技术领域Technical Field
本发明属于类器官技术领域，涉及一种烟雾病血管类器官模型及其构建方法。The present invention belongs to the technical field of organoids and relates to a moyamoya disease vascular organoid model and a construction method thereof.
背景技术Background technique
烟雾病(MMD)是一种慢性脑血管疾病，是导致我国儿童和青壮年脑卒中常见的病因。其特征是颈内动脉及其主要分支的终末部分进行性闭塞或狭窄，在颅底形成异常的烟雾血管网络。由颈内动脉狭窄或闭塞及分支血管破裂引起的脑血管事件是主要临床表现。目前，烟雾病的病因及发病机制尚不清楚，研究显示与遗传、炎症、感染性病变、细胞因子分泌异常和免疫反应等相关。病理表现为内膜纤维细胞增厚，弹性层不规则起伏，中膜变薄，以及细胞外基质异常沉积。Moyamoya disease (MMD) is a chronic cerebrovascular disease and a common cause of stroke in children and young adults in my country. It is characterized by progressive occlusion or stenosis of the terminal parts of the internal carotid artery and its main branches, forming an abnormal moyamoya vascular network at the skull base. Cerebrovascular events caused by stenosis or occlusion of the internal carotid artery and rupture of branch vessels are the main clinical manifestations. At present, the cause and pathogenesis of moyamoya disease are still unclear. Studies have shown that it is related to genetics, inflammation, infectious lesions, abnormal cytokine secretion and immune response. The pathological manifestations are thickening of intimal fibroblasts, irregular undulations of the elastic layer, thinning of the media, and abnormal deposition of extracellular matrix.
2011年，发现烟雾病的第一个易感基因环指蛋白213(ring finger protein 213，RNF213)，该基因是亚洲烟雾病患者最常见的易感基因，RNF213 p.R4810K(rs112735431)突变携带者烟雾病发病风险显著增高。目前对烟雾病的研究未能揭示RNF213突变导致或参与烟雾病发病的关键机制，且现有烟雾病模型并不能模拟烟雾病的发病过程，因此临床标本和动物模型的缺乏阻碍了研究的进展。为了研究烟雾病的发病机制，迫切需要探索新的疾病模型和新的治疗策略。In 2011, the first susceptibility gene for moyamoya disease, ring finger protein 213 (RNF213), was discovered. This gene is the most common susceptibility gene for Asian moyamoya disease patients. Carriers of the RNF213 p.R4810K (rs112735431) mutation have a significantly increased risk of developing moyamoya disease. Current research on moyamoya disease has failed to reveal the key mechanism by which RNF213 mutations cause or participate in the pathogenesis of moyamoya disease, and the existing moyamoya disease models cannot simulate the pathogenesis of moyamoya disease. Therefore, the lack of clinical specimens and animal models has hindered the progress of research. In order to study the pathogenesis of moyamoya disease, it is urgent to explore new disease models and new treatment strategies.
发明内容Summary of the invention
为了弥补现有技术的不足，本发明的目的在于提供一种烟雾病血管类器官模型及其构建方法和用途。In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a moyamoya disease vascular organoid model and a construction method and use thereof.
为了实现上述目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solution:
本发明第一方面提供了一种烟雾病血管类器官模型的构建方法，所述构建方法包括以下步骤：A first aspect of the present invention provides a method for constructing a vascular organoid model of moyamoya disease, the method comprising the following steps:
(1)诱导烟雾病病人来源的iPSC分化为中胚层；(1) Induce iPSCs derived from patients with moyamoya disease to differentiate into mesoderm;
(2)诱导步骤(1)中所述的中胚层产生血管网结构；(2) inducing the mesoderm described in step (1) to produce a vascular network structure;
(3)培养步骤(2)中所述的血管网结构形成血管类器官模型。(3) Cultivating the vascular network structure described in step (2) to form a vascular organoid model.
进一步，所述步骤(1)的具体步骤包括：使用含有WNT激动剂和TGF-β超家族的培养基诱导分化所述iPSC，以获得中胚层。Furthermore, the specific steps of step (1) include: using a culture medium containing a WNT agonist and a TGF-β superfamily to induce differentiation of the iPSC to obtain mesoderm.
进一步，所述WNT激动剂包括Wnt家族、R-spondin家族、Norrin、GSK-抑制剂、RNF43抑制剂或ZNRF3抑制剂中的一种或多种。Furthermore, the WNT agonist includes one or more of the Wnt family, the R-spondin family, Norrin, a GSK-inhibitor, a RNF43 inhibitor or a ZNRF3 inhibitor.
进一步，所述GSK-抑制剂包括SB 216763、SB 415286、GSK-3抑制剂、RNF43抑制剂或ZNRF3抑制剂。Furthermore, the GSK-inhibitor includes SB 216763, SB 415286, a GSK-3 inhibitor, a RNF43 inhibitor or a ZNRF3 inhibitor.
进一步，所述GSK-3抑制剂包括GSK-3α抑制剂或GSK-3β抑制剂。Furthermore, the GSK-3 inhibitor includes a GSK-3α inhibitor or a GSK-3β inhibitor.
进一步，所述GSK-3β抑制剂包括CHIR99021、TD114-2、BIO(6-溴靛玉红-30-丙酮肟)、Kenpaullone、TWS119、CBM1078、SB216763、3F8(TOCRIS)、AR-A014418、FRATide、Indirubin-3’-oxime或L803。Further, the GSK-3β inhibitor includes CHIR99021, TD114-2, BIO (6-bromoindirubin-30-acetone oxime), Kenpaullone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A014418, FRATide, Indirubin-3'-oxime or L803.
进一步，所述GSK-3β抑制剂为CHIR99021。Furthermore, the GSK-3β inhibitor is CHIR99021.
进一步，所述CHIR99021的浓度为12μM。Furthermore, the concentration of CHIR99021 is 12 μM.
进一步，所述TGF-β超家族包括TGF-β蛋白、BMP、GDF、GDNF、激活素、Lefty、Mulllerian抑制物质、抑制素或Nodal。Furthermore, the TGF-β superfamily includes TGF-β protein, BMP, GDF, GDNF, Activin, Lefty, Mulllerian inhibitory substance, Inhibin or Nodal.
进一步，所述BMP包括BMP2、BMP4、BMP6或BMP7。Furthermore, the BMP includes BMP2, BMP4, BMP6 or BMP7.
进一步，所述BMP为BMP4。Furthermore, the BMP is BMP4.
进一步，所述BMP4的浓度为30ng/mL。Furthermore, the concentration of BMP4 is 30 ng/mL.
进一步，所述含有WNT激动剂和TGF-β超家族的培养基还包括基础培养基、血清替代物、谷氨酰胺或其替代物、巯基类还原剂、抗生素。Furthermore, the culture medium containing the WNT agonist and the TGF-β superfamily also includes a basal culture medium, a serum substitute, glutamine or a substitute thereof, a thiol reducing agent, and an antibiotic.
进一步，所述iPSC汇合度选自40-70％。Furthermore, the iPSC confluence is selected from 40-70%.
进一步，所述步骤(1)的培养时间为第0天至第3天。Furthermore, the culture time of step (1) is from day 0 to day 3.
进一步，所述步骤(2)的具体步骤包括：Furthermore, the specific steps of step (2) include:
(a)诱导分化所述的中胚层，获得血管细胞；(a) inducing differentiation of the mesoderm to obtain vascular cells;
(b)将步骤(a)中所述血管细胞包埋在细胞外基质中进行孵育，孵育后使用含有VEGF和FGF的培养基诱导所述血管细胞分化、出芽，获得血管网结构。(b) embedding the vascular cells in step (a) in an extracellular matrix for incubation, and after incubation, using a culture medium containing VEGF and FGF to induce differentiation and sprouting of the vascular cells to obtain a vascular network structure.
