CN116875535A - Smog disease vascular organoid model and construction method thereof - Google Patents

Smog disease vascular organoid model and construction method thereof Download PDF

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CN116875535A
CN116875535A CN202310888004.7A CN202310888004A CN116875535A CN 116875535 A CN116875535 A CN 116875535A CN 202310888004 A CN202310888004 A CN 202310888004A CN 116875535 A CN116875535 A CN 116875535A
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collagen
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smog
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薛艺萌
张谦
赵继宗
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Beijing Tiantan Hospital
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Abstract

The invention discloses a smog blood vessel organoid model and a construction method thereof, wherein the construction method comprises the steps of using a culture medium containing WNT agonist and TGF-beta superfamily to induce differentiation of iPSC from smog patients to obtain mesoderm; inducing differentiation of the mesoderm using a medium containing VEGF and cAMP agonists to obtain vascular cells; and (3) inducing the vascular cells by using a culture medium containing VEGF and FGF to obtain a vascular network structure, and finally successfully constructing the smog vascular organoid model. The smog disease vascular organoid model can observe the three-dimensional environment and self-assembly of tissues, captures important intermediate cell types and genetic information of all development stages, is more comprehensive, is convenient and quick to obtain in vitro, and has wide application prospect.

Description

Smog disease vascular organoid model and construction method thereof
Technical Field
The invention belongs to the technical field of organoids, and relates to a smog vascular organoid model and a construction method thereof.
Background
Smog disease (MMD) is a chronic cerebrovascular disease and is a common cause of cerebral apoplexy in children and young adults in China. It is characterized by progressive occlusion or stenosis of the internal carotid artery and the terminal part of its main branch, forming an abnormal network of aerosol blood vessels at the base of the skull. Cerebrovascular events caused by carotid stenosis or occlusion and branch vessel rupture are the major clinical manifestations. At present, the etiology and pathogenesis of the smog disease are not clear, and research shows that the smog disease is related to heredity, inflammation, infectious lesions, abnormal secretion of cytokines, immune response and the like. Pathologies are manifested by thickening of the intimal fibrocyte, irregular relief of the elastic layer, thinning of the media, and abnormal deposition of extracellular matrix.
In 2011, the first susceptibility gene of smoke disease was found to be ring finger protein 213 (ring finger protein, RNF 213), which is the most common susceptibility gene for asian smoke patients, and the risk of onset of smoke disease in carrier with RNF213 p.r4810k (rs 112735431) mutation was significantly increased. Current studies on smoke disease fail to reveal key mechanisms by which RNF213 mutations lead to or are involved in the pathogenesis of smoke disease, and existing models of smoke disease do not mimic the pathogenesis of smoke disease, so the lack of clinical specimens and animal models has hindered the progress of the study. In order to study the pathogenesis of smoke disease, there is an urgent need to explore new disease models and new therapeutic strategies.
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims to provide a smog disease vascular organoid model, and a construction method and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the first aspect of the invention provides a construction method of a smog vessel organoid model, which comprises the following steps:
(1) Inducing differentiation of ipscs derived from smog patients into mesoderm;
(2) Inducing mesoderm described in step (1) to produce a vascular network structure;
(3) The vascular network structure described in the culturing step (2) is formed into a vascular organoid model.
Further, the specific steps of the step (1) include: differentiation of the ipscs was induced using medium containing WNT agonist and TGF- β superfamily to obtain mesoderm.
Further, the WNT agonist comprises one or more of the WNT family, the R-spondin family, norrin, GSK-inhibitor, RNF43 inhibitor, or ZNRF3 inhibitor.
Further, the GSK-inhibitor comprises SB216763, SB 415286, GSK-3 inhibitor, RNF43 inhibitor or ZNRF3 inhibitor.
Further, the GSK-3 inhibitor comprises a GSK-3 alpha inhibitor or a GSK-3 beta inhibitor.
Further, the GSK-3 ss inhibitors include CHIR99021, TD114-2, BIO (6-bromoindirubin-30-acetoxime), kenpaullone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A014418, FRATide, indirubin-3' -oxide or L803.
Further, the GSK-3 beta inhibitor is CHIR99021.
Further, the concentration of CHIR99021 was 12 μm.
Further, the TGF-beta superfamily includes TGF-beta proteins, BMP, GDF, GDNF, activin, lefty, mulllerian inhibitor, inhibin, or Nodal.
Further, the BMP includes BMP2, BMP4, BMP6, or BMP7.
Further, the BMP is BMP4.
Further, the concentration of BMP4 was 30ng/mL.
Further, the culture medium containing WNT agonist and TGF-beta superfamily also includes basal medium, serum substitute, glutamine or its substitute, sulfhydryl reducing agent, and antibiotic.
