Processing math: 100%
切换中文 维普资讯 中国知网 万方数据
Basic Research | Updated:2024-02-19
    • Construction of hiPSC-derived Excitatory Neural Network-like Tissue

    • SU Qi-song

      ,  

      LI Ge

      ,  

      XU Jin-hai

      ,  

      JIANG Bin

      ,  

      WEI Qing-shuai

      ,  

      ZENG Xiang

      ,  

      ZENG Yuan-shan

      ,  

      ZHU Ping

      ,  
    • Journal of Sun Yat-sen University(Medical Sciences)   Vol. 44, Issue 4, Pages: 625-633(2023)
    • DOI:10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001    

      CLC: R318
    • Published:20 July 2023

      Received:08 February 2023

    Scan for full text

  • Cite this article

    PDF

  • SU Qi-song,LI Ge,XU Jin-hai,et al.Construction of hiPSC-derived Excitatory Neural Network-like Tissue[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(04):625-633. DOI: 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001.

  •  
  •  
    Sections

    Abstract

    Objective

    To construct a neural network-like tissue with the potential of synaptic formation in vitro by seeding human induced pluripotent stem cell-derived neural precursor cells (hiPSC-NPCs) on decellularized optic nerve (DON), so as to provide a promising approach for repair of nerve tissue injury.

    transl

    Methods

    Through directional induction and tissue engineering technology, human induced pluripotent stem cells (hiPSCs) and 3D DON scaffolds were combined to construct neural network-like tissues. Then the hiPSCs were directionally induced into human neural precursor cells (hNPCs) and neurons. Immunofluorescence staining was used to identify cell differentiation efficiency. 3D DON scaffolds were prepared. Morphology and cytocompatibility of scaffolds were identified by scanning electron microscopy and Tunnel staining. Induced hiPSC-NPCs were seeded on DON scaffolds. Immunofluorescence staining, scanning electron microscopy, transmission electron microscopy and patch clamp were used to observe the morphology and functional identification of constructed neural network tissues.

    transl

    Results

    ①The results of immunofluorescence staining suggested that most of hiPSC-NPCs differentiated into neurons in vitro. We had successfully constructed a neural network dominated by neurons. ② The results of scanning electron microscopy and immunohistochemistry suggested that a neural network-like tissue with predominating excitatory neurons in vitro was successfully constructed. ③The results of immunohistochemical staining, transmission electron microscopy and patch clamp indicated that the neural network-like tissue had synaptic transmission function.

    transl

    Conclusion

    A neural network-like tissue mainly composed of excitatory neurons has been constructed by the combination of natural uniform-channel DON scaffold and hiPSC-NPCs, which has the function of synaptic transmission. This neural network plays a significant role in stem cell derived replacement therapy, and offers a promising prospect for repair of spinal cord injury (SCI) and other neural tissue injuries.

    transl

    Keywords

    hiPSC-NPCs; DON; synapse; excitatory neurons; neural network-like tissue

    transl

    心血管手术过程中往往需要短暂地中断中枢神经系统的正常血液供应,这一过程可能会引起脊髓组织的缺血性坏死,导致患者下肢功能障碍甚至瘫痪

    1。目前治疗心血管术后脊髓损伤(spinal cord injury, SCI)的主要方法有手术治疗、药物治疗、高压氧治疗、物理治疗等。然而,由于中枢神经系统的再生能力非常有限,以上这些技术手段难以达到增强内源性再生以补充丢失神经元的目的,治疗效果不尽如人意2。这就要求我们寻找更有效的治疗SCI的新方法。近年来,干细胞治疗和组织工程技术的快速发展为SCI的治疗提供了新的希望。人诱导多能干细胞(human induced pluripotent stem cell, hiPSCs)可直接由供体体细胞重编程而来,具有与胚胎干细胞相似的增殖能力和多能性,解决了神经干细胞、间充质干细胞和胚胎干细胞所涉及的数量、来源、道德、伦理和法律问题3-4。研究表明,hiPSCs在体外可以特异性诱导分化为神经元,为干细胞移植治疗SCI提供了有希望的治疗方法5-7。去细胞视神经(decellularized optic nerve, DON)是一种天然生物材料,具有均匀分布的孔道结构,有利于神经纤维的直行生长。前期研究结果表明,DON支架富含具有生物活性的四型胶原和层粘连蛋白,能够一定程度诱导神经纤维表达整合素并沿支架直行生长,具有引导神经纤维定向生长的效能8。目前,国内外尚未见人诱导多能干细胞源性神经前体细胞(human induced pluripotent stem cell-derived neural precursor cells, hiPSC-NPCs)联合天然生物材料DON构建兴奋性类神经网络组织的相关报道。因此,我们创新性地提出,应用hiPSC-NPCs和DON支架构建一种具有兴奋性突触传递功能的类神经网络组织,为将来进一步在体内运用组织工程技术治疗SCI打下基础。
    transl