进一步，使用含有VEGF和cAMP激动剂的培养基诱导分化所述的中胚层。Furthermore, the mesoderm differentiation is induced using a culture medium containing VEGF and a cAMP agonist.
进一步，所述VEGF包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、PGF中的一种或几种。Furthermore, the VEGF includes one or more of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF.
进一步，所述VEGF为VEGF-A。Furthermore, the VEGF is VEGF-A.
进一步，所述VEGF-A的浓度为100ng/mL。Furthermore, the concentration of VEGF-A is 100 ng/mL.
进一步，所述cAMP激动剂包括forskolin、IBMX、Rolipram、8BrcAMP、PGE2、NKH477、DBcAMP、Sp-8-Br-cAMPs中的一种或多种。Furthermore, the cAMP agonist includes one or more of forskolin, IBMX, Rolipram, 8BrcAMP, PGE2, NKH477, DBcAMP, and Sp-8-Br-cAMPs.
进一步，所述cAMP激动剂为forskolin。Furthermore, the cAMP agonist is forskolin.
进一步，所述forskolin的浓度为2μM。Furthermore, the concentration of forskolin is 2 μM.
进一步，所述FGF包括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中的一种或几种。Furthermore, the FGF includes 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, and FGF-23.
进一步，所述FGF为FGF-2。Furthermore, the FGF is FGF-2.
进一步，所述FGF-2的浓度为100ng/mL。Furthermore, the concentration of FGF-2 is 100 ng/mL.
进一步，所述细胞外基质包括胶原蛋白、Matrigel、明胶、层粘连蛋白、纤连蛋白、透明质酸、壳聚糖中的一种或几种。Furthermore, the extracellular matrix includes one or more of collagen, Matrigel, gelatin, laminin, fibronectin, hyaluronic acid, and chitosan.
进一步，所述细胞外基质为胶原蛋白和Matrigel。Furthermore, the extracellular matrix is collagen and Matrigel.
进一步，所述胶原蛋白包括Collagen-I、Collagen-Ⅱ、Collagen-Ⅲ、Collagen-Ⅳ、Collagen-Ⅴ、Collagen-Ⅵ或Collagen-Ⅶ。Furthermore, the collagen includes Collagen-I, Collagen-II, Collagen-III, Collagen-IV, Collagen-V, Collagen-VI or Collagen-VII.
进一步，所述胶原蛋白为Collagen-I。Furthermore, the collagen is Collagen-I.
进一步，所述Collagen-I的浓度为2.0mg/mL。Furthermore, the concentration of the Collagen-I is 2.0 mg/mL.
进一步，所述Collagen-I的pH值为7.4。Furthermore, the pH value of the Collagen-I is 7.4.
进一步，所述胶原蛋白和Matrigel的混合比例为4:1。Furthermore, the mixing ratio of the collagen and Matrigel is 4:1.
进一步，所述孵育的条件为37℃、2h。Furthermore, the incubation conditions are 37° C. and 2 h.
进一步，所述细胞外基质还与NaOH、DMEM、HEPES、碳酸氢钠、谷氨酰胺、Ham’s F-12混合。Furthermore, the extracellular matrix is also mixed with NaOH, DMEM, HEPES, sodium bicarbonate, glutamine, and Ham's F-12.
进一步，碳酸氢钠的浓度为7.5％。Furthermore, the concentration of sodium bicarbonate was 7.5%.
进一步，所述含有VEGF和cAMP激动剂的培养基还包括基础培养基、血清替代物、谷氨酰胺或其替代物、巯基类还原剂、抗生素。Furthermore, the culture medium containing VEGF and cAMP agonist also includes basal culture medium, serum substitute, glutamine or its substitute, sulfhydryl reducing agent, and antibiotics.
进一步，所述含有VEGF和FGF的培养基还包括SFM培养基、血清。Furthermore, the culture medium containing VEGF and FGF also includes SFM culture medium and serum.
进一步，所述血清包括FBS、小牛血清、马血清、山羊血清或人类血清。Furthermore, the serum includes FBS, calf serum, horse serum, goat serum or human serum.
进一步，所述血清为FBS。Furthermore, the serum is FBS.
进一步，所述步骤(a)的培养时间为第3天至第4天。Furthermore, the culturing time of step (a) is from the 3rd day to the 4th day.
进一步，所述步骤(b)的培养时间为第5天至第10天。Furthermore, the culturing time of step (b) is from the 5th day to the 10th day.
进一步，所述步骤(3)的具体步骤包括：更换为含有VEGF和FGF的培养基培养步骤(2)中所述的血管网结构，以形成血管类器官模型。Furthermore, the specific steps of step (3) include: replacing the culture medium containing VEGF and FGF to culture the vascular network structure described in step (2) to form a vascular organoid model.
进一步，所述基础培养基包括DMEM、IMDM、DMEM/F12、F12、F10、BME、MEM、neurobasal中的一种或几种。Furthermore, the basic culture medium includes one or more of DMEM, IMDM, DMEM/F12, F12, F10, BME, MEM, and neurobasal.
进一步，所述基础培养基为DMEM/F12培养基和neurobasal培养基。Furthermore, the basal culture medium is DMEM/F12 culture medium and neurobasal culture medium.
进一步，所述血清替代物包括KOSR、B27添加剂、N2添加剂、PhysiologixTM XF SR、StemSureTM SerumSubstitute Supplement、KnockoutTM SR中的一种或几种。Furthermore, the serum substitute includes one or more of KOSR, B27 supplement, N2 supplement, Physiologix™ XF SR, StemSure™ SerumSubstitute Supplement, and Knockout™ SR.
进一步，所述血清替代物为B27添加剂和N2添加剂。Furthermore, the serum substitutes are B27 additive and N2 additive.
进一步，所述谷氨酰胺或其替代物包括谷氨酰胺、L-丙氨酰-L-谷氨酰胺、HyCyteTMGluMax、glutaGROTM中的一种或几种。Furthermore, the glutamine or its substitute includes one or more of glutamine, L-alanyl-L-glutamine, HyCyte™ GluMax, and glutaGRO™ .
进一步，所述谷氨酰胺或其替代物为谷氨酰胺。Furthermore, the glutamine or its substitute is glutamine.
进一步，所述巯基类还原剂包括β-巯基乙醇、二硫苏糖醇中的一种或几种。Furthermore, the thiol reducing agent includes one or more of β-mercaptoethanol and dithiothreitol.
进一步，所述巯基类还原剂为β-巯基乙醇。Furthermore, the thiol reducing agent is β-mercaptoethanol.
进一步，所述抗生素包括青霉素-链霉素、庆大霉素、万古霉素中的一种或几种。Furthermore, the antibiotics include one or more of penicillin-streptomycin, gentamicin, and vancomycin.
进一步，所述抗生素为青霉素-链霉素。Furthermore, the antibiotic is penicillin-streptomycin.
进一步，所述烟雾病病人来源的iPSC的获取途径包括商业购买或自己构建。Furthermore, the iPSCs derived from the moyamoya disease patients can be obtained by commercial purchase or self-construction.
进一步，所述烟雾病患者为携带RNF213突变的烟雾病患者。Furthermore, the moyamoya disease patient is a moyamoya disease patient carrying an RNF213 mutation.
本发明第二方面提供了一种烟雾病血管类器官模型，所述烟雾病类器官模型由本发明第一方面所述的构建方法获得。A second aspect of the present invention provides a moyamoya disease vascular organoid model, wherein the moyamoya disease organoid model is obtained by the construction method described in the first aspect of the present invention.
