Human placenta-derived angiogenic stem cells (hPASCs) and application thereof

Abstract

The invention provides human placenta-derived vasogenic stem cells (hPASCs) and application thereof. The human placenta-derived angiogenic stem cells (hPASCs) disclosed by the invention are preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, China; the preservation date is April 2, 2025; the preservation number of the strain is CCTCC (China Center For Type Culture Collection) NO: The hPASCs disclosed by the invention contain angiogenic cell subgroups, have stem cell stemness, have an angiogenesis effect in vivo and in vitro, and can be used for treatment of diseases such as cardiovascular diseases and vascularization construction of artificial organs and tissues.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to human placenta-derived angiogenic stem cells (hPASCs) and applications thereof. Background Art

[0002] End-stage kidney disease (ESKD) is the terminal stage of chronic kidney disease (CKD) characterized by irreversible loss of renal function. ESKD is primarily treated with dialysis and kidney transplantation. Dialysis carries a financial burden and infection risks for patients, while kidney transplantation requires lifelong immunosuppressive medication and is severely limited. In 2010, a report estimated that 4.902 million to 7.083 million ESKD patients worldwide required kidney transplantation (Adv Exp Med Biol 2019, 1165:3-15). However, the number of patients awaiting kidney transplantation far exceeds the number of organ donors, and millions of people die annually from renal failure due to a lack of kidneys (Lancet 2015, 385(9981):1975-1982). Bioengineered kidneys, designed to utilize autologous or allogeneic cells and engineered biomimetic scaffolds to construct replacement tissue in vitro and then implant it to replace, restore, or enhance the function of damaged kidneys, represent one strategy to address this organ shortage.

[0003] Currently, the widely studied scaffold materials for kidney tissue engineering include synthetic polymers such as polycaprolactone (PCL), polylactic acid (PLA) and polyvinyl alcohol (PVA) and natural decellularized scaffolds (Bioengineering (Basel) 2022, 9(10)). Synthetic scaffold materials are difficult to simulate the microenvironment in the body and the mechanical or biochemical properties of natural tissues, overcome immunogenicity, and have limited repair capabilities (Bioact Mater 2022,10:15-31). Kidney decellularized scaffolds remove immunogenic cell components, have good biocompatibility and low rejection rate (Biomater Res 2023,27(1):10), and retain the 3D ultrastructure of blood vessels, glomeruli and tubules, some signals and growth factor proteins, providing a microenvironment for the adhesion, proliferation, migration, differentiation, colonization and growth of implanted cells (Biomaterials 2013, 34(28):6670-6682.). In addition, after in situ transplantation of rat renal decellularized scaffold materials, the exposed extracellular matrix causes thrombosis (Curr Opin Hematol 2016, 23(3):280-287). The lack of parenchymal cell infiltration to fill the scaffold leads to the absorption of the extracellular matrix by inflammatory cell infiltration or protease hydrolysis (Sci Rep 2017, 7:43502). Simply transplanting renal decellularized scaffold materials cannot effectively achieve organ regeneration. Decellularized scaffold material grafts lack blood, oxygen, and nutrient supply in the early stages, and it takes time for the recipient vascular network to grow into the graft tissue. The vascular network plays an important role in the filtration function of the cortical glomeruli and the supply of oxygen and nutrients to maintain the reabsorption and secretion function of the medullary renal tubules. Vascularization is an essential component for the construction of a complete engineered kidney organ. Therefore, the construction of a vascularized tissue-engineered kidney is the fundamental solution to the problem of graft survival (Tissue Eng Part B Rev 2022, 28(1):1-21).

[0004] The vascular system is composed of vessels of varying sizes, anatomy, physiology, and cellular arrangements. Capillaries harbor endothelial progenitor cells, endothelial cells, perivascular cells, smooth muscle cells, vascular macrophages, and vascular wall stem cells. Therefore, cells used for vascularization are divided into two main categories: endothelial cells and supporting cells (Front Bioeng Biotechnol 2023, 11:1103727). Human umbilical vein endothelial cells (hUVECs), or ECs, are currently the most widely used seed cells for vascularization in tissue engineering. hUVECs have been used for vascularization of decellularized scaffolds in organs such as kidney (Nat Med 2013, 19(5):646-651; Front Bioeng Biotechnol 2023, 11:1184408.), liver (Nat Biomed Eng 2020, 4(4):437-445), heart (Biomaterials 2015, 61:279-289), and lung (Ann Surg 2018, 267(3):590-598). However, implanted exogenous ECs cannot further develop into organ-specific vascular phenotypes. Non-organ-specific ECs affect the quality of vascularization, cannot connect with host-specific blood vessels to form a functional vascular network that supports blood circulation, and hinder the regeneration of parenchymal cells in the organ (Trends Biotechnol 2018, 36(8):834-849). In addition, hUVECs do not adhere firmly to decellularized scaffold materials and are easily detached with blood flow, leading to thrombosis (Bioact Mater 2021, 6(8):2557-2568.). As allogeneic antigens, vascular endothelial cells are the preferred targets of autoantibodies in allogeneic tissue or organ transplantation (Front Immunol 2022, 13:). Allogeneic human endothelial cells not only express MHCⅠ and MHCⅡ but also constitutively express costimulatory factors such as PD-L1. They provide costimulatory signals through direct allogeneic recognition to activate T cells and antibody-mediated rejection reactions, activating the complement cascade near allogeneic endothelial cells to form membrane attack complexes (MACs) and local inflammatory mediators, enhancing the direct allogeneic reaction between endothelial cells and T cells, and ultimately leading to strong immune rejection reactions (Trends Biotechnol 2018, 36(8):834-849.). Therefore, directly implanting ECs into renal decellularized scaffold materials to construct vascularized tissue-engineered kidneys cannot effectively achieve vascularization and graft survival. EC construction of vascularized tissue-engineered kidneys has certain limitations.

[0005] To improve the implantation efficiency and function of ECs in decellularized scaffold materials, mesenchymal stem cells (MSCs) were co-transplanted with ECs. Rat glomerular endothelial cells and rat bone marrow mesenchymal stem cells were co-cultured in a renal decellularized scaffold in vitro to achieve microvascularization. MSCs reduced endothelial cell inflammatory damage, reduced cell apoptosis, supported vascular stability, and improved cell viability, cell proliferation, cell migration, and vascularization, resulting in an ECM-derived implantable renal scaffold. However, this study lacked in vivo experiments to verify whether a functional vascular network was successfully constructed (Mater Today Bio 2022, 17:100464). MSCs cannot spontaneously differentiate into endothelial conduits that support blood flow in vivo (Blood 2008, 111(9):4551-4558). MSCs are derived from a wide range of sources, such as adipose tissue and bone marrow, and their multi-lineage differentiation potential is related to their tissue source, i.e., tissue heterogeneity exists (Proc Natl Acad Sci USA 2014, 111(28):10137-10142.). Although some progress has been made in the construction of vascularized decellularized scaffold materials by combined transplantation of MSCs and ECs, a complete vascular network structure and function has not been constructed in vivo, and the tissue-engineered organs formed by in vivo transplantation are significantly different from the regenerated tissues. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present disclosure provides a novel human placenta-derived angiogenic stem cell and its application in the construction of bioengineered organs.

[0007] The first aspect of the present invention provides a human placenta-derived angiogenic stem cell hPASCs Homo sapiens, wherein the hPASCs are deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; deposit date: April 2, 2025; deposit number: CCTCC NO: C202592.

[0008] A second aspect of the present invention provides human placenta-derived angiogenic stem cells (hPASCs), which express markers PAI-1, MECOM, CD201, CD44, CD73, and HLA-ABC, but do not express markers CD34, CD146, CD31, and CD326; preferably, the hPASCs are derived from placental vascular tissue macrophages and / or vascular wall stem cells.

[0009] A third aspect of the present invention provides a method for isolating human placenta-derived angiogenic stem cells (hPASCs), comprising the following steps: 1) digesting human placental tissue; 2) sedimenting the digestion products to obtain a supernatant; 3) filtering the supernatant through a filter, and backflushing the filter to obtain placental microvascular tissue; 4) culturing the placental microvascular tissue in hPASC culture medium; 5) subculturing primary cells; and 6) identifying hPASCs.

[0010] In some embodiments, the diameter of the placental microvascular tissue is 20 μm-100 μm, preferably 40 μm-70 μm.

[0011] In some embodiments, the hPASC culture medium uses peripheral cell culture medium as the basal medium and is supplemented with penicillin / streptomycin dual antibodies.

[0012] In some embodiments, the peripheral cell culture medium is Pericyte Medium, product number 1201, purchased from ScienCell.

[0013] In some embodiments, the serum in the Pericyte Medium is replaced with Platelet Lysate.

[0014] In some embodiments, the identification in step 6) includes defining cells having one or more characteristics selected from the following as hPASCs: a) containing four angiogenic stem cell subpopulations and having stem cell "stemness"; b) being able to form vascular structures in in vitro or in vivo culture; c) surface markers are PAI-1 positive, MECOM positive, CD201 positive, CD44 positive, CD73 positive, HLA-ABC positive, CD34 negative, CD146 negative, CD31 negative and CD326 negative.

[0015] In some embodiments, the angiogenic stem cell subpopulation is obtained by single-cell sequencing analysis.

[0016] In some embodiments, the stem cell "stemness" is determined by CytoTRACE analysis.

[0017] In some embodiments, the ability to form vascular structures in vitro is assessed by in vitro 3D Matrigel or 3D "sandwich" culture.

[0018] The fourth aspect of the present invention provides human placenta-derived angiogenic stem cells (hPASCs) separated according to the separation method described in the third aspect of the present invention.