Further, the iPSC confluence is selected from 40-70%.
Further, the culturing time of the step (1) is from day0 to day 3.
Further, the specific steps of the step (2) include:
(a) Inducing differentiation of the mesoderm to obtain vascular cells;
(b) Embedding the vascular cells in the step (a) in an extracellular matrix for incubation, and inducing the vascular cells to differentiate and bud by using a culture medium containing VEGF and FGF after incubation to obtain a vascular network structure.
Further, the mesoderm was induced to differentiate using medium containing VEGF and cAMP agonist.
Further, the VEGF comprises one or more of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PGF.
Further, the VEGF is VEGF-A.
Further, the concentration of VEGF-A was 100ng/mL.
Further, the cAMP agonists include one or more of forskolin, IBMX, rolipram, 8BrcAMP, PGE2, NKH 477, DBcAMP, sp-8-Br-cAMPs.
Further, the cAMP agonist is forskolin.
Further, the concentration of forskolin is 2 μm.
Further, the FGF comprises one or more of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23.
Further, the FGF is FGF-2.
Further, the FGF-2 concentration is 100ng/mL.
Further, the extracellular matrix comprises one or more of collagen, matrigel, gelatin, laminin, fibronectin, hyaluronic acid and chitosan.
Further, the extracellular matrix is collagen and Matrigel.
Further, the Collagen includes Collagen-I, collagen-II, collagen-III, collagen-IV, collagen-V, collagen-VI or Collagen-VII.
Further, the Collagen is Collagen-I.
Further, the concentration of Collagen-I was 2.0mg/mL.
Further, the pH of the Collagen-I was 7.4.
Further, the mixing ratio of the collagen and Matrigel is 4:1.
Further, the incubation conditions were 37℃for 2h.
Further, the extracellular matrix was also mixed with NaOH, DMEM, HEPES, sodium bicarbonate, glutamine, ham's F-12.
Further, the concentration of sodium bicarbonate was 7.5%.
Further, the culture medium containing VEGF and cAMP agonists also comprises basal medium, serum substitute, glutamine or substitute thereof, sulfhydryl reducing agent, and antibiotics.
Further, the culture medium containing VEGF and FGF also comprises SFM culture medium and serum.
Further, the serum includes FBS, calf serum, horse serum, goat serum or human serum.
Further, the serum is FBS.
Further, the culturing time of the step (a) is from 3 rd day to 4 th day.
Further, the culturing time of the step (b) is from 5 th day to 10 th day.
Further, the specific steps of the step (3) include: the vascular network structure described in step (2) was cultured with a medium containing VEGF and FGF to form a vascular organoid model.
Further, the basal medium comprises one or more of DMEM, IMDM, DMEM/F12, F10 and BME, MEM, neurobasal.
Further, the basal medium is DMEM/F12 medium and neurobasal medium.
Further, the serum replacement comprises KOSR, B27 additive, N2 additive, physiologix TM XF SR、StemSure TM SerumSubstitute Supplement、Knockout TM One or more of the SRs.
Further, the serum replacement is a B27 additive and an N2 additive.
Further, the glutamine or a substitute thereof comprises glutamine, L-alanyl-L-glutamine, hyCyte TM GluMax、glutaGRO TM One or more of them.
Further, the glutamine or a substitute thereof is glutamine.
Further, the sulfhydryl reducing agent comprises one or more of beta-mercaptoethanol and dithiothreitol.
Further, the sulfhydryl reducing agent is beta-mercaptoethanol.
Further, the antibiotics comprise one or more of penicillin-streptomycin, gentamicin and vancomycin.
Further, the antibiotic is penicillin-streptomycin.
Further, the route of acquisition of ipscs from the patient of the smoke disease includes commercial purchase or self-construction.
Further, the smoke patient is a smoke patient carrying a mutation in RNF 213.
In a second aspect the invention provides a smog disease vascular organoid model obtained by the construction method according to the first aspect of the invention.
In a third aspect, the invention provides the use of an organoid model according to the second aspect of the invention for screening for a medicament for the prophylaxis and/or treatment of smog disorders.
In a fourth aspect, the invention provides the use of a smog vessel organoid model according to the second aspect of the invention for the manufacture of a product for evaluating the efficacy of a smog therapeutic drug.
In a fifth aspect, the present invention provides the use of a smoky vascular organoid model according to the second aspect of the invention for evaluating a smoky detection product or method of non-diagnostic interest.
In a sixth aspect, the invention provides the use of a smoke disease organoid model according to the second aspect of the invention for the construction of a smoke disease animal model.