本发明第三方面提供了本发明第二方面所述的类器官模型在筛选预防和/或治疗烟雾病的药物中的应用。The third aspect of the present invention provides use of the organoid model described in the second aspect of the present invention in screening drugs for preventing and/or treating moyamoya disease.
本发明第四方面提供了本发明第二方面所述的烟雾病血管类器官模型在制备评价烟雾病治疗药物的药效的产品中的应用。The fourth aspect of the present invention provides the use of the moyamoya disease vascular organoid model described in the second aspect of the present invention in the preparation of a product for evaluating the efficacy of a drug for treating moyamoya disease.
本发明第五方面提供了本发明第二方面所述的烟雾病血管类器官模型在评价非诊断目的烟雾病检测产品或方法中的应用。The fifth aspect of the present invention provides the use of the moyamoya disease vascular organoid model described in the second aspect of the present invention in evaluating a moyamoya disease detection product or method for non-diagnostic purposes.
本发明第六方面提供了本发明第二方面所述的烟雾病类器官模型在构建烟雾病动物模型中的应用。The sixth aspect of the present invention provides the use of the moyamoya disease organoid model described in the second aspect of the present invention in constructing a moyamoya disease animal model.
本发明的优点和有益效果：Advantages and beneficial effects of the present invention:
本发明提供的一种烟雾病血管类器官模型的构建方法能够成功构建携带易感基因RNF213突变的烟雾病三维血管类器官模型，且本发明构建的烟雾病三维血管类器官模型，可以观察到三维环境和组织的自组装，解决在二维环境中无法观察到的生物学过程；基于患者来源的iPSC细胞构建的烟雾病血管类器官模型，相较于构建基因编辑细胞系，遗传背景信息更加全面；本发明构建的烟雾病血管类器官模型还可以研究血管壁细胞间的相互作用，相较于单独分化某一种细胞，类器官能够更好的模拟体内环境。The method for constructing a moyamoya disease vascular organoid model provided by the present invention can successfully construct a three-dimensional vascular organoid model of moyamoya disease carrying a mutation in the susceptibility gene RNF213, and the three-dimensional vascular organoid model of moyamoya disease constructed by the present invention can observe the three-dimensional environment and self-assembly of tissues, solving biological processes that cannot be observed in a two-dimensional environment; the moyamoya disease vascular organoid model constructed based on patient-derived iPSC cells has more comprehensive genetic background information compared to the construction of gene-edited cell lines; the moyamoya disease vascular organoid model constructed by the present invention can also study the interaction between vascular wall cells, and compared with the differentiation of a single cell, the organoid can better simulate the in vivo environment.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是将皮肤细胞重新编程为iPSC的结果图，其中，1A为皮肤细胞形态(4×)的结果图，1B为在MEF上生长的iPSC克隆(4×)的结果图；FIG1 is a diagram showing the results of reprogramming skin cells into iPSCs, wherein FIG1A is a diagram showing the results of skin cell morphology (4×), and FIG1B is a diagram showing the results of iPSC clones grown on MEFs (4×);
图2是免疫荧光染色鉴定iPSC多能性的结果图；FIG2 is a diagram showing the results of immunofluorescence staining to identify iPSC pluripotency;
图3是成功构建烟雾病血管类器官模型的结果图，其中，3A为iPSC分化流程示意图，3B为构建烟雾病血管类器官模型中各个阶段的形态特征的结果图，3C为免疫荧光技术检测细胞中CD31表达的结果图，3D为免疫荧光技术检测成熟的烟雾病血管类器官中CD31表达(4x)的结果图，3E为免疫荧光技术检测成熟的烟雾病血管类器官中CD31表达(20x)的结果图；Figure 3 is a result diagram of the successful construction of a moyamoya disease vascular organoid model, wherein 3A is a schematic diagram of the iPSC differentiation process, 3B is a result diagram of the morphological characteristics of each stage in the construction of a moyamoya disease vascular organoid model, 3C is a result diagram of the immunofluorescence technique for detecting CD31 expression in cells, 3D is a result diagram of the immunofluorescence technique for detecting CD31 expression in mature moyamoya disease vascular organoids (4x), and 3E is a result diagram of the immunofluorescence technique for detecting CD31 expression in mature moyamoya disease vascular organoids (20x);
图4是单细胞测序实验的结果图，其中，4A为烟雾病和健康对照的单细胞测序UMAP降维可视化和细胞分群注释图，4B为类血管器官中烟雾病和健康对照的细胞分群占比图，4C为内皮细胞标志物PECAM1的基因表达图，4D为内皮细胞标志物CLDN5的基因表达图，4E为内皮细胞标志物SOX17的基因表达图，4F为壁细胞标志物PDGFRB的基因表达图，4G为成纤维细胞标志物DCN的基因表达图，4H为平滑肌细胞标志物PDGFRA的基因表达图；Figure 4 is a graph showing the results of a single-cell sequencing experiment, wherein 4A is a UMAP dimensionality reduction visualization and cell clustering annotation graph of single-cell sequencing of moyamoya disease and healthy controls, 4B is a graph showing the cell clustering proportions of moyamoya disease and healthy controls in vascular organoids, 4C is a gene expression graph of endothelial cell marker PECAM1, 4D is a gene expression graph of endothelial cell marker CLDN5, 4E is a gene expression graph of endothelial cell marker SOX17, 4F is a gene expression graph of parietal cell marker PDGFRB, 4G is a gene expression graph of fibroblast marker DCN, and 4H is a gene expression graph of smooth muscle cell marker PDGFRA;
图5是流式细胞实验的结果图，其中，5A为烟雾病和健康对照的血管类器官流式分析平滑肌标志物CD140b结果图，5B为流式结果统计分析图。注：图2中的DAPI为4’,6-二脒基-2-苯基吲哚，是一种荧光染料。Figure 5 is a graph showing the results of a flow cytometry experiment, where Figure 5A shows the results of flow cytometry analysis of smooth muscle marker CD140b in vascular organoids of moyamoya disease and healthy controls, and Figure 5B shows the statistical analysis of flow cytometry results. Note: DAPI in Figure 2 is 4',6-diamidino-2-phenylindole, a fluorescent dye.
具体实施方式Detailed ways
下文提供了本说明书中使用的一些术语的定义。除非另有说明，本发明中使用的所有技术和科学用语通常具有和本发明所属领域的普通技术人员通常理解的意思相同的意思。The following provides definitions of some terms used in this specification. Unless otherwise defined, all technical and scientific terms used in the present invention generally have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.
在本发明中，术语“烟雾病”或“烟雾病综合征”或“MMD”是指脑动脉的狭窄闭塞性疾病，涉及平滑肌细胞增殖伴有内膜增生，导致韦利斯氏环(circle of Willis)周围的动脉狭窄和闭塞。其涉及在皮层下区域形成类似“烟雾”(“烟雾病”)的新血管。MMD发生在儿童和成人中，有两个高峰—第一个高峰出现在5岁至10岁左右，第二个高峰出现在生命的第三个十年与第五个十年之间。常见症状包含头痛或头晕、四肢或身体一侧无力或瘫痪、言语问题—无法说话或回忆单词，感觉或认知障碍、不自主运动、癫痫发作或意识丧失、视力问题、中风和脑出血。80％的MMD病例是RNF213和/或R4810K突变的携带者。MMD的治疗选择涉及每日使用阿司匹林，改变生活方式以最大化脑灌注，以及外科手术直接或间接旁路以恢复血流。In the present invention, the term "moyamoya disease" or "moyamoya syndrome" or "MMD" refers to a stenotic occlusive disease of the cerebral arteries, involving smooth muscle cell proliferation accompanied by intimal hyperplasia, leading to arterial stenosis and occlusion around the circle of Willis. It involves the formation of new blood vessels resembling "moyamoya" ("moyamoya disease") in the subcortical area. MMD occurs in children and adults, with two peaks - the first peak occurs around 5 to 10 years old, and the second peak occurs between the third and fifth decades of life. Common symptoms include headache or dizziness, weakness or paralysis of the limbs or one side of the body, 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. Treatment options for MMD involve daily use of aspirin, lifestyle changes to maximize cerebral perfusion, and surgical direct or indirect bypass to restore blood flow.
