This flexibility is a significant advantage for research focused on elucidating the temporal aspects of gene function and their effects on differentiation. The inventors' NKX3.1-driven system serves as a versatile tool for in vitro disease modeling and drug discovery, and ultimately, can enable the efficient induction of patient-specific wall cells for precision and regenerative medicine applications.

[0095] In recent years, there has been increasing interest in using inducible TFs for cell differentiation. Among the most significant advantages of TF-driven differentiation is the temporal control it provides. By modulating the expression of NKX3.1 in a time-specific manner, the inventors' system allows for precise analysis of cellular and molecular events occurring during the wall cell specification phase. This provides a novel platform for interrogating processes that form the basis of cell fate determination and is valuable for gaining insights into the detailed mechanisms driving the generation of iMPCs from MePCs.

[0096] An additional advantage of TF-based approaches, such as those described herein, is the potential to develop methods for the simultaneous differentiation of multiple cell types. Simultaneously differentiating human iPSCs into cells from different lineages in a controllable manner is not trivial, as each cell type requires incompatible differentiation conditions. However, orthogonal differentiation approaches dependent on specific TFs can override a wide range of media signals, which enables the simultaneous generation of different cell types. Indeed, recent studies have demonstrated the potential of orthogonal programming in tissue manipulation and organoid systems. For example, the literature [Ng et al .] used a cerebral organoid model and presented the orthogonal differentiation of iPSCs into both neurons and oligodendrocytes through dox-induced transient activation of two TFs, ATOH1 and SOX9, respectively. Literature [Skylar-Scott et al.] used an orthogonal differentiation approach to generate vascular ECs (via ETV2) and neurons (NGN1) from human iPSCs and produced vascularized and patterned cortical organoids within days, demonstrating the applicability of orthogonal programming to vascular structures. However, while the list of TFs supporting efficient cell differentiation into individual cell lineages continues to grow, identifying TFs that can be effectively utilized for parietal cell differentiation within organoid systems remains difficult. Therefore, the inventors also investigated whether inducible activation of NKX3.1 could enable the incorporation of parietal cells into these orthogonal programming efforts within various organoid models.

[0097] The data presented herein, in particular, confirmed the functional qualification of the inventors' iMPCs and their wall cell derivatives. Indeed, the functional properties of perivascular cells are critical to modulating EC behavior and, consequently, to their utility in vascular therapy. Although ECs inherently possess the ability to self-assemble into vascular structures, robust in vivo engraftment and functional vascularization necessarily require perivascular cell support. Traditionally, these helper cells have been supplied from primary perivascular cells, including SMCs, perivascular cells, fibroblasts, and MSCs. However, recent developments have shifted toward the use of pluripotent cells as a personalized and inexhaustible source for perivascular cells.

[0098] Although other protocols have successfully generated functionally qualified perivascular cells from pluripotent stem cells, the inventors' research employs a unique TF-driven approach. Early chemically induced methods relied on culture formation for spontaneous differentiation, but these were subsequently considered non-specific and inefficient. In contrast, more recent 2-D methods, which initially induce differentiation into the intermediate mesodermal stage via the Wnt and activin / Nodal pathways and subsequently transform these cells into perivascular cells through the application of specific growth factors such as PDGF-BB and TGF-β, have proven to be significantly more efficient. Based on these fundamental methods, the inventors' NKX3.1-driven approach provides a streamlined procedure that introduces a reproducible, robust framework to generate functionally qualified iMPCs, bypass the limitations typically associated with culture-induced methods, minimize the risk of off-target effects, and facilitate the standardization of iPSC differentiation into wall cells.

[0099] Transcriptionally, the NKX3.1-induced iMPCs described herein correspond to a progenitor population closely resembling the phenotype of immature perivascular cells. Although some pathways that promote parietal cell differentiation are known, the genetic pathways that guide undifferentiated cells to mature parietal cells are still incompletely elucidated. The inventors' detailed examination of marker expression in iMPCs is CSPG4 , PDGFRB , and DES Expression of, however, contractile proteins, for example ACTA2 , CNN1 , and TAGLNWe identified a phenotype similar to newly formed perivascular cells, indicated by the absence of [unclear]. This phenotype is distinct from that observed in the mature cell population obtained upon interaction with ECs. Instead, iMPCs serve as true precursors capable of fully differentiating into perivascular cells and SMCs after one week of co-culture with ECs. The inventors demonstrated this transformation from MePCs to iMPCs and then to the final wall cell population through their single-cell RNA sequencing and trajectory analysis.

[0100] Furthermore, comparative evaluations demonstrated that the gene expression profiles of the inventors' iMPC-derived SMCs and perivascular cells are substantially consistent with established primary human wall cells and detailed single-cell references from The Tabula Sapiens Consortium, which highlights the relevance of the inventors' differentiation model to its in vivo counterparts.

[0101] Transcriptional and functional analyses have also revealed a close match between iMPCs and primary MSCs, which are widely recognized as wall precursors, but debate regarding their equivalence to perivascular cells persists. Indeed, the inventors' iMPCs exhibit traits consistent with chemically induced mesenchymal precursors identified in previous studies as PDGFRβ+ CD271+ CD73- immature perivascular cells capable of differentiating into mature wall cells. The inventors' work described herein confirms, in particular, that transient activation of NKX3.1 in MePCs is sufficient to generate a population of iMPCs functioning as wall cell precursors exhibiting characteristics consistent with mesenchymal precursors.

[0102] Accordingly, in particular, a powerful and efficient TF-driven methodology for differentiating human iPSCs into functional iMPCs is described herein. By transiently activating NKX3.1 during the intermediate stage of differentiation, the inventors have demonstrated not only remarkable differentiation efficiency but also the functional qualification of the generated iMPCs. Furthermore, the inventors have presented the following two distinct approaches for inducing NKX3.1: a Dox-inducible system and a non-genome footprint modRNA method. Non-viral, non-integrative, and inherently transient, the latter possesses distinct translational advantages. From the perspective of clinical application, the inventors' methods can provide a highly reliable pathway for generating patient-specific wall cells for regenerative medicine and disease modeling. Moreover, the inventors' iMPCs may offer therapeutic potential in conditions characterized by perivascular cell loss, such as diabetic retinopathy and stroke.

[0103] Beyond these immediate translational applications, the research described herein serves as a fundamental platform providing a standardized, reproducible approach for the induction of wall cells from human iPSCs. The inventors' research revealed the exceptional ability of NKX3.1 to rapidly drive the differentiation of human MePCs into functionally qualified iMPCs. This finding simplifies the complexity associated with traditional chemically induced differentiation processes, which rely on a cascade of signaling molecules to achieve cell lineage specificity. The ability to streamline the differentiation process from iPSCs to wall cells into a 4-day window through the transient induction of a single TF has broad implications for both basic research and translational applications.

[0104] iPSC, MePC, & MPC and method for manufacturing MPC

[0105] In general, the present disclosure is based on the finding that NK3 homeobox 1 (NKX3.1) determines the fate of a parietal cell lineage and that NKX3.1 activation in iPSC-derived mesenchymal precursors (MePCs) effectively produced iPSC-derived parietal cell precursors (iMPCs). The present disclosure, in particular, provides a method for preparing and using a parietal cell precursor and a composition comprising a parietal cell precursor.

[0106] The use of cells derived from human pluripotent stem cells, such as human induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), allows cells from any donor to be reprogrammed into a pluripotent, self-renewing state and thus permits the expansion of a homogeneous population of cells from any genetic background. The use of iPSCs overcomes the ethical and political considerations associated with hESCs and can be generated from adult, finally differentiated cells. Briefly, iPSCs were generated by the expression of several key genes presented as required for total reprogramming, namely, combinations of the following: Oct4, Sox2, Klf4, c-Myc, l-Myc, Lin28, and / or Nanog, or one or more factors selected from, for example, Oct4, Sox2, Klf4, c-Myc, l-Myc, Lin28, and / or Nanog. Expression and differentiation analyses suggested that iPSCs are very closely related to ESCs at the molecular level, along with variability between clonal iPSC cultures on a scale similar to that observed when compared to multiple ESC cell lines. iPSCs are a promising and non-invasive approach to obtaining patient-specific wall cells, and they can ultimately lead to cell therapy generated from the patient's own cells in autotransplantation, which can prevent graft rejection.

[0107] Further information comprising additional methods for isolating, manufacturing, and differentiating iPSCs and ESCs is known in the relevant art (e.g., US 20240228951 A1; US ​​20240050483 A1; US ​​2022 / 0243174; US 20200385685 A1; US ​​20200182861 A1; US ​​2018 / 0371422; WO 2015 / 073625; US 2016 / 0002604; US 2014 / 0199274; US 2013 / 0052268; US 2012 / 0128655; and US 2009 / 0226401; as well as US 11,898,169; US 11,001,809; US 10,844,356; US 10,676,165; US 9,657,273; US 9,750,768; US 9,580,689; and US 9,376,664), each of the above is incorporated herein by reference.

[0108] Parietal cells, including perivascular cells and smooth muscle cells (SMCs), are essential components of blood vessels that play a crucial role in vascular development, stability, and function. Perivascular cells are primarily associated with microvessels, such as capillaries, while SMCs are more commonly found in larger vessels, such as arteries and veins. Parietal cells contribute to vascular stabilization, blood flow regulation, endothelial rest, and the integrity of the blood-brain barrier.

[0109] Conventional parietal cell differentiation methods are largely based on angiogenesis and involve the transition of h-iPSCs through two distinct stages. Initially, h-iPSCs differentiate into intermediate mesenchymal cell precursors (MePCs) regulated by Wnt and / or Nodal signaling pathways. Subsequently, the cells undergo SMC specification, driven primarily by TGFβ and PDGF signaling.

[0110] As demonstrated and discussed herein, using TF-based approaches offers several benefits, such as precise temporal control and the potential to develop methods for the simultaneous differentiation of multiple cell types. However, identifying TFs that can be effectively utilized for wall cell differentiation remains difficult.

[0111] NK3 homeobox 1 (NKX3.1; e.g., uniprotKB Q99801-1, uniprotKB Q99801-2, uniprotKB Q99801-3, uniprotKB Q99801-4, uniprotKB Q99801-5, PDB NP_006158.2, and PDB NP_001243268.1) is a TF belonging to the NKX family of homeodomain-containing proteins and plays a pivotal role in the development and maintenance of various tissues, particularly in the differentiation of the prostate and SMC. During SMC differentiation, NKX3.1 interacts with the serum response factor (SRF), which is a critical TF involved in regulating smooth muscle-specific gene expression. In addition, NKX3.1 cooperates with additional TFs and co-activators, such as GATA-6 and myocardin, to further modulate the transcriptional activity of SRFs and promote the expression of smooth muscle-specific genes.

[0112] The data presented herein indicate that transient activation of NKX3.1 in human MePCs strongly drives their differentiation into cell progenitors that can contribute to the parietal cell lineage in terms of their gene expression profiles and functional characteristics. Furthermore, the data suggest that these iPSC-derived parietal cell progenitors (iMPCs) can further mature upon co-culture with endothelial cells (ECs) to generate heterogeneous parietal cell subpopulations, including perivascular cells and SMCs. Importantly, the iMPCs in this study exhibit potent angiogenic capabilities and support EC engraftment in the form of functional blood vessels in vivo, highlighting their therapeutic potential for vascular repair and regenerative medicine applications. By establishing NKX3.1 as a key regulator of parietal cell differentiation from iPSCs and presenting a novel strategy for generating parietal progenitors, the study described herein opens new avenues for understanding parietal cell biology and developing innovative therapeutic approaches for vascular diseases.

[0113] In some embodiments, a method for producing iPSC-derived wall cell precursors (iMPCs) comprises or consists of one or more of the following steps: contacting a population of induced pluripotent stem cells (iPSCs) with a nucleic acid encoding NK3 homeobox 1 (NKX3.1) or a functional variant thereof; converting the iPSCs into mesodermal precursors (MePCs); and inducing the MePCs to express NKX3.1 for a period sufficient to produce iMPCs. In some embodiments, the nucleic acid is a vector (e.g., a piggyback transposon vector or a viral vector). In some embodiments, the vector is a viral vector (e.g., a retrovirus, a lentivirus). In some embodiments, the nucleic acid comprises an inducible promoter that controls the expression of NKX3.1 (e.g., a doxycycline-inducible promoter or other known in the art). In some embodiments, the step of converting an iPSC into a MePC comprises activating the Wnt pathway and / or activating the Nodal pathway for a period sufficient to generate a MePC (e.g., about or at least 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours). In some embodiments, the period sufficient to generate an iMPC is about or at least 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours.

[0114] Also described herein is a method for generating a population of wall cells including perivascular cells, smooth muscle cells, and fibroblasts. The method comprises or consists of one or more of the following steps: generating iMPCs using a method as described herein; and co-culture the iMPCs with a population of endothelial cells (EC or iEC) for a period sufficient to generate a population of wall cells including perivascular cells, smooth muscle cells, and fibroblasts. In some embodiments, the period sufficient to generate a population of wall cells including perivascular cells, smooth muscle cells, and fibroblasts is about or at least 1, 2, 3, 4, 5, 6, or 7 days.

[0115] In some embodiments, the method described herein involves the use of a nucleic acid encoding NKX3.1 (e.g., Uniprot KB Q99801-1, Uniprot KB Q99801-2, Uniprot KB Q99801-3, Uniprot KB Q99801-4, Uniprot KB Q99801-5, PDB NP_006158.2, and PDB NP_001243268.1). In some embodiments, NKX3.1 is a functional variant of NKX3.1. In some embodiments, NKX3.1 is wild-type NKX3.1. In some embodiments, NKX3.1 is human NKX3.1. In some embodiments, the nucleic acid is an exogenous DNA molecule or an exogenous RNA molecule. Non-limiting examples of useful RNA are modified mRNA (modRNA; e.g., US20230364267A1), mRNA, or its functional variants. Non-limiting examples of useful DNA may be vectors, plasmids, transposons, or their functional variants.

[0116] In some embodiments, the method described herein is E26 transform-specific variant 2 ( ETV2It includes the use of nucleic acids encoding ). In some embodiments, ETV2 is a functional variant of ETV2. In some embodiments, ETV2 is wild-type ETV2. In some embodiments, ETV2 is human ETV2. In some embodiments, the nucleic acid is an exogenous DNA molecule or an exogenous RNA molecule. Non-limiting examples of useful RNA are modified mRNA (modRNA; e.g., US20230364267A1), mRNA, or its functional variants. Non-limiting examples of useful DNA may be a vector, plasmid, transposon, or its functional variants.

[0117] Also described herein is a method for preparing vascular organoids (VO) or three-dimensional (3D) cell cultures. The method comprises or consists of one or more of the following steps:

[0118] (i) a step of incubating a first population of iPSC-derived mesenchymal cell precursors ("ETV2 / MePC") containing a nucleic acid encoding ETV2 or its functional variant (optional, exogenous nucleic acid) with a second population of MePCs ("NKX3.1 / MePC") containing a nucleic acid encoding NKX3.1 or its functional variant (optional, exogenous nucleic acid), and

[0119] Here, a step in which the expression of NKX3.1 is controlled by an inducible promoter and the expression of ETV2 is controlled by an inducible promoter;

[0120] (ii) inducing the expression of NKX3.1 in NKX3.1 / MePC to thereby generate iPSC-derived wall cell precursors (iMPCs) and inducing the expression of ETV2 in ETV2 / iPSC to thereby generate iPSC-derived endothelial cells (iECs); and

[0121] (iii) a step of culturing cells for a sufficient period to produce a VO or 3D cell culture, wherein the VO or 3D cell culture comprises iECs that are h-CD31+ and iMPCs that are PDGFRβ+ and h-CD31-.

[0122] In some embodiments, the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are the same (optional, doxycycline inductive promoter). In some embodiments, the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are not the same.