[0019] The fifth aspect of the present invention provides a composition comprising the human placenta-derived angiogenic stem cells hPASCs according to the first, second or fourth aspect of the present invention.

[0020] The sixth aspect of the present invention provides use of the human placenta-derived angiogenic stem cells hPASCs described in the first, second or fourth aspect of the present invention or the composition described in the fifth aspect in the preparation of a medicament for alleviating symptoms or treating diseases.

[0021] In some embodiments, the disease or condition is selected from cardiovascular disease, diabetic complications, neurological diseases, ophthalmic diseases, chronic wounds, and ulcers.

[0022] In some embodiments, the cardiovascular disease is selected from the group consisting of myocardial infarction, chronic ischemic heart disease, peripheral arterial disease, coronary artery restenosis, and heart failure.

[0023] In some embodiments, the diabetic complication is selected from diabetic foot ulcer, diabetic retinopathy, diabetic nephropathy, and diabetic peripheral neuropathy.

[0024] In some embodiments, the neurological disease is selected from ischemic stroke, spinal cord injury, Alzheimer's disease, Parkinson's disease, and multiple sclerosis.

[0025] In some embodiments, the ophthalmic disease is selected from age-related macular degeneration, retinal vein occlusion, corneal neovascular disease, and glaucomatous optic neuropathy.

[0026] In some embodiments, the chronic wound is selected from burn wounds, postoperative refractory wounds and pressure sores.

[0027] In some embodiments, the ulcer is selected from the group consisting of a venous ulcer, an arterial ulcer, and a radiation ulcer.

[0028] A seventh aspect of the present invention provides the use of the human placenta-derived angiogenic stem cells (hPASCs) described in the first, second, or fourth aspects of the present invention, or the composition described in the fifth aspect, in tissue engineering and regenerative medicine. These applications include promoting the vascularization of artificial organs, skin regeneration, bone and cartilage regeneration, neural tissue repair, and reconstruction of complex tissue structures. In some embodiments, these applications are performed in vitro; in some embodiments, these applications are not intended for direct therapeutic purposes.

[0029] The eighth aspect of the present invention provides the use of the human placenta-derived angiogenic stem cells hPASCs described in the first, second or fourth aspect of the present invention or the composition described in the fifth aspect in the preparation of bioengineered artificial organs.

[0030] In some embodiments, the artificial organ includes an engineered kidney, an engineered blood vessel, an engineered liver, a heart patch, an artificial skin, an artificial trachea, an artificial bladder, and an artificial cornea; preferably, the bioengineered artificial organ is a bioengineered kidney.

[0031] The ninth aspect of the present invention provides a method for preparing a bioengineered organ, comprising the step of implanting the human placenta-derived angiogenic stem cells hPASCs and organoids described in the first, second or fourth aspect of the present invention into a decellularized scaffold.

[0032] In some embodiments, the bioengineered organ is selected from an engineered kidney, an engineered blood vessel, an engineered liver, a heart patch, an artificial skin, an artificial trachea, an artificial bladder, and an artificial cornea; preferably, the bioengineered artificial organ is a bioengineered kidney.

[0033] In some embodiments, the bioengineered organ is a bioengineered kidney.

[0034] In some embodiments, the organoid is a kidney organoid.

[0035] In some embodiments, the decellularized scaffold is a kidney decellularized scaffold.

[0036] In some embodiments, the decellularized scaffold is obtained by decellularization of an ex vivo organ.

[0037] In some embodiments, the decellularization treatment includes a perfusion step of administering a decellularized perfusion fluid to the isolated organ; preferably, the perfusion is performed using a peristaltic pump; preferably, the perfusion step is performed once or more; preferably, the perfusion fluid is selected from one or more of heparin sodium solution, TritonX-100 and SDS solution; optionally, a post-perfusion washing step is further included.

[0038] The tenth aspect of the present invention provides a bioengineered organ comprising the human placenta-derived angiogenic stem cells hPASCs as described in the first, second or fourth aspect of the present invention or the composition as described in the fifth aspect of the present invention.

[0039] In some embodiments, the bioengineered organ is obtained according to the method of the eighth aspect of the present invention; preferably, the bioengineered organ is a bioengineered kidney.

[0040] The eleventh aspect of the present invention provides use of the bioengineered organ described in the ninth aspect of the present invention in preparing a drug or medical device for treating a disease; preferably, the bioengineered organ is a bioengineered kidney, and the drug or medical device is used to treat kidney disease.

[0041] The advantages of the present invention over the prior art are:

[0042] 1) This study isolated a novel stem cell line, hPASCs, from the placenta. This cell line contains four angiogenic cell subsets, exhibiting stem cell properties and promoting angiogenesis. hPASCs positively express PAI-1, MECOM, CD201, CD44, CD73, and HLA-ABC; they do not express hematopoietic stem cell and endothelial progenitor cell markers (CD34), pericyte marker (CD146), endothelial cell marker CD31, or epithelial cell marker CD326. Single hPASC cell association analysis with single cells from placental microvascular tissue revealed that hPASCs develop from placental vascular macrophages and / or vascular wall stem cells / or endothelial progenitor cells (EPCs).

[0043] 2) The biomechanical characteristics of hPASCs are different from those of hUVECs and hUCMSCs, and their traction force is between the two.

[0044] 3) hPASCs can form vascular structures when cultured on 3D Matrigel, while hUCMSCs form aggregates but cannot form vascular structures; hPASCs can form vascular structures in 3D "sandwich" culture, while hUVECs and hUCMSCs only form aggregates but cannot form vascular structures.

[0045] 4) hPASCs composited with decellularized scaffold materials can form vascular structures in vitro after in vitro culture, and have stronger adhesion ability to the material than hUVECs and hUCMSCs.

[0046] 5) hPASCs composite acellular scaffold material formed more vascular structures than hUVECs and hUCMSCs in in vivo renal capsule transplantation and in vivo nephrectomy transplantation, and had stronger angiogenic effect and function than the two.

[0047] 6) hPASCs composited with acellular scaffold materials participate in host angiogenesis and differentiate into vascular cells after in vivo renal capsule transplantation and in vivo nephrectomy transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1hPASCs demonstrate stem cell potential and angiogenic molecular signatures. A, hPASC isolation and single-cell sequencing workflow; B, hPASC morphology, scale bar 100 µm; C, hPASC cell type analysis using single-cell RNA sequencing (ScRNA-seq); D, hPASC marker gene analysis using ScRNA-seq; E, ScRNA-seq GO enrichment analysis; F, hPASC stem cell signature analysis and functional phenotype enrichment; G, hPASC stemness analysis using CytoTRACE; H, hPASC stemness potential analysis; I, hPASC stemness relative coefficient; J, hPASC immunofluorescence characterization, scale bar 20 µm; K, hPASC flow cytometry analysis.

[0049] Figure 2 Figure 1 shows single-cell sequencing of human placental vascular tissue and the localization and origin of hPASCs in microvascular tissue. A, Single-cell sequencing analysis of human placental vascular tissue to analyze cell types. B, C, Correlation analysis of hPASCs and human placental vascular tissue single cells. D, Chronological analysis of the correlation between hPASCs and human placental microvascular tissue cell types.

[0050] Figure 3 Figure 3: Angiogenic potential of hPASCs in 3D or 3D "sandwich" cultures. A, Single-cell traction force measurement (TFM) imaging; B, Single-cell root mean square (RMS) and total strain energy (pJ); n = 17, P < 0.05; C, 3D culture model; D, hPASCs in 3D culture exhibit angiogenic potential; E, 3D culture of hUVECs with hUCMSCs or hPASCs at varying ratios; F, Live cell imaging of hPASCs in continuous dynamic 3D culture demonstrates angiogenic potential; G, 3D "sandwich" culture model; HI, hPASCs form vascular networks after 72 h of 3D "sandwich" culture; Scale bar 50 µm.

[0051] Figure 4 hPASCs form vascular structures after in vitro perfusion and culture on a decellularized kidney scaffold. A, B, CD31 and α-SMA immunofluorescence demonstrates hPASCs remodeling vascular structures (arrows). Scale bars: 100 µm, 200 µm. C, Ultrastructure of a vascularized bioengineered kidney: SEM reveals hPASCs forming intercellular connections (arrows). Scale bars: 100 µm, 20 µm. D, TEM demonstrates hPASCs forming connections and adhering to the scaffold, while other groups form necrotic structures (arrows). Scale bars: 1 µm, 2 µm.

[0052] Figure 5 hPASCs implanted into a decellularized scaffold and cultured on the renal capsule promote angiogenesis. A, HE staining; scale bars 50 µm, 100 µm. B, CD31 immunofluorescence; scale bar 200 µm. C, Statistical analysis of vascular density: DKS and DKS+KIO groups (n=3), other groups (n=5); P < 0.05. D, mCherry-hPASCs co-express vascular markers EDH3, CD31, and α-SMA, contributing to host angiogenesis (scale bar 20 µm).

[0053] Figure 6 hPASCs implanted in a decellularized scaffold for culture and transplantation into nephrectomy tissue promote angiogenesis. A, Ultrasound 3D vascular modeling of the implant. B, CD31 immunofluorescence staining, scale bar 200 µm. C, Statistical analysis of vascular density: DKS and DKS+KIO groups (n=4); other groups (n=5), P<0.05. D, Fluorescent tracking of hPASCs with mCherry labeling shows co-expression of hPASCs with the vascular markers EDH3, CD31, and α-SMA, indicating their contribution to host vascular formation. Scale bar 20 µm.