在本发明中，术语“iPSC”或“诱导多能干细胞”或是指这样的干细胞，其能够在体外培养并且有能力分化为构成活体(除了胎盘以外)的任何细胞(三胚层(外胚层，中胚层，内胚层)-来源的组织)(多能性)，并且胚胎干细胞(ES细胞)包括在诱导多能干细胞中。“诱导多能干细胞”获得自受精卵，克隆胚胎，再生性干细胞，和组织中的干细胞。在将若干种基因引入体细胞后具有与胚胎干细胞的分化多能性类似的人工分化多能性的细胞(也称为人工多能干细胞)也包括在多能干细胞中。诱导多能干细胞可以通过本身已知的方法制备。In the present invention, the term "iPSC" or "induced pluripotent stem cell" refers to stem cells that can be cultured in vitro and have the ability to differentiate into any cell (three germ layers (ectoderm, mesoderm, endoderm)-derived tissue) (pluripotency) constituting a living body (except the placenta), and embryonic stem cells (ES cells) are included in induced pluripotent stem cells. "Induced pluripotent stem cells" are obtained from fertilized eggs, cloned embryos, regenerative stem cells, and stem cells in tissues. Cells (also called artificial pluripotent stem cells) that have artificial differentiation pluripotency similar to that of embryonic stem cells after introducing several genes into somatic cells are also included in pluripotent stem cells. Induced pluripotent stem cells can be prepared by methods known per se.
在本发明中，Wnt激动剂包括但不限于Wnt家族、R-spondin家族、R-spondin模拟物、Norrin、GSK-抑制剂、RNF43抑制剂或ZNRF3抑制剂。In the present invention, Wnt agonists include, but are not limited to, Wnt family, R-spondin family, R-spondin mimetics, Norrin, GSK-inhibitors, RNF43 inhibitors or ZNRF3 inhibitors.
在一些实施方案中，WNT激动剂可以包括分泌性糖蛋白(Wnt家族)，所述Wnt家族包括但不限于Wnt-l/Int-1、Wnt-2/Irp(InM-相关蛋白)、Wnt-2b/13、Wnt-3/Int-4、Wnt-3a(R&D系统)、Wnt-4、Wnt-5a、Wnt-5b、Wnt-6(Kirikoshi H等人,2001Biochem Biophys Res Com283 798-805)、Wnt-7a(R&D系统)、Wnt-7b、Wnt-8a/8d、Wnt-8b、Wnt-9a/14、Wnt-9b/14b/15、Wnt-10a、Wnt-10b/12、WnM l和Wnt-16。In some embodiments, the WNT agonist may include a secreted glycoprotein (Wnt family), which includes but is not limited to Wnt-1/Int-1, Wnt-2/Irp (InM-related protein), Wnt-2b/13, Wnt-3/Int-4, Wnt-3a (R&D Systems), Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi H et al., 2001 Biochem Biophys Res Com 283 798-805), Wnt-7a (R&D Systems), Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14, Wnt-9b/14b/15, Wnt-10a, Wnt-10b/12, WnM 1 and Wnt-16.
在一些实施方案中，Wnt激动剂可以包括分泌性蛋白的R-spondin家族，其涉及Wnt信号转导途径的活化和调节并且由4个成员(R-spondin1(NU206,Nuvelo,San Carlos,CA)、R-spondin2((R&D系统)、R-spondin3和R-spondin-4)和Norrin(还称为Nome疾病蛋白或NDP)(R&D系统)组成，其为分泌性调节蛋白，所述蛋白发挥与Wnt蛋白相同的功能，因为其以高亲合性结合于卷曲-4受体并诱导Wnt信号转导途径的活化(Kestutis Planutis等人,(2007)BMC Cell Biol 8 12)。In some embodiments, Wnt agonists may include the R-spondin family of secreted proteins, which are involved in the activation and regulation of the Wnt signaling pathway and consist of four members (R-spondin1 (NU206, Nuvelo, San Carlos, CA), R-spondin2 ((R&D Systems), R-spondin3 and R-spondin-4) and Norrin (also known as Nome disease protein or NDP) (R&D Systems), which is a secreted regulatory protein that performs the same function as Wnt proteins in that it binds to the Frizzled-4 receptor with high affinity and induces activation of the Wnt signaling pathway (Kestutis Planutis et al., (2007) BMC Cell Biol 8 12).
在一些实施方案中，用于本发明的一种或多种Wnt激动剂可以为R-spondin模拟物，例如Lgr5的激动剂，诸如抗-Lgr5抗体。为氨基嘧啶衍生物的Wnt信号转导途径的小分子激动剂是最近被确定的，并且还明确列为Wnt激动剂(Lm等人(2005)AngewChem Int EdEngl 44,1987-90)。In some embodiments, one or more Wnt agonists used in the present invention may be R-spondin mimetics, for example, agonists of Lgr5, such as anti-Lgr5 antibodies. Small molecule agonists of the Wnt signaling pathway that are aminopyrimidine derivatives have recently been identified and are also specifically listed as Wnt agonists (Lm et al. (2005) Angew Chem Int Ed Engl 44, 1987-90).
在一些实施方案中，Wnt激动剂是RNF43或ZNRF3的抑制剂。发明人已发现，RNF43和ZNRF3存在于细胞膜并且负调节膜中Wnt受体复合物的水平，可能通过卷曲蛋白的泛素化。因此，发明人假设用拮抗抗体、RNAi或小分子抑制剂抑制RNF43或ZNRF3会间接刺激Wnt途径。RNF43和ZNRF3具有催化环形域(具有泛素化活性)，其可以在小分子抑制剂设计中用作靶标。多种抗-RNF43抗体和多种抗-ZNRF3抗体是可商购的。在一些实施方案中，这样的抗体在本文情形中是合适的Wnt激动剂。In some embodiments, Wnt agonists are inhibitors of RNF43 or ZNRF3. The inventors have found that RNF43 and ZNRF3 are present in the cell membrane and negatively regulate the level of Wnt receptor complexes in the membrane, possibly through ubiquitination of frizzled protein. Therefore, the inventors assume that inhibiting RNF43 or ZNRF3 with antagonistic antibodies, RNAi or small molecule inhibitors will indirectly stimulate the Wnt pathway. RNF43 and ZNRF3 have catalytic annular domains (with ubiquitination activity), which can be used as targets in the design of small molecule inhibitors. Various anti-RNF43 antibodies and various anti-ZNRF3 antibodies are commercially available. In some embodiments, such antibodies are suitable Wnt agonists in the present context.