[0123] In some embodiments, the period sufficient to produce VO or 3D cell culture is about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0124] In some embodiments, (i) the culture step occurs for about 1 day or 2 days and / or; wherein (i) the culture step occurs for about or at least 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours and / or; wherein the culture occurs for a period sufficient to produce aggregates comprising both NKX3.1 / MePC and ETV2 / MePC.

[0125] In some embodiments, (i) the culture step includes culturing cells using a non-adhesive culture plate and a rotary shaker.

[0126] In some embodiments, a group of NKX3.1 / MePC and a group of ETV2 / MePC are mixed in a ratio of about 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:1 of NKX3.1 / MePC :ETV2 / MePC.

[0127] In some embodiments, a method for preparing a vascular organoid (VO) or a three-dimensional (3D) cell culture is described herein, comprising:

[0128] (i) transfecting a population of iPSC-derived mesenchymal precursors (MePCs) with a nucleic acid (optional, DNA, RNA, mRNA, modRNA) encoding NKX3.1 or a functional variant thereof, thereby generating a population of iPSC-derived wall cell precursors (iMPCs);

[0129] (ii) a step of mixing a population of iMPC and a population of EC (arbitrarily, in a ratio of approximately 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:1 iMPC:EC; and

[0130] (iii) a step of culturing cells for a sufficient period to produce a VO or 3D cell culture, wherein the VO or 3D cell culture comprises iECs that are h-CD31+ and iMPCs that are PDGFRβ+ and h-CD31-.

[0131] In some embodiments, the EC comprises any one or more of iPSC-derived ECs (iECs), human venous endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs, or organ-specific endothelial cells.

[0132] In some embodiments, organ-specific endothelial cells are from organs selected from the following: heart, muscle, kidney, testis, ovary, lymphatic system, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine.

[0133] In some embodiments, the method further comprises the step of transfecting a population of iPSCs with a nucleic acid encoding ETV2 or a functional variant thereof before mixing with a population of iMPCs to thereby generate a population of ECs.

[0134] In some embodiments, VO is of uniform size and / or about 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, or 550 μm diameter and / or; The average diameter size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or; The median size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or;VO or 3D cell cultures contain approximately 1,000, 1,500, 2,000, 2,500, 2,550, 2,600, 2,650, 2,700, 2,750, 2,800, 2,850, 2,900, 2,950, 3,000, 3,050, 3,100, 3,150, 3,200, 3,250, 3,300, 3,350, 3,400, 3,450, 3,500, 4,000, 4,500, or 5,000 cells.;

[0135] In some embodiments, the cells were self-assembled into a network of CD31+ vascular structures containing mature α-SMA+ perivascular wall cells. In some embodiments, VO comprises a network of strengthened blood vessels having apical-basal polarization and / or, wherein VO comprises arteries, veins, and / or capillary ECs. In some embodiments, VO CDH5+ and VWF + EC and ACTA2 +, MYH11 +, TAGLN +, and CNN1 + It includes wall cells. In some embodiments, the method includes the formation of arteries, veins, capillaries, arterioles, venules, or any combination thereof.

[0136] Further information regarding the preparation of cell populations, VOs, and 3D cell cultures is known in the relevant art (see, for example, US 2024 / 0287463 A1; US ​​2024 / 0287463 A1; US ​​2020 / 0182861 A1; US ​​2019 / 0376044 A1; US ​​2020 / 0199541 A1; US ​​2023 / 0287357 A1; US ​​2023 / 0174949 A1; US ​​2023 / 0364267 A1; US ​​Patent No. 11,214,768; WO 2024 / 133285; WO 2022 / 226337; and WO 2023 / 196683), the full text of each of which is incorporated herein by reference.

[0137] Cell composition and vascular organoids (VO)

[0138] In some embodiments, particularly, populations of cells comprising iPSC-derived wall cell precursors (iMPCs) prepared using any of the methods described herein are described herein. In some embodiments, particularly, populations of cells (e.g., iPSCs, MePCs, and iMPCs) comprising nucleic acids encoding NKX3.1 (e.g., UniprotKB Q99801-1, UniprotKB Q99801-2, UniprotKB Q99801-3, UniprotKB Q99801-4, UniprotKB Q99801-5, PDB NP_006158.2, and PDB NP_001243268.1) are described herein. In some embodiments, the nucleic acid is an exogenous DNA molecule or an exogenous RNA molecule. Non-limiting examples of useful RNA are modified mRNA (modRNA; e.g., US20230364267A1), mRNA, or functional variants thereof. Non-limiting examples of useful DNA may be vectors, plasmids, transposons, or their functional variants.

[0139] In addition, a population of cells comprising at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% iPSC-derived wall cell precursors (iMPCs) is described herein. In some embodiments, the iMPCs comprise a nucleic acid encoding NKX3.1 (optional, exogenous nucleic acid) or transiently express NKX3.1 (optional, from a degradable nucleic acid (e.g., mRNA or modRNA)). In some embodiments, the iMPCs express PDGFRβ (CD140b) and aminopeptidase N (CD13). In some embodiments, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1% of the iMPCs express the TRA1-81 antigen. In some embodiments, the iMPCs ACTA2 , CNN1 , TAGLN , MYOCD , MYH11 , CSPG4 , DES , PDE5A , and THY1 It expresses.

[0140] Also described herein is a population of cells generated by co-culturing iMPCs with a population of endothelial cells (ECs or iECs) for a period sufficient to generate a population of wall cells, including perivascular cells, smooth muscle cells, and fibroblasts. In some embodiments, the population of cells comprises a population of wall cells and ECs from iMPCs (optionally, in a ratio of about 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:1 iMPC:EC).

[0141] In some embodiments, the EC comprises any one or more of iPSC-derived ECs (iECs), human venous endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs, or organ-specific endothelial cells.

[0142] In some embodiments, organ-specific endothelial cells are from organs selected from the following: heart, muscle, kidney, testis, ovary, lymphatic system, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine.

[0143] In some embodiments, the method further comprises the step of transfecting a population of iPSCs with a nucleic acid encoding ETV2 or a functional variant thereof before mixing with a population of iMPCs to thereby generate a population of ECs. In some embodiments, a population of cells comprising endothelial cells (ECs):iPSC-derived wall cell precursors (iMPCs) in a ratio of about 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:1 is described herein. In some embodiments, the iMPCs contain a nucleic acid encoding NKX3.1 (optional, exogenous nucleic acid) or transiently express NKX3.1 (optional, from a degradable nucleic acid (e.g., mRNA or modRNA)). In some embodiments, the iMPCs express PDGFRβ (CD140b) and aminopeptidase N (CD13). In some embodiments, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1% of the iMPCs express the TRA1-81 antigen. In some embodiments, the iMPCs ACTA2 , CNN1 , TAGLN , MYOCD , MYH11 , CSPG4 , DES , PDE5A , and THY1It expresses. In some embodiments, the EC comprises any one or more of iPSC-derived ECs (iECs), human vein endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs, or organ-specific endothelial cells. In some embodiments, the organ-specific endothelial cells are from an organ selected from the following: heart, muscle, kidney, testis, ovary, lymphatic system, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine. In some embodiments, the iEC contains a nucleic acid encoding ETV2 (optional, exogenous nucleic acid) or transiently expresses ETV2 (e.g., from a degradable nucleic acid (e.g., mRNA or modRNA)). In some embodiments, NKX3.1 expression is controlled by an inducible promoter and ETV2 expression is controlled by an inducible promoter. In some embodiments, the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are the same (optional, induced by doxycycline). In some embodiments, the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are not the same.

[0144] In some embodiments, the method described herein is E26 transform-specific variant 2 ( ETV2 This includes the use of nucleic acids encoding ) or their functional variants. In some embodiments, the nucleic acid is an exogenous DNA molecule or an exogenous RNA molecule. Non-limiting examples of useful RNA are modified mRNA (modRNA; e.g., US20230364267A1), mRNA, or its functional variants. Non-limiting examples of useful DNA may be a vector, plasmid, transposon, or its functional variants.

[0145] Also described herein are 3D vascular organoids (VO), populations of VO, and 3-dimensional (3D) cell cultures comprising any one or more of the cell populations described herein (e.g., populations of iMPCs containing nucleic acid encoding NKX3.1; iMPCs co-cultured with a population of endothelial cells (EC or iEC); populations of wall cells). iMPCs mediate the formation of functional blood vessels when transplanted together with endothelial cells (EC).

[0146] Also, VO and 3D cell cultures prepared by any one of the methods described herein are described herein. In some embodiments, the VO or 3D cell culture comprises iECs that are h-CD31+ and iMPCs that are PDGFRβ+ and h-CD31-.

[0147] In some embodiments, VO is of uniform size and / or about 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, or 550 μm diameter and / or; Here, the average diameter size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or; Here, the median size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or;Here, the VO or 3D cell culture contains approximately 2,500, 2,550, 2,600, 2,650, 2,700, 2,750, 2,800, 2,850, 2,900, 2,950, 3,000, 3,050, 3,100, 3,150, 3,200, 3,250, 3,300, 3,350, 3,400, 3,450, or 3,500 cells.

[0148] In some embodiments, cells were self-assembled into a network of CD31+ vascular structures containing mature α-SMA+ perivascular wall cells. In some embodiments, the VO and 3D cell culture comprises a network of strengthened blood vessels having apical-basal polarization and / or, wherein VO comprises arteries, veins, and / or capillary ECs. In some embodiments, the VO and 3D cell culture CDH5+ and VWF + EC and ACTA2 +, MYH11 +, TAGLN +, and CNN1 + includes wall cells.

[0149] In some embodiments, the VO and 3D cell cultures comprise CD31+ vascular structures containing mature α-SMA+ perivascular wall cells. In some embodiments, the VO or 3D cell culture comprises a network of strengthened blood vessels having apical-basal polarization and / or arteries, veins, and / or capillary ECs.

[0150] In addition, compositions comprising any of the cell populations and / or VOs described herein are described herein, in particular. In some embodiments, the composition may further comprise one or more of an agent, an excipient, a matrix, or a gel. In some embodiments, any composition may further comprise a gel or matrix comprising a hydrogel. In some embodiments, any composition may further comprise a gel or matrix comprising gelatin, collagen, fibrinogen, thrombin, fibrin, or any combination thereof. In some embodiments, any composition may further comprise a gel or matrix comprising about 1.5 mg / mL collagen, about 30 μg / mL fibrinogen, and about 1 mg / mL human fibronectin. In some embodiments, the matrix may contain collagen and / or fibrin. In some embodiments, fibrin is formed from fibrinogen and thrombin (optional, about 50 μg / mL thrombin). In some embodiments, any composition may further comprise a gel or matrix comprising any one or more of gelatin, collagen, fibrinogen, laminin, entactin, or combinations thereof. In some embodiments, any composition may further comprise a gel or matrix comprising laminin, entactin, and collagen. In some embodiments, any composition may further comprise a gel or matrix comprising about 5.25 mg / mL laminin, about 5.25 mg / mL entactin, and about 0.2 mg / mL collagen IV. In some embodiments, any composition may further comprise a gel, or the matrix is ​​Matrigel™. Matrigel is known in the art (U.S. Patent No. 4,829,000).

[0151] In some embodiments, any of the cell populations, vascular organoids, or compositions may be formulated for administration to a subject (e.g., subcutaneous, intradermal, intramuscular, intralymphatic, intravenous, prostatic, intratumoral, intralymphatic, and intraperitoneal injection). In some embodiments, any of the cell populations, vascular organoids, or compositions may be used to vascularize tissues or organs prior to transplantation into a patient, and thus, the compositions described herein may also include cells and tissues from any one or more of the following: skin, heart, kidney, testis, ovary, bone, lymph, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine.

[0152] Additional information and methods for formulating populations of cells, vascular organoids, or compositions are known in the relevant art (see, for example, US 2024 / 0287463 A1; US ​​2024 / 0287463 A1; US ​​2020 / 0182861 A1; US ​​2019 / 0376044 A1; US ​​2020 / 0199541 A1; US ​​2023 / 0287357 A1; US ​​2023 / 0174949 A1; US ​​2023 / 0364267 A1; US ​​Patent No. 11,214,768; WO 2024 / 133285; WO 2022 / 226337; and WO 2023 / 196683), the full text of each of which is incorporated herein by reference.

[0153] How to use cells and VO

[0154] In particular, a method of administering to a subject a population of cells (e.g., iMPC, iMPC, and EC, including mature wall cell derivatives) and / or any of the VOs described herein is described herein. Useful populations of cells and VOs are described throughout, for example, in the above section.

[0155] In some embodiments, the subject has a disease or disorder associated with vascular disorders (e.g., leaking vessel, narrow vessel, coronary artery disease, peripheral artery disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac rhythm disorders and heart valve disease, cerebral cavernous malformation, hemorrhagic stroke, hereditary hemorrhagic telangiectasia, or arteriovenous malformation), or is at risk of having or developing such a disease or disorder. In some embodiments of any of the methods described herein, the subject has (or has or is at risk of having) any one or more of the following: metabolic disorder, diabetes mellitus, diabetic retinopathy, ischemic injury, vascular disease or disorder, atherosclerosis, age-related macular degeneration (AMD), pulmonary arterial hypertension (PAH), hereditary hemorrhagic telangiectasia (HHT), peripheral arterial disease (PAD), arteriovenous fistula (e.g., in dialysis patients), tumor angiogenesis, tumor metastasis, cancer, metabolic disease, immunological disease, mitochondrial dysfunction, stroke, and / or wound (optional, chronic wound; e.g., diabetic ulcer).

[0156] In some embodiments, a method for treating any one or more of the following, or reducing the risk of causing them, is described herein: vascular disease or disorder, abnormal vascular structure, leaking vessel, narrowed vessel, coronary artery disease, peripheral artery disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiotoxicity (e.g., caused by chemotherapy), cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac rhythm disorder, heart valve disease, cerebral cavernous malformation, hemorrhagic stroke, hereditary hemorrhagic telangiectasia, arteriovenous malformation, metabolic disorder, diabetes mellitus, diabetic retinopathy, ischemic injury, IRI injury, atherosclerosis, age-related macular degeneration (AMD), pulmonary hypertension (PAH), hereditary hemorrhagic telangiectasia (HHT), peripheral artery disease (PAD), arteriovenous fistula (e.g., in dialysis patients), tumor angiogenesis, tumor metastasis, cancer, metabolic disease, stroke, and / or wound (optional, chronic wound; e.g., diabetic ulcer).

[0157] Also described herein are a method for increasing angiogenesis in a subject, a method for vascular cell therapy, and / or a method for angiogenesis or regeneration, comprising the step of administering a therapeutically effective amount of a group of ECs and iMPCs to a subject requiring increased angiogenesis, vascular cell therapy, and / or angiogenesis or regeneration. In some embodiments, the method further comprises the step of identifying a subject requiring increased angiogenesis, vascular cell therapy, angiogenesis, and / or angiogenesis.

[0158] In particular, methods for increasing vascular development, angiogenesis, and cell junctions through the step of administering iMPCs to a subject are described herein. In some embodiments, methods for administering iMPCs and ECs (e.g., therapeutic vascularization) are described herein, and methods for modeling vascular disease (e.g., 3D vascular organoids (VO)) are also described herein. Additionally, methods for tissue manipulation (e.g., small-diameter vascular grafts) are described herein. Tissue-manipulated small-diameter vascular grafts are biomanipulated structures designed to replace damaged or diseased blood vessels. These grafts are typically generated from scaffolds seeded with cells, such as smooth muscle cells (SMCs), which provide structural support and functionality. The cell populations described herein (e.g., NKX3.1-derived iMPCs and their mature wall cell derivatives) may be incorporated into these scaffolds as SMCs, which facilitates the generation of grafts that can be surgically implanted into a patient as vascular replacements. Small-diameter vascular grafts are particularly relevant for clinical applications, such as coronary artery bypass grafts (CABG), peripheral artery disease (PAD), and arteriovenous fistulas for dialysis patients.