[0054] Figure 7 Demonstrating the mechanism by which hPASCs promote angiogenesis in vivo. A, RNA-seq sequencing analysis: GO enrichment analysis; B, KEGG enrichment analysis; n = 3, P < 0.05. C, Western blotting analysis of pAKT and VEGFA proteins in subcapsular renal grafts, n = 3, P < 0.05; D, Western blotting analysis of pAKT and VEGFA proteins in nephrectomy grafts, n = 3, P < 0.05. Detailed Description of the Invention

[0056] Some embodiments according to the present disclosure will be described more fully below. However, various aspects of the present disclosure may be embodied in different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to make the present disclosure thorough and complete and to fully convey the scope of the present disclosure to those skilled in the art. The terms used in the description herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0057] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It should also be understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of this application and the related art, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0058] Unless explicitly stated otherwise, the various features of the present disclosure described herein may be used in any combination. In addition, the present disclosure also contemplates that in an embodiment, any feature or combination of features set forth herein may be excluded or omitted.

[0059] Unless expressly stated otherwise, all references to certain embodiments, features, and terms are intended to include both the recited embodiment, feature, or term and its biological equivalents.

[0060] All references, articles, publications, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any reference, article, publication, patent publication and patent application cited herein is not and should not be taken as an admission or any form of representation that they constitute available prior art or form part of the common general knowledge in any country in the world.

[0061] Each of the embodiments described and illustrated herein has separate components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method may be executed in the order of events recited or in any other order that is logically possible.

[0062] Experimental methods in the following examples disclosed herein, unless otherwise specified, generally follow conventional conditions or those recommended by the manufacturer. The various commonly used chemical and biological reagents used in the examples are commercially available. Unless otherwise noted, this disclosure is not limited to specific materials, reagents, tools, etc.

[0063] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art.

[0064] definition

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0066] As used herein, the term "about" or "approximately" is intended to encompass variations of, in some cases, ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value.

[0067] The terms used in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure. In the present disclosure, it is also possible that steps can be performed in different orders if it is logically feasible.

[0068] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0069] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limits of that range and any other intervening values ​​stated or within that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0070] The terms "comprising," "containing," "including," and "having," and any variations thereof, as used herein, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device. This disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0071] In this disclosure, "plurality" refers to two or more. "And / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0072] With respect to the recitation of numerical ranges herein, each intervening number with equal precision is expressly contemplated. For example, for the range 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0073] As used herein, the term "or" means one, both, or any combination of alternatives, and is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0074] As used herein, the term "stem cell" refers to a type of cell with unlimited or immortal self-renewal capacity, capable of producing at least one type of highly differentiated progeny cell. Most consider stem cells to be cells derived from embryos, fetuses, or adults that, under certain conditions, possess the capacity for unlimited self-renewal, proliferation, and differentiation. They can produce daughter cells with identical phenotypes and genotypes, give rise to specialized cells that comprise tissues and organs, and can also differentiate into progenitor cells.

[0075] As used herein, the term "human placenta-derived angiogenic stem cells (hPASCs)" or "hPASCs" or "human placenta-derived angiogenic stem cells" refers to stem cells isolated and cultured from the human placenta. These cells possess stem cell characteristics and have the potential to differentiate into endothelial cells and smooth muscle cells; they also exhibit angiogenic function when cultured in vitro. The human placenta-derived angiogenic stem cells (hPASCs) used in this invention were deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on April 2, 2025. The deposit number is CCTCC NO: C202592. The hPASCs isolated by the present invention are derived from placental vascular tissue macrophages, vascular wall stem cells and / or vascular endothelial progenitor cells. The hPASCs positively express PAI-1, MECOM, CD201, CD44, CD73 and HLA-ABC; and do not express hematopoietic stem cell and endothelial progenitor cell markers (CD34), pericyte markers (CD146), endothelial cell markers CD31 and epithelial cell markers CD326.

[0076] As used herein, the term "MSCs" (Mesenchymal Stem Cells or Mesenchymal Stromal Cells) refers to a type of adult stem cell / stromal cell with self-renewal and immunomodulatory capabilities. They are widely present in various tissues and participate in tissue repair, immune regulation, and microenvironmental homeostasis by differentiating into specific cell types or secreting bioactive molecules. MSCs are derived from sources such as bone marrow, adipose tissue, Wharton's jelly in the umbilical cord, placenta, dental pulp, and synovium. They can be induced to differentiate into osteoblasts, adipocytes, and chondrocytes ("gold standard" definition), and under certain conditions, they can also differentiate into neuronal cells and endothelial cells.

[0077] As used herein, the term "hUCMSCs" (Human Umbilical Cord Mesenchymal Stem Cells) refers to a type of multipotent mesenchymal stem cell isolated from neonatal umbilical cord tissue (such as Wharton's jelly). These stem cells possess self-renewal capacity, multidirectional differentiation potential, and immunomodulatory properties, making them an important cell source for regenerative medicine and cell therapy. hUCMSCs express mesenchymal stem cell markers (CD73, CD90, and CD105) but lack hematopoietic markers (CD34, CD45) and HLA-DR. They can differentiate into osteoblasts, adipocytes, chondrocytes, and other cells. hUCMSCs can be used for tissue repair and regeneration, immunomodulatory therapy, and anti-fibrosis and anti-aging treatments.

[0078] As described in this article, the term "hUVECs" (Human Umbilical Vein Endothelial Cells) is a primary endothelial cell isolated from the inner wall of the umbilical vein of a newborn. It has typical vascular endothelial properties and is widely used in vascular biology research, drug screening, and the construction of vascularization models in tissue engineering.

[0079] As used herein, the term "bioengineered organ," also known as a biologically engineered organ, is a biologically active organ that is cultivated using cells or tissues from an animal. "Bioengineered organs" are further categorized as xenogeneic and autologous. Xenogeneic organs are human organs cultivated in non-human animals (such as pigs or gene-edited pigs, rabbits, mice, and dogs), while autologous organs are cultivated using the patient's own cells or tissues.

[0080] As used herein, the term "decellularized scaffold" or "DKS" refers to a decellularized matrix scaffold in which cells and some antigenic components that cause immune rejection reactions have been removed from tissues. Its characteristic is that it reduces the ability to cause immune rejection when implanted in the body. After decellularization, the original extracellular matrix structure of tissues and organs, some non-antigenic active components such as polysaccharides, collagen, glycoproteins and fibronectin are retained, and many natural cell binding sites are preserved in the scaffold, which helps the adhesion, proliferation and differentiation of cells during the regeneration process. At present, decellularized scaffolds have been widely used in organ regeneration, tissue repair and reconstruction, bioprinting, disease models and drug screening, and cell culture in whole organ engineering.

[0081] As used herein, the term "organoid" refers to tissue analogs with a defined spatial structure formed by three-dimensional (3D) culture of adult or pluripotent stem cells in vitro. Organoids are not true human organs, but they mimic the structure and function of real organs, closely resembling the in vivo tissue structure and function and capable of long-term, stable culture. Organoids have a wide range of applications in areas such as organ development, regenerative medicine, drug screening, precision medicine, gene editing, and disease modeling.

[0082] As used herein, the term "fluid perfusion culture," "cell perfusion culture," or "fluid perfusion culture" refers to an in vitro culture technique that mimics the dynamic physiological fluid environment of the body in bioengineered organ construction. By continuously or periodically perfusing culture fluid into a three-dimensional biomaterial-cell complex, this technique provides mechanical stimulation (e.g., shear stress), improves material exchange (oxygen, nutrients, metabolic waste), and promotes vascular network formation and tissue functional maturation. "Fluid perfusion culture" can be used for vascularized organ construction, bone and cartilage regeneration, and as a drug testing model.

[0083] As used herein, the term "primary cells" refers to cells cultured immediately after removal from an organism. Primary cells are widely used in molecular, cellular biology, and basic biomedical research, such as proteomics, genomics, cell line research, DNA, RNA, and genetics studies. They are also widely applied in today's hot biopharmaceutical industries, such as drug screening, drug metabolism and toxicology studies, and cancer drug research. The most commonly used primary cell culture methods are tissue explant culture and dispersed cell culture. Tissue explant culture involves directly implanting a fragmented tissue onto the wall of a culture flask and then adding culture medium for culturing. Dispersed cell culture involves removing animal tissue from the body, disaggregating it into single cells, and culturing them in a suitable culture medium to enable the cells to survive, grow, and reproduce.

[0084] As used herein, the term "subject" is intended to include humans and non-human animals. In some embodiments, the subject is a human subject, such as a human patient suffering from a disorder described herein or at risk of suffering from a disorder described herein. The term "non-human animal" includes mammals and non-mammals, such as non-human primates. The cells and compositions described herein are suitable for treating human patients suffering from a disorder described herein. Patients suffering from a disorder described herein include, for example, patients who have developed a disorder described herein but are (at least temporarily) asymptomatic, patients who have exhibited symptoms described herein, and patients suffering from a disorder associated with the disorder.

[0085] As used herein, the term "treatment" refers to a subject (e.g., a human) suffering from a disorder and / or experiencing symptoms of a disorder that, in some embodiments, will suffer less severe symptoms and / or recover faster when treated than if never treated. Treatment can partially or completely alleviate, improve, alleviate, inhibit one or more manifestations of the effects or symptoms, characteristics, and / or causes, or reduce their severity, and / or reduce the incidence, and optionally delay the onset of a disease. In some embodiments, treatment is for subjects who do not show certain signs of a disorder and / or only show early signs of a disorder. In some embodiments, the subject of treatment is a subject who shows one or more established signs of a disorder. In some embodiments, treatment is performed on subjects diagnosed with a disorder.