The invention has the advantages and beneficial effects that:
the method for constructing the smog disease blood vessel organoid model can successfully construct the smog disease three-dimensional blood vessel organoid model carrying the susceptibility gene RNF213 mutation, and the smog disease three-dimensional blood vessel organoid model constructed by the method can observe the self-assembly of a three-dimensional environment and tissues, and solve the biological process which can not be observed in a two-dimensional environment; compared with the construction of a gene editing cell line, the smog vascular organoid model constructed based on patient-derived iPSC cells has more comprehensive genetic background information; the smog disease blood vessel organoid model constructed by the invention can also study the interaction between blood vessel wall cells, and compared with the single differentiation of a certain cell, the organoid can better simulate the in-vivo environment.
Drawings
FIG. 1 is a graph showing the results of reprogramming skin cells to iPSCs, wherein 1A is a graph showing the results of skin cell morphology (4X) and 1B is a graph showing the results of iPSC clones grown on MEFs (4X);
FIG. 2 is a graph of the results of immunofluorescent staining to identify the pluripotency of iPSC;
FIG. 3 is a graph showing the results of a successful construction of a model of a smog vessel organoid, wherein 3A is a schematic diagram of the differentiation flow of iPSC, 3B is a graph showing the results of morphological features of various stages in the construction of a model of a smog vessel organoid, 3C is a graph showing the results of immunofluorescence detection of CD31 expression in cells, 3D is a graph showing the results of immunofluorescence detection of CD31 expression (4 x) in mature smog vessel organoids, and 3E is a graph showing the results of immunofluorescence detection of CD31 expression (20 x) in mature smog vessel organoids;
FIG. 4 is a graph showing the results of a single cell sequencing experiment, wherein 4A is a single cell sequencing UMAP dimension reduction visualization and cell grouping annotation graph of a smoke disease and a healthy control, 4B is a cell grouping duty cycle graph of a smoke disease and a healthy control in a vascular-like organ, 4C is a gene expression graph of an endothelial cell marker PECAM1, 4D is a gene expression graph of an endothelial cell marker CLDN5, 4E is a gene expression graph of an endothelial cell marker SOX17, 4F is a gene expression graph of a wall cell marker PDGFRB, 4G is a gene expression graph of a fibroblast marker DCN, and 4H is a gene expression graph of a smooth muscle cell marker PDGFRA;
fig. 5 is a graph showing the results of a flow cytometry experiment, wherein 5A is a graph showing the results of flow analysis of smooth muscle marker CD140B from vascular organoids of smoke disease and healthy controls, and 5B is a graph showing the flow results of statistical analysis. And (3) injection: DAPI in fig. 2 is 4', 6-diamidino-2-phenylindole, a fluorescent dye.
Detailed Description
The following provides definitions of some of the terms used in this specification. 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 invention belongs.
In the present invention, the term "smoky disease" or "smoky syndrome" or "MMD" refers to stenotic occlusive disease of cerebral arteries involving smooth muscle cell proliferation with intimal hyperplasia, resulting in arterial stenosis and occlusion around the viruse's ring (circle of Willis). It involves the formation of new blood vessels resembling "smoke" ("smoky") in subcortical areas. MMD occurs in children and adults with two peaks-the first peak occurring around the age of 5 to 10 years and the second peak occurring between the third and fifth decade of life. Common symptoms include headache or dizziness, weakness or paralysis of the limbs or body side, speech problems-inability to speak or recall words, sensory or cognitive impairment, involuntary movements, seizures or loss of consciousness, vision problems, stroke and cerebral hemorrhage. 80% of MMD cases are carriers of RNF213 and/or R4810K mutations. The treatment options for MMD involve daily use of aspirin, changing lifestyle to maximize brain perfusion, and surgical direct or indirect bypass to restore blood flow.
In the present invention, the term "iPSC" or "induced pluripotent stem cell" refers to a stem cell which is capable of being cultured in vitro and has the ability to differentiate into any cell (tricdermal (ectodermal, mesodermal, endodermal) -derived tissue) (multipotency) constituting a living body (other than placenta), and embryonic stem cells (ES cells) are included in the induced pluripotent stem cell. "induced pluripotent stem cells" are obtained from fertilized eggs, cloned embryos, regenerative stem cells, and stem cells in tissues. Cells having an artificial differentiation multipotency (also referred to as artificial pluripotent stem cells) similar to the differentiation multipotency of embryonic stem cells after the introduction of several genes into somatic cells are also included in pluripotent stem cells. Induced pluripotent stem cells can be prepared by methods known per se.
In the present invention, wnt agonists include, but are not limited to, wnt family, R-spondin mimetic, norrin, GSK-inhibitor, RNF43 inhibitor or ZNRF3 inhibitor.