[0159] In some embodiments, any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein (e.g., compositions comprising NKX3.1-derived iMPCs and / or their mature wall cell derivatives) are used as cell sources for tissue manipulation applications, particularly in the development of small-diameter vascular grafts. For example, the iMPC-derived cell population described herein may function as smooth muscle cells (SMCs) within the graft scaffold of a small-diameter vascular graft and may be surgically implanted into subjects requiring this (e.g., subjects requiring vascular replacement, subjects receiving or at risk of receiving coronary artery bypass graft (CABG), subjects having or at risk of having peripheral artery disease (PAD), and subjects having or at risk of having an arteriovenous fistula (e.g., dialysis patients)). Unbound by theory, the incorporation of iMPC cells into structural scaffolds enables the creation of functional, living vascular grafts that can be transplanted to repair or replace damaged blood vessels, offering significant potential for clinical scenarios with high demand.

[0160] In some embodiments, a method for transplanting any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein is described herein. In some embodiments, the method comprises the step of administering an effective amount of the cell population, vascular organoid, or composition to a subject.

[0161] In some embodiments, a method for increasing angiogenesis is described herein, comprising the step of administering to a subject requiring increased angiogenesis an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein. In some embodiments, the method comprises: identifying a subject requiring increased angiogenesis; and administering to the subject an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein.

[0162] Also, a method for increasing angiogenesis or vascular regeneration is described herein, comprising the step of administering to a subject in need of increased angiogenesis or vascular regeneration an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein. In some embodiments, the method comprises: identifying a subject in need of increased angiogenesis or vascular regeneration; and administering to the subject an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein.

[0163] In addition, a method for vascular cell therapy is described herein, comprising the step of administering to a subject requiring vascular cell therapy an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein. In some embodiments, the method comprises: identifying a subject requiring vascular cell therapy; and administering to the subject an effective amount of any of the cell populations described herein, any of the VO or 3D cell cultures described herein, and / or any of the compositions described herein.

[0164] In some embodiments of any of the methods described herein, the subject or patient may be receiving an organ transplant, be selected to receive an organ transplant, or require vascularization of an organ. In some embodiments, the organ is selected from the group consisting of skin, heart, kidney, testis, ovary, bone, lymph, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine. In some embodiments of any of the methods described herein, a population of cells, a vascular organoid, or a composition is administered to the subject before, during, or after cell transplantation, tissue transplantation, or organ transplantation.

[0165] In some embodiments of any of the methods described herein, vascularization includes the formation of arteries, veins, capillaries, arterioles, venules, or any combination thereof.

[0166] In some embodiments of any of the methods described herein, the method normalizes and / or corrects an abnormal vascular structure; for example, wherein the vascular structure lacks or has a lack of parietal cells; and / or the vascular structure lacks a stable structure and / or function; and there is leakage or narrowing of the blood vessel, etc. In some embodiments of any of the methods described herein, the subject has a disorder characterized by an abnormal vascular structure and / or a vascular structure lacking or having a lack of parietal cells (e.g., diabetic retinopathy, tumor angiogenesis, tumor metastasis, stroke, ischemic injury, atherosclerosis, age-related macular degeneration (AMD), pulmonary hypertension (PAH), hereditary hemorrhagic telangiectasia (HHT), peripheral artery disease (PAD), arteriovenous fistula (e.g., dialysis patients), and / or wound (e.g., chronic wound; e.g., diabetic ulcer)).

[0167] Without being bound by theory, narrowing of blood vessels results from plaque accumulation and / or chronic inflammation on the walls of the vessels, which may include conditions such as ischemic disease, peripheral artery disease, angina pectoris, heart attack, stroke, Raynaud's disease, Buerger's disease, hypertension, chemotherapy injury, and erectile dysfunction. Furthermore, each of these conditions and vascular diseases frequently results in distal vascular damage and / or dysfunction, which consequently complicates revascularization strategies and recovery from ischemic damage, and in many cases, exacerbates them. The iMPCs, cell populations, VOs, and / or compositions described herein may potentially restore the integrity of vascular structures and / or help stabilize these vessels, or reduce the severity of any damage caused by these conditions, or prevent additional complications from occurring as a result of the condition(s).

[0168] Without being bound by theory, tumors typically exhibit abnormal vascular structures characterized by a lack of adequate wall cell coverage, which leads to leakage and dysfunction of blood vessels. Introducing any of the iMPC, cell population, VO, and compositions of the present invention may help stabilize these blood vessels, improve the delivery of therapeutic agents, and / or reduce metastasis.

[0169] Without being bound by theory, in diabetic retinopathy, loss of perivascular cells leads to a weakened blood-retinal barrier, which results in retinal ischemia and neovascularization. The iMPCs, cell populations, VO, and / or compositions described herein may potentially restore the integrity of retinal vascular structures and / or reduce the progression of the disease.

[0170] Without being bound by theory, after a stroke or ischemic injury, there is usually a loss of vascular integrity and a need for vascular repair. The iMPCs, cell populations, VO, and / or compositions described herein can help re-establish stable blood vessels and / or promote the recovery of diseased tissues.

[0171] Without being bound by theory, in atherosclerosis, vascular stability is impaired due to inflammatory processes and endothelial dysfunction. Parietal cells derived from iMPCs, iMPCs, cell populations, VO, and / or compositions described herein may help strengthen blood vessel walls and / or alleviate the progression of atherosclerotic plaques.

[0172] Without being bound by theory, in wound healing, chronic wounds, such as diabetic ulcers, usually suffer from poor vascularization and lack of wall cell coverage. The iMPCs, cell populations, VO, and / or compositions described herein can promote angiogenesis and vascular stability, thereby facilitating better wound healing outcomes.

[0173] Without being bound by theory, in age-related macular degeneration (AMD), particularly in the wet form, choroidal neovascularization occurs with a lack of perivascular cell support, which leads to fragile and leaking vessels. The iMPCs, cell populations, VO, and / or compositions described herein may help provide the necessary support to these new vessels, reduce leakage, and / or reduce vision loss.

[0174] Without being bound by theory, pulmonary arterial hypertension (PAH) is characterized by abnormal proliferation of pulmonary vascular cells and deficient perivascular cell coverage, which leads to vascular remodeling and hypertension. The iMPCs, cell populations, VO, and / or compositions described herein may stabilize these blood vessels and / or alleviate one or more symptoms (e.g., hypertension).

[0175] Without being bound by theory, hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder that causes abnormal blood vessel formation with deficient wall cell coverage, resulting in hemorrhage and arteriovenous malformations. The iMPCs, cell populations, VO, and / or compositions described herein may potentially normalize these vessels and / or reduce hemorrhagic episodes.

[0176] In some embodiments of any of the methods described herein, a population of cells, vascular organoids, or compositions are administered to a subject by direct injection into a blood vessel or by subcutaneous, intradermal, intramuscular, intralymphatic, intravenous, intraprostatic, intratumoral, intralymphatic, and intraperitoneal injection.

[0177] An effective dose may be administered in one or more doses, applications, or dosages. A person skilled in the art will recognize that certain factors, including but not limited to the severity of the subject's disease or disorder, prior treatment, overall health and / or age, and other existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of the subject with any of the cell therapeutic populations, VO, 3D cell cultures, or compositions described herein with a therapeutic effective dose may include a single treatment or a series of treatments.

[0178] A person skilled in the art will be able to determine and identify a subject or patient suitable for any of the methods described herein (e.g., a subject who has or is at risk of having a stroke, a subject who has or is at risk of having diabetes, a subject who has or is at risk of having diabetic retinopathy, a subject who has or is at risk of having ischemic injury, a subject who has or is at risk of having vascular disease or disorder, and / or a subject who is or is selected to receive a transplant).

[0179] Further information regarding methods for formulating and administering cell populations, VOs, and 3D cell cultures is known in the relevant art (see, for example, US 2024 / 0287463 A1; US ​​2024 / 0287463 A1; US ​​2020 / 0182861 A1; US ​​2019 / 0376044 A1; US ​​2020 / 0199541 A1; US ​​2023 / 0287357 A1; US ​​2023 / 0174949 A1; US ​​2023 / 0364267 A1; US ​​Patent No. 11,214,768; WO 2024 / 133285; WO 2022 / 226337; and WO 2023 / 196683; the full text of each of these is incorporated herein by reference).

[0180] Examples

[0181] The present invention is further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0182] method

[0183] Generation of doxycycline-induced NKX3.1 iPSC cell lines

[0184] Doxycycline-induced NKX3.1 (dox-NKX3.1) cell lines were generated using the Piggyback (PB) transposon and transposase system. A PB transposon vector containing the NKX3.1 ORF was constructed using the Gateway cloning system. The PB super transposase (SBI, PB210PA-1) was purchased from SBI System Biosciences. The PB dox-NKX3.1 transposon and transposase vector were transfected via electroporation into three independent human induced pluripotent stem cell (hiPSC) cell lines generated as previously reported at a 5:1 ratio. 1 microgram of supertransposer and 5 μg of PB transposon were used to transfect 2 million cells using a Neon electroporation system according to the vendor's guidelines (Invitrogen, MPK10096). The electroporation parameters were set to 1150 V for the pulse voltage and 30 ms for the pulse width; two pulses were introduced. For 2 million cells, the inventors used 3 mL of electrolyte buffer and 100 μL of resuspension buffer R in a 100-μL reaction tip. Electroporated cells were seeded onto Matrigel-coated dishes in mTESR Plus medium (STEMCELL Technologies, 100-0276) containing 5 μM Y27632 (Selleckchem, S1049). Positive cells were subsequently selected by adding 0.5 μg / mL of puromycin (InvivoGen, ant-pr-1). iPSC clones were collected by manual selection. The reactivity and pluripotency of clone 9 to doxycycline were verified by qPCR and immunohistochemistry and used in further experiments of this application.

[0185] Differentiation of h-iPSCs into wall cell precursors (S1-NKX3.1)

[0186] To generate wall cell precursors using Dox-NKX3.1-iPSC or modified NKX3.1 RNA, the inventors followed two-stage differentiation over a four-day period. On the first day, the inventors seeded Dox-NKX3.1 or BJ-273 iPSCs on a Matrigel-coated 6-well plate (Corning, cat# 354277) in 5 μM Y27632 in mTESR Plus medium at a seeding density of 200,000 cells / well. On the next day, the medium was exchanged for S1 medium (Sigma-Aldrich, SML1046-25MG) containing 6 μM CHIR99021, formulated with 1X glutamax (Thermo Fisher Scientific, cat# 12634028) and 60 μg / mL ascorbic acid in high-grade DMEM / F-12 (Thermo Fisher Scientific, cat# 12634028). Cells were then continued to be cultured in fresh S1 medium, with fresh CHIR99021 added daily. On the third day, cells were treated with 5 μg / mL doxycycline (Sigma-Aldrich, D9891-10G) in S1 medium and treated with fresh doxycycline by exchanging the medium daily for an additional two days. For modified NKX3.1, on the third day of differentiation, cells were dissociated by TrypLE (Thermo Fisher Scientific, cat# 12563029), and 5 μg of RNA was transfected into 2 million cells by neon electroporation with the same parameter settings as described above. Finally, the electroporated cells were plated on Matrigel-coated 6-well plates in S1 medium. After 4 days of differentiation, iMPCs were maintained in SmGM-2 medium (Lonza, CC-3182) on 1% gelatin-coated plates. iMPCs were subcultured twice a week until passage 2 (P2), after which the frequency was reduced to once a week. The splitting ratio ranged from 1:2 to 1:4 depending on the degree of fusion.

[0187] modRNA synthesis and formulation

[0188] [modRNA(NKX3.1)] chemically encoding NKX3.1 was generated by TriLink BioTechnologies LLC. Briefly, modRNA(NKX3.1) was synthesized in vitro by T7 RNA polymerase-mediated transcription from a linearized DNA template containing 5' and 3' untranslated regions (UTRs) and a poly-A tail. Specifically, NKX3.1 (ORF: ; SEQ ID NO: 39; 705 bp) was cloned into the mRNA expression vector pmRNA containing the T7 RNA polymerase promoter, an unstructured synthetic 5'UTR, multiple cloning sites, and a 3'UTR derived from the mouse <-globin 3' gene. An in vitro transcription reaction (1-ml scale) was performed to generate unmodified mRNA transcripts with wild-type bases and a poly-A tail. Co-transcriptional capping with the CleanCap Cap1 AG trimer generates the naturally occurring Cap1 structure. Deoxyribonuclease treatment was used to remove the DNA template. 5'-triphosphate was removed by phosphatase treatment to reduce the innate immune response. After elution through a silica membrane, the purified RNA was dissolved in ribonuclease-free sodium citrate buffer (1 mM, pH 6.4).

[0189] Isolation and maintenance of human MSCs, ECs, and VSMCs.

[0190] Human mesenchymal stem cells (MSCs) were isolated from bone marrow as previously described in the literature [Lin, R.-Z., Moreno-Luna, R., Li, D., Jaminet, S.-C., Greene, AK, and Melero-Martin, JM (2014). Human endothelial colony-forming cells serve as trophic mediators for mesenchymal stem cell engraftment via paracrine signaling. Proc. Natl. Acad. Sci. 111, 10137-10142] and maintained in MSCGM (Lonza, PT-3001) on 1% gelatin-coated plates. Human primary vascular smooth muscle cells (SMCs) from pulmonary artery tissue were obtained from Lonza (Lonza, CC-2581). SMCs were cultured in SmGM-2 medium (Lonza, CC-3182) on 1% gelatin-coated plates (Sigma-Aldrich, G2500-500G). Endothelial colony-forming cells (ECFC; referred to herein as EC) were isolated from human umbilical cord blood and cultured in ECGM2 (Lonza, CC-3162) supplemented with 20% FBS (Genesee, 25-514) without hydrocortisone. All primary cells were used up to passage 10.

[0191] Flow cytometry analysis

[0192] Cells were dissociated into a single-cell suspension using TrypLE (Thermo Fisher Scientific, cat#12563029) and subsequently washed with FACS buffer formulated in PBS supplemented with 1% bovine serum albumin and 0.2 mM EDTA. For specific experiments, flow cytometry analysis was performed after fixing cells with 4% paraformaldehyde (PFA, Electron Microscope Sciences, cat#15714-S). The staining procedure involved incubating cells with their respective antibodies on ice for 15 minutes. After incubation, cells were washed three times with PBS buffer to remove any unbound antibodies. Flow cytometry analysis was performed using a BD Accuri C6 Plus flow cytometer (BD Biosciences), and the acquired data were analyzed using FlowJo software (Tree Star Inc., Ashland, Oregon). Detailed information regarding the antibodies used in the staining procedure can be found in Table 2.

[0193] Immunofluorescence staining

[0194] The cell is 2x10 4 Number of cells / cm² 2The cells were seeded at a seeding density on tissue culture-treated polymer coverslip 8-well chamber slides (ibidi USA, Fisher Scientific, cat# 50-305-795) or 8-well chamber slides (ibidi USA, Fisher Scientific, NC1535706). On the following day, the cells were fixed with 4% paraformaldehyde (PFA) and permeated with 100% cold methanol at -20°C or 0.2% Triton in PBS for 15 minutes. After blocking with 10% BSA at RT for 30 minutes, the primary antibody was added and the cells were incubated at room temperature for 1 hour or overnight at 4°C. After washing three times with PBS, the cells were incubated with the secondary antibody and DAPI at room temperature for 30 minutes. Slides containing stained cells were fixed using DAKO fluorescent fixation medium (Agilent, S302380-2) or imaged directly without fixation. Images were acquired using an Axio Observer Z1 inverted microscope (Carl Zeiss) and AxioVision Rel. 4.8 software. For phase-contrast imaging, the inventors used an AxioCam MRc5 camera equipped with a 5X or 10X objective lens. Verification of pluripotency was performed using OCT4, NANOG, and SOX2. Detailed information regarding the antibodies used in the staining procedure can be found in Table 2.