[0086] As used in this disclosure, the term "gene" refers to a DNA sequence that includes control sequences and coding sequences for producing RNA (e.g., ribosomal RNA or transfer RNA), polypeptides, or precursors of any of the foregoing substances with non-coding functions. The RNA or polypeptide can be encoded by the full-length coding sequence or by any portion of the coding sequence, as long as the desired activity or function is retained. Therefore, a "gene" refers to a DNA or RNA or portion thereof that encodes a polypeptide or RNA chain that functions in an organism. For the purposes of this disclosure, a gene can be considered to include regions that regulate the production of a gene product, regardless of whether such regulatory sequences are adjacent to the coding sequence and / or transcribed sequence. Therefore, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0087] The terms "protein" (if single-chain), "polypeptide," and "peptide" are used interchangeably herein when referring to a gene product, e.g., when encoded by a coding sequence. "Protein" may also refer to an association of one or more polypeptides. "Gene product" refers to a molecule produced as a result of transcription of a gene. Gene products include RNA molecules transcribed from a gene, as well as proteins translated from such transcripts. As used herein, a "target product" is the product of a gene of interest. DETAILED DESCRIPTION

[0088] The technical solutions of the present disclosure are further illustrated below by way of specific examples. It should be understood by those skilled in the art that the examples are merely to help understand the present disclosure and should not be regarded as specific limitations of the present disclosure.

[0089] Example 1 Isolation and identification of hPASCs

[0090] 1.1 Isolation and culture of hPASCs

[0091] hPASCs were isolated from human placental tissue, digested, and the digestion products were precipitated to obtain the supernatant. The supernatant was filtered through a disposable sterile filter, and the filter membrane was collected and backwashed to obtain placental microvascular tissue (40μm-70μm in diameter). The placental microvascular tissue was cultured in a dedicated culture medium (hPASCs culture medium) to obtain primary cells. The hPASCs culture medium was based on the peripheral cell culture medium (ScienCell TM , Cat.1201) was used as the basal medium, supplemented with penicillin / streptomycin. hPASCs were passaged to passage 5 for analysis and identification. hPASCs were seeded in sterile culture dishes (Jet Biofil, TCD000100) and cultured at 37.5°C in a 5% CO2 incubator. The medium was changed every two days, and passaged every four to five days.

[0092] 1.2 Single-cell sequencing of hPASCs

[0093] Stem cells were collected after passage three and at a confluency of 75%. Their viability was greater than 85% and the cells were filtered through a 40-μm pore size mesh. The single-cell suspension was loaded onto a single-cell microfluidic chip, capturing at least 10,000 cells. The captured mRNA was incubated with a preheated reverse transcription mixture in a thermomixer at 42°C and 1000 rpm for 90 minutes. The captured full-length cDNA was amplified by PCR, purified, fragmented, and adapters were added. The purified cDNA was used for library construction. 150-bp paired-end sequencing was performed on the Illumina NovaSeq platform.

[0094] 1.3 Single-cell sequencing of human placental vascular tissue

[0095] Human placental tissue was digested, and the digestion products were allowed to settle to obtain the supernatant. The supernatant was filtered through a disposable sterile filter, and the filter membrane was collected and backflushed to obtain placental microvascular tissue (40 μm-70 μm in diameter). The placental microvascular tissue was digested into single cells and filtered through a 40-μm pore size mesh. More than 10,000 cells were captured and subjected to single-cell sequencing using the same method as in 1.2.

[0096] 1.4 Single-cell analysis

[0097] Single-cell transcriptome data were processed using CeleScope (version 2.0.4) (https: / / github.com / singleron-RD / CeleScope.Github) and aligned to the human reference genome assembly hg38. Cells were quality-controlled using Seurat (version 4.4.0), excluding cells with fewer than 200 detectable genes, fewer than 500 gene counts, or a mitochondrial gene fraction exceeding 10%. High-quality cells were log-normalized and scaled using the Seurat workflow, and the top 2000 highly variable genes (HVGs) were identified using the FindVariableFeatures function. Principal component analysis (PCA) was performed on these HVGs to extract the 30 principal components (PCs) that explained the most variation. High-quality cells were then visualized in two dimensions using the uniform manifold approximation and projection (UMAP) method. The Wilcoxon rank sum test function FindMarker in Seurat was used to identify differentially expressed genes (DEGs) in a cell cluster compared to other clusters. Cell type annotation was performed using renal gene expression profiles. Without pre-setting the differentiation time or direction, Monocle3 (version 1.3.1) was used for trajectory analysis to predict the differentiation paths between the cell types of each fetal kidney organoid. Although it is difficult to determine the starting point of differentiation due to the lack of prior knowledge, the CytoTRACE software package (version 0.3.3) can assist in predicting the sequence of cell differentiation states and cell stemness. As an open source database, CellphoneDB (version 3.0.0) supports a comprehensive and systematic analysis of intercellular communication molecules by integrating receptor, ligand and their interaction information from databases such as UniProt, Ensembl, PDB, and IUPHAR, thereby studying the interaction and communication mechanisms between different cell types. By calling the statistical analysis function of the CellphoneDB software package, the ligand-receptor relationship represented in the single-cell expression profile was analyzed.

[0098] 1.5 Living Cell Workstation

[0099] hPASCs at passage 3 were cultured in 3D Matrigel and imaged in real time using a live cell workstation CT (Nikon) at 37°C and 5% CO2. Cell images were acquired every 2 hours for 96 hours. Images were then exported to a computer for analysis and integration into a dynamic video.

[0100] 1.6 Flow cytometry analysis of hPASCs

[0101] hPASCs labeled with antibodies were analyzed by flow cytometry (BD, Canto II), and Flow Jo-V10 (version 14.0.0.0) software was used for cell data analysis. The flow cytometry antibodies are as follows: APC Mouse Anti-HumanCD201 (EPCR) (eBioscience™, 17-2018-42), PE-Cyanine7 Human / Mouse CD44 (eBioscience™, 25-0441-81), PE Mouse Anti-Human CD73 (BD ​​Pharmingen™, 550257), PE Mouse Anti-Human HLA-ABC (BD Pharmingen™, 555553), PE Mouse Anti-Human CD34 (BD Pharmingen™, 550761), PerCP-Cy™ 5.5 Mouse Anti-HumanCD146 (BD Pharmingen™, 562134), PE Mouse anti-Human CD31 (BioLegend, 102418), APC Mouse Anti-Human CD326 (BioLegend, 118214).

[0102] 1.7 Immunofluorescence staining of cells

[0103] P3 (passage 3) hPASCs were seeded in 24-well plates (glass slides were mounted in the plates before seeding) until cells reached 70% confluency. The cells were fixed with 4% paraformaldehyde for 15 minutes and washed three times with PBS (Solarbio, T8220, PBST) before staining. The slides were blocked with 5% donkey serum (Solarbio, SL050) for 30-60 minutes. The primary antibody was diluted in 2.5% donkey serum and incubated overnight at 4°C. After washing with PBST, the slides were incubated with a secondary antibody diluted in 2.5% donkey serum for 1 hour. After washing with PBST, the slides were stained with DAPI (Solarbio, S2110), mounted, and observed using a laser confocal microscope (Zeiss, LSM 900). Data were analyzed using ZEN 2.6 software. Primary antibodies: MECOM (ATLAS ANTIBODIES, HPA046537, diluted 1:500), PAI-1 (Abcam, ab222754, diluted 1:1000). Secondary antibodies: Goat anti-Rabbit, FITC (ThermoFisher, F-2765, diluted 1:400).

[0104] 1.8 Results

[0105] The isolation and single-cell sequencing process of hPASCs is as follows Figure 1 As shown in A, hPASCs cells proliferate in vascular cavity-like structures, and the cell morphology is between fibroblast-like and epithelial-like ( Figure 1 B). Single-cell RNA sequencing (scRNA-seq) analysis revealed that third-generation hPASCs could be divided into eight functionally enriched subpopulations, including four pro-angiogenic subpopulations, one proliferative subpopulation, and three immunoregulatory subpopulations ( Figure 1 C and Figure 1 D). Pro-angiogenic subpopulation characteristics: Cluster 1 (Angiogenesis 1): High expression of SERPINE1, ACTG1, ANXA2, and MYL6, associated with macrophage function and angiogenesis; Cluster 2 (Angiogenesis 2): Rich in HTRA3, SPON2, TFPI2, and AKR1B1, promoting perivascular migration and invasion; Cluster 3 (Angiogenesis 3): Specifically expressing pro-angiogenic chemokines such as CXCL1, CXCL3, CXCL8, and CCL2; Cluster 5 (Angiogenesis 4): Containing matrix metalloproteinases (MMP1 and MMP14), STC1, and G0S2, involved in invasion and angiogenesis; Proliferative subpopulation characteristics (Cluster 4): High expression of MT-RNR2, MALAT1, NEAT1, and CALD1, which inhibits cell apoptosis and promotes proliferation. Characteristics of immune regulation subgroups: Cluster 6 (immune regulation 1): marked by interferon-stimulated genes (ISG15 and ISG20), OASL and IFIT1, with innate immune regulation and antiviral functions; Cluster 7 (immune regulation 2): rich in DIO2 and COL12A1, involved in cell migration; Cluster 8 (immune regulation 3): expressing IL1RL1 and CCL20, related to immune regulation and inflammatory response. GO enrichment analysis further confirmed the molecular characteristics of these functional subgroups in angiogenesis, proliferation and immune regulation ( Figure 1 E).

[0106] To verify the stemness characteristics of hPASCs, hPASCs were first divided into three subgroups based on their functions: angiogenic subgroup, immunomodulatory subgroup, and proliferative subgroup ( Figure 1 F); Through the cell trajectory reconstruction analysis of gene counting and expression (CytoTRACE), it was found that hPASCs have the "stemness" of stem cells, that is, the "stemness" between multipotency and unipotency ( Figure 1 G); CytoTRACE's potential score and relative ranking results further confirmed that hPASCs have stem cell potential characteristics ( Figure 1 H, Figure 1 I).

