CN114149971A - 用hPSCs诱导分化得到中脑类器官的方法及中脑类器官 - Google Patents
用hPSCs诱导分化得到中脑类器官的方法及中脑类器官 Download PDFInfo
- Publication number
- CN114149971A CN114149971A CN202111370443.6A CN202111370443A CN114149971A CN 114149971 A CN114149971 A CN 114149971A CN 202111370443 A CN202111370443 A CN 202111370443A CN 114149971 A CN114149971 A CN 114149971A
- Authority
- CN
- China
- Prior art keywords
- hpscs
- culture
- culture solution
- day
- organoids
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002220 organoid Anatomy 0.000 title claims abstract description 59
- 210000001259 mesencephalon Anatomy 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 39
- 230000004069 differentiation Effects 0.000 title claims abstract description 37
- 210000004958 brain cell Anatomy 0.000 title description 2
- 210000004027 cell Anatomy 0.000 claims abstract description 26
- 238000004114 suspension culture Methods 0.000 claims abstract description 11
- 239000003112 inhibitor Substances 0.000 claims abstract description 10
- 238000004113 cell culture Methods 0.000 claims abstract description 7
- 108010082117 matrigel Proteins 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- 238000007664 blowing Methods 0.000 claims abstract description 3
- 238000012258 culturing Methods 0.000 claims abstract description 3
- 230000006698 induction Effects 0.000 claims description 30
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 18
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 17
- 230000008859 change Effects 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 13
- 239000000556 agonist Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 12
- 230000019491 signal transduction Effects 0.000 claims description 12
- 230000001537 neural effect Effects 0.000 claims description 11
- 230000001939 inductive effect Effects 0.000 claims description 10
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 claims description 9
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 claims description 9
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 claims description 9
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 claims description 9
- 229960005070 ascorbic acid Drugs 0.000 claims description 9
- 235000010323 ascorbic acid Nutrition 0.000 claims description 9
- 239000011668 ascorbic acid Substances 0.000 claims description 9
- 108090000031 Hedgehog Proteins Proteins 0.000 claims description 8
- 102000003693 Hedgehog Proteins Human genes 0.000 claims description 8
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 8
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 8
- 210000001728 clone cell Anatomy 0.000 claims description 8