[0195] Intracellular Ca 2+ Flow detection verification

[0196] Intracellular calcium flow was measured and visualized by the Fluo-4 Calcium Imaging Kit (Life Technologies, F10489), and the inventors followed the manual provided by the vendor. pVSMC or Day 4 iMPC were seeded into 8-well chamber slides containing SMGM-2 medium at a cell density of 2000 cells per well. On the following day, the cells were washed with Live Cell Imaging Solution (LCIS) buffer (Thermo Fisher, A14291DJ). The Fluo-4 AM loading solution was prepared by adding 20 mM glucose to Live Cell Imaging Solution (LCIS) buffer containing probenecid, power load, and fluo-4. 200 μL of the loading solution was subsequently added to the cells and incubated at 37°C for 30 minutes, followed by incubation at room temperature for 15 minutes. After washing the cells, the inventors replaced the loading solution with 20 mM glucose-LCIS buffer containing 10 mM carvacol (Millipore Sigma, PHR1511), 0.1 μM endothelin 1 (Millipore Sigma, E7764-10UG), or PBS, and immediately performed imaging. Fluorescence images were obtained using an Axio Observer Z1 inverted microscope (ZEISS) at 5-second intervals for 5 minutes, and the relative fluorescence levels of individual cells were analyzed as average intensity by ImageJ and normalized by (F-F0) / F0.

[0197] Collagen gel shrinkage test

[0198] Prior to the experiment, cells were serum-depleted overnight in basal smooth muscle cell medium (SMCM, ScienceCell, 1101) containing 0.1% FBS in an incubator. The following day, 3 mL of cold collagen solution was prepared by mixing 1.8 mL of 1x DMEM, 0.3 mL of FBS, and 0.75 mL of bovine collagen-1 in a 50 mL Falcon tube. The solution was kept on ice to prevent coagulation while adjusting the pH to 7.4 using 0.1N NaOH. The cell suspension 10 6 The cells were prepared in a collagen solution of 10 cells / ml, and 10 μL of the cell-collagen suspension was plated in triples on angiogenesis μ-slides (EBD, 81506). After plating the collagen-cell suspension, the plates were incubated at 37°C for 30 minutes. Once solidified, 40 μL of SMGM2 medium was added to the top of the gel along with 10 μM U46619 vasoconstrictor. Images were taken after 72 hours of incubation using an Axio Observer Z1 inverted microscope (ZEISS) with a 4X objective and ZEN 3.6 (Blue Edition) software. The surface area of ​​the collagen cells was quantified using ImageJ. The percentage of shrinkage was then calculated by comparing the final area with the initial area on Day 0 using the following formula: Percentage of original gel area = (Final area / Initial area) x 100.

[0199] Fibronectin deposition test

[0200] Total 1x10 4Canine induced wall cell precursors (iMPCs) were seeded in smooth muscle growth medium-2 (SMGM-2) onto 8-well chamber slides (EBD USA, Fisher Scientific, cat# 50-305-795) or 24-well plates. On the following day, the SMGM-2 medium was replaced with fresh SMGM-2 containing 0, 2.5, 10, or 100 ng / mL of transforming growth factor-beta (TGFβ) (Prospec, CYT-716), with or without the TGFβ inhibitor, SB431542 (Santa Cruz Biotech, sc-204265). The cells were then treated for 72 hours. To evaluate fibronectin expression, cells were fixed with 4% PFA and blocked for 30 minutes after being stained with a fibronectin antibody (Abcam, Ab2413) in a blocking solution (1.5% BSA solution) without permeability. Subsequently, after three washes, a goat anti-rabbit-488 secondary antibody was applied to facilitate detection by DAPI. Images were acquired using an Axio Observer Z1 inverted microscope (ZEISS) and ZEN 3.6 (Blue Edition) software, and fibronectin deposition was quantified by measuring green fluorescence intensity and dividing by the number of cells measured by DAPI. Quantification was performed using Image J.

[0201] Wall-endothelial cell co-culture assay

[0202] To perform the endothelial cell co-culture assay, on day 4 of differentiation, induced wall cell progenitors (iMPCs), human mesenchymal stem cells (MSCs), and endothelial colony-forming cells (ECFCs) were collected using 300 μL of TrypLE. A total of 5 x 10⁴ cells were collected. 4Two iMPCs or MSCs and an equal number of ECFCs were seeded onto pre-mixed and 1% gelatin-coated 6-well plates. Co-culture was performed in Endothelial Growth Medium-2 (EGM-2, Lonza, CC-3162) for a duration of 7 days. After the 7-day co-culture period, CD31-positive cells were selectively removed using a magnetic bead-based sorter (Invitrogen, 11155D). CD31-positive endothelial cells (ECs) were separated and discarded using a magnetic separator, while the negative fraction was isolated for further analysis.

[0203] Transwell co-culture assay

[0204] Transwell co-culture experiments were performed using 48-well permeable Transwell plates (Corning, CLS3415) with a pore size of 3 μm. On day 1 prior to co-culture, 1 x 10⁶ 3 Canine endothelial colony-forming cells (ECFCs) were seeded into the bottom wells of the Transwell using K-medium. On the next day, 1 x 10 4 Canine iMPCs were seeded on the top insert in Basal Endothelial Basal Medium-2 (Lonza, EBM-2) supplemented with 5% Fetal Bovine Serum (FBS) on the 4th day of differentiation. After 3 days of co-culture, ECFCs in the bottom wells were fixed and stained with DAPI. Subsequently, four images per well were taken using an Axio Observer Z1 inverted microscope (Carl Zeiss) and AxioVision Rel. 4.8 software, and the images were analyzed using ImageJ software. The analysis included the following functions: threshold at 50, hole filling, water shed, and quantification of stained cells by analyzing particle size larger than 10 pixels.

[0205] Endothelial cell growth assay in conditioned medium

[0206] On the fourth day of differentiation, induced wall cell precursors (iMPCs) were subjected to treatment with 2 mL of basal medium of Endothelial Basal Medium-2 (Lonza, 190860) supplemented with 5% FBS (Genesis, 25-514) on a 6-well tissue culture plate for 24 hours. Subsequently, 12 mL of cultured medium was collected and filtered through a 0.22 μm filter (VWR, 76479-016). The filtered medium was then concentrated using a 3 kDa cut-off centrifuge (Millipore, UFC900324) at 10,000 rpm for 45 minutes. To reconstitute the 2-fold concentrated sample, up to 6 mL of fresh EBM-2 medium was added. For endothelial cell (EC) growth tests, 96-well plates were used, 1000 cells per well were seeded in K-medium, and formulated using Endothelial Cell Growth Medium-2 (ECGM2, Lonza, CC-3162) supplemented with 20% FBS (Genesis, 25-514) without hydrocortisone. The following day, the medium was exchanged for the basal medium or 2X conditioning medium obtained as described above for further experiments.

[0207] Tube formation test

[0208] Human ECFCs on Matrigel were used to perform a tube formation assay. Matrigel (Corning, 354277) was thawed overnight at 4°C, and 200 µL was dispensed into each well of a 24-well plate. After incubation at 37°C for 30 minutes, ECFCs dissociated into TrypLE (Thermo Fisher, 12563029), yielding 2 x 10⁶ per mL. 5 The cells were resuspended in a container, and then 50 µL of the cell suspension was dispensed onto the top of the solidified Matrigel. Basal medium (control) or conditioning medium containing 0.5% FBS was used for culture. After 24 hours of incubation, images were acquired at a phase contrast of 5X and analyzed using the Angiogenesis Analyzer plugin of ImageJ software.

[0209] Wound healing test

[0210] 5x10 4 ECFCs were seeded into each well of a 24-well plate and cultured to 100 percent fusion. Using a 1000P tip, the inventors scratched a row from the center of the well and exchanged the medium with the basal medium of ECM-2 containing 5% FBS or the conditioned medium collected as described in the EC growth assay. After 24 hours of incubation, the scratched area was imaged by 5X phase contrast and the area was analyzed using ImageJ.

[0211] Angiogenesis Array

[0212] The Proteome Profiler kit (R&D Systems, ARY007) was used to analyze the expression profiles of 55 angiogenesis-related proteins. 2-fold concentrated conditioning medium was prepared as described above and according to the instructions in the manufacturer's manual. Briefly, 2 mL of blocking buffer (Array Buffer 7) was added to the membrane and incubated on a vibrating platform shaker for 1 hour. During blocking, 1 mL of each sample was mixed with 0.5 mL of dilution buffer (Array Buffer 4) and 15 µL of detection antibody cocktail and subsequently incubated for 1 hour. After the blocking buffer was removed, the sample-antibody mixture was dispensed and incubated overnight at 4°C on a vibrating shaker. The membrane was washed three times with 20 mL for 10 minutes each. Prior to the addition of streptavidin-HRP, the 4-well multi-plate was washed with distilled water and thoroughly dried. 2 mL of diluted streptavidin-HRP in Array Buffer 5 was added to the membrane of the 4-well plate and incubated on a vibrating shaker for 30 minutes. Subsequently, after three washes, 1 mL of chemical reagent was evenly distributed onto the membrane. Finally, the inventors covered the membrane with a top sheet of plastic protector to remove air bubbles and incubated for 1 minute prior to imaging. For image analysis, array spots were segmented using Cellpose. Expression levels were determined by calculating the product of spot size and intensity. Normalization of values ​​for each experiment was performed by dividing them by the negative control value.

[0213] Angiogenesis Factor Multiplexing Analysis

[0214] The protein concentrations of 17 angiogenesis factors were analyzed using the standard curve of the Human Angiogenesis 17-Flex Discovery Test. The Human Angiogenesis 17-Flex Discovery Test was performed by Eve Technologies (Canada). Luminex xMAP technology was used for multiplexed quantification. Multiplexed analysis was performed by Eve Technologies Corp. (Calgary, Alberta) using the Luminex™ 200 system (Luminex, Austin, Texas, USA). 17 markers were simultaneously measured in samples using Eve Technologies’ Human Angiogenesis & Growth Factor 17-Flex Discovery Test® (MilliporeSigma, Burlington, Massachusetts, USA) according to the manufacturer's protocol. The 17-plex consisted of angiopoietin-2, BMP-9, EGF, endoglin, endothelin-1, FGF-1, FGF-2, polystatin, G-CSF, HB-EGF, HGF, IL-8, leptin, PLGF, VEGF-A, VEGF-C, and VEGF-D. The assay sensitivities of these markers for the 17-plex range from 0.2 to 42.8 pg / mL. Individual analyte sensitivities are available in the MilliporeSigma Milliplex® MAP protocol.

[0215] Quantitative reverse transcription PCR

[0216] RNA was isolated using the RNeasy kit (Qiagen, cat# 74106) or the SYBR™ Green Cells-to-CT™ kit (Thermo Fisher Scientific, cat# 4402954). cDNA was prepared using Reverse Transcription III (Thermo Fisher Scientific, cat# 4368814) or the SYBR™ Green Cells-to-CT™ kit according to the manufacturer's instructions. Quantitative PCR was performed using the SYBR Green Master Mix (Thermo Fisher Scientific, A25776), and detection was achieved using a QuantStudio™ 3 Real-time PCR System, 96-well (Thermo Fisher Scientific, cat# A28567). Expression of the target gene was glyceraldehyde-3-phosphate dehydrogenase ( GAPDH It was normalized for ). Real-time PCR primer sequences are listed in Table 1.

[0217] Statistical Analysis: Unless otherwise specified, data were expressed as mean ± mean SEM. For comparisons between two groups, means were compared using an independent two-sided Student's t-test. Comparisons between multiple groups were performed by analysis of variance (ANOVA) followed by Bonferroni post-hoc analysis. All statistical analyses were performed using GraphPad Prism v.9 software (GraphPad Software Inc.).

[0218] In vitro vascular network formation 'on-a-chip' assay

[0219] The inventors used the idenTx 3 chip and holder from AIM Biotech Pte. Ltd. in accordance with a slightly modified version of the manufacturer's guidelines. ECFC and iMPC were combined in a 3:1 ratio, with 1.2 x 10⁻¹⁴ in 10 μL. 5Cells were prepared and embedded in a hydrogel solution. This hydrogel consisted of 6 mg / mL fibrinogen (Sigma, F8630) in 1x PBS and 50 U / mL thrombin (Sigma, T4648) in 1x PBS at 37°C. The mixture was seeded into the cell / gel channels of the chip. The culture medium was EGM-2 supplemented with 5% FBS and 50 ng / mL VEGF. For cell preparation, cells were suspended in a medium-diluted thrombin solution (4 U / mL) and mixed with the fibrinogen solution to achieve a final concentration of 2 U / mL thrombin and 3 mg / mL fibrinogen. The mixture was then applied to the gel channels of the chip and allowed to polymerize for 30 minutes at 37°C. After polymerization, 15 μL of culture medium was added to both medium channels. To create a flow gradient, the medium volume was adjusted to 70 μL on one side and 50 μL on the other, and the medium was exchanged daily to maintain cell viability. The chip was maintained at 37°C in a 5% CO2 environment.

[0220] animal testing

[0221] All animal experiments were performed at Boston Children's Hospital in accordance with institutional guidelines approved by the Institutional Animal Care and Use Committee (IACUC) Protocol 20-12-4327R. For the assay of in vivo vascular network formation, the inventors purchased 6-week-old athymic nude mice (Foxnl / nu mice) from Envigo and reared them according to the following immunodeficient mouse guidelines.

[0222] In vivo vascular network formation test

[0223] In vivo vascular network formation assay is [Nowak-Sliwinska, P., et al. (2018). Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis 21As previously described in , 425-532. 10.1007 / s10456-018-9613-x], this was performed by co-transplanting human endothelial colony-forming cells (ECFC) or human venous endothelial cells (HUVEC, ATCC, CRL-1730) and wall cells into collagen mixed with fibrinogen gel. Briefly, a collagen / fibrinogen gel solution was prepared on ice by combining 1.5 mg / mL collagen (Trevigen, cat# 3442-050-01), 30 μg / mL fibrinogen (Sigma-Aldrich, F8630-1G), 1 mg / mL human fibronectin (Millipore-Sigma, F0895-2MG) plus 25 mM HEPES and 10% FBS. Two cell types, ECFC or HUVEC (0.8x10⁶ 6 (dogs) and one of the wall cells iMPC, pSMC, bm-MSC (1.2x10⁶ 6 The cells were pre-mixed in 200 μL of pH-neutral gel solution and loaded into a 30G syringe using a 1 mL pipette. Mice were anesthetized with isoflurane, and 50 μL of 50 μg / mL thrombin (Sigma-Aldrich, T4648) was injected subcutaneously, followed by 200 μL of the cell-loaded gel into the same site. The cell-gel grafts were collected after one week to analyze angiogenesis.

[0224] Histology and immunofluorescence staining

[0225] The explanted grafts were fixed overnight in 10% buffered formalin and washed in 70% ethanol. The fixed explanted grafts were embedded in paraffin and incised to a depth of 7 μm. H&E-stained sections were used to evaluate microvascular density. Number of vessels per graft area (vessels / mm²) 2 ) Average number of erythrocyte-filled blood vessels in H&E-stained sections (blood vessels / mm²) 2...was counted as ). For immunostaining, sections were deparaffinized via xylene for 10 minutes, sequentially immersed in ethanol, and subjected to antigen recovery in citric acid buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 95 °C for 30 minutes. Sections were then blocked in 5% BSA for 30 minutes and incubated at RT for 1 hour with primary and secondary antibodies, respectively. Human-specific anti-CD31 antibody was used to stain human blood vessels, and perivascular wall cells were immunostained with anti-aSMA antibody. Anti-GFP or human-specific vimentin antibody was used to track iPSCs. The antibodies are detailed in Table 2.