[0107] The characteristic surface molecular marker spectrum of hPASCs was further established: hPASCs positively expressed PAI-1, MECOM ( Figure 1 J); flow cytometry analysis showed that hPASCs positively expressed CD201, CD44, CD73 and HLA-ABC); did not express hematopoietic stem cell and endothelial progenitor cell markers (CD34), pericyte markers (CD146), endothelial cell marker CD31 and negatively expressed epithelial cell marker CD326 ( Figure 1 K).

[0108] To further trace the origin of hPASCs, single-cell sequencing analysis of human placental vascular tissue was performed. The results showed that human placental vascular tissue contains endothelial progenitor stem cells (EPCs); capillary stem cells (CSCs); pericytes / vascular smooth muscle cells (vSMCs) / mesenchymal stem cells (MSCs); vascular macrophage 2 (vM2); erythrocytes; extraviral trophoblast progenitors (EVT progenitors); T cells; natural killer cells (NK); mononuclear cells (MN); vascular macrophage 1 (vM1); extraviral trophoblast progenitors (EVT); and naive B cells. cell) and 13 subpopulations of syncytiotrophoblast (STB) Figure 2 A). Analysis of the correlation between hPASCs and placental microvascular tissue showed that hPASCs were isolated from most cells in the placental tissue, but some cell populations showed a certain correlation, indicating that hPASCs developed from some cell types in the placental microvascular tissue ( Figure 2 B, Figure 2 C). Further developmental trajectory was identified through pseudo-chronological developmental analysis, and the results showed that hPASCs developed from endothelial progenitor cells (EPCs), vascular wall stem cells (CSCs), and vascular type 2 macrophages (vM2) in the placental microvascular tissue ( Figure 2 D).

[0109] These results demonstrate that hPASCs are a stem cell population with angiogenic potential, distinct from existing stem cell types. Therefore, they are defined as a new type of stem cell: human placenta-derived angiogenic stem cells (hPASCs). The hPASCs obtained in Example 1 were deposited with the China Center for Type Culture Collection (CTCCC), Wuhan University, Wuhan, China, on April 2, 2025; accession number: CCTCC NO: C202592.

[0110] Example 2 Angiogenic Potential of hPASCs Cultured in 3D Matrigel or 3D “Sandwich”

[0111] 2.1 Biomechanical testing of hPASCs

[0112] Acrylamide and bisacrylamide were mixed in a 3%:0.1% ratio to form a polyacrylamide (PA) gel with a stiffness of 1 kPa. The PA gel was coated on a 35 mm glass-bottomed culture dish with silane pretreatment and polymerized at room temperature for 30 minutes. Rhodamine 0.5 μm carboxylic acid-modified fluorescent beads were diluted in water at a ratio of 1:50 and coated on the PA gel for 25 minutes. Fluorescent beads were covalently bound to the gel surface using a solution of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, Invitrogen, 3.8 mg mL⁻¹ in 2-(N-Morpholino)-ethanesulfonic acid (MES, Sigma), pH 5.5, and hydroxy-2,5-dioxopyridine-3-sulfonic acid (Sul-NHS, Sigma, 7.6 mg / mL in MES, pH 5.5) at room temperature for 2 h. The solution was then replaced with PBS (pH 7.4) for 2 h. After activation with sulfonamide-Sanpah (Pierce), vitronectin was functionalized to the gel substrate overnight at 4°C. Cells were seeded on the substrate for 6 h, and 10 μM Y27632 (Y27) or 10 mM Blebbistatin (Bleb) were added during cell differentiation. The cells were incubated for 48 hours to inhibit cytoskeletal contraction. Phase contrast and fluorescence images of single cells were recorded before and after digestion. Digital image correlation (DIC) was used to calculate the displacement field of the substrate caused by cell contraction based on the positional changes of fluorescent beads before and after cell detachment. Traction stresses were reconstructed in MATLAB using a filtering method based on Fourier transform traction cytometry. The root mean square (RMS) traction force and total strain energy (pJ) exerted by individual cells were measured.

[0113] 2.2 hPASCs 3D Matrigel and 3D “sandwich” vascular culture

[0114] 3D culture:

[0115] Material pretreatment: Matrigel (R&D Systems; Cat. No. 3433-005-01) was slowly thawed on ice at 4°C. A 48-well culture plate was preheated to 37.5°C. 1.5 mL microcentrifuge tubes and P200 pipette tips were precooled to 4°C.

[0116] Preparation of the bottom layer: Mix 50% Matrigel (v / v) and 50% culture medium (v / v) evenly; incubate in a 37.5°C, 5% CO2 incubator for 30 minutes to allow it to fully solidify.

[0117] Inoculate the upper layer: add cell suspension (density 5.3×10 6 cells / mL) were resuspended in endothelial cell culture medium and added to the solidified Matrigel layer for culture.

[0118] 3D "sandwich" culture

[0119] Preparation of the matrix lower layer: Mix 100 μL of endothelial cell culture medium and 100 μL of Matrigel in a pre-cooled 1.5 mL centrifuge tube. Transfer the mixture to a pre-heated low-adhesion 48-well plate and incubate at 37.5°C and 5% CO2 for 30 minutes to allow the matrix gel to solidify.

[0120] Cell mesocosm construction: For human umbilical vein endothelial cells (hUVECs), human umbilical cord mesenchymal stem cells (hUCMSCs), human placenta-derived angiogenic stem cells (hPASCs), hUVECs+hUCMSCs co-culture group, and hUVECs+hPASCs co-culture group, 100 μL of cell suspension was mixed with 100 μL of Matrigel (1:1, V / V) and transferred to the solidified matrix layer; the cells were incubated again at 37.5°C for 30 min to allow the matrix gel of the cell layer to solidify.

[0121] Maintenance of upper culture: Add 250 μL of pre-warmed endothelial cell culture medium to each well and replace with fresh medium daily.

[0122] 2.3 Results

[0123] To systematically compare the differences in biomechanical properties of hUVECs, hUCMSCs, and hPASCs, traction force microscopy (TFM) was used to quantitatively analyze the traction force (RMS) and total strain energy (TSE) of single cells. Figure 3A) The RMS traction force of hUCMSCs was significantly higher than that of hUVECs (P<0.0001, n=17) and hPASCs (P<0.0001, n=17), while the RMS traction force of hPASCs was significantly higher than that of hUVECs (P=0.0074, n=17). In terms of total strain energy (pJ), hUCMSCs were also significantly higher than hUVECs (P<0.0001, n=17) and hPASCs (P<0.0001, n=17), but there was no statistical difference between hPASCs and hUVECs ( Figure 3 B). These results indicate that hUCMSCs exhibit the strongest mechanical pulling properties and strain energy.

[0124] To verify the angiogenic ability of hPASCs, an in vitro 3D Matrigel culture system was established. Figure 3 C. First, single culture experiments showed that hUCMSCs formed spherical aggregates after 24 hours, while hUVECs and hPASCs were able to form typical vascular network structures ( Figure 3 D), indicating that hPASCs have intrinsic angiogenic potential. Secondly, through co-culture experiments with different ratios (20:1, 10:1, 5:1, 2:1 and 1:1), it was found that the hUVECs / hUCMSCs co-culture group only formed spherical aggregates, while the hUVECs / hPASCs co-culture group maintained a stable vascular network structure at the 20:1, 10:1 and 5:1 ratios, and also showed significant migration ability at the 2:1 and 1:1 ratios ( Figure 3 E), indicating that hPASCs have superior pro-angiogenic and migratory properties than hUCMSCs.

[0125] Continuous live cell imaging further confirmed that hUVECs, hPASCs, and their 1:1 co-culture group were able to form vascular networks, while hUCMSCs and their co-culture group with hUVECs were completely incapable of this ability ( Figure 3 F).

[0126] In order to more accurately simulate the in vivo microenvironment, the present invention established a 3D "sandwich" Matrigel culture model ( Figure 3 G). To further analyze intercellular interactions, fluorescence labeling technology (mCherry labeled hUCMSCs / hPASCs, GFP labeled hUVECs) was used for observation. The results of this model showed that hPASCs, either alone or co-cultured with hUVECs, could form a three-dimensional vascular network, while hUVECs, hUCMSCs, and their co-culture groups were unable to construct vascular structures ( Figure 3 H and Figure 3I). Notably, the differences in the performance of hUVECs in conventional 3D and "sandwich" models suggest that the latter more accurately mimics the complex spatial microenvironment required for in vivo angiogenesis. Taken together, these results indicate that hPASCs exhibit a unique capacity for spontaneous vascularization, a property not observed in either hUCMSCs or hUVECs.

[0127] Example 3 Vascular structure formation after hPASCs were injected into decellularized kidney scaffold (DKS) and perfused and cultured in vitro

[0128] 3.1 Preparation of rat kidney decellularized scaffold (DKS)

[0129] Rats (SD) were anesthetized with 3% isoflurane (RWD, R510-22-10) and fixed in the supine position. The abdomen was cleaned and hair removed, and a longitudinal incision was made from the pubic symphysis to the xiphoid process. First, 100 ml of sodium heparin solution (BoyunBiotech, BY12126) was injected through the portal vein to remove residual blood from the kidney, and then the perirenal fat and renal capsule were removed. The abdominal aorta and inferior vena cava were bluntly dissected, and the kidney was isolated, retaining the renal artery and 1 cm of the abdominal aorta and the renal vein and 1 cm of the inferior vena cava connected to them. The renal artery and vein were cannulated with a 24G winged indwelling cannula (Huibang) and the vessels were ligated with 4-0 sutures (gold ring). The rat kidney was placed in a 10 cm sterile culture dish (JET, 02023662-TCD010100) with a glass beaker placed underneath to collect the decellularization wastewater. A peristaltic pump (longer pump, equipped with a pump head (DG-4, 10 rollers) was used. A BT300-2J was used to administer decellularization perfusion solution at 5 rpm (5 ml / min). The perfusion process was as follows: first, 500 ml of 1 μg / L heparin sodium solution; second, 500 ml of 1% (v / v) Triton X-100 solution (Beyotime, ST795); third, 800 ml of 8 μg / L SDS solution (BioFroxx, 151-21-3); fourth, after decellularization, the scaffolds were washed with 2000 ml of PBS containing 1% Pen Strep (Gibco, 15140-122) at 1.5 rpm (1.5 ml / min); finally, after washing, the decellularized kidney scaffolds were placed in a 50 ml centrifuge tube containing PBS (containing 1% Pen Strep) and stored at -80°C.