    1 材料与方法

    1.1 DON支架的制备

    DON支架的制备如前期实验方案所述

    8:将猪眼球从- 80 ℃超低温冰箱中取出,解冻后从猪眼球后方取下视神经,剥离视神经上的硬脑膜和蛛网膜,用刀片切成1 cm的小段组织。将组织浸泡在含2 %青霉素-链霉素(15140122, Gibco, NY, USA)、100 μg/mL庆大霉素(R01510, Gibco)和2.5 μg/mL两性霉素 (R01510, Gibco)的超纯水中,于4 ℃摇床(250 r/min; 奥盛, 杭州, 中国)过夜。后转移至常温摇床(250 r/min ; 奥盛, 杭州, 中国)放置2 h。将离心管内液体换成含1 %青霉素-链霉素(Gibco)的超纯水,浸泡6 h(每2 h换1次液)。组织用含1 %青霉素-链霉素和3%TritonX-100 (0694-1L, Amresco, OH, USA)的超纯水于常温摇床上浸泡12 h,用无菌超纯水洗3次(每次10 min)后,继续用含4 %脱氧胆酸钠(30970, Sigma-Aldrich, St. Louis, MO, US)的超纯水于常温摇床上浸泡24 h。以上步骤反复进行3次,以去除组织中的细胞。用50 U/mL的DNA酶(D4513, Sigma-Aldrich)和10 μg/mL 的RNA酶(R6513, Sigma-Aldrich)于常温摇床上浸泡组织3 h以去除核残留物。用无菌超纯水洗3次(每次20 min)。用超纯水浸泡去细胞组织,冻干后备用。用前用刀片将其切成厚度为2 mm的圆柱体。
    transl

    1.2 hiPSC-NPCs的构建及其诱导分化

    将2 × 105个复苏的 hiPSCs (中盛溯源, 安徽, 中国)接种至Matrigel® Matrix(354277, Corning, NY, USA)包被的孔板中,每天更换 mTeSRTM1 培养液(85850, Stemcell, Vancouver, Canada)。当细胞密度增长至90%左右时进行传代或冻存。将2 × 105个复苏的 hiPSCs接种至Matrigel® Matrix包被的孔板中,第2天开始加入 STEMdiff SMADi Neural induction medium(08581, Stemcell),每天换液。14天后,使用 STEMdiff Neural progenitor medium (05833, Stemcell)进行半换液,第2天使用STEMdiff Neural progenitor medium全换液,每天换液。当细胞密度增长至90 %左右时进行传代或冻存。将1 × 104个hiPSC-NPCs种植于Matrigel® Matrix包被的孔板中,加入 STEMdiff Neural progenitor medium培养2 d后,加入神经元诱导培养基500 μL 进行培养,每天半换液。神经元诱导培养基由1:1 DMEM/F12(10565-018, Gibco)和Neuronbasal A(10888022, Gibco)混合配制而成,内含0.5 % N2(17502-048, Gibco)、1 % B27(17504-044, Gibco)、20 ng/mL BDNF(248-BDB/CF, R&D, MN, USA)、2 μmol/L db-cAMP(D0627, Sigma-Aldrich)。

    transl

    1.3 hiPSC源性类神经网络组织的构建

    在种植细胞前将DON支架放在Matrigel® Matrix中包被过夜,以促进hiPSC-NPCs的黏附。使用0.05 % Trypsin-EDTA(25300-054, GIBCO)于细胞培养箱中消化hiPSC-NPCs 3min,335.4×g离心后吸弃上清,加入STEMdiff Neural progenitor medium重悬。之后使用10 μL培养基重悬5 × 105个细胞接种于1个DON生物支架上。孵育15 min后,每个孔加入500 μL STEMdiff Neural progenitor medium培养2 d后,神经元诱导培养基500 μL进行培养,每天半换液。

    transl

    1.4 多色免疫荧光染色

    使用10 %的山羊血清对体外培养的细胞爬片或类神经网络组织切片进行封闭后,使用含0.3 % TritonX-100 (Amresco, OH, USA)的PBS配备合适浓度的一抗溶液:anti-Nanog(Abcam, 1: 200)、anti-Oct4(Abcam, 1: 200)、anti-Ki67(Abcam, 1: 200)、anti-Sox1(Abcam, 1: 100)、anti-Nestin(Sigma–Aldrich, 1: 100)、anti-Oilgo2(Abcam, 1: 200)、anti-GFAP(Abcam, 1: 1 000)、anti-NF(Sigma–Aldrich, 1: 500)、anti-Map2(Abcam, 1: 1 000)、anti-SYP(Abcam, 1: 500)、anti-PSD95(Abcam, 1: 500)、anti-ChAT(Merck Millipore, 1: 200)、anti-Glutamate(Abcam, 1: 100)、anti-GAD67(Merck Millipore, 1: 500)。于4℃冰箱孵育过夜。然后用对应种属的二抗溶液于37 ℃烘箱中孵育60 min。最后使用Hoechst33342(Hoe, H21492, Invitrogen, NY, USA)对细胞核进行染色15 min。使用80%甘油封片后,在显微镜下观察并拍照。