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical group C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 8
- JNGZXGGOCLZBFB-IVCQMTBJSA-N compound E Chemical compound N([C@@H](C)C(=O)N[C@@H]1C(N(C)C2=CC=CC=C2C(C=2C=CC=CC=2)=N1)=O)C(=O)CC1=CC(F)=CC(F)=C1 JNGZXGGOCLZBFB-IVCQMTBJSA-N 0.000 claims description 7
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 claims description 6
- 108010007093 dispase Proteins 0.000 claims description 6
- -1 cAMP Proteins 0.000 claims description 5
- 235000003170 nutritional factors Nutrition 0.000 claims description 5
- 239000002243 precursor Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 108010031318 Vitronectin Proteins 0.000 claims description 4
- 102100035140 Vitronectin Human genes 0.000 claims description 4
- 230000008410 smoothened signaling pathway Effects 0.000 claims description 4
- JMIFGARJSWXZSH-UHFFFAOYSA-N DMH1 Chemical compound C1=CC(OC(C)C)=CC=C1C1=CN2N=CC(C=3C4=CC=CC=C4N=CC=3)=C2N=C1 JMIFGARJSWXZSH-UHFFFAOYSA-N 0.000 claims description 3
- 102000002254 Glycogen Synthase Kinase 3 Human genes 0.000 claims description 3
- 108010014905 Glycogen Synthase Kinase 3 Proteins 0.000 claims description 3
- 210000005036 nerve Anatomy 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000013589 supplement Substances 0.000 claims description 3
- 239000012580 N-2 Supplement Substances 0.000 claims description 2
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 230000001464 adherent effect Effects 0.000 claims description 2
- 239000007640 basal medium Substances 0.000 claims description 2
- 239000003797 essential amino acid Substances 0.000 claims description 2
- 235000020776 essential amino acid Nutrition 0.000 claims description 2
- 230000003760 hair shine Effects 0.000 claims description 2
- 210000005064 dopaminergic neuron Anatomy 0.000 abstract description 18
- 238000011160 research Methods 0.000 abstract description 6
- 238000007877 drug screening Methods 0.000 abstract description 4
- 238000010874 in vitro model Methods 0.000 abstract description 3
- 210000000133 brain stem Anatomy 0.000 abstract description 2
- 230000005714 functional activity Effects 0.000 abstract description 2
- 230000001575 pathological effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 13
- 239000003550 marker Substances 0.000 description 8
- 208000018737 Parkinson disease Diseases 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 230000036982 action potential Effects 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 230000022256 midbrain development Effects 0.000 description 3
- 230000009456 molecular mechanism Effects 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 102100037293 Atrial natriuretic peptide-converting enzyme Human genes 0.000 description 2
