CN120574767A

CN120574767A - Human placenta-derived angiogenic stem cells (hPASCs) and their applications

Info

Publication number: CN120574767A
Application number: CN202510757426.XA
Authority: CN
Inventors: 徐军军; 陈信如
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2025-06-06
Filing date: 2025-06-06
Publication date: 2025-09-02

Abstract

The present disclosure provides human placenta-derived vascular stem cells hPASCs and uses thereof. The human placenta-derived vascular stem cells hPASCs of the present disclosure are preserved in China center for type culture Collection, with addresses of China, the university of Wuhan, and the preservation date of 2025, 4 months and 2 days, and with preservation number of CCTCC NO: C202592. hPASCs of the present disclosure contain a subpopulation of angiogenic cells and have stem cell "stem" properties, both in vivo and in vitro, which can be used for the treatment of cardiovascular disease and other diseases and for the vascularization construction of artificial organs and tissues.

Description

Human placenta source vascular stem cell hPASCs and application thereof

Technical Field

The disclosure relates to the field of biotechnology, in particular to human placenta-derived vascular stem cells hPASCs and application thereof.

Background

End stage renal disease (End-STAGE KIDNEY DISEASE, ESKD) is the irreversible loss of kidney function to the final stage of Chronic kidney disease (Chronic KIDNEY DISEASE, CKD). ESKD is mainly treated by dialysis and kidney transplantation, patient dialysis bears economic burden and risk of infection, kidney transplantation requires life-long administration of immunosuppressive drugs and serious shortage of kidney sources, and patients of 490.2-708.3 ten thousand ESKD in the world are reported to require kidney transplantation treatment in 2010 (Adv Exp Med Biol 2019, 1165:3-15), but wait for the number of kidney transplantation patients to far exceed that of organ donors, millions of people die from renal failure due to lack of kidney sources in the year (Lancet 2015, 385 (9981): 1975-1982). The bioengineered kidney organ aims at utilizing autologous or allogeneic cells of a patient and an engineering bionic scaffold to construct and implant a substitute tissue in vitro to replace, restore or enhance the function of damaged kidney, and is one of strategies for solving the problem of organ source shortage.

Renal tissue engineering scaffold materials currently under widespread investigation include synthetic polymers such as Polycaprolactone (PCL), polylactic acid (PLA) and polyvinyl alcohol (PVA) and natural decellularized scaffolds, etc. (Bioengineering (Basel) 2022, 9 (10)). Synthetic scaffold materials have difficulty simulating the mechanical or biochemical properties of the microenvironment and natural tissue in an organism and have limited ability to overcome immunogenicity and repair (Bioact Mater 2022, 10:15-31). The kidney decellularized scaffold removes immunogenic cellular components, has good biocompatibility and low rejection rate (Biomater Res 2023,27 (1): 10), and retains vascular, glomerular and tubular 3D ultrastructions, partial signaling and growth factor proteins, providing an adhesion, proliferation, migration, differentiation, colonization and growth microenvironment for implanted cells (Biomaterials 2013, 34 (28): 6670-6682.). In addition, rat kidney decellularized scaffold material is thrombogenic with naked extracellular matrix after in situ transplantation (Curr Opin Hematol 2016, 23 (3): 280-287), lacks a parenchymal cell infiltration filling scaffold, inflammatory cell infiltration or proteolytic extracellular matrix uptake (Sci Rep 2017, 7:43502.). Organ regeneration cannot be effectively realized by simply transplanting the kidney acellular stent material, the early stage of the acellular stent material is lack of blood, oxygen and nutrition supply, and the recipient vascular network needs time to grow into the graft tissue. The vascular network plays an important role in playing a role in filtering the cortical glomeruli, supplying oxygen and nutrition, maintaining the reabsorption and secretion functions of the medullary tubules, and vascularizing the components necessary for constructing the complete engineering kidney organ. Therefore, the construction of vascularized tissue engineering kidneys only fundamentally solves the graft survival problem (Tissue Eng Part B Rev 2022, 28 (1): 1-21).

The vascular system consists of vessels of different sizes, anatomies, physiology and cell arrangement, capillaries with endothelial progenitor cells, endothelial cells, perivascular cells, smooth muscle cells, vascular macrophages, vascular wall stem cells, etc. Cells used for vascularization therefore fall into two broad categories, endothelial cells and supporting cells (Front Bioeng Biotechnol 2023, 11:1103727.). The seed cells widely used in tissue engineering vascularization at present are human umbilical vein endothelial cells hUVECs (human Umbilical Vein Endothelial Cells), abbreviated as ECs. hUVECs has been applied to vascularization of decellularized scaffolds of organs such as kidneys (Nat Med 2013, 19 (5): 646-651;Front Bioeng Biotechnol 2023, 11:1184408.), livers (Nat Biomed Eng 2020, 4 (4): 437-445), hearts (Biomaterials 2015, 61:279-289), lungs (Ann Surg 2018, 267 (3): 590-598). However, implantation of exogenous ECs does not develop further into organ-specific vascular phenotypes, non-organ-specific ECs affect vascularization quality, fail to communicate with host-specific vessels to form a functional vascular network supporting blood circulation and impede regeneration of parenchymal cells within the organ (Trends Biotechnol 2018, 36 (8): 834-849), and furthermore, hUVECs does not adhere well to decellularized scaffold materials and is prone to shedding with blood flow to thrombosis (Bioact Mater 2021, 6 (8): 2557-2568.). Vascular endothelial cells are the preferred targets of autoantibodies in allogenic tissue or organ transplantation (Front Immunol 2022, 13:), allogenic humanized endothelial cells not only express MHCI and MHCII and constitutively express co-stimulatory factors such as PD-L1, but also provide co-stimulatory signals through direct allogenic recognition to activate T cells and antibody-mediated rejection, so that complement cascade near allogenic endothelial cells is activated to form a tapping membrane complex (MAC) and local inflammatory mediators, the direct allogenic reaction between endothelial cells and T cells is enhanced, and finally strong immune rejection is caused (Trends Biotechnol 2018, 36 (8): 834-849.). Therefore, the vascularized tissue engineering kidney constructed by directly implanting ECs into the kidney acellular scaffold material can not effectively realize vascularization and graft survival, and the vascularized tissue engineering kidney constructed by EC has a certain limitation.

In order to improve the implantation efficiency and function of ECs in decellularized scaffold materials, mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs) are transplanted in combination with ECs. Rat glomerular endothelial cells and rat bone marrow mesenchymal stem cells are co-cultured in vitro in a kidney decellularized scaffold to realize micro-vascularization, MSCs reduce endothelial cell inflammatory injury, reduce apoptosis, support vascular stabilization, improve cell viability, cell proliferation, cell migration and vascularization, and an ECM-derived implantable kidney scaffold is prepared, but the study lacks in vivo experiments to verify whether or not functional vascular network is successfully constructed (Mater Today Bio 2022, 17:100464). MSCs cannot spontaneously differentiate into endothelial vessels supporting Blood flow in vivo (Blood 2008, 111 (9): 4551-4558). And MSCs are widely available, such as fat, bone marrow, and their multi-lineage differentiation potential is related to their tissue origin, i.e., the presence of tissue heterogeneity (Proc NATL ACAD SCI USA 2014, 111 (28): 10137-10142.). Although MSCs and ECs are combined to graft and construct vascularized decellularized scaffold materials, complete vascular network structures and functions cannot be constructed in vivo, and tissue engineering organs formed by in vivo transplantation have great differences from the recombinant tissues.

Disclosure of Invention

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 bioengineering organ construction.

The first aspect of the invention provides human placenta-derived vascular stem cells hPASCs Homo sapiens, wherein the hPASCs is preserved in China center for type culture Collection, and has the addresses of China, wuhan, university of Wuhan, and the preservation date of 2025, month 4 and 2, and the preservation number of CCTCC NO: C202592.

In a second aspect, the invention provides a human placenta-derived pro-vascular stem cell hPASCs, wherein hPASCs expresses markers PAI-1, MECOM, CD201, CD44, CD73 and HLA-ABC, and does not express markers CD34, CD146, CD31 and CD326, and preferably wherein hPASCs is derived from placental vascular tissue macrophages and/or vascular wall stem cells.

The third aspect of the invention provides a separation method of human placenta-derived vascular stem cells hPASCs, which comprises the following steps of 1) digesting human placenta tissues, 2) settling digested products to obtain a supernatant, 3) filtering the supernatant by a filter screen, backflushing the filter screen to obtain placenta microvascular tissues, 4) culturing the placenta microvascular tissues in a hPASC culture medium, 5) subculturing primary cells, and 6) identifying hPASC.

In some embodiments, the placental microvascular tissue has a diameter of 20 μm to 100 μm, preferably 40 μm to 70 μm.

In some embodiments, the hPASC medium is based on peripheral cell culture medium and penicillin/streptomycin diabodies are added.

In some embodiments, the peripheral cell culture medium is Pericyte Medium, cat# 1201, purchased from scientific.