    transl

    1.5 Tunnel染色

    类神经网络组织在体外培养28 d后,使用4 %PFA固定30 min,用PBS溶液洗3次,每次10 min。进行Tunnel染色前,使用含0.5 %TritonX-100的PBS溶液室温孵育5 min。按照Tunnel试剂盒(C1086, 碧云天, 上海, 中国)的使用说明,配制适当量的Tunnel检测液。PBS溶液洗3次,每次10 min。在组织切片上加上适量的Tunnel检测液,37 ℃避光孵育1 h。PBS溶液洗3次,每次10 min。使用80 %甘油封片后,在显微镜下观察并拍照。

    transl

    1.6 扫描电镜

    hiPSC源性类神经网络组织在体外培养28 d后,先使用40 g/L多聚甲醛固定30 min,再使用2.5 %戊二醛后固定30 min。使用冷冻干燥机干燥24 h。喷金颗粒120 s镀膜后,于扫描电镜下观察细胞在生物材料上的生长状况。

    transl

    1.7 透射电镜

    体外培养28 d后,使用含40 g/L多聚甲醛和2.5 %戊二醛的固定液固定hiPSC源性类神经网络组织30 min。用1 %锇酸固定1 h后,进行酒精和丙酮梯度脱水,使用Epon包埋样品,于60 ℃烘箱中聚合72 h。使用RM2065切片机(Leica)进行半薄切片后,行甲苯胺蓝染色,定位后再进行超薄切片,经醋酸铀和柠檬酸双重染色后,在电子显微镜(Philips, Eindhoven, Holland)下观察。

    transl

    1.8 全细胞膜片钳

    进行全细胞膜片钳记录时采用集成钳制放大器(integrated patch-clamp amplifier, IPA )和Igor8软件进行信号采集,过滤频率为1 kHz,采样频率为50 kHz。将培养28 d的hiPSC源性类神经网络组织置于孵育槽中持续灌流氧饱和的人工脑脊液,灌流速度为0.3~0.5 mL/min。记录用的外液为人工脑脊液,其成分为(mmol/L):124 NaCl、3 KCl、1.25 NaH2PO4、2 CaCl2·2H2O、1 MgCl2·6H2O、26 NaHCO3、10 glucose。玻璃电极电阻为4-6 MΩ,钳制细胞电流小于-100 pA 纳入统计,监测系列电阻(series resistance, Rs)、输入电阻(input resistance, Ri)和膜电容(membrane capacitance)大小。记录动作电位(action potential, AP):注入负电流钳制细胞处于-70 mV,从0 pA 开始,逐步增加地注入电流,到400 pA结束,步进电流为25 pA,即依次注入0 pA,25 pA,50 pA至375 pA,400 pA。相邻两次注入电流的时间间隔为15 s。记录微小兴奋性突触后电流(miniature excitatory postsynaptic current, mEPSC):将细胞钳制在- 70 mV,加入1 μmol/L TTX,20 μmol/L PTX与100 μmol/L D,L-APV,分别阻断钠通道、GABAa受体和NMDA受体。记录微小抑制性突触后电流(miniature inhibitory postsynaptic current, mIPSC):将细胞钳制在- 60 mV,加入1 μmol/L TTX,20 μmol/L CNQX与100 μmol/L D,L-APV,分别阻断钠通道,AMPA受体和NMDA受体。

    transl

    1.9 统计学方法

    在200倍显微镜下对每张行免疫荧光化学染色的hiPSCs、hiPSC-NPCs爬片或构建的类神经网络组织的组织切片进行五个视野的选取,对各视野中的各个标志物的阳性率进行计数统计,每个独立实验重复5次。实验数据以平均百分率±标准差(ˉx±s)表示。