- 101710133555 Atrial natriuretic peptide-converting enzyme Proteins 0.000 description 2
- 102100029284 Hepatocyte nuclear factor 3-beta Human genes 0.000 description 2
- 102100030634 Homeobox protein OTX2 Human genes 0.000 description 2
- 101001062347 Homo sapiens Hepatocyte nuclear factor 3-beta Proteins 0.000 description 2
- 101000584400 Homo sapiens Homeobox protein OTX2 Proteins 0.000 description 2
- 101000979001 Homo sapiens Methionine aminopeptidase 2 Proteins 0.000 description 2
- 101000969087 Homo sapiens Microtubule-associated protein 2 Proteins 0.000 description 2
- 101001109698 Homo sapiens Nuclear receptor subfamily 4 group A member 2 Proteins 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 102100021118 Microtubule-associated protein 2 Human genes 0.000 description 2
- 102100022676 Nuclear receptor subfamily 4 group A member 2 Human genes 0.000 description 2
- 206010034010 Parkinsonism Diseases 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229920006324 polyoxymethylene Polymers 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 208000014644 Brain disease Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 101001094700 Homo sapiens POU domain, class 5, transcription factor 1 Proteins 0.000 description 1
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000001654 germ layer Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 210000004558 lewy body Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000021317 sensory perception Effects 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种用hPSCs诱导分化得到中脑类器官的方法及中脑类器官,方法具体步骤如下:将hPSCs贴壁培养于室温下基质胶包被的培养皿中,待稳定贴壁后,再加入各抑制剂等培养液,分化第8天时,再于CO2孵箱中孵育,再将克隆细胞群轻吹下,转移至离心管中加入培养液后进行离心,吸取上清,将离心管底部的克隆细胞群转移至细胞培养瓶,加入各培养液悬浮培养,再多次换液加入不同培养液进行悬浮培养,第45天时进行半换液,得到目标中脑类器官。一种中脑类器官,即为本发明的方法制得的包含大量多巴胺能神经元的3D中脑类器官模型。建立了3D中脑类器官模型,包含大量多巴胺能神经元,具备高功能活性。可作为未来药物筛选,病理研究的人源性体外模型。
Description
技术领域
本发明涉及一种用hPSCs诱导分化得到中脑类器官的方法及中脑类器官,属于生物医学和发育生物技术领域。
背景技术
中脑是哺乳动物中枢神经系统最重要的组成部分,主要参与感官知觉、运动、学习、语言等中枢神经系统的指令,部位受损是造成神经退行性疾病帕金森综合症的主要原因。
然而,对中脑以及帕金森病研究的限速因素之一就是体外模型的缺乏。hPSCs技术的出现,为中脑发育、帕金森综合征的发病机制和分子机理的研究,以及个体用药筛选提供了可用的细胞模型。
hPSCs包括人胚胎干细胞(human embryonic stem cells,hESCs)和人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)。第一株hESCs是从早期胚胎的内细胞团中分离出来的,而hiPSCs则是Shinya Yamanaka用逆转录病毒将包含人Oct3/4,Sox2, Klf4, c-Myc的四个因子转入人皮肤成纤维细胞后重编程后得到的。hiPSCs在形态、增殖、表面抗原、基因表达、端粒酶活性展现出了与hESCs非常相似的特性。hPSCs具有独特的性质,它们不但能进行自我更新,而且具有分化为三个胚层的能力。研究证明,在特定条件下hPSCs可以分化为任何一种细胞类型,如神经前体细胞,心肌细胞,肝细胞等。它们可作为一种再生资源治疗神经类疾病,如I型糖尿病,帕金森疾病,心血管疾病,肝脏疾病。hPSCs的出现,将为再生医学中应用细胞治疗提供不可估量的前景。
大量研究数据表明,相较于2D培养体系,3D培养体系所得的类器官可以更好的模拟脑内三维空间上的结构以及由各种因子和信号所组成的复杂的微环境,并更加准确概括细胞与细胞之间、细胞与细胞外基质的交互作用。因此,中脑类器官可以更好的作为中脑发育研究的模型。此外,2D培养体系无法模拟帕金森路易小体的形成,传统小鼠模型仅可模拟帕金森病晚期表型,无法重现帕金森病的发展过程。