In some embodiments, the serum in Pericyte Medium is replaced with PLATELET LYSATE.

In some embodiments, the identifying in step 6) comprises defining hPASCs cells having one or more characteristics selected from the group consisting of a) containing 4 subpopulations of angiogenic stem cells and having stem cell "stem properties", b) being capable of forming vascular structures in vitro or in vivo, c) having a PAI-1 positive, MECOM positive, CD201 positive, CD44 positive, CD73 positive, HLA-ABC positive, CD34 negative, CD146 negative, CD31 negative, and CD326 negative surface markers.

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

In some embodiments, the stem cell "stem property" is obtained by CytoTRACE analysis.

In some embodiments, the ability of the in vitro to form vascular structures is assessed by in vitro 3D Matrigel or 3D "candwick" culture.

According to a fourth aspect of the present invention, there is provided human placenta-derived vascular stem cells hPASCs isolated according to the isolation method of the third aspect of the present invention.

In a fifth aspect, the present invention provides a composition comprising the human placenta-derived pro-vascular stem cell hPASCs of the first, second, or fourth aspects of the present invention.

In a sixth aspect, the present invention provides the use of the human placenta-derived vascular stem cell hPASCs described in the first, second, or fourth aspects or the composition described in the fifth aspect, in the preparation of a medicament for the alleviation of a disorder or the treatment of a disease.

In some embodiments, the disease or disorder is selected from the group consisting of cardiovascular disease, diabetic complications, neurological disease, ophthalmic disease, chronic wounds, and ulcers.

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

In some embodiments, the diabetic complication is selected from the group consisting of a diabetic foot ulcer, a diabetic retinopathy, a diabetic nephropathy, and a diabetic peripheral neuropathy.

In some embodiments, the neurological disease is selected from the group consisting of ischemic stroke, spinal cord injury, alzheimer's disease, parkinson's disease, and multiple sclerosis.

In some embodiments, the ophthalmic disease is selected from the group consisting of age-related macular degeneration, retinal vein occlusion, corneal neovascularization disease, and glaucoma optic neuropathy.

In some embodiments, the chronic wound is selected from the group consisting of burn wounds, postoperative refractory wounds, and pressure sores.

In some embodiments, the ulcer is selected from venous ulcers, arterial ulcers, and radioactive ulcers.

The seventh aspect of the present invention provides the use of the human placenta-derived vascular stem cell hPASCs of the first, second or fourth aspect of the present invention or the composition of the fifth aspect in tissue engineering and regenerative medicine. Among these applications are promotion of vascularization of artificial organs, skin regeneration, bone and cartilage regeneration, nerve tissue repair and reconstruction of complex tissue structures. In some embodiments, the use is performed in vitro, and in some embodiments, the use is for indirect therapeutic purposes.

According to an eighth aspect of the present invention, there is provided the use of the human placenta-derived vascular stem cell hPASCs of the first, second or fourth aspects of the present invention or the composition of the fifth aspect for the preparation of a bioengineered artificial organ.

In some embodiments, the artificial organ comprises an engineered kidney, an engineered blood vessel, an engineered liver, a cardiac patch, an artificial skin, an artificial trachea, an artificial bladder, and an artificial cornea, preferably the bioengineered artificial organ is a bioengineered kidney.

In a ninth aspect, the present invention provides a method for preparing a bioengineered organ, comprising the step of implanting human placenta-derived vascular stem cells hPASCs and organoids of the first, second or fourth aspects of the invention into a decellularized scaffold.

In some embodiments, the bioengineered organ is selected from the group consisting of an engineered kidney, an engineered blood vessel, an engineered liver, a cardiac patch, an artificial skin, an artificial trachea, an artificial bladder and an artificial cornea, preferably the bioengineered artificial organ is a bioengineered kidney.

In some embodiments, the bioengineered organ is a bioengineered kidney.

In some embodiments, the organoid is a kidney organoid.

In some embodiments, the decellularized scaffold is a kidney decellularized scaffold.

In some embodiments, wherein the decellularized scaffold is obtained from an ex vivo organ by decellularization treatment.

In some embodiments, wherein the decellularized treatment comprises a perfusion step of administering a decellularized perfusate to the isolated organ, preferably with a peristaltic pump, preferably the perfusion step is performed one or more times, preferably the perfusate is selected from one or more of a heparin sodium solution, triton x-100, and SDS solution, optionally further comprising a post-perfusion washing step.

In a tenth aspect, the invention provides a bioengineered organ comprising human placenta-derived pro-vascular stem cells hPASCs according to the first, second or fourth aspects of the invention or a composition according to the fifth aspect of the invention.

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

In an eleventh aspect, the invention provides the use of a bioengineered organ according to the ninth aspect of the invention for the manufacture of a medicament or medical device for the treatment of a disease, preferably the bioengineered organ is a bioengineered kidney for the treatment of kidney diseases.

The invention has the advantages compared with the prior art that:

1) The invention provides a novel stem cell line hPASCs which is obtained by separating from placenta, contains 4 angiogenesis cell subsets, has stem cell 'stem property' and has angiogenesis promoting function. hPASCs positive expression of PAI-1, MECOM, CD201, CD44, CD73 and HLA-ABC, no expression of hematopoietic stem cell and endothelial progenitor cell marker (CD 34), no expression of pericyte marker (CD 146), no expression of endothelial cell marker CD31 and epithelial cell marker CD326.hPASCs single cells and placenta microvascular tissue single cells are subjected to association analysis and tracing, and hPASCs is developed from placenta vascular tissue macrophages and/or vascular wall stem cells and/or vascular Endothelial Progenitor Cells (EPCs).

2) HPASCs are different from hUVECs and huchmscs in their biomechanical characteristics and their traction forces are intermediate.

3) HPASCs on 3D Matrigel, while hUCMSCs form aggregates that cannot form vascular structures, hPASCs on 3D "sandwich" culture, while hUVECs and hUCMSCs form aggregates only, and cannot form vascular structures.

4) The hPASCs composite decellularized scaffold material can form a vascular structure in vitro after in-vitro culture, and has stronger adhesion capability to materials than hUVECs and hUCMSCs.

5) HPASCs the composite decellularized scaffold material forms more vascular structures than hUVECs and hUCMSCs in-vivo renal capsule transplantation and in-vivo nephrectomy transplantation, and has stronger angiogenesis promoting effect and function compared with the two.

6) HPASCs composite decellularized scaffold material in vivo renal capsule transplantation and in vivo nephrectomy transplantation participate in host angiogenesis, and vascular cells are formed by differentiation.

Drawings

FIG. 1 shows hPASCs has the "stem" potential of stem cells and angiogenic molecular features. A, hPASCs separation and single cell sequencing procedure, B, hPASCs cell morphological characteristics, a scale of 100 μm, C, hPASCs single cell RNA sequencing (ScRNA-seq) analysis hPASCs cell types, D, scRNA-seq analysis HPASCS MARKER genes, E, scRNA-seq GO enrichment analysis, F, hPASCs stem cell characteristic analysis functional phenotype enrichment, G, hPASCs CytoTRACE 'dryness' analysis, H, hPASCs 'dryness' potential analysis, I, hPASCs 'dryness' relative coefficients, J, hPASCs immunofluorescence identification, a scale of 20 μm, K, hPASCs flow analysis.

FIG. 2 shows single cell sequencing of human placental vascular tissue and hPASCs cellular microvascular tissue localization sources. A, single cell sequencing and analyzing cell types of human placenta-derived vascular tissues. B-C, hPASCs single cell and human placenta vascular tissue single cell correlation analysis. D, hPASCs and human placenta microvascular tissue cell type correlation simulated clockwise development analysis.

FIG. 3 shows hPASCs D or 3D "sandwich" culture angiogenic potential. A single cell Traction (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 3D culture with angiogenic potential, E, hUVECs and hUCMSCs or hPASCs different proportion 3D culture, F continuous dynamic 3D culture hPASCs living cell imaging with angiogenic potential, G,3D "sandwich" culture model, H-I, hPASCs forming vascular network in 3D "sandwich" culture 72H with a proportion of 50 μm.

FIG. 4 shows the vascular structure formation following hPASCs in vitro infusion of kidney decellularized scaffold material perfusion culture. a-B, CD31 and a-SMA immunofluorescence display hPASCs reconstruct vascular structures (indicated by arrows), scale bars 100 μm,200 μm. C, vascularized bioengineered kidney ultrastructure SEM shows hPASCs forming connection structure between cells (arrow indication), scale 100 μm,20 μm, D, TEM shows hPASCs forming connection structure and adhering to stent material, other group forming necrosis structure (arrow indication), scale 1 μm,2 μm.

FIG. 5 shows that hPASCs implantation of decellularized scaffold material promotes angiogenesis after culture by renal capsule membrane transplantation. A, HE staining, scale bar 50 μm,100 μm. B, CD31 immunofluorescence, 200 μm scale. C, vascular density statistical analysis, DKS and dks+kio groups (n=3), other groups (n=5), P < 0.05.D, mCherry-hPASCs and vascular markers EDH3, CD31, alpha-SMA are co-expressed to participate in host angiogenesis, with a scale of 20 μm.