In some embodiments, WNT agonists may include secreted glycoproteins (Wnt family) including, but not limited to, wnt-l/Int-1, wnt-2/Irp (InM-related protein), wnt-2b/13, wnt-3/Int-4, wnt-3a (R & D system), wnt-4, wnt-5a, wnt-5b, wnt-6 (Kirikoshi H et al, 2001Biochem Biophys Res Com 283 798-805), wnt-7a (R & D system), wnt-7b, wnt-8a/8D, wnt-8b, wnt-9a/14, wnt-9b/14b/15, wnt-10a, wnt-10b/12, wnM l, and Wnt-16.
In some embodiments, wnt agonists may include the R-spondin family of secreted proteins that are involved in the activation and modulation of the Wnt signaling pathway and consist of 4 members (R-spondin 1 (NU 206, nuvelo, san Carlos, CA), R-spondin2 ((R & D system), R-spondin3 and R-spondin-4) and Norrin (also known as Nome disease protein or NDP) (R & D system), which are secreted regulatory proteins that function the same as Wnt proteins in that they bind to the frizzled-4 receptor with high affinity and induce activation of the Wnt signaling pathway (Kestutis Planutis et al, (2007) BMC Cell Biol 8 12).
In some embodiments, the one or more Wnt agonists used in the invention may be R-spondin mimetics, e.g., agonists of Lgr5, such as anti-Lgr 5 antibodies. Small molecule agonists of the Wnt signaling pathway, which are aminopyrimidine derivatives, have been recently identified and are also explicitly listed as Wnt agonists (Lm et al (2005) AngewChem Int Ed Engl 44,1987-90).
In some embodiments, the Wnt agonist is an inhibitor of RNF43 or ZNRF 3. The inventors have found that RNF43 and ZNRF3 are present in the cell membrane and down regulate the level of Wnt receptor complex in the membrane, possibly through ubiquitination of frizzled proteins. Thus, the inventors hypothesize that inhibition of RNF43 or ZNRF3 with antagonistic antibodies, RNAi or small molecule inhibitors would indirectly stimulate the Wnt pathway. RNF43 and ZNRF3 have catalytic loop domains (with ubiquitination activity) that can be used as targets in small molecule inhibitor design. Various anti-RNF 43 antibodies and various anti-ZNRF 3 antibodies are commercially available. In some embodiments, such antibodies are suitable Wnt agonists in the context herein.
In some embodiments, the Wnt agonist may be a GSK-inhibitor. Known GSK-inhibitors include small interfering RNAs (siRNA), lithium (Sigma), and FRAT-family members and FRAT-derived peptides that inhibit GSK-3 interactions with axins. In the present invention, the Wnt agonist is a GSK-3 inhibitor, and the GSK-3 inhibitor includes but is not limited to a GSK-3 alpha inhibitor or a GSK-3 beta inhibitor. Examples of GSK-3β inhibitors include, but are not limited to CHIR99021, TD114-2, BIO (6-bromoindirubin-30-acetoxime), kenpaullone, TWS119, CBM1078, SB216763, 3F8 (tocis), AR-a014418, FRATide, indirubin-3' -oxime or L803. In specific embodiments of the invention, CHIR99021 is added to the medium until the final concentration is 50nM to 100 μm, e.g., 100nM to 50 μm, 1 μm to 30 μm, 1 μm to 15 μm, 5 μm, or 15 μm. Preferably, the concentration of CHIR99021 is 12 μm.
In the present invention, examples of the TGF- β superfamily include, but are not limited to, TGF- β proteins (including TGF- β3), bone Morphogenic Proteins (BMPs) (including BMP2, BMP4, BMP6, or BMP 7), growth Differentiation Factors (GDF), glial-derived neurotrophic factors (GDNF), activin, lefty, mulllerian inhibitory substances (Mu lllerian Inhibiting Substance, MIS), inhibin, or Nodal. In a specific embodiment of the invention, the TGF- β superfamily is BMP4. The final concentration of BMP4 is 1-100ng/mL, e.g., 1-80ng/mL, 1-60ng/mL, 1-40ng/mL, 10-40ng/mL, 20-40ng/mL; further, the final concentration of BMP4 was 30ng/mL.
In the present invention, the term "culture medium" is well-known in the art and generally refers to any substance or formulation used to culture living cells. The term "medium" as used in reference to cell culture includes components of the surrounding environment of the cell. The medium may be solid, liquid, gas or a mixture of phases and materials. The medium includes liquid growth medium and liquid medium that does not sustain cell growth. The medium also includes gel-like media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous media include a gas phase to which cells grown on a petri dish or other solid or semi-solid support are exposed. The term "medium" also refers to a material intended for cell culture, even though the material has not been in contact with cells. In other words, the nutrient-rich liquid to be used for the culture is the medium. The term "basal medium" refers to a medium that promotes the growth of many types of microorganisms that do not require any special nutritional supplements. Most basal media generally comprise four basic chemical groups: amino acids, carbohydrates, inorganic salts and vitamins. Basal media are generally used as the basis for more complex media to which supplements such as serum or serum substitutes, buffers, growth-promoting factors, antibiotics, etc. are added. Examples of basal media include, but are not limited to, one or more of Eagle Basal Medium (BME), minimal Essential Medium (MEM), duibecco's Modified Eagle Medium (DMEM), medium 199, nutrient mixtures HamsF10 (F10) and HamsF12 (F12), mcCoy's 5A, duIbecco ' sMEM/F-12 (DMEM/F12), RPMI 1640, iscove's Modified Duibecco's Medium (IMDM), L-15 medium, neurobasal. In a specific embodiment of the invention, the basal medium is DMEM/F12 medium and neurobasal medium.