[0130] 3.2 Kidney Organoid (KIO) Preparation

[0131] Kidney organoids were constructed from human kidney donations or biopsies. Tissue was processed using a 1:1 mixture of collagenase I (Gibco, 171018029) and collagenase II (Gibco, 171701015) at a concentration of 1 mg / ml. Digestion was performed at 37°C for 30 minutes and terminated by adding DMEM / F-12 medium (Sigma; D8537-500 ml) supplemented with 10% fetal bovine serum (FBS). Cellular components were then pelleted by centrifugation at 800 × g for 5 minutes. Prior to 3D "sandwich" culture, 48-well plates (Jet Biofil) were prewarmed in a CO2 incubator. Matrigel (R&D Systems; 3433-005-01) was kept on ice throughout the process to maintain its biological activity. This 3D "sandwich" culture system consists of three layers: a bottom layer consisting of a mixture of 50% Matrigel (v / v) (R&D Systems, 3433-005-01) and 50% PM-hKOCM (v / v). This mixture is added to a prewarmed plate and gently tilted to spread the Matrigel evenly. The plate is then solidified in a 37°C CO2 incubator for 30 minutes. A primary kidney cell suspension is added to the middle layer of a 30% Matrigel and 70% PM-hKOCM mixture, mixed thoroughly, and then added to the solidified bottom layer. The mixture is solidified at 37°C for 30 minutes. Finally, 300 µL of PM-hKOCM complete culture medium is slowly added to the surface of the solidified middle layer. The entire culture system is maintained in a CO2 incubator, with fresh culture medium replaced daily. This yields kidney organoids (KIOs).

[0132] Among them, the culture medium composition of PM-hKOCM is: pericyte medium (ScienCell TM, Cat.1201), supplemented with: 1% Pen Strep (V / V), 1% GlutaMAX-1 (V / V), 1% MEM NEAA (V / V) (Gibco, 11140-050), 10 mM HEPES (Solarbio, H1095), 1% Insulin-Transferrin-Selenium-X (V / V) (Gibco, 51500-056), 0.1% 2-mercaptoethanol (V / V) (Gibco,21985-023), 2% B-27(V / V) (Gibco,17504044), 1% KnockOut SR XenoFree medium(KSR) (Gibco,12618013), 100 ng / mL R-spondin 1 (R&D systems, 4645-RS-025), 25ng / mL Wnt3a (R&D systems, 5036-WNP), 3µM CHIR-99021 (Selleck, S1263), 50ng / mLEGF (Peprotech, 100-47), 50ng / mL FGF10 (Peprotech, 100-26), 50 ng / mL KGF (Peprotech, 100-19), 0.2 µM A 83-01 (MedChemExpress, HY-10432), 50 ng / mL GDNF (Peprotech, 450-10), 0.2 µM LDN193189 (MedChemExpress, HY-12071), 5µM SB202190 (MedChemExpress, HY-10295), 0.1µM TTNPB (MedChemExpress, HY-15682), 10µM Y-27632 (Selleck; S6390) and 0.2% Primocin™ (V / V) (Invivogen, ant-pm).

[0133] 3.3 hPASCs injected into renal decellularized scaffolds in vitro for recellularization and perfusion

[0134] Resuspend 2 × 10 cells in 150 μl of cell culture medium 7To prepare the recellularized kidney scaffold, a 1 ml syringe (Tansoole, 02026616-TS069-004) was used to draw up the cell suspension and inject an equal amount through the indwelling cannulas connected to the artery and vein of the decellularized kidney scaffold. A 50 ml centrifuge tube was placed in a mobile phase bottle, and 20 ml of cell culture medium was added. The arterial (A) and venous (V) silicone tubing of the peristaltic pump were connected to the indwelling cannulas at the arterial and venous ends of the recellularized kidney scaffold, respectively, through the mobile phase bottle cap. The kidney scaffold was immersed in the cell culture medium. The speed was adjusted to 17 rpm (1 ml / min), and the medium flow direction was adjusted from end A to end V. The peristaltic pump was started and, after 12 hours, the speed was adjusted to 20 rpm (1.2 ml / min). The cell culture medium was changed every 2-3 days.

[0135] After hPASCs were inoculated into a renal decellularized scaffold and cultured for 4 days in vitro, the renal organoids were resuspended in culture medium to 150 μL. Following the same injection method as previously described, the renal organoids were injected into the renal scaffold and cultured for 24 hours before harvesting and renal capsule transplantation. Prior to transplantation, one tissue block was fixed in 4% paraformaldehyde (Shanghai Lingfeng, 20100601) for paraffin embedding, and another was fixed in pre-chilled 2.5% glutaraldehyde (Alfa Aesar, 111-30-8) for scanning and transmission electron microscopy. The remaining tissue was used for subcapsular transplantation in rat renal tissue.

[0136] 3.4 Scanning electron microscope (SEM)

[0137] The specimens, approximately 4 mm × 3 mm × 3 mm, were immersed in 2.5% glutaraldehyde (EMS, 16020) at 4°C for at least 12 h. The specimens were washed three times with deionized water for 15 min each time and placed in a Petri dish (Jet, TCD010100) with double-distilled water to completely submerge the tissue. The specimens were then frozen at −80°C for 24 h and dried in a vacuum freeze dryer (Labconco, USA) for 24 h. The specimens were then fixed to a copper plate with conductive adhesive and gold-sprayed using a vacuum sprayer (HITACHI, Japan) for 2 min. Images were acquired using a scanning electron microscope (HITACHI, Japan).

[0138] 3.5 Transmission electron microscope (TEM)

[0139] Sample volume is about 1mm 3The tissues were washed with 0.1% sodium chloride solution (Zuoba Health Technology Co., Ltd., C19091502), fixed with 2.5% glutaraldehyde (EMS, 16020) at 4°C for 2 h, washed three times with 0.1 M phosphate buffer (Yubo Biotechnology, YB160232), fixed with 1% osmium acid (tedpella, 18459) in the dark for 1 h, washed twice with phosphate buffer and purified water, stained with 1% uranyl acetate (Syntechem, 541-09-3) for 2 h, dehydrated in 70% acetone, 80% acetone, 90% acetone, and 100% acetone, and then soaked in a 1:1 mixture of acetone and epoxy resin (SPI, 1220324) and placed in a 37°C oven for 1 h. They were then placed in a 1:4 mixture of acetone and epoxy resin in a 37°C oven overnight and in a 45°C oven with epoxy resin. The tissue was then placed in an oven for 2 h, oriented and embedded, and then placed in a 45°C oven for 3 h and a 65°C oven for 48 h for polymerization. An ultramicrotome (Ultramicrotome, USA) was used to cut tissue slices less than 100 nm in size and observed with a transmission electron microscope (HITACHI, Japan), with images captured at 15,000× and 30,000× magnifications.

[0140] 3.6 Results

[0141] Fluorescently labeled hUCMSCs, hPASCs, and hUVECs were further used to investigate their angiogenesis ability in decellularized scaffolds (DKS). The DKS+hPASCs+KIO group formed vascular structures in which hPASCs expressed CD31, indicating that hPASCs were able to differentiate into endothelial cells and form vascular lumen structures. The DKS+hUVECs+KIO group had vascular structures, but hUVECs and CD31 did not completely overlap, indicating that hUVECs had lost their endothelial cell characteristics. In the DKS+hPASCs+hUVECs+KIO group, vascular structures formed in which hUVECs adhered to the outside of hPASCs, indicating that hPASCs were more likely to adhere to the scaffold than endothelial cells. In the DKS+hUCMSCs+KIO and DKS+hUCMSCs+hUVECs+KIO groups, cells aggregated without a specific cell distribution. No vascular structures or CD31 expression were observed in the DKS and DKS+KIO groups. Figure 4 A). Further α-SMA staining showed similar results to the above CD31 ( Figure 4 B). SEM results showed that the spatial structure of the scaffolds in the DKS+KIO and DKS+hPASCs+KIO groups was well maintained during continuous dynamic culture in vitro, while the spatial structures of the other groups collapsed to varying degrees; cell-connected structures appeared in the DKS+hPASCs+KIO group ( Figure 4C). TEM results showed that cells in the DKS+hPASCs+KIO group extended pseudopodia to attach to the scaffold and formed connecting structures between cells; while cell debris, vacuoles and apoptotic bodies were observed in the other groups ( Figure 4 D). In summary, hPASCs have strong adhesion ability and are able to form vascular structures.

[0142] Example 4 hPASCs injected into decellularized scaffold material for culture and then transplanted into renal capsule to promote angiogenesis

[0143] 4.1 Subcapsular renal transplantation:

[0144] Rats were anesthetized with 2% isoflurane inhalation and immobilized in the right lateral decubitus position. A 1.5-2 cm longitudinal incision was made in the left renal region, and both kidneys were exposed under sterile conditions. A 2 mm incision was made at the inferior pole of the renal capsule. The graft was implanted into the subcapsular space and then advanced to the superior pole using microforceps. After the kidney was returned to the peritoneal cavity, the incision was closed in layers with 4-0 sutures. Rats were sacrificed 14 days after surgery for specimen collection.