因此,建立一种高效可重复的hPSCs定向分化为中脑类器官的方法尤为重要。理想状态下,通过分化获得的中脑类器官应包含大量多巴胺能神经元,且展现成熟的电生理活性。在本专利中,我们找到了一种高效分化hPSCs至中脑类器官的方法,为中脑发育、帕金森综合征的发病机制和分子机理的研究,以及个体用药筛选提供了可用的细胞模型。
发明内容
发明目的:针对上述现有存在的问题和不足,本发明的目的是提供一种用hPSCs诱导分化得到中脑类器官的方法及中脑类器官,建立了3D中脑类器官模型,包含大量多巴胺能神经元,具备很好的功能活性。可以很好的作为未来药物筛选,病理研究的人源性体外模型。
技术方案:为实现上述发明目的,本发明采用以下技术方案:
一种用hPSCs诱导分化得到中脑类器官的方法,
步骤1:将hPSCs贴壁培养于室温下以Vitronectin基质胶包被的培养皿中;
步骤2:待hPSCs稳定贴壁后,将培养液换为神经诱导培养液Neural inductionmedium,分化1天;
步骤3:往步骤2中的培养液中加入1-10μM的诱导因子BMP信号通路抑制剂 DMH1、1-10μM的TGF-β信号通路抑制剂SB431542、1-1000ng/mL的Sonic Hedgehog信号通路激动剂SHH和0.1-10μM的GSK-3信号通路抑制剂 CHIR-99021,再分化7天;
步骤4:分化第8天时,再加入1U/mL的Dispase覆盖住hPSCs,于37℃,5%的CO2孵箱中孵育,当克隆细胞群的边缘发亮,并微微卷起时,将Dispase吸除,再用DMEM/F12培养液轻洗,之后吸走DMEM/F12培养液;
步骤5:用DMEM/F12培养液将步骤4得到的克隆细胞群轻吹下,转移至离心管中加入DMEM/F12培养液后进行离心,吸取上清,将离心管底部的克隆细胞群转移至细胞培养瓶,加入神经诱导培养液Neural induction medium、0.1-10μM的诱导因子CHIR-99021、1-1000ng/mL的Sonic Hedgehog信号通路激动剂SHH和0.1-10μM的SAG进行悬浮培养2天;
步骤6:第10天形成球状神经前体细胞后进行半换液,到第12天时再进行半换液;
步骤7:待第13天时进行全换液,在培养液中加入诱导因子FGF8b和SonicHedgehog信号通路激动剂SHH,并在培养液中加入诱导因子FGF8b和SAG,进行悬浮培养,再培养至第18天,进行半换液;
步骤8:至第19天,进行全换液,再加入诱导因子FGF8b和SHH进行悬浮培养,至第33天,进行半换液;
步骤9:至第34天,进行全换液,并加入营养因子GDNF、BDNF、cAMP、TGF-β3、compound E和ascorbic acid进行悬浮培养,第45天时进行半换液,得到目标中脑类器官。
进一步的,所述步骤1中的培养液为体积比为50:1的Essential 8TM Basal MediumDMEM/F12(Ham)(1:1)和Essential 8TM Supplement (50X)。
进一步的,步骤2和步骤5中所述的神经诱导培养液Neural induction medium为DMEM/F12培养基、非必须氨基酸NEAA和无血清的添加剂N2 supplement组成,三种成分的浓度比例为98:1:1。
进一步的,所述步骤5中使用15ml的离心管,设置800rpm,1min进行离心。
进一步的,步骤7在培养液中加入1-1000ng/mL的诱导因子FGF8b和1-100ng/mL的Sonic Hedgehog信号通路激动剂SHH,并在培养液中加入1-1000ng/mL的诱导因子FGF8b和1-10μM的SAG。
进一步的,所述诱导因子FGF8b为100ng/mL,Sonic Hedgehog信号通路激动剂SHH为20ng/mL,并在培养液中加入100ng/mL的诱导因子FGF8b和0.5μM的SAG。
进一步的,步骤8中加入1-1000ng/mL的诱导因子FGF8b和1-100ng/mL的SHH。
进一步的,所述诱导因子FGF8b为100ng/mL, 所述SHH为20ng/mL。
进一步的,步骤9中加入营养因子GDNF、BDNF、cAMP、TGF-β3、compound E和ascorbic acid进行培养。
进一步的,步骤9中的营养因子GDNF为1-100ng/mL、BDNF为1-100ng/mL、cAMP为1-10μM、TGF-β3为1-100ng/mL、compound E为1-10μM和ascorbic acid为1-1000μM。
进一步的,所述营养因子GDNF为10ng/mL、BDNF为10ng/mL、cAMP为1μM、TGF-β3为10ng/mL、compound E为1μM和ascorbic acid 为200μM。
一种中脑类器官,其由前述的任一方法所得。
有益效果:与现有技术相比,本发明具有以下优点:这是一种新式的用hPSCs诱导分化得到中脑类器官的方法及中脑类器官,找到了一种高效分化hPSCs至中脑类器官的方法,为中脑发育、帕金森综合征的发病机制和分子机理的研究,以及个体用药筛选提供了可用的细胞模型。
附图说明
图1是本发明诱导hPSCs分化至中脑类器官的示意图;
图2是本发明的实施例初期荧光结果示意图,
图中:a为实施例在诱导分化的第1天,b为实施例在诱导分化的第9天;
图3是本发明的实施例中期荧光结果示意图,
图中:c为实施例在诱导分化的第19天,d为实施例在诱导分化的第34天;
图4是本发明的实施例在诱导分化第34天,中脑类器官表达的中脑标记物OTX2、FOXA2、Corin、幼稚神经元标记物TUJ1的荧光示意图;
图5是本发明的在实施例在诱导分化的第34天,中脑类器官表达多巴胺能神经元标记物TH、成熟多巴胺能神经元标志物NURR1和成熟神经元标志物MAP2的荧光示意图;
图6是本发明的实施例中类器官内的多巴胺能神经元诱导的钠电流示意图;
图7是本发明的实施例中类器官内的多巴胺能神经元诱导出较高频率及振幅的动作电位示意图;
图8是本发明的实施例中类器官内的多巴胺能神经元产生自发性动作电位指标示意图。
具体实施方式