In the present invention, the serum replacement in the medium has a meaning well known to those skilled in the art, and refers to a composition or formulation used as a serum replacement in the course of culturing pluripotent stem cells while maintaining an undifferentiated state. That is, the serum replacement is capable of supporting the growth of embryonic stem cells or undifferentiated pluripotent stem cells without the need for serum supplementation. In certain exemplary embodiments, the serum replacement comprises: one or more amino acids, one or more vitamins, one or more trace metal elements. In some cases, the serum replacement may further comprise one or more components selected from the group consisting of: albumin, reduced glutathione, transferrin, insulin, and the like. Non-limiting examples of serum substitutes include, but are not limited to, knockOutTM SR (abbreviated as KOSR), KOSR CTS, N2 additive, CTS N2 additive, B27 additive, physiologix TM XF SR、StemSure TM SerumSubstitute Supplement、Knockout TM One or more of the SRs. In a specific embodiment of the invention, the serum replacement is a B27 additionAdditives and CTS N2 additives.
In the present invention, glutamine or a substitute thereof in a medium includes, but is not limited to: glutamine, L-alanyl-L-glutamine, L-glycyl-L-glutamine, N-acetyl-L-glutamine, hyCyte TM GluMax、glutaGRO TM Or a combination thereof. In a specific embodiment of the invention, the glutamine or a substitute thereof is glutamine.
In the present invention, antibiotics in the medium include, but are not limited to, one or more of Bacitracin (Bacitracin), neomycin (Neomycin), erythromycin (Erythromycin), chloramphenicol (chlormphenicol), penicillin-streptomycin, gentamicin, vancomycin. Additional antibiotic substances for use in the present invention will be apparent to those of ordinary skill in the art.
In the present invention, examples of Vascular Endothelial Growth Factor (VEGF) in the medium include, but are not limited to, one or more of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PGF; preferably, the VEGF is VEGF-A. The concentration of VEGF-A may be, for example, 1-500ng/mL,1-400ng/mL,1-300ng/mL,1-200ng/mL. In Sub>A specific embodiment of the invention, the concentration of VEGF-A is 100ng/mL.
In the present invention, examples of Fibroblast Growth Factor (FGF) in the medium include, but are not limited to, one or more of FGF-1, FGF-2 (bFGF), FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23. In a specific embodiment of the invention, FGF is FGF-2. The concentration of FGF-2 may be, for example, 1-500ng/mL,1-400ng/mL,1-300ng/mL,1-200ng/mL. In a specific embodiment of the invention, the concentration of FGF is 100ng/mL.
In the present invention, cAMP activators in the medium may include one or more of forskolin, IBMX, rolipram, 8BrcAMP, prostaglandin E2 (PGE 2), NKH 477, dibutyl-cAMP (DBcAMP), sp-8-Br-cAMPs. The concentration of a cAMP activator inhibitor such as forskolin may be, for example, 0.1. Mu.M to 100. Mu.M, for example, 1. Mu.M to 50. Mu.M, for example, 5. Mu.M to 20. Mu.M. In a specific embodiment of the invention, the concentration of forskolin is 2 μm.
In the present invention, the term "extracellular matrix (ECM)" is secreted by connective tissue cells and comprises a variety of polysaccharides, water, elastin, and glycoproteins, including Collagen (Collagen), fibronectin, entactin, and laminin. Different types of ECM are known, which include different compositions, such as containing different types of glycoproteins or different combinations of glycoproteins. Examples of cells producing extracellular matrix are chondrocytes mainly producing collagen and proteoglycans, fibroblasts mainly producing type IV collagen, laminin, procollagen and fibronectin, and colon myofibroblasts mainly producing collagen (I, III and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin and myoglycoprotein C. The polysaccharides, elastin, glycoproteins, collagen, fibronectin, entactin and laminin contained in ECM are various polysaccharides, elastin, glycoproteins, collagen, fibronectin, entactin and laminin contained in ECM known in the art. For example, collagen may be Collagen-I, collagen-II, collagen-III, collagen-IV, collagen-V, collagen-VI or Collagen-VII, which are well known in the art to be incorporated into native ECMs.