[0145] 4.2 Histomorphological staining

[0146] Tissue engineering specimens were fixed in 4% paraformaldehyde (Shanghai Lingfeng, 20100601) for 24 hours, embedded in paraffin, and serially sectioned at 4 μm (LEICA). The sections were baked at 60°C for 1 hour and immersed in xylene I (JIANI, 20130115), xylene II (JIANI, 20130115), anhydrous ethanol (Luquan, 20220401), 95%, 90%, 85%, and 80% ethanol for 10 minutes each. Finally, the sections were dewaxed and stained by immersion in distilled water for 5 minutes. Hematoxylin-eosin staining (Solarbio, G1120) and Masson's trichrome staining (Solarbio, G1343) were performed according to the manufacturer's instructions.

[0147] 4.3 Immunofluorescence staining

[0148] Tissue sections were immersed in PBS buffer (Solarbio, P1010) for 10 minutes. Sections were permeabilized with 0.25% Triton X-100 (Beyotime, P0096) for 5–15 minutes, followed by washing with PBS containing 0.1% Tween-20 (Solarbio, T8220, PBST). Blocking was performed with 5% donkey serum (Solarbio, SL050) for 30–60 minutes. Primary antibodies were diluted in 2.5% donkey serum and incubated overnight at 4°C. After washing in PBST, sections were incubated with secondary antibodies diluted in 2.5% donkey serum for 1 hour and 15 minutes. After washing in PBST, sections were stained with DAPI (Solarbio, S2110), mounted, and observed using a laser confocal microscope (Zeiss, LSM 900). Data were analyzed using ZEN 2.6 software. Primary antibodies: CD31 (Abcam; ab281583, diluted 1:100); α-SMA (Abcam; ab7817, diluted 1:150); EHD antibody (Proteintech; 25320-1-AP, diluted 1:200).

[0149] 4.4 Statistical methods

[0150] Continuous data were expressed as mean ± standard deviation (SD) if they followed a normal distribution, with α = 0.05. Comparisons between two groups were performed with the two-sample t-test if the variances were equal, and with the Welch two-sample t-test if the variances were unequal. Comparisons among multiple groups were performed with one-way ANOVA with Turkey's post hoc test if the variances were equal, and with one-way ANOVA with Tamhane's post hoc test if the variances were unequal. ， Data were analyzed using the T2 test. A two-sided P < 0.05 was considered statistically significant. Continuous data not normally distributed were expressed as median with interquartile range (α = 0.05). Comparisons between two groups were performed using the Mann-Whitney U test, and multiple group comparisons were performed using the Kruskal-Wallis rank-sum test. Differences were considered statistically significant when the two-sided P < 0.05 was considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics 25 software, and all statistical graphs were generated using GraphPad Prism.

[0151] 4.5 Results

[0152] To determine the angiogenic effect of hPASCs renal capsule transplantation, HE staining showed that the number of vascular structures in the DKS+hPASCs+KIO and DKS+hPASCs+hUVECs+KIO groups was higher than that in the other groups ( Figure 5 A). Further CD31 staining showed that the CD31 in the DKS+hPASCs+KIO group + There were significantly more blood vessels than in other groups ( Figure 5 B); Statistical analysis showed that CD31 + The vascular density was significantly higher than that in the DKS (n=3, P=0.002), DKS+KIO (n=3, P=0.046), DKS+hUCMSCs+KIO (n=5, P=0.004) and DKS+hUVECs+KIO (n=5, P=0.001) groups, while there was no statistical difference in the CD31+ vascular density between the DKS+hUVECs+KIO, DKS+hUCMSCs+KIO, DKS+KIO and DKS groups ( Figure 5 C) The results indicate that hPASCs have a stronger angiogenic effect than hUVECs and hUCMSCs, while co-implantation of hUCMSCs or hPASCs with hUVECs did not significantly promote angiogenesis in the renal capsule. To further verify whether hPASCs participate in the formation of host blood vessels under the renal capsule, mCherry-hPASCs were tracked, and the results showed that mCherry co-expressed with EDH3, CD31 and α-SMA in renal vascular endothelial cells ( Figure 5 D) These results indicate that hPASCs participate in host angiogenesis after implantation into decellularized scaffolds.

[0153] Example 5 hPASCs injected into decellularized scaffolds for culture and then transplanted into the kidney to promote angiogenesis

[0154] 5.1 Partial Nephrectomy and Transplantation

[0155] Rats were anesthetized with 2% isoflurane and placed in the right lateral decubitus position. The left kidney was aseptically exposed through a transverse incision (1.5-2 cm) dorsal to the renal region; the right kidney remained untouched. After blunt dissection of the left renal artery and vein, blood flow was occluded with a non-invasive microvascular clamp. The left kidney exhibited cyanosis after clamping, and the clamp was maintained for 35 minutes to prevent ischemia-reperfusion injury. After resection of the lower third of the kidney, the remaining kidney was equidistantly sutured to a trimmed stent (similar in size to the resected kidney) using absorbable 5-0 sutures (Jinhuan Medical Supplies Co., Ltd., R516), with sutures spaced approximately 2 mm apart for a total of 8-9 stitches. Following transplantation, the vascular clamp was removed and perfusion restored, resulting in immediate redness of the kidney. After the left kidney was returned to the peritoneal cavity, the incision was closed in layers using non-absorbable 4-0 sutures (Jinhuan Medical Supplies Co., Ltd., F402). Fourteen days postoperatively, the rats were sacrificed and the left kidneys were harvested.

[0156] 5.2 Ultrasonic testing

[0157] Renal ultrasound was performed 14 days after partial nephrectomy and transplantation using a high-resolution animal ultrasound system (VisualSonics, Vevo 3100). Rats were anesthetized with 2% isoflurane and placed in the right lateral decubitus position. The left renal region was examined using an MX250, 20 MHz transducer. The transducer was slowly moved from the ventral aspect to the dorsal aspect near the spine, parallel to the long axis of the kidney, to scan the entire kidney. The size, morphology, location, and blood flow of the host kidney and transplanted area were recorded.

[0158] 5.3 Results

[0159] Ultrasound 3D vascular modeling of nephrectomy and transplantation at D14 showed that the vascular density of the DKS+hPASCs+KIO implantation group was higher than that of the other groups, while the vascular density of the DKS+KIO, DKS+hUVECs+KIO, and DKS+hUCMSCs+hUVECs+KIO grafts was relatively low ( Figure 6 A). Fluorescence staining showed that DKS+hPASCs+KIO transplants had CD31 + A relatively large number of blood vessels ( Figure 6 B), Statistical analysis showed that the vascular density of DKS+hPASCs+KIO grafts was higher than that of DKS+hUVECs+KIO (n=5, P=0.032), DKS+hUCMSCs+KIO (n=5, P=0.003), DKS+KIO (n=4, P=0.001) and DKS (n=4, P=0.000037) groups, and there was no significant difference among other groups; the vascular density of DKS+hPASCs+KIO grafts was higher than that of DKS+hUVECs+hPASCs+KIO group (n=5, P=0.024) ( Figure 6C); Statistical results showed that hPASCs had a stronger ability to promote angiogenesis in the nephrectomy model than hUCMSCs or hUVECs, and co-transplantation with hUVECs had no significant effect on host angiogenesis. Again, hPASCs fluorescent mCherry labeling and tracking results showed that hPASCs co-expressed vascular surface markers EDH3, CD31, and α-SMA ( Figure 6 D) These results indicate that hPASCs participate in host angiogenesis.

[0160] Example 6 Mechanism of hPASCs promoting angiogenesis in vivo

[0161] 6.1 RNA sequencing analysis

[0162] Tissue samples (20-50 mg) were placed in RNase-free EP tubes (Axygen, MCT-150-C) and stored at -80°C. Homogenize the sample in 1 ml of TRIzol (Ambion, 15596026) using a tissue grinder (Shanghai Jingxin, JXFSPTPR-24) at 60 Hz for 50 seconds. After standing at room temperature for 5 minutes, the homogenate was transferred to a fresh tube. 200 μl of chloroform (Aldrich, 485403-500MG) was added, the mixture was mixed, and the mixture was allowed to stand for 3 minutes. The mixture was then centrifuged (12,000 rpm, 15 minutes, 4°C). The aqueous phase was collected, and 5 μl of AcrylCarrier (Solarbio, SA1020) and 500 μl of isopropanol (Titan, G75885B) were added. The mixture was mixed, allowed to stand for 10 minutes, and then centrifuged (12,000 rpm, 10 minutes, 4°C). The supernatant was discarded, 1 ml of 75% ethanol was added, and the mixture was mixed and centrifuged (7,500 rpm, 2 minutes, 4°C). The remaining supernatant was dried for 5 minutes and then resuspended in 30 μl of RNase-free dH2O (Takara, RR047A). RNA concentration and quality were measured using NanoDropOne (Thermo Fisher Scientific), with a target A260 / A280 ratio of 1.9-2.0. Total RNA purity and integrity were further assessed using a K5500 spectrophotometer and an RNA Nano 6000 Assay Kit (Agilent Technologies, 5067-1511). mRNA was purified using oligo-dT magnetic beads and fragmented with a divalent cation solution, then used as a template for first-strand cDNA synthesis. The complementary strand was then synthesized to form double-stranded cDNA. Library fragments were purified using the QiaQuick PCR Purification Kit (Qiagen, 28104), end-repaired, A-tailed, and adapter-ligated. The target product was amplified by PCR to construct a library. RNA library clusters were generated using the HiSeq PE Cluster Kit v4-cBot-HS (Illumina) and sequenced on the Illumina platform, generating 150-bp paired-end reads. After sequencing, differentially enriched genes were analyzed for highly expressed signaling pathways and validated by Western blotting. Three biological replicates were performed, and a P value < 0.05 was considered statistically significant.