在一些实施方案中，Wnt激动剂可以为GSK-抑制剂。已知的GSK-抑制剂包括小干扰RNA(siRNA,Cell Signaling)、锂(Sigma)以及FRAT-家族成员和抑制GSK-3与轴蛋白相互作用的FRAT-衍生的肽。在本发明中，所述Wnt激动剂为GSK-3抑制剂，GSK-3抑制剂包括但不限于GSK-3α抑制剂或GSK-3β抑制剂。GSK-3β抑制剂的实例包括但不限于CHIR99021、TD114-2、BIO(6-溴靛玉红-30-丙酮肟)、Kenpaullone、TWS119、CBM1078、SB216763、3F8(TOCRIS)、AR-A 014418、FRATide、Indirubin-3’-oxime或L803。在本发明的具体实施例中，向培养基添加CHIR99021直至最终浓度为50nM至100μM，例如100nM至50μM、1μM至30μM、1μM至15μM、5μM或15μM。优选地，CHIR99021的浓度为12μM。In some embodiments, the Wnt agonist can be a GSK-inhibitor. Known GSK-inhibitors include small interfering RNA (siRNA, Cell Signaling), lithium (Sigma), and FRAT-family members and FRAT-derived peptides that inhibit the interaction of GSK-3 with axin. 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-3α inhibitor or a GSK-3β inhibitor. Examples of GSK-3β inhibitors include but are not limited to CHIR99021, TD114-2, BIO (6-bromoindirubin-30-acetone oxime), Kenpaullone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A 014418, FRATide, Indirubin-3'-oxime or L803. In a specific embodiment of the present invention, CHIR99021 is added to the culture medium to a final concentration of 50 nM to 100 μM, such as 100 nM 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.
在本发明中，TGF-β超家族的实例包括但不限于TGF-β蛋白(包括TGFβ3)、骨形态发生蛋白(BMP)(包括BMP2、BMP4、BMP6或BMP7)、生长分化因子(GDF)、神经胶质衍生的神经营养因子(GDNF)、激活素、Lefty、Mulllerian抑制物质(Mülllerian Inhibiting Substance，MIS)、抑制素或Nodal。在本发明的具体实施例中，TGF-β超家族为BMP4。BMP4的最终浓度为1-100ng/mL，例如1-80ng/mL、1-60ng/mL、1-40ng/mL、10-40ng/mL、20-40ng/mL；进一步，BMP4的最终浓度为30ng/mL。In the present invention, examples of the TGF-β superfamily include, but are not limited to, TGF-β proteins (including TGFβ3), bone morphogenetic proteins (BMPs) (including BMP2, BMP4, BMP6, or BMP7), growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), activins, Lefty, Mulllerian inhibitory substances (MIS), inhibins, or Nodal. In a specific embodiment of the present invention, the TGF-β superfamily is BMP4. The final concentration of BMP4 is 1-100 ng/mL, for example, 1-80 ng/mL, 1-60 ng/mL, 1-40 ng/mL, 10-40 ng/mL, 20-40 ng/mL; further, the final concentration of BMP4 is 30 ng/mL.
在本发明中，术语“培养基”在本领域中是公认的并且一般指用于培养活细胞的任何物质或制剂。如用于提及细胞培养的术语“培养基”包括细胞周围环境的组分。培养基可为固体、液体、气体或相和材料的混合物。培养基包括液体生长培养基以及不维持细胞生长的液体培养基。培养基还包括凝胶状培养基，诸如琼脂、琼脂糖、明胶和胶原蛋白基质。示例性气态培养基包括气相，在培养皿或其他固体或半固体支持物上生长的细胞暴露于该气相。术语“培养基”还指旨在用于细胞培养的材料，即使该材料尚未与细胞接触。换句话说，准备用于培养的富含营养的液体是培养基。术语“基础培养基”指促进不需要任何特殊营养补充剂的许多类型微生物生长的培养基。大多数基础培养基一般包含四种基本化学组：氨基酸、碳水化合物、无机盐和维生素。基础培养基一般用作更复杂培养基的基础，向其中添加补充剂，诸如血清或血清替代物、缓冲液、促生长因子、抗生素等。基础培养基的实施例包括但不限于Eagle基础培养基(BME)、最低必需培养基(MEM)、DuIbecco改良的Eagle培养基(DMEM)、培养基199、营养混合物HamsF10(F10)和HamsF12(F12)、McCoy’s 5A、DuIbecco’sMEM/F-12(DMEM/F12)、RPMI 1640、Iscove改良的DuIbecco培养基(IMDM)、L-15培养基、neurobasal中的一种或多种。在本发明的具体实施例中，基础培养基为DMEM/F12培养基和neurobasal培养基。In the present invention, the term "culture medium" is generally recognized in the art and generally refers to any substance or preparation used to culture living cells. The term "culture medium" as used to refer to cell culture includes components of the environment surrounding the cells. The culture medium can be a solid, liquid, gas, or a mixture of phases and materials. Culture medium includes liquid growth medium and liquid culture medium that does not maintain cell growth. Culture medium also includes gel-like culture medium, such as agar, agarose, gelatin and collagen matrix. Exemplary gaseous culture medium includes a gas phase, and cells grown on a culture dish or other solid or semi-solid support are exposed to the gas phase. The term "culture medium" also refers to a material intended for cell culture, even if the material has not yet been contacted with the cells. In other words, a nutrient-rich liquid prepared for cultivation is a culture medium. The term "basal medium" refers to a culture medium that promotes the growth of many types of microorganisms that do not require any special nutritional supplements. Most basal culture media generally contain four basic chemical groups: amino acids, carbohydrates, inorganic salts and vitamins. Basal culture media are generally used as the basis for more complex culture media, to which supplements such as serum or serum substitutes, buffers, growth factors, antibiotics, etc. are added. Examples of basal culture media include, but are not limited to, Eagle basal medium (BME), minimum essential medium (MEM), DuIbecco's modified Eagle medium (DMEM), medium 199, nutrient mixture HamsF10 (F10) and HamsF12 (F12), McCoy's 5A, DuIbecco'sMEM/F-12 (DMEM/F12), RPMI 1640, Iscove's modified DuIbecco medium (IMDM), L-15 medium, neurobasal, and one or more thereof. In a specific embodiment of the present invention, the basal culture medium is DMEM/F12 medium and neurobasal medium.
在本发明中，培养基中的血清替代物具有本领域技术人员公知的含义，其是指在维持未分化状态的情况下对多能干细胞进行培养的过程中，作为血清替代物而使用的组合物或调配物。也即，血清替代物能够支持胚胎干细胞或未分化多能干细胞的生长而无需补充血清。在某些示例性实施方案中，所述血清替代物包含：一种或多种氨基酸、一种或多种维生素、一种或多种微量金属元素。在一些情况下，血清替代物可以进一步包含一种或多种选自下列的成分：白蛋白、还原型谷胱甘肽、转铁蛋白、胰岛素等。血清替代物的非限制性实例包括但不限于KnockOutTM SR(简称为KOSR)、KOSR CTS、N2添加剂、CTS N2添加剂、B27添加剂、PhysiologixTM XF SR、StemSureTM SerumSubstitute Supplement、KnockoutTM SR中的一种或多种。在本发明的具体实施例中，血清替代物为B27添加剂和CTS N2添加剂。In the present invention, the serum substitute in the culture medium has a meaning known to those skilled in the art, which refers to a composition or formulation used as a serum substitute in the process of culturing pluripotent stem cells while maintaining an undifferentiated state. That is, the serum substitute can support the growth of embryonic stem cells or undifferentiated pluripotent stem cells without supplementing serum. In certain exemplary embodiments, the serum substitute comprises: one or more amino acids, one or more vitamins, one or more trace metal elements. In some cases, the serum substitute may further comprise one or more components selected from the following: albumin, reduced glutathione, transferrin, insulin, etc. Non-limiting examples of serum substitutes include, but are not limited to, one or more of KnockOutTM SR (abbreviated as KOSR), KOSR CTS, N2 additive, CTS N2 additive, B27 additive, PhysiologixTM XF SR, StemSureTM SerumSubstitute Supplement, and KnockoutTM SR. In a specific embodiment of the present invention, the serum substitute is B27 additive and CTS N2 additive.