FIG. 6 shows that the nephrectomy graft promotes angiogenesis after hPASCs injections of decellularized scaffold material for culture. A, ultrasonic 3D vessel modeling in implants. B, CD31 immunofluorescence staining, scale 200 μm. C, vascular density statistical analysis, DKS group and dks+kio group (n=4), other group (n=5), P <0.05. The tracking of the D, hPASCs fluorescent mCherry marker showed that hPASCs co-expressed with the vascular marker EDH3, CD31, alpha-SMA indicated hPASCs to be involved in host angiogenesis, with a scale of 20. Mu.m.

FIG. 7 shows hPASCs in vivo promotion of the angiogenic mechanism. A, RNA-seq sequencing analysis GO ENRICHMENT ANALYSIS, B, KEGG ENRICHMENT ANALYSIS, n=3, P <0.05.C, western blotting of kidney membranous graft to pAKT and VEGFA protein, n=3, P <0.05, and D, western blotting of kidney excision model graft to pAKT and VEGFA protein, n=3, P <0.05.

Detailed Description

Some embodiments according to the present disclosure are described more fully below. However, aspects of the present disclosure may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Unless defined otherwise, 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 will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

The various features of the disclosure described herein may be used in any combination unless specifically indicated. Furthermore, the present disclosure also contemplates that in embodiments, any feature or combination of features set forth herein may be excluded or omitted.

All specified embodiments, features and terms are intended to include the recited embodiments, features or terms and their biological equivalents unless specifically indicated otherwise.

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 references, articles, publications, patent publications, and patent applications cited herein is not, and should not be taken as, an admission or any form of suggestion that they constitute a part of the effective prior art or form part of the common general knowledge in any country in the world.

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

The experimental methods of specific conditions not specified in the examples below are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various commonly used chemical and biological reagents used in the examples are all commercially available products. Unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, tools, etc.

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.

Definition of the definition

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.

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

The terminology used in the description of the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. It is also possible in the present disclosure that steps may be performed in a different order where logically possible.

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.

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 limit of that range and any other stated or intervening value in 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 limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

The terms "having," "containing," "including," and "having" and any variations thereof herein are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device. The present disclosure also contemplates other embodiments of "comprising," "consisting of," and "consisting essentially of," the embodiments or elements presented herein, whether or not explicitly stated.

References to "a plurality" in this disclosure refer to two or more. "and/or" describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate that there are three cases of a alone, a and B together, and B alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.

With respect to recitation of ranges of values herein, each intervening value, having the same degree of precision therebetween, is specifically contemplated. For example, for the range 6-9, the numbers 7 and 8 are considered in addition to 6 and 9, 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 have been explicitly considered.

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

As used herein, the term "stem cell" is a class of cells having an unlimited or immortal ability to self-renew, capable of producing at least one type of highly differentiated daughter cell. Most stem cells are considered to be a type of cells from the embryo, fetus or adult body that have the ability to self-renew and proliferate and differentiate without restriction under certain conditions, and are capable of producing daughter cells of the same phenotype as the genotype and themselves, and also capable of producing specialized cells that make up tissues and organs of the body, while also differentiating into progenitor cells.

As used herein, the term "human placenta-derived pro-vascular stem cell hPASCs" or "hPASCs" or "human placenta-derived pro-vascular stem cell" refers to stem cells obtained by isolated culture of a human placenta. The cells have stem cell characteristics, have the differentiation potential to vascular endothelial cells and smooth muscle cells, and have the function of forming blood vessels through in vitro space culture. The human placenta-derived vascular stem cells hPASCs which are specifically used in the invention are preserved in China center for type culture Collection (China, wuhan, university of Wuhan, and the preservation date is 2025, 4 and 2, and the preservation number is CCTCC NO: C202592 at the 4 and 2 days of 2025. The hPASCs isolated by the invention is derived from placenta vascular tissue macrophages, vascular wall stem cells and/or vascular endothelial progenitor cells, hPASCs positively expresses PAI-1, MECOM, CD201, CD44, CD73 and HLA-ABC, does not express hematopoietic stem cell and endothelial progenitor cell markers (CD 34), does not express pericyte markers (CD 146), and does not express endothelial cell markers CD31 and epithelial cell markers CD326.

As used herein, the term "MSCs", MESENCHYMAL STEM CELLS or MESENCHYMAL STROMAL CELLS, mesenchymal stem cells/mesenchymal stromal cells, is a class of adult stem cells/stromal cells with self-renewing ability and immunoregulatory function, widely present in a variety of tissues, involved in tissue repair, immunoregulation and maintenance of microenvironment homeostasis by differentiation into specific cell types or secretion of bioactive molecules. MSCs are derived from bone marrow, adipose tissue, wharton's jelly of umbilical cord, placenta, dental pulp, synovium, etc. Can be induced to differentiate into osteoblast, adipocyte, chondrocyte (defined by "gold standard"), and can differentiate into nerve-like cells, vascular endothelial cells, etc. under some conditions.

As described herein, the term "huchtmscs", human Umbilical Cord MESENCHYMAL STEM CELLS, human umbilical cord mesenchymal stem cells, is a type of multipotent mesenchymal stem cells isolated and extracted from neonatal umbilical cord tissue (e.g., wharton's jelly), has self-renewal capacity, multipotent differentiation potential and immunoregulatory properties, and is an important cell source for regenerative medicine and cell therapy. The hUCMSCs express mesenchymal stem cell markers (CD 73, CD90 and CD 105), do not express hematopoietic markers (CD 34 and CD 45) and HLA-DR, and can be differentiated into osteoblasts, adipocytes, chondrocytes and the like. hUCMSCs can be used for tissue repair and regeneration, immunoregulatory treatment, anti-fibrosis, anti-aging and the like.

As described herein, the term "hUVECs", human Umbilical Vein Endothelial Cells, human umbilical vein endothelial cells are primary endothelial cells isolated from the inner wall of the umbilical vein of a neonate, have typical vascular endothelial properties, and are widely used in vascular biological research, drug screening, and vascularization model construction in tissue engineering.

As used herein, the term "bioengineered organs," also known as biological artificial organs, are organs or tissues that are cultured to form biological activities using cells or tissues on an animal. The "bioengineered organs" are also divided into foreign and autologous artificial organs. The foreign artificial organ refers to a human organ cultivated on a non-human animal (such as pig or gene-edited pig, rabbit, mouse, dog, etc.), while the autologous artificial organ is a human organ cultivated by using cells or tissues of the patient themselves.

As used herein, the term "decellularized scaffold" or "DKS" refers to a decellularized matrix scaffold from which cells and a portion of the antigen components that cause immune rejection are removed from tissue, characterized by a reduced ability to cause immune rejection upon implantation in vivo. The original extracellular matrix structure of the tissue organ after decellularization, some non-antigen active ingredients such as polysaccharide, collagen, glycoprotein, fibronectin and the like are preserved, and a plurality of natural cell binding sites are preserved in the scaffold to help the adhesion, proliferation and differentiation of cells during the regeneration process. The decellularized scaffold is widely applied to aspects of organ regeneration, tissue repair and reconstruction, biological printing, disease model and drug screening, cell culture and the like in whole organ engineering at present.

As used herein, the term "organoid" refers to a tissue analog having a spatial structure formed by in vitro three-dimensional (3D) culture using adult stem cells or pluripotent stem cells. Organoids are not truly human organs, but can structurally and functionally mimic real organs, maximally mimic in vivo tissue structure and function, and enable long-term stable subculture. Organoids are widely used in the fields of organ development, regenerative medicine, drug screening, precision medicine, gene editing, disease modeling, and the like.

As used herein, the term "fluid circulation perfusion culture" or "cell perfusion culture" or "fluid perfusion culture" is an in vitro culture technique that mimics the dynamic environment of physiological fluids in vivo in bioengineered organ construction by continuously or periodically perfusing a culture fluid into a three dimensional biomaterial-cell complex, providing mechanical stimulation (such as shear stress), improving mass exchange (oxygen, nutrients, metabolic waste), and promoting vascular network formation and tissue functional maturation. The fluid circulation perfusion culture can be used for vascularized organ construction, bone and cartilage regeneration and drug testing models.

As used herein, the term "primary cells" refers to cells that are cultured immediately after removal from the body. The primary cells are widely applied to basic researches of molecules, cell biology and biomedicine, such as proteomics, genomics, cell strain (line) research, DNA, RNA, genetics research and the like, and can be applied to the current hot biomedical industry such as drug screening, drug metabolism and toxicology research, cancer drug research and the like. The most commonly used cultures of primary cells are tissue mass cultures and discrete cell cultures. The tissue block culture is to directly transplant the sheared tissue block on the wall of a culture bottle, and culture the tissue block after adding a culture medium. The dispersive cell culture is to take out animal tissue from body and disperse it into single cell, and culture it in proper culture medium to make the cell survive, grow and reproduce.