ECMs suitable for use herein are available from commercial sources. Examples of commercially available extracellular matrices include extracellular matrix proteins (Invitrogen, R&D systems) and matrigel (Matrixgel TM BD Biosciences), and the like. In particular embodiments of the invention, the extracellular matrix includes collagen and Matrigel; further, the Collagen is Collagen-I.
In the present invention, the term "serum-free medium" or "SFM medium" is a medium that is serum-free (e.g., fetal Bovine Serum (FBS), calf serum, horse serum, goat serum, human serum, etc.) and is generally indicated by the letter SFM. Exemplary but non-limiting serum-free media familiar to those skilled in the art include HuMEC basal serum-free media, KNOKOKOUTTM CTSTM exotic ESC/iPSC media, STEMPROTM-34SFM media, STEMPROTM NSC media, ESSENTIALTM-8 media, media 254, media 106, media 131, media 154, media 171, media 200, media 231, heptoZYME-SFM, human endothelial-SFM, FREESTYLETM 293 expression media, media 154CF/PRF, media 154C, media 154CF, media 106, media 200PRF, media 131, essentialTM-6 media, STEMPROTM-34 media astrocyte medium, AIM medium CTSTM, AMINOMAXTM C-100 basal medium, AMINOMAXTM-II complete medium, CD FORTICHOTM medium, CDCHO AGT medium, CHO-S-SFM medium, FREESTYLETM CHO expression medium, CD OPTICHOTM medium, CD CHO medium, CDDG44 medium, SF-900TM medium, EXPI293TM expression medium, LHC basal medium, LHC-8 medium, 293SFM medium, CD 293 medium, AEM growth medium, per. Cell medium, AIM medium, culture medium, keratinocyte-SFM medium, LHC-8 medium, LHC-9 medium, and any derivatives or modifications thereof.
In the present invention, the term "treatment" refers to (1) preventing the appearance of symptoms or disease in a subject who is susceptible to or has not yet displayed a condition; (2) inhibiting the disease or preventing its progression or recurrence; or (3) ameliorating or causing regression of the disease or condition. As understood in the art, "treatment" is a method for achieving a beneficial or desired result, including clinical results. For the purposes of this technology, a beneficial or desired result can include, but is not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a disorder (including a disease), stabilized (i.e., not worsening) state of a disorder (including a disease), delay or slowing of a disorder (including a disease), progression, amelioration or palliation of the disorder (including a disease), state, and remission (whether partial or total), whether detectable or not.
The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. Simple modifications of the invention in accordance with the essence of the invention are all within the scope of the invention as claimed.
EXAMPLE 1 construction of Induced Pluripotent Stem Cells (iPSC) from patients with smog disease
1. Experimental method
Skin tissue from patients with RNF213 mutations were selected, approximately 4mm x 4mm, isolated and cultured as dermal fibroblasts (MEFs), and reprogrammed to ipscs using sendai virus reprogramming kit. Cells were plated onto MEFs for co-culture following Sendai virus infection. After infection, typical human embryonic stem cell-like clones are picked, mechanically separated and gently blown off into small cell clusters, passaged onto new feeder cells, mechanically passaged for more than two generations, and then continuously passaged by collagenase, the cell clones are kept in an undifferentiated state and are typical in morphology, and the construction of iPSC is completed. Expression of stem cell pluripotency markers (nuclear transcription factor OCT4, NANOG) was detected by immunofluorescent staining.
2. Experimental results
The results of the iPSC construction experiments are shown in fig. 1 and demonstrate successful reprogramming of skin tissue from patients with RNF213 mutant smoke to iPSC.
The immunofluorescence detection experiment results are shown in fig. 2, and the results show that the iPS cell line positively expresses nuclear transcription factors OCT4 and NANOG, which indicate that the skin tissues of patients carrying RNF213 mutant smog are successfully reprogrammed to iPSC.
Example 2 cultivation of vascular organoids Using smog-infected patient-derived iPSCs
1. Experimental method
Stage 1: induction of differentiation of iPSC into mesoderm (Day 0-3)
Differentiation can be started when the confluence of the iPSC reaches 40-70%. Starting on Day0 (Day 0), WNT signaling was activated by addition of CHIR99021 and incubated with BMP4 for 3 days. The culture medium was mixed DMEM/F12 medium, neurobasal medium, B27 additive, N2 additive, glutamine, β -mercaptoethanol (1:100), penicillin-streptomycin, 12 μm CHIR99021 and 30ng/mL BMP-4, and the aggregates were transferred to low adhesion plate cultured cells, induced to differentiate into mesoderm.
