【作者】 李彬；

【导师】 陈璞；

【作者基本信息】 武汉大学 ， 生物医学工程， 2022， 硕士

【摘要】 背景:神经系统疾病每年影响全球数百万人的健康。为了揭示这些疾病的发病机制,促进神经药物的开发,迫切需要开发一种能够高度模拟人脑的体外研究模型。近年来,人源性大脑类器官被神经科学界认为是更符合人类生理的大脑模型。人源性大脑类器官是以诱导多能干细胞（human induced pluripotent stem cell,hiPSC）为种子细胞,基于发育生物学原理,在体外构建的具有与人脑高度类似的细胞构成、发育轨迹和神经网络的体外“迷你大脑”。大脑类器官由Lancaster于2013年首次构建成功,该模型目前已被广泛应用于脑研究的各个领域,如神经发育疾病,神经退行性疾病,遗传性神经系统疾病等。然而,目前人源性大脑类器官培养的金标准——Lancaster法,存在着一些弊端:1、均一性差。缺乏精确的微环境控制,导致人源性大脑类器官形态存在较大的异质性;2、操作复杂性高。在不同的培养阶段,需要对人源性大脑类器官进行多次转移,操作复杂性高,污染的风险大;3、多个类器官易融合。悬浮培养的方法可能会导致类器官融合;4、难以进行原位观察成像。为了解决这些技术难题,我们拟结合生物工程方法,实现hiPSC的图案化,从而精确控制人源性大脑类器官形态和大小,构建高度均一的人源性大脑类器官。方法:我们使用软光刻法制作具有微柱阵列的SU-8阳模;在SU-8阳模上浇注并压制聚二甲氧基硅氧烷（polydimethylsiloxane,PDMS）预聚物,制备PDMS穿孔薄膜;将制备好的PDMS穿孔薄膜铺在24孔板的底部,并包被一层2%（v/v）Matrigel;将hiPSC悬液以2×10⁵个细胞每孔的密度接种到PDMS薄膜上。细胞贴壁后,移除PDMS薄膜,留下单层图案化的hiPSC。hiPSC依次在拟胚体形成培养基中培养6天,在神经诱导培养基中培养5天。然后用Matrigel包被后在神经扩张培养基中培养3天。最后,在神经成熟培养基中分化为成熟的图案化大脑类器官。结果:研究结果显示,图案化hiPSC经历了细胞增殖、拟胚体形成、神经花环形成和神经成熟。该类器官的形态较Lancaster法形成的类器官更为均一,其面积变异系数比Lancaster法形成的类器官小3倍。此外,图案化大脑类器官的发育与早期人脑的发育相似。我们对培养至28天的图案化大脑类器官与18天的大脑类器官进行了转录水平分析,基因本体论分析显示,与培养至18天的图案化大脑类器官相比,大脑发育相关基因在第28天显著上调。同时,差异基因表达分析显示,端脑和皮质特异性基因在图案化大脑类器官中表达上调。此外,免疫荧光结果显示,第28天的图案化大脑类器官包含多种脑特异性细胞类型,包括神经干细胞、神经元以及星形胶质细胞。为了证明该类器官的效用,我们使用构建的图案化大脑类器官评估了Aβ₄₂O的神经毒性。5%Aβ₄₂O处理72h后,q PCR分析显示MAP2和TUJ基因表达显著降低。此外,TUNEL检测进一步显示图案化大脑类器官中细胞凋亡增加,表明Aβ₄₂O能够诱导图案化大脑类器官中神经元的丢失。结论:在本研究中,我们结合工程技术,创新性的构建了图案化大脑类器官。与Lancaster法相比,图案化大脑类器官存在以下优势:1、均一性高。实现了对类器官形态和大小的精确调控,降低了类器官的异质性,提高了类器官的均一性;2、简化培养流程。不需要转移,简化了人源性大脑类器官的培养流程,降低了潜在污染风险;3、避免融合。贴壁培养大脑类器官,有效防止类器官间的接触,避免了类器官融合;4、易于原位观察成像。实现培养中的类器官实时观察,解决了传统方法中无法实时观察的局限。另一方面,图案化大脑类器官与传统方法培养的大脑类器官类似,具有与人脑类似的多种神经细胞种群,且对神经毒性药物具有与其他模型相类似的细胞毒性响应。综上所述,图案化方法解决了传统大脑类器官培养中面临的均一性差的挑战。我们相信,图案化大脑类器官将在神经发育、神经毒性和神经疾病等不同的领域得到广泛的应用。更多还原