As used herein, the term "subject" is intended to include both human and non-human animals. In some embodiments, the subject is a human subject, e.g., a human patient having or at risk of having 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 a human patient suffering from the disorders described herein. Patients suffering from the disorders described herein include, for example, patients who have developed the disorders described herein but are (at least temporarily) asymptomatic, patients who have exhibited the symptoms described herein, and patients suffering from disorders associated with the disorders.

As used herein, the term "treat" 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 when untreated. The treatment may partially or completely reduce, ameliorate, alleviate, inhibit one or more manifestations of an effect or symptom, feature, and/or cause, or reduce the severity thereof, and/or reduce the incidence of, and optionally delay the onset of, a disease. In some embodiments, the treatment is for a subject that does not exhibit certain signs of a certain disorder and/or a subject that exhibits only early signs of a certain disorder. In some embodiments, the subject is a subject exhibiting one or more determined signs of the disorder. In some embodiments, the treatment is performed on a subject diagnosed with a disorder.

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

When referring to a gene product, e.g., encoded by a coding sequence, the terms "protein" (if single-stranded), "polypeptide" and "peptide" are used interchangeably herein. "protein" may also refer to the association of one or more polypeptides. "Gene product" refers to a molecule that results from transcription of a gene. Gene products include RNA molecules transcribed from the gene, as well as proteins translated from such transcripts. Herein "target product" is the product of a gene of interest.

Detailed Description

The technical solution of the present disclosure is further described below by means of specific embodiments. It should be apparent to those skilled in the art that the examples are merely provided to aid in the understanding of the present disclosure and should not be construed as a specific limitation on the present disclosure.

Example 1 hPASCs isolation and identification

1.1 HPASCs isolation culture

HPASCs is separated from human placenta tissue, the human placenta tissue is digested, the digested product is settled to obtain a supernatant, the supernatant is filtered by a disposable sterile filter screen, a filter membrane is collected and is backflushed to obtain placenta microvascular tissue (the diameter is 40-70 mu m), and the placenta microvascular tissue is cultured in a special culture medium (hPASCs culture medium) to obtain primary cells. hPASCs Medium A peripheral cell culture medium (science ^TM, cat.1201) was used as basal medium, and penicillin/streptomycin diabodies were added. And carrying out correlation analysis and identification after hPASCs th passage. hPASCs were inoculated into sterile petri dishes (Jet Biofil, TCD 000100), incubated in 5% CO ₂ incubator at 37.5 ℃. The liquid is changed every 2 days, and the culture is passaged every 4-5 days.

1.2 HPASCs Single cell sequencing

Stem cells were collected at 75% confluency for 3 passages. The survival rate is more than 85%, a 40-micrometer aperture screen is used for filtering, and the single cell suspension is loaded on a single cell microfluid chip to capture at least 10000 cells. The captured mRNA was incubated with the pre-heated reverse transcription mixture in a thermal mixer for 90min at 42℃and 1000 rpm ℃. The captured full-length cDNA was amplified using PCR, purified and cleaved, and a linker was added. The purified cDNA was used for library construction. Paired-end sequencing of 150 bp was performed using Illumina NovaSeq platforms.

1.3 Single cell sequencing of human placenta vascular tissue

Digesting human placenta tissue, settling digested product to obtain supernatant, filtering the supernatant with disposable sterile filter screen, collecting filter membrane, back flushing the filter membrane to obtain placenta microvascular tissue (diameter 40-70 μm), and filtering with 40 μm pore size screen to obtain greater than 10000 cells, and sequencing single cell by the same method as 1.2.

1.4 Single cell analysis

Single cell transcriptome data was processed using CeleScope (version 2.0.4) (https:// gitsub.com/singleron-RD/celescope. Gitsub) and aligned with the reference genome assembly hg 38. Cells were screened for quality control by Seurat (version 4.4.0) and cells with fewer than 200 detectable genes, less than 500 gene counts, or more than 10% mitochondrial gene. The high quality cells were log normalized and scaled using Seurat workflow and the first 2000 hypervariable genes were screened for FindVariableFeatures function (HVGs). Based on these hypervariable genes, principal Component Analysis (PCA) was performed to extract 30 Principal Components (PCs) that explain the greatest variation, and then high quality cells were visualized in two dimensions using the Uniform Manifold Approximation and Projection (UMAP) method. Differentially expressed genes of one cell cluster compared to other cell clusters were identified by Wilcoxon rank sum test function FINDMARKER in Seurat (DEGs). Cell types were annotated in conjunction with renal gene expression profiles. Track analysis was performed using Monocle (version 1.3.1) to predict the differentiation pathway between each fetal kidney organoid cell type without presetting the differentiation time or direction. Although it is difficult to determine the differentiation onset without a priori knowledge, cytoTRACE software package (version 0.3.3) can aid in predicting the order of cell differentiation status and cell stem properties. CellphoneDB (version 3.0.0) is used as an open source database, and by integrating the receptor, ligand and interaction information thereof in the database such as UniProt, ensembl, PDB, IUPHAR, the comprehensive system analysis of intercellular communication molecules is supported, so that interaction and communication mechanisms among different cell types are studied. Analyzing the ligand-receptor relationship characterized in the single cell expression profile by calling the statistical analysis function of CellphoneDB software package

1.5 Live cell workstation

Generation 3 hPASCs was cultured in 3D Matrigel and imaged in real time using live cell workstation CT (Nikon) at 37 ℃ under 5% co ₂. Cell images were collected every 2 hours and recorded for 96 hours. The images are then exported to a computer for analysis and integration into a dynamic video.

1.6 HPASCs flow authentication analysis

The antibody-labeled hPASCs was analyzed using a Flow cytometer (BD, canto II) and cell data analysis was performed using Flow Jo-V10 (version 14.0.0.0) software. Flow antibodies were as follows ：APC Mouse Anti-Human CD201 (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-Human CD146 (BD Pharmingen™, 562134), PE Mouse anti-Human CD31 (BioLegend, 102418), APC Mouse Anti-Human CD326 (BioLegend, 118214).

1.7 Cell immunofluorescent staining

P3 (3 rd generation) hPASCs was seeded in 24 well plates (slides were seeded in well plates before cell seeding) and cells reached 70% confluency, fixed with 4% paraformaldehyde for 15min, washed 3 times with PBS (Solarbio, T8220, PBST) for staining analysis. Blocking was performed with 5% donkey serum (Solarbio, SL 050) for 30-60 min. Primary antibodies were incubated overnight at 4 ℃ after dilution with 2.5% donkey serum. After PBST wash, secondary antibody diluted with 2.5% donkey serum was incubated for 1 hour. After PBST washing, DAPI dye (Solarbio, S2110) was added, and after sealing, the wafer was observed using a confocal laser microscope (Zeiss, LSM 900). Data were analyzed using ZEN 2.6 software. One anti-information is MECOM (ATLAS ANTIBODIES, HPA046537, diluted 1:500), PAI-1 (Abcam, ab222754, diluted 1:1000). Secondary antibody information Goat anti-Rabbit, FITC (ThermoFisher, F-2765,diluted 1:400).

1.8 Results

HPASCs isolation and Single cell sequencing As shown in FIG. 1A, hPASCs cells proliferated in a vascular luminal-like structure, with cell morphology between that of a fibroblast and an epithelial sample (FIG. 1B). By single cell RNA sequencing (scRNA-seq) analysis, the third generation hPASCs can be divided into 8 functionally enriched subpopulations including 4 pro-angiogenic subpopulations, 1 proliferative subpopulation and 3 immunomodulatory subpopulations (fig. 1C and 1D). The pro-angiogenic subgroup features Cluster 1 (angiogenesis 1) which expresses SERPINE1, ACTG1, ANXA2 and MYL6 highly and is related to macrophage function and angiogenesis, cluster 2 (angiogenesis 2) which is rich in HTRA3, SPON2, TFPI2 and AKR1B1 and promotes perivascular migration and invasion, cluster 3 (angiogenesis 3) which expresses specific pro-angiogenic chemokines such as CXCL1, CXCL3, CXCL8 and CCL2, cluster 5 (angiogenesis 4) which contains matrix metalloproteinases (MMP 1 and MMP 14), STC1 and G0S2 and participates in invasion and angiogenesis, and proliferation subgroup features (Cluster 4) which expresses MT-RNR2, MALAT1, NEAT1 and CALD1 highly and has the effects of inhibiting apoptosis and promoting proliferation. The immunoregulation subgroup is characterized by Cluster 6 (immunoregulator 1) taking interferon stimulus genes (ISG 15 and ISG 20), OASL and IFIT1 as markers and having the functions of innate immunoregulation and antiviral, cluster 7 (immunoregulator 2) which is rich in DIO2 and COL12A1 and participates in the cell migration process, and Cluster 8 (immunoregulator 3) which expresses IL1RL1 and CCL20 and is related to immunoregulation and inflammatory response. GO enrichment analysis further confirmed the molecular characteristics of these functional subgroups in terms of angiogenesis, proliferation and immunomodulation (fig. 1E).