Stage 2: induction of vascular network structure (Day 3-10)
The mesoderm obtained in stage 1 was switched to vascular induction medium containing VEGF-A for 2 days (Day 3-4). The culture medium was Sub>A mixed DMEM/F12 medium, sub>A neurobasal medium, sub>A B27 supplement, an N2 supplement, glutamine, betSub>A-mercaptoethanol (1:100), penicillin-streptomycin, 100ng/mL VEGF-A, and 2. Mu.M forskolin (forskolin).
Day5 (Day 5), cell aggregates were embedded in a 3D collagen I-Matrigel matrix in 12-well plates, as follows: 2.0mg/mL type I Collagen solution (Collagen I) was prepared, and 0.1N NaOH, 10 XDMEM, HEPES, 7.5% sodium bicarbonate, glutamine (Glutamax) and Ham's F-12 were mixed, and the pH of the Collagen I solution was measured rapidly with a pH indicator strip and found to be 7.4. Mixing the Collagen I-Matrigel according to the ratio of 4:1, and planting the aggregates in the Collagen I-Matrigel matrix to ensure that the aggregates are uniformly distributed; the gel was allowed to set by incubation at 37℃for 2h. The medium was human endothelial SFM medium, 15% FBS, 100ng/mL VEGF-A and 100ng/mL FGF-2, and vascular differentiation and sprouting were induced.
Stage 3: formation of vascular organoids
The whole matrigel was free from the bottom of the well using the rounded end of sterile forceps. The gel was transferred to the lid of a 10cm dish under aseptic conditions using a sterile laboratory spoon. Two sterile 30 gauge needles were used to sever the single vascular network and minimize the amount of excess matrix around the organoids. Single organoids were transferred to 6-well low-attachment plates, and organoids were cultured by adding human endothelial SFM medium, 15% FBS, 100ng/mL VEGF-A, and 100ng/mL FGF-2.
Immunofluorescence techniques detect the expression of cell-specific markers (endothelial cell CD31 markers, smooth muscle cell markers).
2. Experimental results
The experimental results are shown in fig. 3, and the results show that the vascular organoid model is successfully constructed.
Example 3 blood vessel organoid function verification experiment
1. Experimental method
The vascular organoids cultured in example 2 were verified to be smoke disease organoids using single cell sequencing experiments and flow cytometry experiments, as follows:
single cell sequencing method: 40-50 organoids were collected from each group, washed with DPBS and minced with a scalpel. Organoids were transferred to DPBS solutions containing neutral protease enzyme and collagenase. Incubate at 37℃for 20 to 30 minutes. And centrifuged at 300g for 5 minutes at 4 ℃. The cell pellet was resuspended in HBSS, filtered through a cell filter, and centrifuged again at 300g for 5 minutes at 4 ℃ and resuspended in 2% FBS, the cells counted and diluted to the appropriate concentration of 10,000 cells for 10 x single cell sequencing.
The flow experimental method comprises the following steps: the organoid was digested with Ackutase cell dissociation solution to form a single cell suspension. Approximately 1X 10≡6 single cells per group were incubated with Alexa 488-labeled CD31 antibody (1:1000 dilution, BD) for 30 min. The results were analyzed using FlowJo software.
2. Experimental results
The experimental results are shown in fig. 4 and 5, and the results show that the cell annotation is carried out by single cell sequencing, the proportion of smooth muscle in the smoke disease group is higher than that of the healthy control group, and the results are verified by flow analysis to be consistent with the increase of smooth muscle which is the pathological feature of the smoke disease, and the disease phenotype is simulated.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. A method for constructing a smog vessel organoid model, which is characterized by comprising the following steps:
(1) Inducing differentiation of ipscs derived from smog patients into mesoderm;
(2) Inducing mesoderm described in step (1) to produce a vascular network structure;
(3) The vascular network structure described in the culturing step (2) is formed into a vascular organoid model.