在本发明中，培养基中的谷氨酰胺或其替代物包括但不限于：谷氨酰胺、L-丙氨酰-L-谷氨酰胺、L-甘氨酰-L-谷氨酰胺、N-乙酰-L-谷氨酰胺、HyCyteTM GluMax、glutaGROTM或它们的组合。在本发明的具体实施例中，谷氨酰胺或其替代物为谷氨酰胺。In the present invention, glutamine or its substitute in the culture medium includes but is not limited to: glutamine, L-alanyl-L-glutamine, L-glycyl-L-glutamine, N-acetyl-L-glutamine, HyCyte™ GluMax, glutaGRO™ or a combination thereof. In a specific embodiment of the present invention, glutamine or its substitute is glutamine.
在本发明中，培养基中的抗生素包括但不限于杆菌肽(Bacitracin)、新霉素(Neomycin)、红霉素(Erythromycin)、氯霉素(Chloramphenicol)、青霉素-链霉素、庆大霉素、万古霉素中的一种或几种。另外的本发明中使用的抗生物质对于本领域的普通技术人员而言是显而易见的。In the present invention, the antibiotics in the culture medium include, but are not limited to, one or more of bacitracin, neomycin, erythromycin, chloramphenicol, penicillin-streptomycin, gentamicin, and vancomycin. Other antibiotics used in the present invention are obvious to those of ordinary skill in the art.
在本发明中，培养基中的血管内皮生长因子(VEGF)的实例包括但不限于VEGF-A、VEGF-B、VEGF-C、VEGF-D、PGF中的一种或几种；优选地，所述VEGF为VEGF-A。VEGF-A的浓度可以是，例如1-500ng/mL，1-400ng/mL，1-300ng/mL，1-200ng/mL。在本发明的具体实施例中，VEGF-A的浓度为100ng/mL。In the present invention, examples of vascular endothelial growth factor (VEGF) in the culture medium include, but are not limited to, one or more of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF; preferably, the VEGF is VEGF-A. The concentration of VEGF-A can be, for example, 1-500 ng/mL, 1-400 ng/mL, 1-300 ng/mL, 1-200 ng/mL. In a specific embodiment of the present invention, the concentration of VEGF-A is 100 ng/mL.
在本发明中，培养基中的成纤维细胞生长因子(FGF)的实例包括但不限于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中的一种或几种。在本发明中的具体实施例中，FGF为FGF-2。FGF-2的浓度可以是，例如1-500ng/mL，1-400ng/mL，1-300ng/mL，1-200ng/mL。在本发明的具体实施例中，FGF的浓度为100ng/mL。In the present invention, examples of fibroblast growth factor (FGF) in the culture 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, and FGF-23. In a specific embodiment of the present invention, FGF is FGF-2. The concentration of FGF-2 can be, for example, 1-500 ng/mL, 1-400 ng/mL, 1-300 ng/mL, 1-200 ng/mL. In a specific embodiment of the present invention, the concentration of FGF is 100 ng/mL.
在本发明中，培养基中的cAMP激活剂可以包括forskolin、IBMX、Rolipram、8BrcAMP、Prostaglandin E2(PGE2)、NKH 477、dibutyryl-cAMP(DBcAMP)、Sp-8-Br-cAMPs中的一种或多种。cAMP激活剂抑制剂如forskolin的浓度可以是，例如0.1μM至100μM，例如1μM至50μM，例如5μM至20μM。在本发明的具体实施例中，forskolin的浓度为2μM。In the present invention, the cAMP activator in the culture medium may include one or more of forskolin, IBMX, Rolipram, 8BrcAMP, Prostaglandin E2 (PGE2), NKH 477, dibutyryl-cAMP (DBcAMP), Sp-8-Br-cAMPs. The concentration of the cAMP activator inhibitor, such as forskolin, may be, for example, 0.1 μM to 100 μM, such as 1 μM to 50 μM, such as 5 μM to 20 μM. In a specific embodiment of the present invention, the concentration of forskolin is 2 μM.
在本发明中，术语“细胞外基质(ECM)”由结缔组织细胞分泌，包含多种多糖、水、弹性蛋白和糖蛋白，其中糖蛋白包括胶原蛋白(Collagen)、纤连蛋白、巢蛋白和层粘连蛋白。不同类型的ECM是已知的，其包括不同的组成，如含有不同类型的糖蛋白或糖蛋白的不同组合。产细胞外基质的细胞的实例是主要产胶原蛋白和蛋白聚糖的软骨细胞，主要产IV型胶原蛋白、层粘连蛋白、间质前胶原蛋白和纤连蛋白的成纤维细胞，和主要产胶原蛋白(I、III和V型)、硫酸软骨素蛋白聚糖、透明质酸、纤连蛋白和肌糖蛋白C的结肠成肌纤维细胞。ECM中所含的多糖、弹性蛋白、糖蛋白、胶原蛋白、纤连蛋白、巢蛋白和层粘连蛋白为本领域周知的ECM中所含的各种多糖、弹性蛋白、糖蛋白、胶原蛋白、纤连蛋白、巢蛋白和层粘连蛋白。例如，胶原蛋白可以是本领域周知的包含于天然ECM中的Collagen-I、Collagen-Ⅱ、Collagen-III、Collagen-Ⅳ、Collagen-Ⅴ、Collagen-Ⅵ或Collagen-Ⅶ。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, wherein glycoproteins include collagen, fibronectin, entactin and laminin. Different types of ECM are known, and they include different compositions, such as containing different types of glycoproteins or different combinations of glycoproteins. Examples of cells producing extracellular matrix are chondrocytes that mainly produce collagen and proteoglycans, fibroblasts that mainly produce type IV collagen, laminin, interstitial procollagen and fibronectin, and colon myofibroblasts that mainly produce collagen (I, III and V types), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin and myoglycoprotein C. 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, the collagen may be Collagen-I, Collagen-II, Collagen-III, Collagen-IV, Collagen-V, Collagen-VI or Collagen-VII which are contained in natural ECM and are well known in the art.
适用于本文的ECM可从市售途径获得。可商购的细胞外基质的实例包括细胞外基质蛋白(Invitrogen，R&D systems)和基质胶(MatrixgelTM，BD Biosciences)等。在本发明的具体实施例中，细胞外基质包括胶原蛋白和Matrigel；进一步，胶原蛋白为Collagen-I。ECM suitable for use herein can be obtained from commercial sources. Examples of commercially available extracellular matrices include extracellular matrix proteins (Invitrogen, R&D systems) and matrix gel (MatrixgelTM , BD Biosciences), etc. In a specific embodiment of the present invention, the extracellular matrix includes collagen and Matrigel; further, the collagen is Collagen-I.