[0226] Bulk RNA sequencing

[0227] RNA extraction, library preparation, and sequencing were performed at Azenta Life Sciences (South Plainfield, NJ, USA) as follows: Total RNA was extracted from fresh frozen cell pellet samples using the Qiasymphony RNA kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

[0228] Library preparation and Illumina sequencing with poly-A selection

[0229] RNA samples were quantified using a Qubit 2.0 fluorescence spectrometer (Life Technologies, Carlsbad, California, USA), and RNA integrity was checked using an Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, California, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, Ipswitch, Massachusetts, USA) in accordance with the manufacturer's instructions. Briefly, mRNA was initially enriched with oligod(T) beads. The enriched mRNA was fragmented at 94°C for 15 minutes. First-strand and second-strand cDNA were subsequently synthesized. The cDNA fragments were end-recovered at the 3' end and adenylated; after a universal adapter was ligated to the cDNA fragments, an index was added, and the library was enriched by PCR using limited cycles. The sequencing library was validated on an Agilent Tape Station (Agilent Technologies, Palo Alto, California, USA) and quantified using a Qubit 2.0 fluorescence spectrometer (Invitrogen, Carlsbad, California) as well as by quantitative PCR (KAPA Biosystems, Wilmington, Massachusetts, USA). The sequencing library was clustered on two flow cells. After clustering, the flow cells were loaded onto an Illumina Instrument 4000 according to the manufacturer's instructions. The samples were sequenced using a 2x150 bp two-sided (PE) configuration. Image analysis and base extraction were performed by the Control software. The raw sequence data (.bcl files) generated by the sequencer were converted to fastq files and demultiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence verification.

[0230] Analysis of RNA sequencing data

[0231] After examining the quality of the raw data, the sequence reads were trimmed using Trimmomatic v.0.36 to remove possible adapter sequences and nucleotides of poor quality. The trimmed reads were then aligned using STAR aligner v.2.5.2b to the Homo sapiens available in ENSEMBL ( Homo sapiens ) was mapped against the reference genome. The STAR aligner is a splice-aligner that detects splice junctions and incorporates them to help align the entire read sequence. A BAM file was generated as a result of this step. Unique gene hit counts were calculated using feature counts from the Subread package v.1.5.2. Only unique reads belonging to exon regions were counted.

[0232] After the extraction of gene hit factors, the gene hit factor table was used for downstream differential expression analysis. Using DESeq2, comparisons of gene expression between groups of samples were performed. Wald's test was used to generate p-values ​​and Log2-fold changes. Genes with adjusted p-values ​​< 0.05 and absolute Log2-fold changes > 1 were extracted as differentially expressed genes for each comparison. Gene ontology analysis was performed on statistically significant sets of genes by running the software GeneSCF. The goa_human or mouse GO list was used to cluster sets of genes based on their biological processes and determine their statistical significance. PCA analysis was performed using the "plotPCA" function within the DESeq2 R package. The plot presents samples on a 2D plane spanned by their first two principal components. The top 500 genes, selected by the highest row variance, were used to generate the plot.

[0233] Single-cell RNA sequencing

[0234] cell and Library manufacturing

[0235] After treating cells with 300 μL of TrypLE at 37°C for 3 minutes, they were pipetted several times using a p1000 pipette. Subsequently, the dissociated cells were filtered through a FACS filter cell strainer. Single-cell RNA samples were prepared using Chromium Next 10X Genomics technology (10xGenomics, PN-1000128, PN-1000127) according to the manufacturer's protocol. Quantification of DNA samples was performed using a Qubit 2.0 fluorescence spectrometer, and quality control evaluations were conducted using an Agilent TapeStation D5000 at the Harvard Core Facility. RNA sequencing libraries were subsequently prepared using a dual index kit (10xGenomics, PN-1000213) according to the manufacturer's instructions. Validation of the sequencing library was performed at the Harvard core facility using an Agilent Tape Station D1000.

[0236] Illumina Sequencing

[0237] Illumina sequencing was performed by Medgenome (California, USA). The library was sequenced using an Illumina Novaseq 6000 sequencer (Illumina, San Diego, California). 150 PE reads were generated for a total of ~503 GB of data. Illumina raw BCL sequencing files were processed using CellRanger software (10x Genomics) to generate FASTQ files and count matrices (support.10xgenomics.com / single-cell-gene-expression / software / overview / welcome). Feature-barcode matrices were obtained from "cellranger count" for all samples.

[0238] Dataset Quality Management

[0239] Single-cell sequencing datasets were processed using the 10x Genomics Cell Ranger (version 7.1.0) toolset. For each sample, the "cellranger count" pipeline was used to quantify gene expression from FASTQ files, which included read alignment, filtering, barcode scanning, and UMI counting. During this step, the GRCh38 human genome (version refdata-gex-GRCh38-2020-A) served as a reference. Subsequently, the filtered feature barcode matrix files were imported into the Seurat package (version 4.1.0) for quality control, analysis, and exploration. To remove doublets, the DoubletFinder package (version 2.0.3) was applied. Only cells expressing more than 200 and fewer than 9,000 unique genes, with a mitochondrial percentage of less than 20%, were retained for further analysis.

[0240] Cell clustering

[0241] Dataset normalization, scaling, dimensionality reduction, cell clustering, and differential expression gene (DEG) identification were performed using the Seurat package (version 4.1.0). Briefly, the global-scaling normalization method "LogNormalize" was applied to normalize feature expression measurements for each cell, followed by the "ScaleData" function being used for linear transformation. The top 2,000 most variable genes were identified using the "FindVariableFeatures" function, which performed Principal Component Analysis (PCA). The "FindNeighbors" function was used to construct a K-Nearest Neighbors (KNN) graph based on the first 15 principal components (PC), and the "FindClusters" function was performed to cluster cells into different populations based on the graph. DEGs for each population were identified using the "FindAllMarkers" function with default parameters.

[0242] Trajectory Inference

[0243] To describe cell differentiation trajectories, Monocle3 (version 1.3.4) was used for single-cell trajectory analysis. Throughout the analysis, the PCA dimensionality reduction algorithm and log normalization methods were used in the preprocessing step. The "reduce_dimension" function was subsequently applied to dimensionality reduction using the UMAP algorithm. To cluster cells, the "cluster_cells" function was used with the Leiden clustering method. After preprocessing, dimensionality reduction, and cell clustering, trajectories were constructed by the "learn_graph" function. The generated trajectory structures were visualized by the "plot_cells" function.

[0244] Comparative transcriptome analysis of iMPC-derived wall cell and air-available wall cell data

[0245] The inventors used the "Scanorama" algorithm to compare iMPC-derived perivascular cells / SMCs with air-available wall cell data (Hie, B., et. al., (2019) Nat. Biotechnol. 37 , 685-691). The reference dataset used was "TS_Vasculature" from The Tabula Sapiens Consortium (Consortium*, TS, Jones, RC, Karkanias, J., Krasnow, MA, Pisco, AO, Quake, SR, Salzman, J., Yosef, N., Bulthaup, B., Brown, P., et al. (2022). The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans. Science 376 , eabl4896). For each perivascular cell / SMC in the inventors' scRNA-seq dataset, the inventors identified the cell type most similar to the reference dataset and counted their occurrences. Using edgeR (version 4.0.16) for pseudo-bulk differential expression analysis, the inventors calculated Spearman counts to evaluate the similarity between iMPC-derived and primary perivascular cells / SMCs. The results were visualized in Pheatmap (version 1.0.12) using ECFCs as a negative control (Robinson, MD, et. al., (2009) Bioinformatics 26 , 139-140).

[0246] Cell-cell interaction analysis

[0247] The inventors used the R packages CellChat (version 1.6.1) and Monocle (version 2.22.0), respectively, to profile cell-cell communication and cell trajectories (Jin, S., et. al., (2021) Nat Commun 12 , 1088).

[0248] Gene regulatory factor network analysis

[0249] The R package hdWGCNA was used to construct specific co-expression networks across cell layers (Morabito, S., et. al., (2023) Cell Rep. Methods 3 , 100498).

[0250] Statistical analysis

[0251] Unless specifically stated otherwise, data were presented as mean ± standard error of the mean (SEM). When comparing two groups, mean values ​​were compared using an independent two-sided Student's t-test. Multiple group comparisons were performed using analysis of variance (one-way ANOVA) followed by Bonferroni correction. No exclusion criteria were applied to any of the analyses. All statistical calculations were performed using GraphPad Prism v.9 software (GraphPad Software Inc.). Statistical significance was set at P < 0.05.

[0252] Table 1. Sequences of primers used in RT-qPCR

[0253]

[0254]

[0255] Table 2. List of Antibodies

[0256]

[0257] Example 1: Transient NKX3.1 activation efficiently differentiates MePCs into iMPCs

[0258] The inventors initially identified NKX3.1 in screenings conducted in collaboration with Dr. George Church of Harvard (Ng, AHM et al. A comprehensive library of human transcription factors for cell fate engineering. Nat Biotechnol 39, 510-519 (2020)). To evaluate the differentiation potential of NKX3.1, the inventors first genetically engineered human iPSCs to express NKX3.1 in response to doxycycline (Dox) using a piggyback transposon system. Puromycin-selected clones were screened for the homologous expression of NKX3.1 upon administration of Dox (Figs. 1j-1o). The engineered clones (named iPSC-Dox-NKX3.1) remained pluripotent and maintained the expression of pluripotency markers OCT4, SOX2, and NANOG at levels comparable to their parental iPSC counterparts (Figs. 1j-1o). Next, the inventors engineered human iPSC cell lines with dox-induced NKX3.1 transgenes and developed a novel feeder-free protocol (Figs. 1a and 1i). The first 48-hour step converts iPSCs into mesodermal precursors (MePCs) via the Wnt and Nodal pathways. The second step transiently activates NKX3.1 for 48 h to generate iMPCs. Flow cytometry confirmed the presence of wall cells by analyzing PDGFRβ (CD140b) and aminopeptidase N (CD13) expression. Puromycin-selected clones were screened for NKX3.1 isoexpression upon administration of Dox (Figs. 1j–1m). The engineered clone (named iPSC-Dox-NKX3.1) remained pluripotent and maintained the expression of pluripotency markers OCT4, SOX2, and NANOG at levels comparable to its parental iPSC counterpart (Fig. 1m).As discussed above, the two-dimensional, feeder-free, and chemically defined protocol relies on the timely transfer of iPSCs through two distinct steps, each lasting 48 hours (Figs. 1a and 1i). The first step, involving the conversion of iPSCs to the intermediate MePC, is mediated by the activation of the Wnt signaling pathway using the glycogen synthase kinase 3 inhibitor CHIR99021 and is characterized by the transient activation of TF TBXT (Fig. 2a, bottom panel). Again, the second step involves the activation of NKX3.1 through the provision of Dox in the absence of any growth factor for 48 hours (Figs. 1g and 15a). Subsequently, the generated cells, designated herein as iMPCs, were grown in serum-containing smooth muscle growth medium (SMGM) for further passage.

[0259] During 4 days of differentiation, the inventors observed significant morphological changes in cells that gradually resembled mesenchymal cell types (Fig. 1h). Furthermore, iMPCs were morphologically similar to parietal cells during culture, displaying characteristic stellate features (Fig. 1h). The inventors tracked the presence of the parietal population during differentiation by analyzing the expression of PDGFRβ (CD140b), a common parietal cell marker, and aminopeptidase N (CD13), known to be expressed in vivo in parietal cells, using flow cytometry.

[0260] The protocol effectively converted iPSCs into CD140b+ / CD13+ iMPCs (~99% efficiency; Fig. 1b), and less than 1% of cells expressed the TRA1-81 antigen, indicating minimally undifferentiated iPSCs (Fig. 1c). After one passage in culture, iMPCs expressed wall-specific contractile proteins, including alpha-smooth muscle actin (α-SMA), calponin, transgelin (SM22), and vimentin (Fig. 1d). Comparison of iMPC mRNA expression with control human primary pulmonary vascular SMCs and bone marrow-derived mesenchymal stem cells (MSCs) confirmed wall cell specification (Figs. 1e-1f). Expression of SMC markers in iMPCs was comparable to or higher than that of SMCs and MSCs (Fig. 1e). Specifically, expression of selected smooth muscle markers in iMPCs was the same as or (e.g., ACTA2 , TPM1 ) or was significantly higher ( CNN1 , TAGLN , MYOCD , MYH11 ) (Fig. 1e). Perivascular cell marker expression is higher in MSCs, PDGFRB Except for, it is similar or upscaled in iMPC ( CSPG4 , DES , NDUFA4L2 , PDE5 A, and THY1 ) (Fig. 1f).

[0261] This effective conversion of MePC to PDGFRβ+ iMPC through the activation of NKX3.1 was reproducible across iPSC cell lines from three distinct cell origins (Fig. 1n-1o).

[0262] After differentiation, indirect immunofluorescence confirmed the expression of wall-specific contractile cytoskeletal proteins, including alpha-smooth muscle actin (α-SMA), calponin, transgelin (SM22), and vimentin, in iMPCs (Fig. 1d). The expression of these wall cell markers was highly uniform (>90%) and reproducible across iMPCs derived from three distinct iPSC cell lines (Figs. 1n-1o and 16a-16c). Importantly, in the absence of doxycycline, the expression levels of wall cell markers were significantly lower, which supports the role of NKX3.1 in wall cell specification (Figs. 1n-1o and 15b).

[0263] It is important to note that during the differentiation of MePCs into iMPCs, the expression of NKX3.1 was only transient (Figs. 1g, 2a, and 15a). This transient expression enabled the possibility of developing non-genome footprint protocols using chemically modified mRNA (modRNA). Indeed, transfection of unmodified iPSCs with modRNA encoding NKX3.1 enabled potent transient expression of NKX3.1 (Figs. 3a–3g). Furthermore, activation of NKX3.1 with modRNA in MePCs efficiently produced iMPCs that were indistinguishable from those generated by Dox-induced protocols, including potent expression of wall cell markers (Figs. 3a–3g).

[0264] Example 2. Comparison of the inventors' TF-induced method and a standard chemically-induced differentiation protocol

[0265] The inventors compared the iMPCs generated via the inventors' NKX3.1-induced protocol with the chemical induction protocol originally reported by Patsch et al. in 2015. This protocol generates SMCs (referred to herein as iSMCs) from the same MePCs but uses PDGF-BB (10 ng / mL) and Activin A (2 ng / mL) for 48 h (Fig. 2a). Both iMPCs and iSMCs exhibited transient NKX3.1 expression in a similar pattern (Fig. 2a). During both the NKX3.1-induced and chemically-induced protocols, the cells TBXT and NKX3.1 It exhibited a similar sequential pattern of transient expression, which corresponds to their transition through the mesoderm and parietal cell differentiation stages, respectively (Fig. 2a).

[0266] SMC marker expression is significantly higher in iSMC, ACTA2 and MYOCD Except for [unclear], the results were consistent in both iMPC and iSMC (Fig. 2b). Conversely, perivascular cell markers were more prevalent in iMPC (Fig. 2c). In short, both protocols successfully generate wall cells; however, the chemically induced approach predominantly generates cells with an SMC phenotype, whereas NKX3.1 induction likely generates a more diverse iMPC population including both SMC and perivascular cells.

[0267] It is important to note that the inventors' protocol for generating iSMCs was inspired by, rather than directly following, the protocol described by [Patsch et al.]. To enable a comparable mesoderm stage and to effectively compare the results of the inventors' NKX3.1 induction protocol with those of a chemically induced protocol, the inventors adopted only the aspect of differentiation from mesoderm to parietal cells (i.e., the use of PDGF-BB and activin A) of their protocol.

[0268] Together, these data suggest that while both protocols are effective in generating wall cells, the chemically-induced method preferentially produces cells matching the SMC phenotype, whereas the NKX3.1-induced protocol generates iMPCs that possess characteristics of both SMCs and perivascular cells.

[0269] In summary, transient activation of NKX3.1 expression in MePCs (via Dox-inducible systems or modRNA) effectively and efficiently converted human iPSCs into cells exhibiting a distinct wall cell phenotype.

[0270] Example 3. A non-genome footprint approach to inducing iMPC with modified mRNA

[0271] NKX3.1 expression is required only transiently (Fig. 2a), which creates an opportunity to use chemically modified mRNA (modRNA) and non-genome footprint protocols (modRNA is always transient) (Figs. 3a and 3f). To this end, the inventors designed a modRNA (Trilink) encoding NKX3.1 and confirmed that transfection of unmodified iPSCs with this modRNA resulted in significant transient expression of NKX3.1 (Fig. 3b). The use of modRNA to generate iMPCs was effective (Figs. 3a-3g).