下面结合附图和具体实施例,进一步阐明本发明,应理解这些实施例仅用于说明本发明而不用于限制本发明的范围,在阅读了本发明之后,本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
实施例:
一种新式的用hPSCs诱导分化得到中脑类器官的方法及中脑类器官,本实施例步骤如下:
步骤1)将hPSCs贴壁培养于在室温下用基质胶Vitronectin提前2小时包被过的培养皿中;hPSCs所用培养液为Essential 8,由体积比为50:1的Essential 8TM BasalMedium DMEM/F12(Ham)(1:1)与Essential 8TM Supplement (50X)配制而成;hPSCs在培养皿中稳定贴壁后,将培养液替换成神经诱导培养液(Neural induction medium, NIM)。其中,神经诱导培养液NIM为DMEM/F12培养基,NEAA,N2以体积比98:1:1配制而成。
步骤2)分化第1天在神经诱导培养液中加入诱导因子1-10μM BMP信号通路抑制剂DMH1、1-10μM TGF-β信号通路抑制剂SB、1-1000ng/mL Sonic Hedgehog信号通路激动剂SHH、0.1-10μM GSK-3信号通路抑制剂 CHIR 99021。分化第1天至分化第8天,每天半换液。
步骤3)分化第9天,加入1ml Dispase(1U mL-1)于37°C,5%CO2的孵箱中孵育10-20分钟,移至显微镜下观察,克隆边缘发亮,并微微卷起时,将Dispase吸除;用DMEM/F12轻洗细胞,10-20s后吸走,再加入DMEM/F12轻轻将克隆吹下,同时将已吹下的克隆从液体中吸出转移至15ml离心管中;将装有细胞和DMEM/F12的离心管离心,800rpm,1min;吸除上清,将离心管底部的细胞转移至细胞培养瓶,换上神经诱导培养液(Neural induction medium,NIM)进行悬浮培养,并加入诱导因子0.1-10μM CHIR 99021以及Sonic Hedgehog信号通路激动剂1-1000ng/mL SHH和0.1-10μM SAG。
步骤4)分化第13天,进行全换液,并在培养液中加入诱导因子10-100ng/mL FGF8b和0.1-10μM SAG。分化第14天到分化第18天,在细胞培养瓶中悬浮培养,隔天进行半换液。
步骤5)分化第19天,进行全换液,并在培养液中加入诱导因子10-100ng/mL FGF8b和1-1000ng/mL SHH。分化第20天到分化第33天,在细胞培养瓶中悬浮培养,隔天进行半换液。
步骤6)分化第34天,进行全换液,并在培养液中加入营养因子10ng/mL GDNF、10ng/mL BDNF、1μM cAMP、10ng/mL TGF-β3、1μMcompound E、200μM ascorbic acid。分化第35天到分化第45天,在细胞培养瓶中悬浮培养,隔天进行半换。
步骤7)在细胞分化的第19天后根据中脑类器官表达因子的时间固定细胞,进行细胞免疫荧光染色鉴定。其具体步骤为:将中脑类器官用1mL枪头吸出,放置于1.5mL 离心管内,加入polyformaldehyde(PFA)固定2-4后吸走,加入Phosphate Buffered Saline(PBS)清洗3次后加入20%的蔗糖脱水,4℃静置过夜。第二天吸弃20%的蔗糖,加入20%的蔗糖,再次静置脱水,直至类器官沉入离心管底部。之后,将类器官利用OCT包埋,并进行切片。进一步地,利用PBS对类器官切片进行清洗3次,每次10min;加入终浓度为1% Triton和5% Donkey-Serum打孔封闭1hour;加入0.2% Triton与5% Donkey-Serum配制的一抗,4°C过夜。用PBS洗3次,每次10min,加入二抗,用5% Donkey-Serum配制,室温孵育1h,再用PBS洗3次,每次10min。用封片剂封片,观察荧光结果。
如图1所示为诱导hPSCs分化至中脑类器官的流程。如图2的a图所示,为分化第一天的荧光结果示意图,结合图1和图2可知,细胞传代后第二天于基质胶Vitronectin稳定贴壁。如图2的b图可见,分化9天后,形成内部致密的克隆。如图3的c图所示,分化19天,克隆吹起后悬浮培养,形成球状神经前体细胞Neurospheres(NS)。如图3的d图所示,分化34天,NS内部前体细胞增殖,NS体积增加,包含大量多巴胺能神经元等细胞。形成中脑类器官。标尺=100μm。
图4显示,在分化在诱导分化的D34天,中脑类器官可大量表达中脑标记物OTX2(红)、FOXA2(红)、Corin(绿色)、幼稚神经元标记物TUJ1(绿色)。图5显示,在分化在诱导分化的D34天,中脑类器官可大量表达多巴胺能神经元标记物TH、成熟多巴胺能神经元标志物NURR1(红)以及成熟神经元标志物MAP2(绿)。标尺=100μm。
图6显示类器官内的多巴胺能神经元可被诱导出超过2nA的钠电流。图7显示类器官内的多巴胺能神经元可被诱导出较高频率及振幅的动作电位。图8显示类器官内的多巴胺能神经元可产生自发性动作电位指标。
实验结果证实,本发明所述的诱导分化方法可构建包含大量多巴胺能神经元的中脑类器官,且分化至45天,多巴胺能神经元即可于功能性成熟。
Claims (12)
1.一种用hPSCs诱导分化得到中脑类器官的方法,其特征在于:
步骤1:将hPSCs贴壁培养于室温下以Vitronectin基质胶包被的培养皿中;
步骤2:待hPSCs稳定贴壁后,将培养液换为神经诱导培养液Neural inductionmedium,分化1天;
步骤3:往步骤2中的培养液中加入1-10μM的诱导因子BMP信号通路抑制剂 DMH1、1-10μM的TGF-β信号通路抑制剂SB431542、1-1000ng/mL的Sonic Hedgehog信号通路激动剂 SHH和0.1-10μM的GSK-3信号通路抑制剂 CHIR-99021,再分化7天;
步骤4:分化第8天时,再加入1U/mL的Dispase覆盖住hPSCs,于37℃,5%的CO2孵箱中孵育,当克隆细胞群的边缘发亮,并微微卷起时,将Dispase吸除,再用DMEM/F12培养液轻洗,之后吸走DMEM/F12培养液;