在本发明中，术语“无血清培养基”或“SFM培养基”是不含血清(例如胎牛血清(FBS)、小牛血清、马血清、山羊血清、人类血清等)的培养基且一般通过字母SFM表示。熟练的业内人士熟悉的示例性但非限制性无血清培养基包括HuMEC基础无血清培养基、KNOCKOUTTM CTSTM无外来物ESC/iPSC培养基、STEMPROTM-34SFM培养基、STEMPROTM NSC培养基、ESSENTIALTM-8培养基、培养基254、培养基106、培养基131、培养基154、培养基171、培养基171、培养基200、培养基231、HeptoZYME-SFM、人类内皮-SFM、FREESTYLETM 293表达培养基、培养基154CF/PRF、培养基154C、培养基154CF、培养基106、培养基200PRF、培养基131、EssentialTM-6培养基、STEMPROTM-34培养基、星形胶质细胞培养基、AIM培养基CTSTM、AMINOMAXTM C-100基础培养基、AMINOMAXTM-II完全培养基、CD FORTICHOTM培养基、CDCHOAGT培养基、CHO-S-SFM培养基、FREESTYLETM CHO表达培养基、CD OPTICHOTM培养基、CD CHO培养基、CDDG44培养基、SF-900TM培养基、EXPI293TM表达培养基、LHC基础培养基、LHC-8培养基、293SFM培养基、CD 293培养基、AEM生长培养基、PER.细胞培养基、AIM培养基、培养基、角质细胞-SFM培养基、LHC培养基、LHC-8培养基、LHC-9培养基和其任何衍生物或修改型。In the present invention, the term "serum-free medium" or "SFM medium" is a medium that does not contain serum (eg, fetal bovine serum (FBS), calf serum, horse serum, goat serum, human serum, etc.) and is generally represented by the letters SFM. Exemplary but non-limiting serum-free culture media familiar to those skilled in the art include HuMEC Basal Serum-Free Medium, KNOCKOUT™ CTS™ Xeno-Free ESC/iPSC Medium, STEMPRO™-34 SFM Medium, STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254, Medium 106, Medium 131, Medium 154, Medium 171, Medium 171, Medium 200, Medium 231, HeptoZYME-SFM, Human Endothelial-SFM, FREESTYLE™ 293 Expression Medium, Medium 154CF/PRF, Medium 154C, Medium 154CF, Medium 106, Medium 200PRF, Medium 131, Essential™-6 Medium, STEMPRO™-34 Medium, Astrocyte Medium, AIM Medium CTS™, AMINOMAX™ C-100 Basal Medium, AMINOMAX™-II Complete Medium, CD FORTICHOTM medium, CDCHOAGT 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, medium, keratinocyte-SFM medium, LHC medium, LHC-8 medium, LHC-9 medium, and any derivatives or modifications thereof.
在本发明中，术语“治疗”是指(1)防止易感或尚未显示病症的受试者出现症状或疾病；(2)抑制疾病或阻止其发展或复发；或(3)改善或导致疾病或病症消退。如本领域所理解的，“治疗”是用于获得有益的或期望的结果(包括临床结果)的方法。出于本技术的目的，有益的或期望的结果可以包括但不限于减轻或改善一个或多个症状、病症(包括疾病)程度的减轻、病症(包括疾病)状态的稳定(即，不恶化)，病症(包括疾病)的延迟或减缓、病症(包括疾病)、状态和缓解(无论是部分还是全部)的进展、改善或缓解，无论是否可检测。In the present invention, the term "treatment" refers to (1) preventing the onset of symptoms or disease in a subject who is susceptible or has not yet shown symptoms; (2) inhibiting a disease or preventing its development or recurrence; or (3) ameliorating or causing regression of a disease or condition. As understood in the art, "treatment" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of the present technology, beneficial or desired results may include, but are not limited to, alleviation or improvement of one or more symptoms, alleviation of the degree of a condition (including a disease), stabilization of the condition (including a disease) state (i.e., not worsening), delay or mitigation of a condition (including a disease), progression, improvement or relief of a condition (including a disease), state, and relief (whether partial or complete), whether detectable or not.
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。根据本发明的实质对本发明进行的简单改进都属于本发明要求保护的范围。The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention made according to the essence of the present invention all fall within the scope of protection claimed in the present invention.
实施例1构建烟雾病病人来源的诱导多能干细胞(iPSC)Example 1 Construction of induced pluripotent stem cells (iPSCs) from patients with moyamoya disease
1、实验方法1. Experimental methods
选取携带RNF213突变烟雾病患者的皮肤组织，约4mm×4mm，分离培养为皮肤真皮成纤维细胞(MEFs)，利用仙台病毒重编程试剂盒将其重编程为iPSC。细胞在仙台病毒感染后转铺在MEFs上进行共培养。感染后，挑取典型的人胚胎干细胞样的克隆，机械法分离并轻轻吹散为小的细胞团块，传代至新的饲养层细胞上，机械法连续传代两代以上后，使用胶原酶法继续传代，细胞克隆保持未分化的状态，形态典型，完成构建iPSC。通过免疫荧光染色检测干细胞多能性标志物(核转录因子OCT4、NANOG)的表达。Skin tissues of patients with moyamoya disease carrying RNF213 mutations, approximately 4 mm × 4 mm, were selected and cultured as skin dermal fibroblasts (MEFs), which were reprogrammed into iPSCs using the Sendai virus reprogramming kit. After infection with Sendai virus, the cells were transferred to MEFs for co-culture. After infection, typical human embryonic stem cell-like clones were picked, mechanically separated and gently blown into small cell clumps, and passaged to new feeder cells. After mechanical passage for more than two generations, the collagenase method was used to continue passage. The cell clones remained undifferentiated and had typical morphology, completing the construction of iPSCs. The expression of stem cell pluripotency markers (nuclear transcription factors OCT4 and NANOG) was detected by immunofluorescence staining.
2、实验结果2. Experimental results
构建iPSC实验结果如图1所示，结果表明，成功将携带RNF213突变烟雾病患者的皮肤组织重新编程为iPSC。The results of the iPSC construction experiment are shown in Figure 1, and the results show that the skin tissue of patients with moyamoya disease carrying RNF213 mutations was successfully reprogrammed into iPSCs.
免疫荧光检测实验结果如图2所示，结果显示，iPS细胞系阳性表达核转录因子OCT4、NANOG，表明成功将携带RNF213突变烟雾病患者的皮肤组织重编程为iPSC。The results of the immunofluorescence detection experiment are shown in Figure 2. The results showed that the iPS cell line positively expressed nuclear transcription factors OCT4 and NANOG, indicating that the skin tissue of the moyamoya disease patient carrying the RNF213 mutation was successfully reprogrammed into iPSC.
实施例2利用烟雾病病人来源的iPSCs培养血管类器官Example 2 Cultivation of vascular organoids using iPSCs from patients with moyamoya disease
1、实验方法1. Experimental methods
阶段1：诱导iPSC分化为中胚层(Day0-3)Stage 1: Inducing iPSCs to differentiate into mesoderm (Day 0-3)
待iPSC汇合度达40-70％，可启动分化。第0天(Day0)开始，通过加入CHIR99021来激活WNT信号并与BMP4一起培养3天。培养基为混合DMEM/F12培养基、neurobasal培养基、B27添加剂、N2添加剂、谷氨酰胺、β-巯基乙醇(1:100)、青霉素-链霉素、12μM CHIR99021和30ng/mL BMP-4，并将聚集体转移至低附着板培养细胞，诱导分化为中胚层。When the iPSC confluence reaches 40-70%, differentiation can be initiated. Starting from Day 0, WNT signaling is activated by adding CHIR99021 and cultured with BMP4 for 3 days. The culture medium is a mixture of DMEM/F12 medium, neurobasal medium, B27 supplement, N2 supplement, glutamine, β-mercaptoethanol (1:100), penicillin-streptomycin, 12μM CHIR99021 and 30ng/mL BMP-4, and the aggregates are transferred to low attachment plates to culture cells and induce differentiation into mesoderm.
阶段2：诱导产生血管网结构(Day3-10)Stage 2: Inducing the formation of vascular network structure (Day 3-10)
将阶段1获得的中胚层切换到含有VEGF-A的血管诱导培养基中培养2天(Day3-4)。培养基为混合DMEM/F12培养基、neurobasal培养基、B27添加剂、N2添加剂、谷氨酰胺、β-巯基乙醇(1:100)、青霉素-链霉素、100ng/mL VEGF-A和2μM毛喉素(forskolin)。The mesoderm obtained at stage 1 was switched to a vascular induction medium containing VEGF-A for 2 days (Day 3-4). The culture medium was a mixture of DMEM/F12 medium, neurobasal medium, B27 supplement, N2 supplement, glutamine, β-mercaptoethanol (1:100), penicillin-streptomycin, 100 ng/mL VEGF-A and 2 μM forskolin.