[0272] Indeed, activating NKX3.1 in MePCs generated iMPCs with high efficiency (approximately 95% conversion as measured by flow cytometry; Fig. 3c), which were indistinguishable from those produced by the Dox-induced protocol and exhibited potent expression of wall cell markers (Fig. 3d). Collectively, activating NKX3.1 (via Dox or modRNA) efficiently converts human MePCs into iMPCs displaying a potent wall cell phenotype. Furthermore, activation of NKX3.1 with modRNA in MePCs efficiently produced iMPCs that were indistinguishable from those produced by the Dox-induced protocol, including potent expression of wall cell markers (Figs. 3a-3g).

[0273] In summary, transient activation of NKX3.1 expression in MePCs (via the Dox-inducible system or modRNA) effectively and efficiently converted human iPSCs into cells exhibiting a distinct wall cell phenotype (Fig. 1a-3g).

[0274] Example 4. Contraction and secretion qualification of iMPC

[0275] The inventors evaluated several functional properties of their NKX3.1-induced iMPCs, focusing on wall cell-associated features such as calcium influx, contractility, extracellular matrix synthesis, and EC interactions. iMPC contractility was tested using vasoconstrictive agents. Calcium imaging suggested that endothelin-1 and carvacol increased intracellular calcium in iMPCs, similar to control primary MSCs and SMCs (Figs. 4a, 4f, and 4g). A three-dimensional collagen contractility assay further confirmed their response to vasoconstrictive stimuli (U46619; a thromboxane A2 (TXA2) analog acting as a potent vasoconstrictor) as iMPCs contracted similarly to MSCs and iSMCs (Fig. 4b). These findings indicate that iMPCs can respond to vasoconstrictive stimuli, and thus suggest that iMPCs in vivo It confirms that it exhibits significant functional characteristics of wall cells.

[0276] Extracellular fibronectin deposition in iMPCs was evaluated after TGF-β treatment. TGF-β caused significant fibronectin production, which was inhibited by the TGF-β signaling inhibitor SB31542 (Figs. 4c, 4h, 4i, and 4j). This increase in fibronectin production is evident at both protein (Figs. 4c, 4h, and 4i) and mRNA (Fig. 4j) levels. Additionally, the introduction of a small molecule (SB431542) that inhibits TGF-β signaling effectively prevented fibronectin production, thereby confirming that fibronectin deposition in iMPCs is mediated by TGF-β. The ability to deposit extracellular fibronectin represents a key functional characteristic of wall cells.

[0277] Finally, the inventors also explored the ability of iMPCs to interact with ECs by producing angiogenic factors. Their ability to interact with ECs by producing angiogenic factors is pivotal for parietal cell function. The inventors investigated the ability of iMPCs to modulate EC behavior through the secretion of parasecretory angiogenic-inducing factors and compared this with that of SMCs and MSCs by examining their respective conditioning media using an angiogenic protein array and a quantitative luminex assay (Figs. 4d-4e). The angiogenic protein array (Fig. 4d) and luminex protein assay (Fig. 4e) of the conditioning media from iMPCs, SMCs, and MSCs revealed that iMPCs secreted several angiogenic-inducing factors. In particular, iMPCs secreted various angiogenesis-inducing factors, including VEGF-A, PLGF, HB-EGF, HGF, several members of the IGFBP family, as well as members of the protein serine protease inhibitor (serpin) superfamily (Serpin E1 and Serpin F1) and urokinase-type plasminogen activator (uPA). Some factors were more abundant in iMPCs compared to primary SMCs (e.g., PLGF), while others were less prominent in iMPCs (e.g., VEGF-A and FGF2). Nevertheless, the overall angiogenesis-inducing secretory profile of iMPCs was consistent with that expected for parietal cells.

[0278] Together, these results suggest that the inventors' NKX3.1-induced iMPCs mimic some of the functional properties typically associated with parietal cells, including contractile response, the ability to deposit fibronectin, and the secretion of angiogenic factors.

[0279] Example 5. Modification of EC function by iMPC: In vitro and in vivo assays

[0280] The inventors investigated the effect of iMPC-secreted proteins on EC activity using human umbilical cord blood-derived endothelial colony-forming cells (ECFC, referred to herein as EC) in three in vitro assays (Figs. 5a-5d). Indirect co-culture with iMPC (Fig. 5a) and exposure to iMPC-conditioned medium (CM-(iMPC)) for 72 hours (Fig. 5b) both promoted EC proliferation, which is comparable to the results obtained with CM-(SMC) and CM-(MSC). In both assays, the number of ECs exposed to CM-(iMPC) for 72 hours was significantly higher than the number observed when cells were exposed to the basal control medium. Furthermore, CM-(iMPC) enhanced the ability of ECs to migrate and re-endothelialize scratched monolayers (scratch assay; Fig. 5c), as well as to assemble capillary-like structures in three-dimensional cultures (Fig. 5d). Collectively, the inventors' data indicate that iMPCs effectively modulate in vitro EC function through the secretion of parasecretory factors, and that their ability to influence EC activity is comparable to that of control-walled SMCs and MSCs.

[0281] To evaluate a more physiologically relevant assay, the inventors utilized an established in vitro model of directly co-culturing iMPCs with ECs in a three-dimensional (3D) hydrogel. This microphysiological system—a microfluidic 'on-a-chip' model—facilitates the dynamic interaction of cells and the formation of microvascular networks through angiogenesis. First, the inventors combined GFP-labeled iMPCs and DsRed-labeled ECs within a fibrin gel and examined the ability of iMPCs to enable angiogenesis (Fig. 5j). This setup resulted in the formation of vascular structures lined by DsRed+ ECs within two days (Fig. 5j). Furthermore, immunofluorescence staining confirmed the formation of a vascular network within the chip, and a continuous endothelial lining was indicated by CD31 and VE-cadherin, and the presence of α-SMA+ and SM22+ iMPCs acted as perivascular cells adjacent to parts of the EC-lined lumen (Fig. 5k). This 'on-a-chip' model confirmed the potential of iMPCs to assemble complex vascular networks when co-cultured with ECs, thus supporting their functionality as wall cells.

[0282] In vivo functional iMPCs (e.g., functioning as perivascular cells and in vivo To evaluate the support for angiogenesis, the inventors subcutaneously transplanted ECs along with iMPCs into immunodeficient mice using the inventors' established model (Figs. 5e-5l). One week after transplantation, the inventors removed the grafts and analyzed them for the formation of human-specific vascular networks. Grafts containing parietal cells (SMC, MSC, or iMPC) showed evidence of blood perfusion (Fig. 5e), and histological examination revealed the presence of perfused vessels containing murine erythrocytes (Fig. 5f); there were no signs of hemorrhage or thrombosis (i.e., platelet aggregation and uniform fibrin deposition), indicating proper functionality. Indeed, H&E staining confirmed that all grafts seeded with parietal cells formed numerous perfused vessels containing murine erythrocytes (Fig. 5f), whereas grafts containing ECs alone failed to form perfused vessels. There were no significant differences in mean microvascular density across grafts with different parietal cell populations (Fig. 5g). This quantification of mean microvascular density at Day 7 confirmed that there were no statistically significant differences between grafts prepared with each of the different parietal cell populations (Fig. 5g). The ability of iMPCs to support in vivo vascular networks was demonstrated using another source of primary ECs. Grafts containing human venous endothelial cells (HUVECs) and iMPCs produced mature and perfused vessels, as evidenced by human-specific CD31 staining and the presence of human perivascular cells (Figs. 17a-17d).

[0283] Most of the blood vessels within the graft stained positive for human-specific CD31, confirming that they were lined by the transplanted human EC (Fig. 5h). The human blood vessels were perfused, indicating that they were connected to the murine host blood vessels (Fig. 5h). Furthermore, α-SMA-positive perivascular cells surrounded all human blood vessels within the graft (Fig. 5h), confirming the contribution of iMPCs to the perivascular compartment of the blood vessels.

[0284] The formation of EC-lined vascular structures depended on the presence of parietal cells. Perfused vessels stained positive for human-specific CD31, indicating that the newly formed human vascular structures established functional anastomosis with the murine host vessels (Fig. 5i). Perivascular involvement of α-SMA-expressing iMPCs was confirmed by human-specific vimentin staining observed in cells surrounding human EC-lined microvessels (Figs. 5i-5j). In the specified experiments, the inventors used GFP-labeled iMPCs to track their in vivo localization. Double staining of GFP and α-SMA revealed that after 7 days in vivo, GFP-expressing iMPCs were detected primarily in proximity to and immediately adjacent to the luminal structures (Fig. 5j), indicating their structural involvement in the perivascular compartment of the newly formed vessels. Quantification of parietal cell coverage revealed that the majority (>90%) of human blood vessels exhibited perivascular coverage, and a significant proportion of these vessels are covered by transplanted iMPCs (Fig. 5l).

[0285] In conclusion, the inventors' results demonstrate that iMPCs can modulate EC function equivalently to control wall SMCs and MSCs, both in vitro and in vivo.

[0286] Example 6. Maturation of iMPC upon interaction with EC

[0287] During angiogenesis, the maturation of parietal cell precursors depends significantly on their interactions with ECs. The interactions between parietal cell precursors and ECs play a pivotal role in angiogenesis, maturation, and stabilization. Simultaneously, these interactions drive the maturation of parietal cell precursors into the ultimately differentiated parietal cell type. The inventors sought to determine whether their iMPCs could mature similarly upon co-culture with ECs. Therefore, the inventors co-cultured iMPCs with ECs for 7 days to produce co-iMPCs. Subsequently, the inventors isolated the iMPCs (referred to herein as co-iMPCs) as CD31-negative cells from the co-cultured ECs and MACS and subjected them to gene expression analysis via bulk RNA-seq (Fig. 6a). On a global scale, hundreds of differentially expressed genes were found between iMPCs and co-iMPCs. Upon closer examination, comparisons revealed thousands of differentially expressed genes between iMPCs and co-iMPCs (Fig. 6a). Principal component analysis of these differentially expressed genes suggested that co-iMPCs exhibited transcriptional proximity to primary wall cells (specifically, SMCs and MSCs) more closely than iMPCs did before co-culture with ECs (Fig. 6b). This finding suggested the occurrence of the wall cell maturation process.

[0288] Furthermore, hierarchical clustering analysis suggested that co-iMPC aligned more closely with primary SMC and MSC enterprise-wide than iMPC (Fig. 6i). Pairwise correlation (Fig. 6e) and principal component analysis (Fig. 6b) further confirmed this hierarchical association.

[0289] To further understand the transcriptional differences between iMPCs and co-iMPCs, the inventors performed gene ontology (GO) enrichment analysis. In particular, the inventors observed significant enrichment of genes associated with mature parietal cell function in co-iMPCs (Fig. 6c). These included genes associated with extracellular matrix organization, regulation of angiogenesis, regulation of angiogenesis, smooth muscle contraction, connective tissue development, and response to TGFβ (Fig. 6c). Additionally, qPCR analysis confirmed significant upregulation of several parietal cell-associated genes in co-iMPCs, including both SMC and perivascular cell markers (Figs. 6d, 6f, and 6g). These were SMC-associated genes, e.g. ACTA2 , CNN1 , TAGLN , MYOCD , and TPM1 (Figs. 6d and 6f), as well as CSPG4 and PDE5A It included perivascular cell-related genes such as (Figs. 6d and 6g). Notably, control iMPCs cultured in the same medium for 7 days without ECs did not exhibit the upregulation of maturation wall markers observed when co-cultured with ECs (Fig. 18). This overall upregulation pattern in co-iMPCs reflected that observed in primary MSCs after 7 days of co-culture with ECs, highlighting the widely recognized precursor role of MSCs.

[0290] Furthermore, immunofluorescence staining of co-iMPCs confirmed the distinct presence of both 3G5+ perivascular cells (where 3G5 ganglioside antigens are expressed on the cell surface of perivascular cells) and α-SMA+ / 3G5- SMCs (Fig. 6h). This 3G5 ganglioside antibody has previously been verified to accurately label perivascular cells in both culture and clinical samples. Furthermore, studies have demonstrated that 3G5 is not found in vascular SMCs and have utilized the 3G5 antibody for the identification and isolation of perivascular cells across various tissues, including human skin and mouse hearts. Therefore, the 3G5 ganglioside is recognized as a highly reliable marker for identifying perivascular cells.

[0291] Finally, it is important to note that iMPCs exhibited only minimal MYH11 expression (a marker of mature SMC) at both mRNA and protein levels prior to co-culture with ECs (Figs. 6f and 6j). This is consistent with well-documented observations that MYH11 expression is generally suppressed in SMCs when cultured alone. Instead, potent expression of MYH11 is typically reported in vivo, in freshly isolated cells, or in co-culture systems that facilitate interaction with ECs. Indeed, upon 7-day co-culture of iMPCs and ECs, the inventors observed that some cells displayed high levels of both MYH11 and α-SMA, while others showed high MYH11 but low α-SMA (Fig. 6j), suggesting a heterogeneous mixture of wall cell phenotypes. This protein-level evidence supports the presence of MYH11+ SMCs among the generated wall cells and reinforces the context-dependent nature of MYH11 expression in SMCs.

[0292] Collectively, these findings were consistent with the concept of iMPC maturation into parietal cells upon interaction with ECs. These results indicate that NKX3.1-induced iMPCs are not passive regulators of EC function. Instead, they display an active, dynamic response to EC interactions, which leads to significant maturation into parietal cells. This differentiating ability, characterized by distinct upregulation of mature parietal cell-associated genes, highlights the precursor nature of iMPCs.

[0293] Example 7. iMPC maturation and wall cell heterogeneity

[0294] Next, the inventors performed single-cell RNA sequencing (scRNA-seq) to investigate whether EC co-culture facilitates iMPC maturation and reveals parietal cell heterogeneity. These experiments utilized scRNA-seq to investigate the extent to which iMPCs can reproduce parietal cell heterogeneity and to examine the differentiation of iPSCs into iMPCs, as well as the interactions between iMPCs and ECs (Figs. 7a-7c and 11a-11h). Cells were examined at various points in the differentiation protocol (Day 0, Day 2, and Day 4) and after 7 days of co-culture with ECs (Day 11) (Figs. 7a and 11a). Specifically, the inventors sampled four critical steps of the inventors' differentiation protocol corresponding to Day 0 (iPSC), Day 2 (MePC after mesodermal differentiation), Day 4 (iMPC after NKX3.1 activation), and Day 11 after 7 days of co-culture of iMPC and EC (Figs. 7a-7c, 11a-14, and 19a-20). The goal was to determine whether co-culture with EC promotes iMPC maturation and diversifies the parietal cell population into recognizable perivascular cell types.

[0295] After co-culture, ECs and iMPCs were denoted as co-ECs and co-iMPCs, respectively. Using the inventors' 10X Genomics platform, the inventors generated data for 10,000 cells per time point (at each differentiation stage). Seurat v325 facilitated subsequent cell clustering into normalization and integrated analysis between time points (Figs. 7b and 11b). This analysis generated 19 distinct clusters, 15 of which were manually annotated into 8 to 9 distinct groups, which are iPSCs ( OCT4 , NANOG , SOX2 Commented by the expression of), MePC ( TBXT , MIXL1 ), iMPC ( CSPG4 , PDGFRB ), EC and Joint-EC ( PECAM1 , CDH5 ), SMC ( ACTA2 , CNN1 , TAGLN ), perivascular cells ( NT5E , TAGLN , CSPG4 , PDGFRB ), and fibroblasts ( COL1A1 , COL1A2 , DAY It includes (see UMAP plot, Figs. 7b and 11b). Distinct cell populations were identified based on the expression of characteristic cell markers (Figs. 7b, 11b, 11d, 12–14, and 19a–20). For example, iMPCs (cluster #3 in Figs. 11b and 11c) showed high levels NKX3.1 Represents, CSPG4 , PDGFRB , and THE expressed, but CD73 ( NT5E ) and contractile proteins ( ACTA2 , CNN1 , and DAY They had reduced expression of the gene coding for ), and the fibroblast-like population (cluster #4 in Figs. 11b and 11c) had high NKX3.1 , PDGFRB , ACTA2 , and PDGFRA Expression was characterized (Figs. 7b-7c, 11b-11g, 12-14, and 19a-20). The annotated populations were also iPSCs (cluster #1 in Figs. 11b and 11c, OCT4 , NANOG , and SOX2 Indicated by) and MePC (cluster #2 in Figs. 11b and 11c, TBX6 , MSGN1 , MIXL1 and TBXT It included (expressing) (Figs. 7b-7c, 11b-11g, 12-14, and 19a-20). Notably, PDGFRA The expression of was prominent in cluster #4 compared to other annotated parietal cell populations, which is consistent with conventional criteria used in the field of identifying fibroblasts (Lendahl, U., et. al., (2022) Nat. Commun. 13 , 3409).