To verify the stem characteristics of hPASCs, hPASCs was first functionally divided into three sub-vascular, immunoregulatory and proliferative populations (FIG. 1F), hPASCs was found to have stem cell "stem" i.e. "stem" multipotency to single potency (FIG. 1G) by gene counting and analysis of expressed cell trabecular reconstruction (CytoTRACE), and the potency score and relative ordering results of CytoTRACE further demonstrated that hPASCs has stem cell potency characteristics (FIG. 1H, FIG. 1I).

A characteristic surface molecular marker lineage of hPASCs was further established, hPASCs positive for PAI-1, MECOM (FIG. 1J), while hPASCs positive for CD201, CD44, CD73 and HLA-ABC, non-expression of hematopoietic stem cell and endothelial progenitor markers (CD 34), non-expression of pericyte markers (CD 146), non-expression of endothelial cell markers CD31 and negative for epithelial cell markers CD326 (FIG. 1K) were analyzed by flow through analysis.

To further trace hPASCs, single cell sequencing analysis of human placental vascular tissue revealed that human placental vascular tissue contained vascular endothelial progenitor cells (Edothelial progenitor STEM CELLS, EPCs), vascular wall stem cells (CAPILLARY STEM CELLS, CSCs), pericytes (Pericyte)/vascular smooth muscle cells (vascular Smooth musle cells, vSMCs)/Mesenchymal Stem Cells (MSC), vascular type 2 macrophages (vascular Macrophage, vM 2), erythrocytes (Erythrocyte), extraplacental trophoblast progenitor cells (extravillous trophoblast progenitor, evtpropgonitors), T cells (T cells), natural killer cells (Nature KILLER CELLS, NK), mononuclear cells (mononuclear cell, MN), vascular type 1 macrophages (vascular Macrophage 1, vm1), extraplacental trophoblast progenitor cells (extravillous trophoblast, EVT) thereof, naive B cells (Na ve B cell) and syntonic trophoblast cells (Syncytiotrophoblast, STB) 13 subsets (fig. 2A). hPASCs and placental microvascular tissue correlation analysis showed that hPASCs was isolated from most cells of placental tissue, but that part of the cell population had some correlation, indicating that hPASCs developed from part of the cell types of placental microvascular tissue (fig. 2B, fig. 2C). Further, the development trace was identified by pseudo-clockwise development analysis, and the result shows that hPASCs was developed from placental microvascular tissue with vascular Endothelial Progenitor Cells (EPCs), vascular wall stem cells (CSCs) and vascular type 2 macrophages (vM 2) (fig. 2D).

The above results indicate that hPASCs is a population of stem cells with angiogenic potential and is different from the existing stem cell types, and therefore is defined as a novel stem cell, namely human placenta-derived pro-vascular stem cell (human PLACENTA DERIVED angiogenic STEM CELLS), abbreviated as hPASCs. The human placenta-derived vascular stem cells hPASCs obtained in example 1 were preserved in China center for type culture Collection (China, university of Wuhan, and CCTCC NO: C202592) at 4/2 of 2025.

Example 2 hPASCs 3D Matrigel or 3D "sandwich" cultured angiogenic potential

2.1 HPASCs biomechanical assay

Acrylamide and bisacrylamide were mixed in a ratio of 3% to 0.1% to form a Polyacrylamide (PA) gel with a stiffness of 1 kpa. The PA gel is coated on a 35mm glass bottom culture dish, is pretreated by combining silane, and is polymerized for 30min at room temperature. Rhodamine 0.5 μm carboxylic acid modified fluorescent beads were diluted with water at a 1:50 ratio and coated on the PA gel surface 25 min. 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide, hydrochloric acid (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 (sulo-nhs, sigma,7.6 mg/mL MES, pH 5.5) solutions were allowed to covalently bind fluorescent beads to the gel surface at room temperature for 2h, PBS (pH 7.4) was used instead for 2h after activation with sulfa-Sanpah (Pierce), vitronectin 4℃overnight functionalized gel substrate cells were seeded on the substrate for 6h, 10 μ M Y27632 (Y27) or 10mM Blebb statin (Bleb) were added during cell differentiation to suppress shrinkage of the cytoskeleton, single cell images (phase contrast) and fluorescent images were recorded before and after cell shrinkage were recorded using Digital Image Correlation (DIC) techniques, and the total strain-to measure the total strain-to-transfer the traction-induced by the cell-to-frame-cell-deflection-reference strain transformation in the Fourier transform method was applied to the matrix-dependent strain-cell strain-change in the site-dependent fluorescent bead.

2.2 HPASCs 3D Matrigel and 3D "sandwich" cultured vessels

3D culture:

material pretreatment, namely placing Matrigel (R & D Systems; product No. 3433-005-01) on 4 ℃ ice for slow thawing, preheating a 48-hole culture plate to 37.5 ℃, and pre-cooling a 1.5 mL microcentrifuge tube and a P200 pipette tip to 4 ℃ in advance.

The bottom layer is prepared by mixing 50% Matrigel (V/V) and 50% culture medium (V/V) uniformly, and incubating in 37.5 deg.C and 5% CO ₂ incubator for 30min to solidify thoroughly.

Upper layer inoculation cell suspensions (density 5.3X10 ⁶ cells/mL) were resuspended in endothelial cell culture medium and added to the coagulated matrigel for upper layer culture.

3D "sandwich" culture

Preparation of matrix lower layer 100 μl of endothelial cell culture medium was mixed with 100 μ L MATRIGEL in a pre-chilled 1.5 mL centrifuge tube, the mixture was transferred to a pre-warmed low adsorption 48 well plate and incubated for 30min at 37.5 ℃ with 5% CO ₂ to solidify the matrigel.

The cell middle layer is constructed by mixing 100 mu L of cell suspension and 100 mu L MATRIGEL (1:1, V/V) respectively with human umbilical vein endothelial cells (hUVECs), human umbilical mesenchymal stem cells (hUCMSCs), human placenta-derived vascular stem cells (hPASCs), hUVECs +hUCMSCs co-culture group and hUVECs + hPASCs co-culture group, transferring to the coagulated matrix layer, and incubating again at 37.5 ℃ for 30 minutes to coagulate the cell layer matrigel.

The upper layer culture was maintained by adding 250. Mu.L of pre-warmed endothelial cell medium per well, changing fresh medium daily.

2.3 Results

For systematic comparison hUVECs, hUCMSCs and hPASCs of differences in biomechanical properties, single cell traction (RMS) and Total Strain Energy (TSE) were quantitatively analyzed using a traction microscope (TFM). Single cell RMS profile analysis showed that the traction RMS of humscs was significantly higher than hUVECs (P <0.0001, n=17) and hPASCs (P <0.0001, n=17), while the traction RMS of hPASCs was significantly higher than hUVECs (p=0.0074, n=17), and that humscs were also significantly higher than hUVECs (P <0.0001, n=17) and hPASCs (P <0.0001, n=17) in terms of total strain energy (pJ), but there was no statistical difference between hPASCs and hUVECs (fig. 3B). These results indicate that hucss exhibit the strongest mechanical traction characteristics and strain energy.

To verify hPASCs's angiogenic ability, an in vitro 3D Matrigel culture system was established, and the 3D culture pattern was as shown in figure 3C. First, culture experiments alone showed that huchscs formed spherical aggregates after 24 hours, whereas hUVECs and hPASCs both formed a typical vascular network structure (fig. 3D), suggesting that hPASCs has intrinsic angiogenic potential. Second, it was found by co-culture experiments at different ratios (20:1, 10:1, 5:1, 2:1, and 1:1) that the hUVECs/huchfcs co-culture group formed only globular aggregates, while the hUVECs/hPASCs co-culture group maintained stable vascular network structure at the 20:1, 10:1, and 5:1 ratios, and also exhibited significant migration capacity at the 2:1 and 1:1 ratios (fig. 3E), indicating hPASCs had pro-angiogenic and migration properties superior to huchfcs.

Continuous live cell imaging further demonstrated that hUVECs, hPASCs and its 1:1 co-culture group formed vascular network, whereas the huchscs and their co-culture group with hUVECs did not possess this capability at all (fig. 3F).

To more accurately simulate in vivo microenvironments, the present invention established a 3d "sandwich" matrigel culture model (fig. 3G). To further resolve the intercellular interactions, observations were made using fluorescent labeling techniques (mCherry labeled huchcss/hPASCs, GFP labeled hUVECs). The model results showed that hPASCs alone or co-cultured with hUVECs formed a spatial three-dimensional vascular network, whereas hUVECs, hUCMSCs and its co-cultured group failed to construct vascular structures (fig. 3H and 3I). Notably, the differences in performance of hUVECs in conventional 3D and "sadwick" models suggest that the latter more accurately mimic the complex spatial microenvironment required for angiogenesis in vivo. Taken together, hPASCs exhibited unique spontaneous angiogenic capacity, a property not observed in both hcmscs and hUVECs.