步骤5:用DMEM/F12培养液将步骤4得到的克隆细胞群轻吹下,转移至离心管中加入DMEM/F12培养液后进行离心,吸取上清,将离心管底部的克隆细胞群转移至细胞培养瓶,加入神经诱导培养液Neural induction medium、0.1-10μM的诱导因子CHIR-99021、1-1000ng/mL的Sonic Hedgehog信号通路激动剂SHH和0.1-10μM的SAG进行悬浮培养2天;
步骤6:第10天形成球状神经前体细胞后进行半换液,到第12天时再进行半换液;
步骤7:待第13天时进行全换液,在培养液中加入诱导因子FGF8b和Sonic Hedgehog信号通路激动剂SHH,并在培养液中加入诱导因子FGF8b和SAG,进行悬浮培养,再培养至第18天,进行半换液;
步骤8:至第19天,进行全换液,再加入诱导因子FGF8b和SHH进行悬浮培养,至第33天,进行半换液;
步骤9:至第34天,进行全换液,并加入营养因子GDNF、BDNF、cAMP、TGF-β3、compound E和ascorbic acid进行悬浮培养,第45天时进行半换液,得到目标中脑类器官。
2.根据权利要求1所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:所述步骤1中的培养液为体积比为50:1的Essential 8TM Basal Medium DMEM/F12(Ham)(1:1)和Essential 8TM Supplement (50X)。
3.根据权利要求1所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:步骤2和步骤5中所述的神经诱导培养液Neural induction medium为DMEM/F12培养基、非必须氨基酸NEAA和无血清的添加剂N2 supplement组成,三种成分的浓度比例为98:1:1。
4.根据权利要求1所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:所述步骤5中使用15ml的离心管,设置800rpm,1min进行离心。
5.根据权利要求1所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:步骤7在培养液中加入1-1000ng/mL的诱导因子FGF8b和1-100ng/mL的Sonic Hedgehog信号通路激动剂SHH,并在培养液中加入1-1000ng/mL的诱导因子FGF8b和1-10μM的SAG。
6.根据权利要求5所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:所述诱导因子FGF8b为100ng/mL,Sonic Hedgehog信号通路激动剂SHH为20ng/mL,并在培养液中加入100ng/mL的诱导因子FGF8b和0.5μM的SAG。
7.根据权利要求1所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:步骤8中加入1-1000ng/mL的诱导因子FGF8b和1-100ng/mL的SHH。
8.根据权利要求7所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:所述诱导因子FGF8b为100ng/mL, 所述SHH为20ng/mL。
9.根据权利要求1所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:步骤9中加入营养因子GDNF、BDNF、cAMP、TGF-β3、compound E和ascorbic acid进行培养。
10.根据权利要求9所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:步骤9中的营养因子GDNF为1-100ng/mL、BDNF为1-100ng/mL、cAMP为1-10μM、TGF-β3为1-100ng/mL、compound E为1-10μM和ascorbic acid为1-1000μM。
11.根据权利要求10所述的用hPSCs诱导分化得到中脑类器官的方法,其特征在于:所述营养因子GDNF为10ng/mL、BDNF为10ng/mL、cAMP为1μM、TGF-β3为10ng/mL、compound E为1μM和ascorbic acid 为200μM。
12.一种中脑类器官,其特征在于:其由权利要求1-11所述的任一方法所得。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111370443.6A CN114149971A (zh) | 2022-01-12 | 2022-01-12 | 用hPSCs诱导分化得到中脑类器官的方法及中脑类器官 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111370443.6A CN114149971A (zh) | 2022-01-12 | 2022-01-12 | 用hPSCs诱导分化得到中脑类器官的方法及中脑类器官 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114149971A true CN114149971A (zh) | 2022-03-08 |
Family
ID=80457036
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111370443.6A Pending CN114149971A (zh) | 2022-01-12 | 2022-01-12 | 用hPSCs诱导分化得到中脑类器官的方法及中脑类器官 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114149971A (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114457015A (zh) * | 2022-03-11 | 2022-05-10 | 中国科学院广州生物医药与健康研究院 | 纹状体类脑器官及其培养基、培养方法和应用 |