[0296] Next, the inventors evaluated the appearance of each cluster over time (Figs. 7c and 11c). The temporal evolution of these clusters coincided with the progression from iPSC (Day 0) to MePC (Day 2) and then to iMPC (Day 4) (Figs. 7c and 11c).

[0297] Additionally, the inventors [described] the axial mesoderm ( TBX6 , MSGN1 ), segment ( FOXC2 , MEOX2 , TCF15 ), and tibial segment ( PAX9 , SOX9 , NKX3.2 The inventors analyzed scRNA-seq data for markers associated with ) (Figs. 19a-19d). At the mesoderm stage (Day 2 MePC, before NKX3.1 activation), TBX6 and MSGN1This was detectable, which is consistent with their expected expression during early mesoderm differentiation. However, after NKX3.1 activation on Day 4 (iMPC), these markers were not significantly expressed, suggesting a transition from a general mesoderm identity toward a more defined lineage. Markers associated with somite and tibial differentiation showed negligible expression on Day 2, and only FOXC2 and SOX9 Only iMPC showed partial expression on day 4 (Figs. 19a-19d). This pattern indicates a minimal effect of NKX3.1 activation on inducing somatic or tibial identities directly from MePC.

[0298] The inventors also analyzed the interaction between iMPCs and ECs. After 7 days of co-culture with ECs, iMPCs matured into three distinct wall cell subpopulations (clusters #5, #6, and #7 on day 11; Figs. 7b, 7c, 11b, 11c). These wall cell clusters were no longer NKX3.1 Although it did not express (its transient activation was confirmed), general perivascular Marker PDGFRB and NT5E (CD73) was uniformly expressed. Notably, while iMPCs resembled neovascular perivascular cells, the wall cell clusters after co-culture with ECs resembled mature perivascular cells, including perivascular cells (cluster #5), contracted SMCs (c-SMC; cluster #6), and synthetic SMCs (s-SMC; cluster #7). These clusters, PDGFRB While sharing expression, significant differences were observed, particularly with respect to genes associated with contractile proteins and ECM production (Fig. 11e). Indeed, a direct comparison of differentially expressed genes revealed cell contractility in SMCs (e.g., compared to perivascular cells (cluster #5)). ACTA2 , CNN1 , DAY ) and ECM proteins (e.g., FN1 , COL1A1 , COL1A2 We revealed significant upregulation in the gene coding for ) (Fig. 11e), which is consistent with their perivascular role in vivo. Meanwhile, a direct comparison between the two clusters of SMCs revealed the contraction of c-SMC and s-SMC, respectively (e.g., ACTA2 , MYL9 , DAY Upregulation of) and synthesis (e.g., FN1 , COL5A1 , COL4A1 It revealed a clear distinction between the phenotypes (Fig. 11e), which is consistent with the previous description of these two types of SMC signs.

[0299] Additionally, the inventors extended their comparative analysis to evaluate the similarity between their iMPC-derived wall cells (i.e., after co-culture with ECs) and primary wall cells. First, the inventors performed a comparative analysis with a publicly available bulk RNA dataset to provide a more precise context. Specifically, the inventors compared their scRNA-seq data from cells characterized as SMCs (Figs. 7a-7c; clusters #6 and #7 in Figs. 11a-11h) with human aortic SMCs from the public dataset. Similarly, the inventors' cells identified as perivascular cells (Figs. 7a-7c; cluster #5 in Figs. 11a-11h) were compared with the public dataset of primary human brain perivascular cells. Pearson correlation analysis of these comparisons demonstrated a strong correlation (correlation coefficient ~0.6, p < 0.001) for both comparison sets, indicating significant transcriptional agreement between the inventors' derived wall cells and the aerial dataset (Figs. 21a-21b).

[0300] Furthermore, to establish additional unbiased criteria, the inventors used the comprehensive Tabula Sapiens consortium vascular structure dataset. This dataset includes various arrays of vascular endothelial and parietal cell types. By overlaying the inventors' scRNA-seq data from day 11 (i.e., parietal cells generated from iMPCs after 7 days of co-culture with ECs), the inventors observed that the inventors' cells identified as SMC-like clusters (Figs. 7a-7c; clusters #6 and #7 in Figs. 11a-11h) exhibited significant overlap with reference SMCs (4,832 of the inventors' SMCs matched reference Tabula Sapiens SMCs; Fig. 21c). Similarly, most of the inventors' cells classified as perivascular cells (Figs. 7a-7c; Cluster #5 in Figs. 11a-11h) closely matched reference perivascular cells (808 of the inventors' perivascular cells matched reference Tabula Sapiens perivascular cells; Fig. 21d). This analysis indicates that most of the inventors' SMCs strongly match established reference SMCs. This also suggests that cells from the inventors' perivascular cell clusters show more similarity to reference perivascular cells than to SMCs or fibroblasts.

[0301] These comparative evaluations demonstrate that the gene expression profiles of the inventors' iMPC-derived SMCs and perivascular cells show significant agreement with established primary human wall cells and detailed single-cell references from The Tabula Sapiens Consortium, which suggests the relevance of the inventors' differentiation model to its in vivo counterparts.

[0302] To gain further insight into the signaling involved in parietal cell differentiation and maturation, the inventors examined cell-non-autonomous signals derived from ECs that promote parietal cell maturation. Using CellChat to analyze the inventors' scRNA-seq data, the inventors identified several key signaling pathways, particularly NOTCH and TGF-β, known to be implicated in angiogenesis and parietal cell development (Figs. 22a-22b). To confirm the functional importance of these pathways, the inventors performed an in vitro assay in which iMPCs were co-cultured with ECs in the presence of specific pathway inhibitors (Fig. 22c). Inhibition of NOTCH signaling to DAPT significantly impaired the maturation of iMPCs in both perivascular and SMC phenotypes, confirming the role of NOTCH in parietal cell maturation (Fig. 22d). Similarly, inhibition of TGF-β signaling to SB431542 selectively destroyed SMC maturation while preserving the perivascular cell-like phenotype, indicating its pivotal role in this process (Fig. 22e).

[0303] The inventors' analysis also included examining differential gene expression profiles between nascent and more mature perivascular cells (Cluster #5 on Day 4 and Day 11, respectively). The inventors identified distinct genetic markers that differentiate early-stage perivascular cells from their mature counterparts. Analysis of the GO pathways in these DEGs revealed that Day 11 perivascular cells exhibited significant enrichment in genes associated with extracellular matrix organization, cell adhesion, and the TGF-β signaling pathway, which are characteristic of mature wall cell phenotypes (Figs. 23a-23c). In contrast, Day 4 perivascular cells exhibited enriched expression in genes linked to cell proliferation, regulation of cell differentiation, and the Wnt signaling pathway, reflecting their developmental stage which is more closely linked to mesodermal precursors (see DEG). Furthermore, GO and KEGG analysis of differential gene expression between perivascular cells (cluster #5) and c-SMCs (cluster #6) confirmed significant enrichment in extracellular matrix organization, cell-matrix adhesion, cell contractility, and functions associated with various signaling pathways related to TGF-β signaling in c-SMCs (Figs. 23a-23c), which indicates contractile and structural roles typically associated with SMCs.

[0304] Next, the inventors performed an in-depth analysis of their scRNA-seq data to identify the gene regulatory networks (GRNs) driving the differentiation of their parietal cell population from MePCs into iMPCs, perivascular cells, and SMCs (Figs. 24a-24b). Using a combination of transcription factor motif enrichment analysis and gene expression correlation mapping, the inventors identified distinct GRNs governing progression from MePCs to mature parietal cells, including perivascular cells and SMCs (Fig. 24a). This approach utilized regulatory elements predicted to be active in each cell state, providing a dynamic perspective on transcriptional control that shapes cell fate determination. For example, in early-stage MePCs, these networks included pivotal regulators, such as MIXL1 and MSX1, during mesodermal specification (GRN9, Fig. 24b). Subsequently, several GRNs were highly active in iMPCs compared to mature SMCs and perivascular cells (GRN4 and GRN12, Fig. 24b), suggesting that genes contained in these GRNs, such as TIMP1, TGFB1, JAK, and PIEZO1, may be influenced by NKX3.1. As the cells progress toward a more defined wall cell fate, the inventors observed a transition in active GRN10, accompanied by increased indications of motifs related to TGF-β signaling (TGFB2), ECM production, and contractile function—key aspects of the mature wall cell phenotype (Fig. 24b).

[0305] Finally, the inventors' trajectory analysis with pseudo-time plots (Fig. 11f) provided additional insights into the temporal evolution from iPSCs to MePCs and subsequently to iMPCs (Fig. 11g). This analysis also confirmed that iMPC interactions with ECs promoted the development of various mature wall cell subpopulations, starting as perivascular cells and progressing to c-SMCs and s-SMCs (Fig. 11g). Further pseudo-time analysis provided a more precise visualization of the developmental trajectories and maturation stages of different cell subsets derived from iMPCs (Fig. 25). This pseudo-time trajectory analysis indicated that perivascular cell clusters appear to be temporally closer to iMPCs than SMC clusters, suggesting an earlier stage in perivascular cell development. This is consistent with the view that iMPCs resemble nascent perivascular cells. On day 11, the perivascular cell cluster (#5) appears earlier than the synthetic SMC cluster (#7) (Fig. 25), highlighting the developmental hierarchy. Furthermore, the inventors' analysis revealed insights into the origin of s-SMCs during the co-culture maturation stage. Pseudo-chronological trajectory analysis suggests that synthetic SMCs (s-SMCs) represent a late stage of parietal cell differentiation, emerging from contractile SMCs (c-SMCs) under the influence of successive endothelial interactions, highlighting the dynamic interaction of cell-autonomous and non-autonomous signals in parietal cell diversification (Fig. 25).

[0306] As expected, the cell populations on days 0, 2, and 4 were relatively homogeneous, reflecting the simultaneous transition from iPSCs to MePCs and iMPCs (Figs. 7c and 11c). Notably, analysis of co-iMPCs on day 11 indicated that after co-culture with ECs, iMPCs matured into three distinct parietal cell types: SMCs, perivascular cells, and fibroblasts (Figs. 7c and 11c). These data were consistent with the view of iMPC maturation upon interaction with ECs, which results in parietal cell heterogeneity.

[0307] Therefore, iMPCs act as true parietal cell precursors; after one week of co-culture with ECs, iMPCs diversified into distinct perivascular parietal cell subpopulations, including perivascular cells and SMCs. This finding suggests that EC interactions are pivotal in maturation in iMPCs, allowing for a strong re-enactment of parietal cell heterogeneity (carton illustration in Fig. 11h).

[0308] Example 8. Effective co-differentiation of iPSCs into iECs and iMPCs in a 3D vascular organoid (VO) model

[0309] Utilizing the inventors' previously established method for generating iECs via ETV2, the inventors explored the simultaneous differentiation of iPSCs into iECs and iMPCs using ETV2 and NKX3.1, respectively. This resulted in the development of a novel 3D VO model (Fig. 8a). Briefly, the inventors used their genetically engineered dox-ETV2-iPSC and dox-NKX3.1-iPSC cell lines. After conversion to MePC over three days, cells from these two iPSC cell lines (a 1:1 mixture) were aggregated into 3D using non-adhesive culture plates and a rotary shaker. Subsequently, the cells readily differentiated into iECs (h-CD31+) and iMPCs (h-CD31- / PDGFRβ+) upon exposure to Dox for three days (Fig. 8c). Furthermore, the cells self-assembled into a robust network of CD31+ vascular structures containing mature α-SMA+ perivascular wall cells (Fig. 8d). This method can rapidly generate numerous VOs of uniform size (~200 μm) (Fig. 8b), which exhibit properties consistent with proper vascular development, including a robust network of strengthened vessels with appropriate apical-basal polarization and diverse endothelial heterogeneity with arterial, venous, and capillary ECs (not presented).

[0310] Example 9. VO engraftment and formation of a perfused vascular network

[0311] The inventors evaluated the ability of the inventors' VO to form functional blood vessels in vivo. To this end, the inventors used a new capsule model of transplantation into immunodeficient NSG mice (1,000 VO / mouse; Figs. 8e-8g). On day 14, examination of the explant suggested robust vascularization of the graft. Indeed, histological (H&E) analysis revealed that the graft possessed an extensive network of perfused vessels (Fig. 8f). The microvessels were lined primarily by h-iECs (Fig. 8g), as confirmed by the expression of human-specific CD31, and contained mouse erythrocytes, indicating the formation of functional anastomosis with the host circulatory system. Furthermore, the human vessels were surrounded by α-SMA+ perivascular wall cells, which are an indication of vascular maturation and stability (Fig. 8g). In summary, the inventors developed a novel and efficient method for the co-differentiation of endothelial and wall cells in a VO model. When implanted in vivo, VO forms a robust, functional vascular network. This VO model will aid the inventors in their research to determine the underlying mechanisms of iMPC maturation.

[0312] Example 10. Maturation of iMPC and iEC in the inventors' VO model

[0313] The inventors explored the maturation of VO-derived iMPCs and iECs (referred to as VO-iMPCs and VO-iECs) compared to 2D single-culture differentiated iMPCs and iECs. After enzymatic digestion of VO (Day 5) and MACS sorting, the inventors obtained CD31+ VO-iECs and CD31- VO-iMPCs (Fig. 9a). qPCR comparison of core endothelial and parietal cell marker expressions showed various EC markers in VO-iECs ( CDH5 ; VWF ) (Fig. 9b) and several wall cell markers in VO-iMPC ( ACTA2 , MYH11 , DAY , CNN1Significant upregulation of ) (Fig. 9c) was revealed.

[0314] This supports the view that orthogonal co-differentiation of endothelial and parietal cells in the inventors' VO model, accompanied by the activation of ETV2 and NKX3.1, promotes the maturation of generated iMPCs and iECs.

[0315] Example 11. Transplantation of VO into ischemic tissue

[0316] To evaluate the efficacy of VO engraftment in an ischemic state, the inventors used the inventors' murine model of hind limb ischemia. Immuno-deficient, athymic nude mice aged 10 weeks were induced to become diabetic by a single intraperitoneal injection of streptozotocin (STZ; 220 mg / kg).

[0317] One week after STZ injection, diabetic mice were anesthetized using isoflurane. After achieving anesthesia, 7-0 acromion sutures were used to ligate the proximal and deep femoral artery and vein, and intervascular vessels were resected to completely occlude blood flow. Subsequently, 1,000 VO2 molecules, each containing a luciferase reporter in iEC, were resuspended in 50 μL of Matrigel and injected intramuscularly at the site of femoral artery and vein ligation (Fig. 10a). Engraftment of VO2 in the ischemic hind limbs was successfully visualized via bioluminescence on days 1 and 7 post-injection (Fig. 10b). Significantly, VO2 engraftment resulted in improved blood flow in the ischemic limbs, and laser Doppler imaging indicated a 50% recovery of blood flow at two weeks post-injection (Fig. 10c). Furthermore, engraftment prevented the development of necrotic tissue in mice treated with VO (Fig. 10d). In stark contrast, untreated control mice exhibited severe necrosis and impaired blood flow in their ischemic legs. In summary, the inventors' data suggest the potential of engraftment and the role of VO as an in vivo therapeutic agent that prevents necrosis and restores blood flow in a hindlimb ischemia model of diabetic mice.