2. The construction method according to claim 1, wherein the specific steps of step (1) include: inducing differentiation of the ipscs using a medium containing WNT agonist and TGF- β superfamily to obtain mesoderms;
preferably, the WNT agonist comprises one or more of the WNT family, the R-spondin family, norrin, GSK-inhibitor, RNF43 inhibitor or ZNRF3 inhibitor;
preferably, the GSK-inhibitor comprises a GSK-3 inhibitor;
preferably, the GSK-3 inhibitor comprises a GSK-3 a inhibitor or a GSK-3 β inhibitor;
preferably, the GSK-3 ss inhibitor comprises CHIR99021, TD114-2, BIO, kenpaullone, TWS119, CBM1078, SB216763, TOCRIS, AR-A014418, FRATide, indirubin-3' -oxide or L803;
preferably, the GSK-3β inhibitor is CHIR99021;
preferably, the concentration of CHIR99021 is 12 μm;
preferably, the TGF- β superfamily comprises TGF- β proteins, BMP, GDF, GDNF, activin, lefty, mulllerian inhibitor, inhibin, or Nodal;
preferably, the BMP comprises BMP2, BMP4, BMP6, or BMP7;
preferably, the BMP is BMP4;
preferably, the concentration of BMP4 is 30ng/mL;
preferably, the medium containing WNT agonist and TGF- β superfamily further comprises basal medium, serum replacement, glutamine or its replacement, sulfhydryl reducing agent, antibiotic;
preferably, the specific step of the step (2) includes:
(a) Inducing differentiation of the mesoderm to obtain vascular cells;
(b) Embedding the vascular cells in the step (a) in an extracellular matrix for incubation, and inducing differentiation and sprouting of the vascular cells by using a culture medium containing VEGF and FGF after incubation to obtain a vascular network structure;
preferably, the mesoderm is induced to differentiate using a medium containing VEGF and cAMP agonists;
preferably, the VEGF comprises one or more of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PGF;
preferably, the VEGF is VEGF-A;
preferably, the concentration of VEGF-A is 100ng/mL;
preferably, the cAMP agonist comprises one or more of forskolin, IBMX, rolipram, 8BrcAMP, PGE2, NKH 477, DBcAMP, sp-8-Br-cAMPs;
preferably, the cAMP agonist is forskolin;
preferably, the concentration of forskolin is 2 μm;
preferably, the FGF comprises one or more of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23;
preferably, the FGF is FGF-2;
preferably, the FGF-2 is provided at a concentration of 100ng/mL;
preferably, the extracellular matrix comprises one or more of collagen, matrigel, gelatin, laminin, fibronectin, hyaluronic acid, and chitosan;
preferably, the extracellular matrix is collagen and Matrigel;
preferably, the Collagen comprises Collagen-I, collagen-II, collagen-III, collagen-IV, collagen-V, collagen-VI or Collagen-VII;
preferably, the Collagen is Collagen-I;
preferably, the concentration of the Collagen-I is 2.0mg/mL;
preferably, the pH of the Collagen-I is 7.4;
preferably, the mixing ratio of the collagen to Matrigel is 4:1;
preferably, the incubation conditions are 37 ℃ for 2 hours;
preferably, the extracellular matrix is further mixed with NaOH, DMEM, HEPES, sodium bicarbonate, glutamine, ham's F-12;
preferably, the concentration of sodium bicarbonate is 7.5%; preferably, the culture medium containing VEGF and cAMP agonists further comprises basal medium, serum replacement, glutamine or its replacement, sulfhydryl reducing agent, antibiotics;
preferably, the culture medium containing VEGF and FGF also comprises SFM culture medium and serum;
preferably, the serum comprises FBS, calf serum, horse serum, goat serum or human serum;
preferably, the serum is FBS;
preferably, the specific step of the step (3) includes: the vascular network structure described in step (2) was cultured with a medium containing VEGF and FGF to form a vascular organoid model.
3. The method of claim 2, wherein the basal medium comprises one or more of DMEM, IMDM, DMEM/F12, F10, BME, MEM, neurobasal;
preferably, the basal medium is DMEM/F12 medium and neurobasal medium.
4. The construction method according to claim 2, wherein the serum replacement comprises KOSR, B27 additive, N2 additive, physiologix TM XF SR、StemSure TM SerumSubstitute Supplement、Knockout TM One or more of the SRs;
preferably, the serum replacement is a B27 additive and an N2 additive;
preferably, the glutamine or a substitute thereof comprises glutamine, L-alanyl-L-glutamine, hyCyte TM GluMax、glutaGRO TM One or more of the following;
preferably, the glutamine or a substitute thereof is glutamine;
preferably, the sulfhydryl reducing agent comprises one or more of beta-mercaptoethanol and dithiothreitol;
preferably, the sulfhydryl reducing agent is beta-mercaptoethanol;
preferably, the antibiotics comprise one or more of penicillin-streptomycin, gentamicin and vancomycin;
preferably, the antibiotic is penicillin-streptomycin.
5. The method of claim 1, wherein the pathway for obtaining ipscs of smoky patient origin comprises commercial purchase or self-construction;
preferably, the smoke patient is a smoke patient carrying a mutation in RNF 213.
6. A smog disease vascular organoid model, characterized in that it is obtained by a construction method according to any one of claims 1-5.
7. Use of the organoid model of claim 6 for screening for a medicament for the prevention and/or treatment of smog disorders.
8. Use of a smog disease vascular organoid model of claim 6 in the manufacture of a product for evaluating the efficacy of a smog disease treatment drug.
9. Use of a smoky vascular organoid model according to claim 6 for evaluating a smoky detection product or method of non-diagnostic interest.
10. Use of the smoke disease organoid model of claim 6 for constructing a smoke disease animal model.
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