【Abstract】 Background:Neurological diseases affect the health of millions of people globally every year.High-fidelity brain models are urgently demanded to reveal the underlying mechanism of these diseases and promote neurological drug discovery.Emerging human brain organoids（h BOs）has been increasingly recognized as a more human physiologically relevant brain model to address this issue.However,the currently gold standard of h BOs culture,namely Lancaster method,suffers from several disadvantages.First,precise microenvironmental control is lacking,resulting in great heterogeneity in h BOs morphology.Second,multiple-step h BOs transfer is requested during the different culture stages,which is labor-intensive and potentially increases the risk of contamination.Third,suspension culture in this method may cause organoid fusion and is also inaccessible for in-situ observation.To address these technical challenges,we here present a novel method to generate uniform brain organoids using hiPSC micropatterning technique（HMPT）.Method:Briefly,SU-8 masters with microcolumn array were fabricated using the standard photolithography method.PDMS perforating films were fabricated by pouring and pressing PDMS prepolymer on the SU-8 master.The tailored PDMS films were laid on the bottom of a 24-well plate and coated with 2%Matrigel.The hiPSC suspension was seeded onto the PDMS films at a density of 2500 cells mm^-2.After cell attachment,the PDMS films were removed to leave monolayer micropatterned hiPSCs.The hiPSCs were cultured in EB formation medium for 6 days and induction medium for 5 days in sequence.Then,formed neuroectoderm were coated with Matrigel and cultured in neural expansion medium for 3 days.Finally,the micropatterned brain organoids（MBOs）were formed in the neural maturation medium.The micropatterned hiPSCs experienced cell stratification,EB formation,rosette formation,and neural maturation,and ultimately formed MBOs on Day 25.The MBOs displayed a more uniform morphology,and the coefficient of variation of MBOs area was three times smaller compared to that in the Lancaster method.The formation of MBOs is reminiscent of the early neurodevelopmental stage of brain.The GO analysis indicated that brain development-relevant genes were significantly up-regulated on day28 compared with those on day 18.Simultaneously,differential gene expression analysis showed that telencephalon and cortical brain-specific genes were up-regulated in MBOs.Furthermore,immunofluorescence analysis demonstrated that MBOs on day28 contained multiple brain-specific cell types.To demonstrate the utility of MBOs,we assessed the neurotoxicity of Aβ₄₂O.After 72-h Aβ₄₂O treatment,q PCR analysis showed that the gene expression of MAP2 and TUJ significantly decreased.Additionally,TUNEL assays further displayed increased cell apoptosis in MBOs,revealing neurotoxicity-induced neuron loss.Previously,HMPTs have been demonstrated to generate adherent boundary-constrained embryonic tissues for studying the development and morphogenesis of cardiac tissue,neural tube and neuroepithelium.Here,we for the first time employed HMPT to generate h BOs.Compared to the Lancaster method,h BOs formation using HMPT doesn’t request multiple-step h BOs transfers and suspension culture,thus avoiding potential contamination and organoid fusion.Additionally,the HMPT permits long-term in-situ observation of neurodevelopment of h BOs,which could not be conducted in the Lancaster method.Overall,HMPT method has addressed the long-lasting homogeneity challenge faced by the conventional h BOs culture method.We expect MBOs will find broad applications in diverse fields not limited to neurodevelopment,neurotoxicity and neurological disease studies.更多还原