[0318] Cited references

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[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

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[0329] Other embodiments

[0330] Although the invention has been described in conjunction with its detailed description, it should be understood that the description is intended to illustrate, not limit, the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and variations are within the scope of the following claims.

Claims

Claim 1 A method for producing iPSC-derived wall cell precursors (iMPCs), comprising the following steps: contacting a population of induced pluripotent stem cells (iPSCs) with a nucleic acid encoding NK3 homeobox 1 (NKX3.1) or a functional variant thereof; converting the iPSCs into mesodermal precursors (MePCs); and inducing the MePCs to express NKX3.1 for a period sufficient to produce iMPCs. Claim 2 The method of claim 1, wherein the nucleic acid is a DNA molecule (optional, a vector (e.g., a piggyback transposon vector or a viral vector)) or RNA (e.g., mRNA or modified mRNA). Claim 3 In paragraph 2, the method wherein the vector is a viral vector (e.g., retrovirus, lentivirus). Claim 4 A method according to any one of claims 1 to 3, wherein the nucleic acid comprises an inducible promoter (e.g., a doxycycline-inducible promoter) that controls the expression of NKX3.

1. Claim 5 A method according to any one of claims 1 to 4, wherein the step of converting the iPSC into a MePC comprises activating the Wnt path and / or activating the Nodal path for a period sufficient to generate the MePC (e.g., about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours). Claim 6 A method according to any one of claims 1 to 5, wherein the period sufficient to generate iMPC is about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours. Claim 7 A method for producing iPSC-derived wall cell precursors (iMPCs), comprising the step of inducing MePCs to express NK3 homeobox 1 (NKX3.1) or a functional variant thereof for a period sufficient to generate iMPCs. Claim 8 A method according to claim 7, wherein MePC comprises a nucleic acid encoding NKX3.1 or a functional variant thereof. Claim 9 A method according to claim 7, wherein the induction comprises transfecting a MePC with a nucleic acid encoding NKX3.1 or a functional variant thereof. Claim 10 A method according to claim 8 or 9, wherein the nucleic acid is DNA (optional, plasmid, vector, viral vector) or RNA molecule (optional, mRNA or modified RNA (modRNA)). Claim 11 A method according to any one of claims 8 to 10, wherein the nucleic acid comprises an inducible promoter (e.g., a doxycycline-inducible promoter) that controls the expression of NKX3.

1. Claim 12 A method according to claim 11, wherein the induction comprises contacting the cell with an agent that activates an inducible promoter (e.g., doxycycline). Claim 13 A method according to any one of claims 7 to 12, wherein the period sufficient to generate iMPC is about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours. Claim 14 A method for generating a population of wall cells including perivascular cells, smooth muscle cells, and fibroblasts, comprising: generating iMPCs using the method of any one of claims 1 to 13; and co-culture the iMPCs with a population of endothelial cells (ECs) for a period sufficient to generate a population of wall cells including perivascular cells, smooth muscle cells, and fibroblasts. Claim 15 A method according to claim 14, wherein the period sufficient to generate a population of wall cells including perivascular cells, smooth muscle cells, and fibroblasts is about 1, 2, 3, 4, 5, 6, or 7 days. Claim 16 A method for increasing angiogenesis in a subject, comprising the step of administering a therapeutically effective amount of EC and iMPC to the subject. Claim 17 A group of cells produced by the method of any one of paragraphs 1 to 13. Claim 18 A population of cells containing at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% iPSC-derived wall cell precursors (iMPCs). Claim 19 In paragraph 18, a population of cells in which the iMPC contains a nucleic acid encoding NKX3.1 (optional, exogenous nucleic acid) or transiently expresses NKX3.1 (optional, from a degradable nucleic acid (e.g., mRNA or modRNA)). Claim 20 In claim 18 or 19, a population of cells in which iMPCs express PDGFRβ (CD140b) and aminopeptidase N (CD13). Claim 21 A population of cells according to any one of claims 18 to 20, wherein less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the iMPCs express the TRA1-81 antigen. Claim 22 In any one of paragraphs 18 through 21, iMPC ACTA2 , CNN1 , TAGLN , MYOCD , MYH11 , CSPG4 , DES , PDE5A , and THY1 A group of cells that express Claim 23 A population of cells containing endothelial cells (EC):iPSC-derived wall cell precursors (iMPCs) in a ratio of approximately 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:

1. Claim 24 In paragraph 23, a population of cells in which iMPC contains a nucleic acid encoding NKX3.1 (optional, exogenous nucleic acid) or transiently expresses NKX3.1 (optional, from a degradable nucleic acid (e.g., mRNA or modRNA)). Claim 25 A population of cells in which iMPCs express PDGFRβ (CD140b) and aminopeptidase N (CD13), in claim 23 or 24. Claim 26 A population of cells according to any one of claims 23 to 25, wherein less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the iMPCs express the TRA1-81 antigen. Claim 27 In any one of paragraphs 23 through 26, iMPC ACTA2 , CNN1 , TAGLN , MYOCD , MYH11 , CSPG4 , DES , PDE5A , and THY1 A group of cells that express Claim 28 A population of cells according to any one of paragraphs 23 through 27, wherein the EC comprises any one or more of iPSC-derived ECs (iECs), human venous endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs, or organ-specific endothelial cells. Claim 29 In paragraph 28, the organ-specific endothelial cells are from an organ selected from the following: heart, muscle, kidney, testis, ovary, lymphatic system, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine. Claim 30 A population of cells according to claim 28 or 29 in which the iEC contains a nucleic acid encoding ETV2 (optional, exogenous nucleic acid) or transiently expresses ETV2 (optional, from a degradable nucleic acid (e.g., mRNA or modRNA)). Claim 31 A population of cells in which, in any one of claims 28 to 30, NKX3.1 expression is controlled by an inducible promoter and ETV2 expression is controlled by an inducible promoter. Claim 32 In paragraph 31, a population of cells in which the inducible promoter for NKX3.1 and the inducible promoter for ETV2 are the same (optional, doxycycline). Claim 33 A population of cells in which the inducible promoter for NKX3.1 and the inducible promoter for ETV2 are not identical, as in paragraph 31. Claim 34 A vascular organoid (VO) or three-dimensional (3D) cell culture comprising a population of cells according to any one of claims 18 to 33. Claim 35 A method for preparing an vascular organoid (VO) or a three-dimensional (3D) cell culture comprising: (i) a step of culturing a first population of iPSC-derived mesenchymal cell precursors ("ETV2 / MePC") containing a nucleic acid encoding ETV2 or a functional variant thereof (optional, exogenous nucleic acid) with a second population of MePCs ("NKX3.1 / MePC") containing a nucleic acid encoding NKX3.1 or a functional variant thereof (optional, exogenous nucleic acid), wherein the expression of NKX3.1 is controlled by an inducible promoter and the expression of ETV2 is controlled by an inducible promoter; (ii) inducing the expression of NKX3.1 in NKX3.1 / MePC to thereby produce iPSC-derived wall cell precursors (iMPCs) and inducing the expression of ETV2 in ETV2 / MePC to thereby produce iPSC-derived A step of generating endothelial cells (iEC); and (iii) a step of culturing the cells for a period sufficient to generate a VO or 3D cell culture, wherein the VO or 3D cell culture comprises iECs that are h-CD31+ and iMPCs that are PDGFRβ+ and h-CD31-. Claim 36 In paragraph 35, the method in which the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are the same (optional, doxycycline). Claim 37 A method in which, in paragraph 35, the inductive promoter for NKX3.1 and the inductive promoter for ETV2 are not identical. Claim 38 A method according to any one of claims 35 to 37, wherein the period sufficient to produce VO or 3D cell culture is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. Claim 39 A method according to any one of claims 35 to 38, wherein (i) the culture step occurs for about 1 day or 2 days and / or; (i) the culture step occurs for about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours and / or; and the culture occurs for a period sufficient to produce aggregates comprising both NKX3.1 / MePC and ETV2 / MePC. Claim 40 A method according to any one of claims 35 to 39, wherein (i) the culture step comprises culturing cells using a non-adhesive culture plate and a rotary shaker. Claim 41 A method according to any one of claims 35 to 40, wherein a group of NKX3.1 / MePC and a group of ETV2 / MePC are mixed in a ratio of approximately 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:1 of NKX3.1 / MePC:ETV2 / MePC. Claim 42 A method for producing a vascular organoid (VO) or a three-dimensional (3D) cell culture comprising: (i) transfecting a population of iPSC-derived mesenchymal precursors (MePCs) with a nucleic acid (optional, RNA, mRNA, modRNA) encoding NKX3.1 or a functional variant thereof to thereby produce a population of iPSC-derived wall cell precursors (iMPCs); (ii) mixing a population of iMPCs with a population of ECs (optional, in a ratio of about 1:3, 1:2, 2:3, 1:1, 3:2, 1:2, or 3:1 iMPCs:ECs); and (iii) culturing the cells for a period sufficient to produce a VO or 3D cell culture, wherein the VO or 3D cell culture comprises iECs that are h-CD31+ and iMPCs that are PDGFRβ+ and h-CD31-. Claim 43 In paragraph 42, the method wherein the EC comprises any one or more of iPSC-derived ECs (iECs), human venous endothelial cells (HUVECs), endothelial colony-forming cells (ECFCs), adipose tissue-derived ECs, or organ-specific endothelial cells. Claim 44 In paragraph 42, the organ-specific endothelial cells are from an organ selected from the following: heart, muscle, kidney, testis, ovary, lymphatic system, liver, pancreas, brain, lung, bone marrow, spleen, large intestine, and small intestine. Claim 45 A method according to claim 42, further comprising the step of transfecting a population of iPSCs with a nucleic acid encoding ETV2 or a functional variant thereof before mixing with a population of iMPCs, thereby generating a population of ECs. Claim 46 In any one of paragraphs 35 to 45, VO is of uniform size and / or approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, or 550 μm diameter and / or; The average diameter of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or; The median size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or;A method in which a VO or 3D cell culture comprises approximately 1,000, 1,500, 2,000, 2,250, 2,500, 2,550, 2,600, 2,650, 2,700, 2,750, 2,800, 2,850, 2,900, 2,950, 3,000, 3,050, 3,100, 3,150, 3,200, 3,250, 3,300, 3,350, 3,400, 3,450, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, or 5,000 cells.; Claim 47 A method according to any one of claims 35 to 46, wherein the cells are self-assembled into a network of CD31+ vascular structures containing mature α-SMA+ perivascular wall cells. Claim 48 A method according to any one of claims 35 to 47, wherein VO comprises a network of reinforced blood vessels having apical-basal polarization and / or VO comprises arteries, veins, and / or capillary ECs. Claim 49 In any one of paragraphs 35 through 48, VO CDH5+ and VWF + EC and ACTA2 +, MYH11 +, TAGLN +, and CNN1 A method that includes +wall cells. Claim 50 VO or 3D cell culture produced by the method of any one of claims 35 to 49. Claim 51 In paragraph 50, the VO or 3D cell culture is of uniform size and / or; VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, or 550 μm diameter and / or; The average diameter of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or; The median size of the VO or 3D cell culture is approximately 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μm in diameter and / or;A VO or 3D cell culture comprising approximately 1,000, 1,500, 2,000, 2,250, 2,500, 2,550, 2,600, 2,650, 2,700, 2,750, 2,800, 2,850, 2,900, 2,950, 3,000, 3,050, 3,100, 3,150, 3,200, 3,250, 3,300, 3,350, 3,400, 3,450, 3,500, 3,750, 4,000, 4,250, 4,500, or 5,000 cells.; Claim 52 A VO or 3D cell culture according to claim 50 or 51, wherein the VO or 3D cell culture comprises a CD31+ vascular structure containing mature α-SMA+ perivascular wall cells. Claim 53 A VO or 3D cell culture according to any one of claims 50 to 52, wherein the VO or 3D cell culture comprises a network of reinforced blood vessels having apical-basal polarization and arteries, veins, and / or capillary ECs. Claim 54 In any one of paragraphs 50 to 53, VO or 3D cell culture is an EC marker in VO-iEC ( CDH5 ; VWF ) (Fig. 9b) and several wall cell markers in VO-iMPC ( ACTA2 , MYH11 , TAGLN , CNN1 VO or 3D cell culture containing ). Claim 55 A composition comprising a group of cells according to any one of claims 17 to 33 or a VO or 3D cell culture according to any one of claims 34 and 50 to 54. Claim 56 A composition according to claim 55, further comprising any one or more of an agent, an excipient, a matrix, or a gel. Claim 57 A composition according to claim 56, wherein the gel or matrix comprises a hydrogel. Claim 58 A composition according to claim 56, wherein the gel or matrix comprises gelatin, collagen, fibrinogen, thrombin, fibrin, or a combination thereof. Claim 59 A composition according to claim 56, wherein the gel or matrix comprises about 1.5 mg / mL collagen, about 30 μg / mL fibrinogen, and about 1 mg / mL human fibronectin. Claim 60 A composition according to claim 56, wherein the gel or matrix comprises any one or more of gelatin, laminin, entactin, collagen, fibrinogen, and combinations thereof. Claim 61 A composition according to claim 56, wherein the gel or matrix comprises laminin, entactin, and collagen. Claim 62 A composition according to claim 56, wherein the gel or matrix comprises about 5.25 mg / mL laminin, about 5.25 mg / mL entactin, and about 0.2 mg / mL collagen IV. Claim 63 A composition according to claim 56, wherein the gel or matrix is ​​Matrigel™. Claim 64 A method for implanting a population of cells according to any one of claims 17 to 33, a vascular organoid according to any one of claims 34 and 50 to 54, or a composition according to any one of claims 55 to 63 into a subject, comprising the following: administering an effective amount of the population of cells, the vascular organoid, or the composition to the subject. Claim 65 A method for increasing angiogenesis in a subject, comprising: identifying a subject requiring increased angiogenesis; and administering to the subject an effective amount of a population of cells of any one of claims 17 to 33, a vascular organoid of any one of claims 34 and 50 to 54, or a composition of any one of claims 55 to 63. Claim 66 A method for increasing angiogenesis or vascular regeneration in a subject, comprising: identifying a subject requiring angiogenesis or vascular regeneration; and administering to the subject an effective amount of a population of cells according to any one of claims 17 to 33, a vascular organoid according to any one of claims 34 and 50 to 54, or a composition according to any one of claims 55 to 63. Claim 67 A method for vascular cell therapy comprising: a step of identifying a subject requiring vascular cell therapy; and a step of administering to the subject an effective amount of a population of cells according to any one of claims 17 to 33, a vascular organoid according to any one of claims 34 and 50 to 54, or a composition according to any one of claims 55 to 63. Claim 68 A method according to any one of paragraphs 16 and 64 through 67, wherein the subject has (or has or is at risk of having) any one or more of the following: diabetes mellitus, diabetic retinopathy, ischemic injury, vascular disease or disorder, atherosclerosis, age-related macular degeneration (AMD), pulmonary hypertension (PAH), hereditary hemorrhagic telangiectasia (HHT), peripheral arterial disease (PAD), arteriovenous fistula (e.g., in dialysis patients), tumor angiogenesis, tumor metastasis, stroke, and / or wound (optional, chronic wound; e.g., diabetic ulcer). Claim 69 A method according to any one of claims 16 and 64 through 68, wherein a population of cells, a vascular organoid, or a composition is administered to a subject before, during, or after cell transplantation, tissue transplantation, or organ transplantation. Claim 70 A method according to any one of claims 16 and 64 to 69, wherein a population of cells, a vascular organoid, or a composition is administered to a subject by direct injection into a blood vessel or by subcutaneous, intradermal, intramuscular, intralymphatic, intravenous, prostatic, intratumoral, intralymphatic, and intraperitoneal injection.

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