[0163] 6.2 Western blotting experiment

[0164] Transplanted kidney tissue was lysed with RIPA lysis buffer (Beyotime, P1048), homogenized, and incubated in an ice-water bath for 10 minutes. The supernatant was collected by centrifugation at 12,000 rpm for 10 minutes at 4°C. BCA working solution and standards (ThermoScientific, 23225) were prepared. Absorbance at 562 nm was measured, and a standard curve was constructed to calculate sample concentration. 5× loading buffer (Fude Biological Technology, FD002) was added at a ratio of 1:4 (v / v), boiled at 99°C for 10 minutes, and stored at -20°C. The sample and a protein marker (ThermoScientific, REF26617) were loaded onto a PAGE gel (YAMAY BIOTECH, PG21) and electrophoresed at 80 V for 30 minutes, then switched to 100 V until bromophenol blue reached the bottom of the gel. PVDF membranes (Immobilon, ISEQ00010) were activated with methanol (Hongsheng Fine Chemical, CY20211010) for 5 minutes and then transferred at 300 mA for 1.5 hours. The membranes were blocked with 5% blocking buffer (Beyotime, P0216) at room temperature for 1 hour and incubated with primary antibodies overnight at 4°C. The primary antibodies used included rabbit polyclonal antibodies against AKT (abclonal, A11016), rabbit polyclonal antibodies against phospho-AKT (abclonal, AP0274), rabbit polyclonal antibodies against VEGFA (abclonal, A5708), and a rabbit monoclonal antibody against β-actin (abclonal, AC026). Secondary antibodies (goat anti-rabbit IgG, HRP-conjugated antibody, CST, 7074) were incubated for 1 hour at room temperature. Images were acquired using a multifunctional gel imaging system (BIO-RAD, USA), and grayscale values ​​were analyzed using Image Lab and ImageJ 3.0 software.

[0165] 6.3 Results

[0166] RNA sequencing and GO enrichment analysis showed that compared with the DKS+hUVECs+KIO group, the vascular development pathway in the DKS+hPASCs+KIO group was significantly upregulated; and compared with the DKS+hUCMSCs+KIO group, the angiogenesis regulatory pathway was significantly upregulated ( Figure 7 A). KEGG analysis further showed that the expression level of PIP3 / AKT signaling pathway in the DKS+hPASCs+KIO group was higher than that in the DKS+hUVECs+KIO group ( Figure 7B), suggesting that hPASCs transplantation can activate angiogenesis-related pathways. Western blotting verification results showed that in the subrenal capsule transplantation model, the expression of pAKT in the DKS+hPASCs+KIO group was significantly higher than that in the DKS+hUVECs+KIO group (n=3, P=0.0246) and the DKS+hUCMSCs+KIO group (n=3, P=0.0032); the expression of VEGFA in the DKS+hPASCs+KIO group was significantly higher than that in the DKS+hUVECs+KIO group (n=3, P=0.0398) ( Figure 7 C). In the rat model of partial nephrectomy combined with bioengineered kidney suture, the expression of pAKT in the DKS+hPASCs+KIO group was significantly higher than that in the DKS+hUVECs+KIO group (n=3, P=0.0297); the expression of VEGFA in the DKS+hPASCs+KIO group was significantly higher than that in the DKS+hUVECs+KIO group (n=3, P=0.0456) and the DKS+hUCMSCs+KIO group (n=3, P=0.0044) ( Figure 7 D). Therefore, hPASCs promote angiogenesis by activating the AKT signaling pathway.

Claims

1. Human placenta-derived angiogenic stem cells (hPASCs), characterized by: The deposit number of the hPASCs is CCTCC NO: C202592.

2. Human placenta-derived angiogenic stem cells (hPASCs), characterized by: The hPASCs are markers PAI-1 positive, MECOM positive, CD201 positive, CD44 positive, CD73 positive, HLA-ABC positive, CD34 negative, CD146 negative, CD31 negative and CD326 negative; preferably, the hPASCs are derived from placental vascular tissue macrophages, vascular wall stem cells and / or endothelial progenitor cells.

3. A method for isolating human placenta-derived angiogenic stem cells (hPASCs), characterized in that: The method comprises the following steps: 1) digesting human placental tissue; 2) sedimenting the digestion product to obtain a supernatant; 3) filtering the supernatant through a filter, and backflushing the filter to obtain placental microvascular tissue; 4) culturing the placental microvascular tissue in hPASC culture medium to obtain primary cells; 5) Primary cell subculture; 6) hPASC identification; Preferably, the diameter of the placental microvascular tissue is 20 μm-100 μm, preferably 40 μm-70 μm; Preferably, the hPASC culture medium uses peripheral cell culture medium as the basal medium and is supplemented with penicillin / streptomycin dual antibodies; Preferably, the peripheral cell culture medium is Pericyte Medium, product number 1201, purchased from ScienCell; Preferably, the serum in the Pericyte Medium is replaced by Platelet Lysate; Preferably, the identification includes defining cells having one or more characteristics selected from the following as hPASCs: a) containing four angiogenic stem cell subpopulations and having stem cell "stemness"; b) being able to form vascular structures in in vitro or in vivo culture; c) being PAI-1 positive, MECOM positive, CD201 positive, CD44 positive, CD73 positive, HLA-ABC positive, CD34 negative, CD146 negative, CD31 negative and CD326 negative for surface markers.

4. Human placenta-derived angiogenic stem cells (hPASCs) obtained by the isolation method according to claim 3.

5. A composition, characterized in that Comprising the human placenta-derived angiogenic stem cells hPASCs according to any one of claims 1, 2 and 4.

6. Use of the human placenta-derived angiogenic stem cells (hPASCs) according to any one of claims 1, 2, and 4, or the composition according to claim 5, in the preparation of a medicament for alleviating symptoms or treating diseases, wherein the disease or condition is selected from cardiovascular disease, diabetic complications, nervous system diseases, ophthalmic diseases, chronic wounds, and ulcers.

7. The use according to claim 6, wherein the cardiovascular disease is selected from myocardial infarction, chronic ischemic heart disease, peripheral arterial disease, coronary artery restenosis and heart failure; and / or the diabetic complications are selected from diabetic foot ulcers, diabetic retinopathy, diabetic nephropathy and diabetic peripheral neuropathy; and / or the nervous system diseases are selected from ischemic stroke, spinal cord injury, Alzheimer's disease, Parkinson's disease and multiple sclerosis; and / or the ophthalmic diseases are selected from age-related macular degeneration, retinal vein occlusion, corneal neovascular disease and glaucomatous optic neuropathy; and / or the chronic wounds are selected from burn wounds, postoperative refractory wounds and pressure sores; and / or the ulcers are selected from venous ulcers, arterial ulcers and radiation ulcers.

8. Use of the human placenta-derived angiogenic stem cells hPASCs according to any one of claims 1, 2 and 4 or the composition according to claim 5 in tissue engineering and regenerative medicine, characterized in that: The applications include promoting vascularization of artificial organs, skin regeneration, bone and cartilage regeneration, nerve tissue repair and complex tissue structure reconstruction; preferably, the applications are performed in vitro.

9. Use of the human placenta-derived angiogenic stem cells hPASCs according to any one of claims 1, 2 and 4 or the composition according to claim 5 in preparing bioengineering artificial organs, wherein: The bioengineered artificial organs include engineered kidneys, engineered blood vessels, engineered livers, heart patches, artificial skin, artificial trachea, artificial bladders and artificial corneas; preferably, the bioengineered artificial organ is a bioengineered kidney.

10. A method for preparing a bioengineered organ, comprising the step of implanting the human placenta-derived angiogenic stem cells (hPASCs) and organoids according to any one of claims 1, 2 and 4 into a decellularized scaffold.

11. The method according to claim 10, wherein the bioengineered organ is selected from an engineered kidney, an engineered blood vessel, an engineered liver, a heart patch, an artificial skin, an artificial trachea, an artificial bladder and an artificial cornea; preferably, the bioengineered artificial organ is a bioengineered kidney; preferably, the bioengineered organ is a bioengineered kidney, and / or the organoid is a kidney organoid, and / or the decellularized scaffold is a kidney decellularized scaffold. 12 . The method according to claim 10 , wherein the decellularized scaffold is obtained by decellularizing an isolated organ.

13. The method according to claim 12, wherein the decellularization treatment comprises a perfusion step of administering a decellularized perfusate to the isolated organ; preferably, the perfusion is performed using a peristaltic pump; preferably, the perfusion step is performed once or more; preferably, the perfusate is selected from one or more of heparin sodium solution, Triton X-100 and SDS solution; optionally, the method further comprises a post-perfusion washing step.

14. A bioengineered organ comprising the human placenta-derived angiogenic stem cells (hPASCs) according to any one of claims 1, 2 and 4, or the composition according to claim 5; preferably, the bioengineered organ is obtained according to the method according to any one of claims 10 to 13; preferably, the bioengineered organ is selected from a bioengineered kidney, a bioengineered blood vessel, a bioengineered liver, a heart patch, an artificial skin, an artificial trachea, an artificial bladder and an artificial cornea, preferably a bioengineered kidney.

15. Use of the bioengineered organ according to claim 14 in preparing a drug or medical device for treating a disease; preferably, the bioengineered organ is a bioengineered kidney, and the drug or medical device is used to treat kidney disease.

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