第5天(Day5)，将细胞聚集体包埋在12孔板的3D胶原I-Matrigel基质中，具体步骤如下：配制2.0mg/mL I型胶原蛋白溶液(Collagen I)，混合0.1N NaOH、10×DMEM、HEPES、7.5％碳酸氢钠、谷氨酰胺(Glutamax)和Ham’s F-12，快速用pH指示条测量胶原I溶液的pH值，pH值为7.4。按4:1比例混合Collagen I-Matrigel，将聚集体种在Collagen I-Matrigel基质中，使聚集体分布均匀；在37℃下孵育2h，使凝胶凝固。培养基为人内皮SFM培养基、15％ FBS、100ng/mL VEGF-A和100ng/mL FGF-2，诱导血管分化、出芽。On day 5, the cell aggregates were embedded in the 3D collagen I-Matrigel matrix in a 12-well plate. The specific steps were as follows: 2.0 mg/mL type I collagen solution (Collagen I) was prepared, mixed with 0.1N NaOH, 10×DMEM, HEPES, 7.5% sodium bicarbonate, glutamine (Glutamax) and Ham’s F-12, and the pH value of the collagen I solution was quickly measured with a pH indicator strip, and the pH value was 7.4. Collagen I-Matrigel was mixed in a 4:1 ratio, and the aggregates were seeded in the Collagen I-Matrigel matrix to make the aggregates evenly distributed; incubated at 37°C for 2 hours to solidify the gel. The culture medium was human endothelial SFM culture medium, 15% FBS, 100 ng/mL VEGF-A and 100 ng/mL FGF-2 to induce vascular differentiation and sprouting.
阶段3：形成血管类器官Stage 3: Formation of vascular organoids
使用无菌镊子的圆端从孔底游离整个基质胶。在无菌条件下，用无菌实验室勺子将凝胶转移到10cm培养皿的盖子上。使用两根无菌30号针切断单个血管网络，并尽量减少类器官周围过多基质的数量。将单个类器官，转移到6孔低附着板上，加入人内皮SFM培养基、15％ FBS、100ng/mL VEGF-A和100ng/mL FGF-2，培养类器官。Use the rounded end of sterile forceps to free the entire matrix gel from the bottom of the well. Under sterile conditions, transfer the gel to the lid of a 10 cm culture dish using a sterile laboratory spoon. Use two sterile 30-gauge needles to cut off individual vascular networks and minimize the amount of excess matrix surrounding the organoids. Transfer individual organoids to a 6-well low attachment plate and add human endothelial SFM medium, 15% FBS, 100 ng/mL VEGF-A, and 100 ng/mL FGF-2 to culture the organoids.
免疫荧光技术检测细胞特异性标志物(内皮细胞CD31标志物、平滑肌细胞标志物)的表达。Immunofluorescence technology was used to detect the expression of cell-specific markers (endothelial cell CD31 marker, smooth muscle cell marker).
2、实验结果2. Experimental results
实验结果如图3所示，结果表明成功构建血管类器官模型。The experimental results are shown in Figure 3, which show that the vascular organoid model was successfully constructed.
实施例3血管类器官功能验证实验Example 3 Vascular organoid function verification experiment
1、实验方法1. Experimental methods
使用单细胞测序实验和流式细胞实验验证实施例2中培养的血管类器官为烟雾病类器官，具体方法如下：Single cell sequencing experiments and flow cytometry experiments were used to verify that the vascular organoids cultured in Example 2 were moyamoya disease organoids. The specific methods were as follows:
单细胞测序方法：每组收集40-50个类器官，用DPBS洗涤后，用解剖刀切碎类器官。将类器官转移到含有中性蛋白酶酶和胶原酶的DPBS溶液中。37℃下孵育20至30分钟。并在4℃下以300g的速度离心5分钟。将细胞沉淀重悬于HBSS中，通过细胞过滤器过滤，并在4℃下以300g再次离心5分钟，用2％ FBS重悬，细胞计数并稀释至10，000个细胞的适当浓度，进行10×单细胞测序。Single-cell sequencing method: 40-50 organoids were collected from each group, washed with DPBS, and minced with a scalpel. The organoids were transferred to a DPBS solution containing neutral protease and collagenase. Incubated at 37°C for 20 to 30 minutes. Centrifuged at 300g for 5 minutes at 4°C. The cell pellet was resuspended in HBSS, filtered through a cell strainer, and centrifuged again at 300g for 5 minutes at 4°C, resuspended with 2% FBS, and the cells were counted and diluted to an appropriate concentration of 10,000 cells for 10× single-cell sequencing.
流式实验方法：用Accutase细胞解离溶液消化类器官，形成单细胞悬液。每组约1×10^6个单细胞与Alexa 488标记的CD31抗体(1:1000稀释，BD)一起孵育30分钟。使用FlowJo软件对结果进行分析。Flow cytometry: Organoids were digested with Accutase cell dissociation solution to form a single cell suspension. Approximately 1×10^6 single cells in each group were incubated with Alexa 488-labeled CD31 antibody (1:1000 dilution, BD) for 30 minutes. The results were analyzed using FlowJo software.
2、实验结果2. Experimental results
实验结果如图4、图5所示，结果显示，通过单细胞测序进行细胞注释，在烟雾病组中，平滑肌的比例高于健康对照组，并通过流式分析验证了这一结果，和烟雾病的病理特征平滑肌增多一致，模拟了该疾病表型。The experimental results are shown in Figures 4 and 5. The results showed that through single-cell sequencing for cell annotation, the proportion of smooth muscle in the moyamoya disease group was higher than that in the healthy control group, and this result was verified by flow cytometry, which was consistent with the pathological feature of moyamoya disease, the increase in smooth muscle, and simulated the phenotype of the disease.
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。The above-mentioned embodiments are only used to understand the method and core idea of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.
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| CN117143807A (en)* | 2023-10-30 | 2023-12-01 | 北京大学 | Vascular organoid, preparation method thereof, cell treatment composition and application thereof in ischemic diseases |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111065731A (en)* | 2017-06-16 | 2020-04-24 | Imba-莫利库尔生物技术研究所 | Vascular organoids, methods of producing and using the same |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11629340B2 (en)* | 2017-03-03 | 2023-04-18 | Obsidian Therapeutics, Inc. | DHFR tunable protein regulation |
| WO2019107535A1 (en)* | 2017-11-30 | 2019-06-06 | 公立大学法人横浜市立大学 | Formation of three-dimensional organ from pluripotent stem cells |
| CN109576155B (en)* | 2018-12-18 | 2021-12-07 | 大连理工大学 | Universal nervous system chip |
| WO2021076617A1 (en)* | 2019-10-14 | 2021-04-22 | The Regents Of The University Of California | Broad spectrum anti-cancer compounds |
| CN115851578B (en)* | 2022-12-23 | 2023-11-21 | 华南理工大学 | Kit for continuously amplifying hepatic progenitor cell organoids and/or hepatocyte organoids through 3D suspension induction and application of kit |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111065731A (en)* | 2017-06-16 | 2020-04-24 | Imba-莫利库尔生物技术研究所 | Vascular organoids, methods of producing and using the same |
| Publication number | Publication date |
|---|---|
| CN116875535A (en) | 2023-10-13 |
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