Example 3 hPASCs vascular Structure formation after in vitro infusion of kidney acellular scaffold Material (DKS) perfusion culture

3.1 Preparation of rat kidney decellularized scaffold material (DKS)

A3% isoflurane (RWD, R510-22-10) of a rat (SD) is inhaled for anesthesia, a supine position is used for fixing and cleaning the abdomen, a longitudinal incision is made from pubic symphysis to the xiphoid process, firstly, 100 ml heparin sodium solution (Boyun Biotech, BY 12126) is injected into the liver portal vein to remove residual blood in the kidney, then perirenal fat and kidney capsule are removed, the abdominal aorta and inferior vena cava are subjected to blunt separation, the kidney artery and the kidney vein are reserved, 1 cm abdominal aorta and the vein are connected with the kidney vein, and the inferior vena cava of 1 cm are connected with the vein, 24G strip needle cannula (Vigorboom) is used for cleaning the kidney artery and vein, 4-0 suture (gold ring) is used for carrying out ligation fixation, a glass-taking off waste liquid is placed under the rat kidney in a 10cm sterile culture dish (JET, 02023662-TCD 010100), a peristaltic pump (longe R pump, 300-2J) is used for removing cells BY using an assembly pump head (DG-4, 10 rolers) at a rotation speed of 5rpm (5 ml/min), 1% of the blood is firstly, 1G abdominal aorta and kidney vein and the lower vena vein are reserved for 1G, a 1G strip needle cannula (35F) is placed in a three-96 DEG, a carrier (35.35-35.35.35.35.1% saline solution is filled in a three-96% (35 DEG, and a solution is filled in a three-35.35% carrier (35.35.35.1% saline) for a solution, and a solution is filled for washing solution for a three-35.35% saline carrier (35.35.35.5% and 500.5% saline-35.5% and finally, and a solution is placed in the carrier is filled for a carrier for a solution).

3.2 Kidney organoids (KIO) preparation

The kidney organoid constructed tissue is derived from human kidney donation or biopsy tissue. Tissue treatment employed 1:1 mix of collagenase I (Gibco, 171018029) and collagenase II (Gibco, 171701015) at a concentration of 1 mg/ml. Digestion was carried out at 37℃for 30 minutes, and the reaction was stopped by adding DMEM/F-12 medium (Sigma; D8537-500 ml) containing 10% Fetal Bovine Serum (FBS). The cell fraction was then pelleted by centrifugation at 800 Xg for 5 minutes. Prior to 3D "sadwick" culture, 48 well plates (Jet Biofil) were placed in a CO ₂ incubator for preheating. Matrigel (R & D Systems; 3433-005-01) was stored on ice throughout to maintain its bioactivity. The 3D 'sandwich' culture system consists of a three-layer structure, wherein the bottom layer is a mixed solution of 50% Matrigel (V/V) (R & D Systems, 3433-005-01) and 50% PM-hKOCM (V/V), the Matrigel is uniformly spread by adding a preheating pore plate and then is placed in a 37 ℃ CO ₂ incubator to be solidified for 30 minutes, the middle layer is a mixed system of 30% Matrigel and 70% PM-hKOCM, primary kidney cell suspension is mixed into the medium, the mixture is added onto the solidified bottom layer after being fully mixed, the mixture is solidified for 30 minutes at 37 ℃, and finally 300 mu L of PM-hKOCM complete culture medium is slowly added to the surface of the solidified middle layer. The whole culture system is maintained in a CO ₂ incubator, and fresh culture medium is replaced every day. Kidney organoids (KIO) were obtained.

Wherein PM-hKOCM medium composition is pericyte medium (scientific ^TM, cat.1201), and supplementary ingredients are ：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/mL EGF (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 SB 202190 (MedChemExpress, HY-10295), 0.1µM TTNPB (MedChemExpress, HY-15682),10µM Y-27632（Selleck;S6390） and 0.2% PrimocinTM (V/V) (Invivogen, ant-PM) are added.

3.3 HPASCs in vitro injection of kidney acellular scaffold material for circulatory perfusion and cell-like culture

150 Μl of cell culture medium is used for resuspension of 2×10 ⁷ cells, a 1ml injector (Tansoole, 02026616-TS 069-004) is used for sucking the cell suspension, equal injection is carried out through an indwelling needle sleeve connected with the artery and vein of the decellularized kidney stent, a 50ml centrifuge tube is placed in a mobile phase bottle, 20 ml cell culture medium is added, a peristaltic pump silicone tube artery end (A end) and a peristaltic pump silicone tube vein end (V end) are respectively connected with the indwelling needle sleeve at the artery end and the vein end of the decellularized kidney stent through a mobile phase bottle cap, the kidney stent is immersed in the cell culture medium, the rotating speed is adjusted to be 17rpm (1 ml/min), the flowing direction of the culture medium is adjusted to be from the A end to the V end, the peristaltic pump is started, the rotating speed is adjusted to be 20 rpm (1.2 ml/min) after 12 hours, and liquid is replaced every 2-3 days.

HPASCs in vitro injecting kidney decellularized scaffold for 4 days, re-suspending kidney organoid to 150 μl with culture medium, injecting kidney organoid into kidney scaffold material according to the same injection method, culturing to 24: 24h, and transplanting kidney capsule. One tissue mass was fixed in 4% paraformaldehyde (Shanghai Lingfeng, 20100601) for paraffin embedding and the other tissue mass was fixed in pre-chilled 2.5% glutaraldehyde (AlfaAesar, 111-30-8) for scanning electron microscopy and transmission electron microscopy prior to implantation, with the remaining tissue for rat kidney subintimal implantation.

3.4 Scanning electron microscope (Scanning electron microscope, SEM)

Sample volume of about 4 mm x 3 x mm x 3 mm,2.5% glutaraldehyde (EMS, 16020) soaked above 12 and h ℃ at 4 ℃, washed 3 times with deionized water, 15 min each time, placed in petri dishes (Jet, TCD 010100) and double distilled water added to completely submerge the tissue, placed in-80 ℃ refrigerator 24h, vacuum freeze dryer (Labconco, USA) dry 24h, copper plate fixed to conductive adhesive, vacuum spray plating (HITACHI, japan) spray plating 2 and min, scanning electron microscope (HITACHI, japan) collect images.

3.5 Transmission electron microscope (Transmission electron microscope TEM)

Sample volume was about 1mm ³, 0.1% sodium chloride solution (leva health technologies, C19091502) washed, 2.5% glutaraldehyde (EMS, 16020) fixed at 4oC for 2h, 0.1M phosphate buffer (Yubo biology, YB 160232) washed 3 times, 1% osmium acid (tedpella, 18459) fixed in the dark for 1 h, phosphate buffer, pure water washed 2 times each, 1% uranium acetate (SYNTECHEM, 541-09-3) dyed for 2h,70% acetone, 80% acetone, 90% acetone, 100% acetone sequentially dehydrated, the tissue sequentially soaked in acetone and epoxy (SPI, 1220324) 1:1 mixture, placed in 37 ℃ oven 1 h, placed in acetone and epoxy 1:4 mixture overnight at 37 ℃ oven, placed in 45 ℃ oven 2h after the epoxy was directionally embedded, sequentially placed in 45 ℃ oven 3h,65 ℃ oven 48h polymerized. Microtomes (Ultramicrotome, USA) cut tissue slices smaller than 100nm, observed with a transmission electron microscope (HITACHI, japan) and imaged at 15000×,30000×.

3.6 Results

Fluorescent markers huchcss, hPASCs and hUVECs further trace the angiogenic capacity in decellularized scaffolds (DKS). The dks+ hPASCs +kio group formed hPASCs expressed CD31 vascular structure, indicating that hPASCs was able to differentiate into endothelial cells and form vascular lumen structures, the dks+ hUVECs +kio group had vascular structures, but hUVECs did not fully overlap with CD31, indicating that hUVECs lost endothelial cell characteristics, the dks+ hPASCs + hUVECs +kio group formed vascular structure hUVECs attached to the outside of hPASCs, indicating that hPASCs was more prone to attach to scaffolds than endothelial cells hUVECs, the cells aggregated without specific cell distribution in the dks+hsmscs+ hUVECs +kio group, and the DKS and dks+kio groups did not see vascular structures and CD31 expression (fig. 4A). Further alpha-SMA staining showed similar results to CD31 described above (fig. 4B). SEM results show that the in vitro continuous dynamic culture of DKS+KIO and DKS+ hPASCs +KIO groups has well maintained spatial structure, while other groups have different degrees of collapse of spatial structure, and DKS+ hPASCs +KIO groups have cell connection structure (figure 4C). TEM results indicated that the DKS+ hPASCs +KIO group cells extended pseudopodia attached to the scaffold and formed a connection structure between cells, while cell debris, vacuoles and apoptotic bodies were observed in the other groups (FIG. 4D). In summary, hPASCs has strong adhesion capability and can form vascular structures.