CN115322965A (zh) * | 2022-08-19 | 2022-11-11 | 同济大学 | 一种体外获得后脑底板细胞的方法、成套培养基及应用 |
CN117778313A (zh) * | 2024-02-23 | 2024-03-29 | 成都云测医学生物技术有限公司 | 脑类器官获得间充质干细胞分化方法和应用 |
CN117778313B (zh) * | 2024-02-23 | 2024-05-24 | 成都云测医学生物技术有限公司 | 脑类器官获得间充质干细胞分化方法和应用 |
-
2022
- 2022-01-12 CN CN202111370443.6A patent/CN114149971A/zh active Pending
Non-Patent Citations (2)
Title |
---|
JUNGHYUN JO等: "Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons", 《CELL STEM CELL》 * |
Y. CHEN等: "Chemical Control of Grafted Human PSC-Derived Neurons in a Mouse Model of Parkinson’s Disease", 《CELL STEM CELL》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114457015A (zh) * | 2022-03-11 | 2022-05-10 | 中国科学院广州生物医药与健康研究院 | 纹状体类脑器官及其培养基、培养方法和应用 |
CN115322965A (zh) * | 2022-08-19 | 2022-11-11 | 同济大学 | 一种体外获得后脑底板细胞的方法、成套培养基及应用 |
CN115322965B (zh) * | 2022-08-19 | 2024-01-30 | 同济大学 | 一种体外获得后脑底板细胞的方法、成套培养基及应用 |
CN117778313A (zh) * | 2024-02-23 | 2024-03-29 | 成都云测医学生物技术有限公司 | 脑类器官获得间充质干细胞分化方法和应用 |
CN117778313B (zh) * | 2024-02-23 | 2024-05-24 | 成都云测医学生物技术有限公司 | 脑类器官获得间充质干细胞分化方法和应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2021072818A (ja) | 終脳又はその前駆組織の製造方法 | |
CN114149971A (zh) | 用hPSCs诱导分化得到中脑类器官的方法及中脑类器官 | |
CN109136185B (zh) | 一种类脑器官器的制备方法和应用 | |
CN110484506B (zh) | 胶质母细胞瘤类器官模型的构建方法和应用 | |
US20230002725A1 (en) | Small mobile stem cells (sms) and uses thereof | |
WO2021232830A1 (zh) | TGF-β抑制剂在诱导神经干细胞及类器官形成中的应用 | |
CN113136367B (zh) | 一种纹状体类器官的制备方法和应用 | |
Dehdilani et al. | Improved survival and hematopoietic differentiation of murine embryonic stem cells on electrospun polycaprolactone nanofiber | |
JP2021524253A (ja) | バイオリアクターでの細胞培養システム | |
Li et al. | Generation and staging of human retinal organoids based on self-formed ectodermal autonomous multi-zone system | |
CN112852709B (zh) | 小鼠肺类器官培养方法 | |
KR101896803B1 (ko) | 인간 만능줄기세포로부터 중간엽 줄기세포로의 분화 유도 생산율을 증가시키는 방법 및 이에 의해 생성된 중간엽 줄기세포 | |
US20220145247A1 (en) | Generation of human pluripotent stem cell derived artificial tissue structures without three dimensional matrices | |
CN107164325B (zh) | MSCs来源的少突胶质细胞的制备方法及试剂盒 | |
JP7198524B2 (ja) | スフェロイドの製造方法および多能性幹細胞マーカーを発現させる方法 | |
CN110656089B (zh) | 一种将iPS细胞定向诱导分化到成熟神经元的方法 | |
CN114058587A (zh) | hPSCs诱导分化制备的基底前脑胆碱能类器官及其制备方法 | |
CN114107206A (zh) | 基于hPSCs诱导分化得到的纹状体类器官及hPSCs诱导分化方法 | |
CN114807034A (zh) | 一种人多能干细胞来源的Müller细胞的制备方法 | |
CN114292804A (zh) | 一种血管化脂肪类器官培养方法 | |
CN114958746B (zh) | 一种多能干细胞诱导产生3d类脑体的方法及试剂盒 | |
CN115354029B (zh) | 神经类器官的制备方法和神经类器官 | |
CN112553160B (zh) | 一种化学诱导皮质神经元的方法及培养基 | |
KR20170125180A (ko) | 신경줄기세포의 분화 방법 | |
CN116983479A (zh) | 一种利用3d打印制备人工血管实现人脑类器官血管化的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220308 |