EXAMPLE 4 hPASCs renal capsule transplantation after injection of the decellularized scaffold Material for culture promotes angiogenesis

4.1 Kidney subintimal transplantation:

Rats were fixed in the right lateral position after inhalation anesthesia with 2% isoflurane. A1.5-2 cm column incision was made in the left kidney region, exposing the bilateral kidneys under sterile conditions. After implantation of the graft into the subcapsular space, a 2mm incision was made in the subrenal electrode, which was pushed to the upper electrode with micro forceps. After the kidneys still receive the abdominal cavity, the incision is sutured in layers with 4-0 sutures. The rats were sacrificed 14 days after surgery for material selection.

4.2 Tissue morphology staining

Tissue engineering samples were fixed with 4% paraformaldehyde (Shanghai Lingfeng, 20100601) at 24: 24 h, paraffin embedded, and serial 4 μm tissue sections (LEICA). Baking at 60deg.C to obtain 1 h, sequentially adding into xylene I (JIA NI, 20130115), xylene II (JIA NI, 20130115), absolute ethanol (spa, 20220401), 95%,90%,85%, 80% ethanol solution, soaking 10min respectively, and finally soaking 5min in distilled water for dewaxing to water, and dyeing. H & E staining (Hematoxylin-eosin staining) (Solarbio, G1120), masson trichromatic staining (Masson's trichrome staining) (Solarbio, G1343) are described in the specification.

4.3 Immunofluorescent staining

The tissue sections were immersed in PBS buffer (Solarbio, P1010) for 10 min. Permeabilization with 0.25% Triton X-100 (Beyotime, P0096) for 5-15 min followed by washing with PBS containing 0.1% Tween-20 (Solarbio, T8220, PBST). Blocking was performed with 5% donkey serum (Solarbio, SL 050) for 30-60 min. Primary antibodies were incubated overnight at 4 ℃ after dilution with 2.5% donkey serum. After PBST wash, secondary antibody diluted with 2.5% donkey serum was incubated for 1 hour 15 minutes. After PBST washing, DAPI dye (Solarbio, S2110) was added, and after sealing, the wafer was observed using a confocal laser microscope (Zeiss, LSM 900). Data were analyzed using ZEN 2.6 software. Primary antibody information, CD31 (Abcam; ab281583,1:100 dilution), alpha-SMA (Abcam; ab7817,1:150 dilution), EHD antibody (Proteintech; 25320-1-AP, diluted 1:200).

4.4 Statistical method

The continuous data is expressed by Mean ± standard deviation (Mean ± SD), α=0.05, the comparison between two groups uses two independent sample t-tests if the variances are uniform, a Welch two-sample t-test if the variances are not uniform, the comparison between multiple groups uses one-factor analysis of variance and Turkey's multiple analysis (one-way ANOVA with Turkey's post hoc test) if the variances are uniform, and the analysis is performed by one-factor analysis of variance and Tamhane ^, s T2 test if the variances are not uniform. Differences were considered statistically significant at double sided P < 0.05. If the continuous data do not follow normal distribution, the continuous data are expressed by median and quartile spacing (MEDIAN WITH interquartile range), alpha=0.05, the comparison between two groups uses Mann-Whitney U test, and the comparison between multiple groups uses Kruskal-Wallis rank sum test. Differences were considered statistically significant at double sided P < 0.05. All statistical analyses were performed using IBM SPSS STATISTICS software and all statistical plots were GRAPHPAD PRISM.

4.5 Results

To determine hPASCs kidney capsule graft pro-angiogenic effects, HE staining showed higher numbers of vascular structures in the dks+ hPASCs +kio and dks+ hPASCs + hUVECs +kio groups than in the other groups (fig. 5A). Further by staining with cd31, the dks+ hPASCs +kio group showed significantly more blood vessels than the other groups (fig. 5B), statistical analysis showed that the cd31 ⁺ vascular density in the dks+ hPASCs +kio implant was significantly higher than that in the DKS (n=3, p=0.002), dks+kio (n=3, p=0.046), dks+hmscs+kio (n=5, p=0.004) and dks+ hUVECs +kio (n=5, p=0.001), whereas there was no statistical difference in the dks+ hUVECs +kio, dks+hmscs+kio, dks+kio to cd31+ vascular density in the DKS group (fig. 5C), which indicated hPASCs had a stronger pro-angiogenic effect compared to hUVECs and hmmscs, whereas the hmscs or hPASCs co-implantation with hUVECs did not significantly promote renal capsule graft angiogenesis. To further verify whether hPASCs is involved in host angiogenesis under the kidney capsule, mCherry-hPASCs was followed and found to co-express mCherry with kidney vascular endothelial cell EDH3, CD31 with α -SMA (fig. 5D), indicating that hPASCs is involved in host angiogenesis after implantation of the decellularized scaffold material.

EXAMPLE 5 hPASCs Kidney resected graft promotion of angiogenesis after injection of acellular scaffold Material culture

5.1 Partial kidney resection and implantation

Rats remained in the right lateral position after 2% isoflurane anesthesia. Left kidney was aseptically exposed through a lateral dorsal incision (1.5-2 cm) in the kidney area, and right kidney was left untreated. After blunt separation of the left renal artery and vein, blood flow was blocked with a non-invasive minimally vascular clamp. After clamping, the left kidney is in cyanosis, and the clamping is maintained for 35 minutes to prevent ischemia reperfusion injury. After removal of the lower pole 1/3 of the kidney, the remaining kidney was sutured equidistant from the trimmed stent (volume similar to the removed kidney) using absorbable 5-0 sutures (gold ring medical Co., ltd., R516), spaced about 2mm apart, for a total of 8-9 needles. After implantation, the vascular clamps are removed to restore blood perfusion, and the kidneys immediately restore redness. After the left kidney was returned to the abdominal cavity, the incision was sutured in layers using a non-absorbable 4-0 suture (gold ring medical supplies limited, F402). Rats were sacrificed 14 days post-surgery to obtain left kidney specimens.

5.2 Ultrasonic detection

Kidney B-ultrasonography was performed on day 14 after renal segmental resections and transplants using a high resolution animal ultrasound system (visualsonic, vevo 3100). After anesthetizing the rats with 2% isoflurane and placing in the right lateral decubitus position, the left kidney area was examined using a MX250:20 MHz probe. The probe is slowly moved from the ventral side to the dorsal side near the spine, parallel to the long axis of the kidneys, to scan the entire kidneys. The size, morphology, location and blood flow of the host kidney and the transplanted area were recorded.

5.3 Results

Nephrectomy transplanted in D14 ultrasound 3D vascular modeling showed that dks+ hPASCs +kio implanted groups had higher vascular densities than other groups, while dks+kio, dks+ hUVECs +kio and dks+hcmscs+ hUVECs +kio graft vascular densities were relatively low (fig. 6A). Fluorescence staining showed relatively high numbers of vessels in dks+ hPASCs +kio graft CD31 ⁺ (fig. 6B), statistical analysis showed that dks+ hPASCs +kio graft vessel density was higher than dks+ hUVECs +kio (n=5, p=0.032), dks+hcmscs+kio (n=5, p=0.003), there was no significant difference between dks+kio (n=4, p=0.001) and DKS (n=4, p= 0.000037) groups, and other groups, dks+ hPASCs +kio graft vessel density was higher than dks+ hUVECs + hPASCs +kio group (n=5, p=0.024) (fig. 6C), statistical results showed that hPASCs had a stronger pro-angiogenic capacity in the kidney excision model relative to hcmscs or hUVECs, and implantation in combination with hUVECs had no significant effect on host angiogenesis. Again, hPASCs fluorescent mCherry marker tracking results showed that hPASCs was co-expressed with the vascular surface-associated surface markers EDH3, CD31 and α -SMA (fig. 6D), indicating that hPASCs was involved in host angiogenesis.

Example 6 hPASCs in vivo mechanism for promoting angiogenesis

6.1 RNA sequencing analysis

Tissue samples (20-50 mg) were placed in an RNase-free EP tube (Axygen, MCT-150-C) and stored at-80 ℃. The homogenate was homogenized in 1ml TRIzol (Ambion, 15596026) using a tissue mill (Shanghai Jingxin, JXFSPTPR-24) at 60 Hz frequency for 50 seconds, and after standing at room temperature for 5 minutes, the homogenate was transferred to a fresh tube. 200 μl chloroform (Aldrich, 485403-500 MG) was added, mixed well and left to stand for 3 minutes, and centrifuged (12,000 rpm,15 minutes, 4 ℃). The aqueous phase was collected, added 5. Mu. L ACRYL CARRIER (Solarbio, SA 1020) and 500. Mu.l of isopropanol (Titan, G75885B), mixed well, left to stand for 10 minutes and centrifuged (12,000 rpm,10 minutes, 4 ℃). The supernatant was discarded, 1ml of 75% ethanol was added, and the mixture was centrifuged (7,500 rpm,2 minutes, 4 ℃). After the residual supernatant was dried for 5 minutes, it was resuspended with 30. Mu.l of RNase dH ₂ O (Takara, RR 047A). RNA concentration and quality were measured using a Nano Drop One (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 RNA Nano 6000 detection kit (Agilent Technologies, 5067-1511). mRNA was purified using oligo-dT magnetic beads, and after fragmentation in a divalent cation solution, first strand cDNA was synthesized as a template, and complementary strand was synthesized to form double-stranded cDNA. Library fragments were purified using the qiagquick PCR purification kit (Qiagen, 28104), end repaired, tailed a and ligated. The library was constructed by amplifying the target product by PCR. RNA library cluster generation was performed using HiSeq PE Cluster Kit v4-cBot-HS (Illumina), and sequencing was performed on the Illumina platform to obtain a 150 bp double-end read length. And (5) analyzing a high-expression signal path enriched by differential genes after sequencing, and verifying by Western blotting. The 3 biological replicates were set and P values <0.05 were considered statistically significant.

6.2 Western blotting experiment

Transplanted kidney tissue was lysed with RIPA lysate (Beyotime, P1048), homogenized and incubated in an ice water bath for 10 min. The supernatant was obtained by centrifugation at 12,000 rpm for 10 minutes at 4 ℃. BCA working fluid and standard (ThermoScientific, 23225) were prepared, the absorbance of 562 nm was measured and a standard curve was drawn to calculate the sample concentration. 5 Xloading buffer (Fude Biological Technology, FD 002) was added at 1:4 (v/v), and the mixture was boiled at 99℃for 10 minutes and stored at-20 ℃. Samples and protein markers (ThermoScientific, REF 26617) were loaded onto a PAGE gel (YAMAY BIOTECH, PG 21), 80V electrophoresed for 30min, and changed to 100V until bromophenol blue reached the bottom of the gel. After activation of PVDF membrane (Immobilon, ISEQ 00010) with methanol (Hongsheng FINE CHEMICAL, CY 20211010) for 5 min, 300: 300 mA was transferred for 1.5 h. The primary antibody was incubated overnight at 4℃at room temperature for 1 hour with 5% blocking solution (Beyotime, P0216). The primary antibodies used include AKT rabbit polyclonal antibody (abclonal, A11016), phosphorylated AKT rabbit polyclonal antibody (abclonal, AP 0274), VEGFA rabbit polyclonal antibody (abclonal, A5708) and internal reference beta-Actin rabbit monoclonal antibody (abclonal, AC 026). Secondary antibodies (goat anti-rabbit IgG, HRP-labeled antibody, CST, 7074) were incubated for 1 hour at room temperature. Images were acquired using a multi-functional gel imaging system (BIO-RAD, usa), image Lab and ImageJ 3.0 software to analyze gray values.

6.3 Results

RNA sequencing and GO enrichment analysis showed that the vascular development pathway was significantly up-regulated in DKS+ hPASCs +KIO group compared to DKS+ hUVECs +KIO group, while the angiogenic regulation pathway was significantly up-regulated compared to DKS+hUCMSCs+KIO group (FIG. 7A). KEGG analysis further showed that PIP3/AKT signaling pathway expression levels were higher in the dks+ hPASCs +kio group than in the dks+ hUVECs +kio group (fig. 7B), suggesting that hPASCs could activate angiogenesis-related pathways after transplantation. Western blotting verification results show that the expression level of the DKS+ hPASCs +KIO group pAKT is significantly higher than that of the DKS+ hUVECs +KIO group (n=3, P=0.0246), the DKS+hUCMSCs+KIO group (n=3, P=0.0032), and the expression level of the DKS+ hPASCs +KIO group VEGFA is significantly higher than that of the DKS+ hUVECs +KIO group (n=3, P=0.0398) in the kidney subintimal transplantation model (FIG. 7C). In the rat model with combined bioengineering and kidney suturing for renal partial resection, the pAKT expression level of DKS+ hPASCs +KIO group was significantly higher than that of DKS+ hUVECs +KIO group (n=3, P=0.0297), and the VEGFA expression level of DKS+ hPASCs +KIO group was significantly higher than that of DKS+ hUVECs +KIO group (n=3, P= 0.0456) and DKS+hUCMSCs+KIO group (n=3, P=0.0044) (FIG. 7D). Therefore hPASCs promotes angiogenesis by activating AKT signaling pathways.

Claims

1. The human placenta-derived vascular stem cell hPASCs is characterized in that the preservation number of the human placenta-derived vascular stem cell hPASCs is CCTCC NO: C202592.

2. Human placenta-derived pro-vascular stem cells hPASCs, characterized in that said hPASCs is positive for the markers PAI-1, MECOM positive, CD201 positive, CD44 positive, CD73 positive, HLA-ABC positive, CD34 negative, CD146 negative, CD31 negative and CD326 negative, preferably said hPASCs is derived from placental vascular tissue macrophages, vascular wall stem cells and/or vascular endothelial progenitor cells.

3. The separation method of human placenta-derived vascular stem cells hPASCs is characterized by comprising the following steps of 1) digesting human placenta tissues, 2) settling digestion products to obtain supernatant, 3) filtering the supernatant by a filter screen, and backflushing the filter screen to obtain placenta microvascular tissues, 4) culturing the placenta microvascular tissues in a hPASC culture medium to obtain primary cells, 5) subculturing the primary cells, and 6) identifying hPASC;

preferably, wherein the placental microvascular tissue has a diameter of 20 μm to 100 μm, preferably 40 μm to 70 μm;

preferably, the hPASC culture medium takes a peripheral cell culture medium as a basal medium, and penicillin/streptomycin double antibody is added;

Preferably, the peripheral cell culture medium is Pericyte Medium, cat No. 1201, purchased from scientific;

Preferably, the serum in Pericyte Medium is replaced by PLATELET LYSATE;

Preferably, the identifying comprises defining cells having one or more characteristics selected from the group consisting of a) containing 4 sub-populations of angiogenic stem cells and having stem cell "stem properties", b) being capable of forming vascular structures in vitro or in vivo culture, c) surface markers being PAI-1 positive, MECOM positive, CD201 positive, CD44 positive, CD73 positive, HLA-ABC positive, CD34 negative, CD146 negative, CD31 negative and CD326 negative as hPASCs.

4. A human placenta-derived pro-vascular stem cell hPASCs obtained by the isolation method of claim 3.

5. A composition comprising the human placenta-derived vascular stem cell hPASCs of any one of claims 1,2, and 4.

6. Use of the human placenta-derived pro-vascular stem cell hPASCs of any one of claims 1,2, and 4 or the composition of claim 5 in the manufacture of a medicament for the alleviation of a disorder or treatment of a disease, wherein the disease or disorder is selected from the group consisting of cardiovascular disease, diabetic complications, neurological disease, ophthalmic disease, 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 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 neurological disease is selected from ischemic cerebral apoplexy, spinal cord injury, alzheimer's disease, parkinson's disease and multiple sclerosis, and/or the ophthalmic disease is selected from age-related macular degeneration, retinal vein occlusion, corneal neovascularization disease and glaucomatous optic neuropathy, and/or the chronic wounds are selected from burn wounds, post-operative difficult 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 pro-vascular stem cell hPASCs of any one of claims 1,2 and 4 or the composition of claim 5 in tissue engineering and regenerative medicine, wherein the use comprises promoting vascularization of artificial organs, skin regeneration, bone and cartilage regeneration, nerve tissue repair and reconstruction of complex tissue structures, preferably the use is performed in vitro.

9. Use of the human placenta-derived pro-vascular stem cell hPASCs or the composition of claim 5 according to any one of claims 1,2 and 4 in the preparation of a bioengineered artificial organ, wherein the bioengineered artificial organ comprises an engineered kidney, an engineered blood vessel, an engineered liver, a cardiac patch, an artificial skin, an artificial trachea, an artificial bladder and an artificial cornea, preferably the bioengineered artificial organ is a bioengineered kidney.

10. A method of preparing a bioengineered organ comprising the step of implanting the human placenta-derived vascular stem cell hPASCs and organoids of any one of claims 1, 2 and 4 into a decellularized scaffold.

11. The method of claim 10, wherein the bioengineered organ is selected from the group consisting of an engineered kidney, an engineered blood vessel, an engineered liver, a cardiac 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 or 11, wherein the decellularized scaffold is obtained from an ex vivo organ by decellularization treatment.

13. The method of claim 12, wherein the decellularized treatment comprises a perfusion step of administering a decellularized perfusate to the isolated organ, preferably with a peristaltic pump, preferably one or more times, selected from the group consisting of heparin sodium solution, triton x-100 and SDS solution, optionally further comprising a post-perfusion washing step.

14. A bioengineered organ comprising human placenta-derived pro-vascular stem cells hPASCs according to any one of claims 1,2 and 4 or a composition according to claim 5, preferably obtainable according to the method of any one of claims 10 to 13, preferably selected from bioengineered kidneys, bioengineered blood vessels, bioengineered liver, cardiac patches, artificial skin, artificial trachea, artificial bladder and artificial cornea, preferably bioengineered kidneys.

15. The use of a bioengineered organ according to claim 14 for the manufacture of a medicament or medical device for the treatment of a disease, preferably the bioengineered organ is a bioengineered kidney for the treatment of kidney disease.