    2 结 果

    2.1 hiPSCs的培养及鉴定

    复苏的hiPSCs的形态学观察和各标志物的表达情况如图1所示。在倒置荧光显微镜下观察到复苏的hiPSCs呈克隆样生长,细胞团具有清晰的轮廓,单个细胞呈圆形,核仁明显,核浆比较大(图1A)。Nanog、Oct4为细胞多能性标志物,Ki67为细胞增殖活力指标,两者在细胞中表达越高,表明细胞全能性越高,增殖活力越强。免疫细胞荧光化学染色显示复苏的hiPSCs具有(96.60± 0.50)% Nanog(图1B)和(96.23 ± 0.85) % Oct4(图1C)多能性标记物阳性率,具有(96.75 ± 1.15) % Ki67(图1D)增殖能力标志物阳性率。以上结果提示复苏的hiPSCs维持了干细胞的全能性,并具有良好的增殖能力。

    transl

    fig

    图1  hiPSCs的形态学观察和各标志物的表达

    Fig. 1  Morphological observation of hiPSCs and expression of the representative markers

    A: Cell morphology of hiPSCs; B-C: Nanog, Oct4 expressed by hiPSCs; D: Ki67 expressed by hiPSCs. Scale bars in A-D = 50 μm.

    icon Download:  Full-size image | High-res image | Low-res image

    2.2 hiPSC-NPCs的诱导与鉴定

    hiPSC-NPCs的形态学观察和各标志物的表达如图2所示。倒置荧光显微镜下可观察到构建的hiPSC-NPCs具有花环样形态,并具有细胞突起(图2A)。免疫细胞荧光化学染色显示构建的hiPSC-NPCs具有 Pax6(图2B)、Sox1(图2C)和Nestin(图2D)神经前体细胞标志物的高表达。其中,Pax6阳性率为(96.47 ± 1.84)%,Sox1阳性率为(96.62 ± 0.86) %,Nestin阳性率为(96.09 ± 0.35)%。此外,观察到构建的hiPSC-NPCs具有Ki67表达(图2E),这提示构建的hiPSC-NPCs维持了一定的细胞增殖能力,但并没有在免疫细胞荧光化学染色中观察到hiPSCs阳性标志物Oct4(图2F)。以上结果提示构建的hiPSC-NPCs为高纯度的,不具有hiPSCs的残留。

    transl

    fig

    图2  hiPSC-NPCs的形态学观察和各标志物的表达

    Fig. 2  Morphological observation of hiPSC-NPCs and expression of the representative markers

    A: Cell morphology of hiPSC-NPCs; B-D: Pax6, Sox1, Nestin expressed by hiPSC-NPCs; E: Ki67 expressed by hiPSC-NPCs; F: Oct4 expressed by hiPSC-NPCs. Scale bars in A-F = 50 μm.

    icon Download:  Full-size image | High-res image | Low-res image

    2.3 hiPSC-NPCs的分化及其鉴定

    hiPSC-NPCs在体外培养7 d后的形态学观察和各标志物的表达如图3所示。倒置荧光显微镜下可观察到hiPSC-NPCs聚团生长,细胞团与细胞团间长突起明显(图3A)。免疫细胞荧光化学染色显示hiPSC-NPCs在体外培养7 d后具有(90.28 ± 0.36)% Map2阳性(图3B,D),(1.46 ± 0.41)% GFAP阳性(图3C,D)。以上结果提示hiPSC-NPCs在体外培养7 d后大部分向神经元方向分化,只有少量hiPSC-NPCs向星形胶质细胞方向分化。

    transl

    fig

    图3  hiPSC-NPCs分化后的细胞形态学观察和各标志物的表达

    Fig. 3  Morphological observation and expression of the representative markers after differentiation of hiPSC-NPCs

    A: Cell morphology of hiPSC-NPCs after 7 days of induction; B: Map2 expressed by hiPSC-NPCs after differentiation; C: GFAP expressed by hiPSC-NPCs after differentiation; D: Quantitative analysis of Map2 and GFAP. Scale bars in A-C = 50 μm.

    icon Download:  Full-size image | High-res image | Low-res image

    2.4 hiPSC源性类神经网络组织的构建

    DON支架及类神经网络组织的形态学观察和各标志物的表达情况如图4所示。DON支架是一个直径约2~3 mm的类圆柱体(图4A)。扫描电镜下能够观察到DON支架具有一个个较为均匀的圆形孔道(图4B)。将hiPSC-NPCs种植于DON上,在体外培养28 d后,能够在扫描电镜下观察到在DON的表面及其孔道内都具有细胞的生长,且细胞与细胞之间能形成长突起(图4C)。行免疫组织化学染色发现,构建的类神经网络组织在体外培养14 d后只有(3.32 ± 1.11)%的细胞具有Tunnel凋亡细胞染色阳性(图4D)。同时,观察到种植于DON材料上的hiPSC-NPCs聚团生长,具有神经丝(neurofilament,NF)阳性,细胞团与细胞团之间可以通过长突起进行连接(图4E)。体外培养14 d后的分化鉴定结果显示大部分细胞具有Map2阳性,只有少部分细胞具有GFAP和Oligo2阳性(图4F-H)。其中,Map2阳性率为(80.86 ± 1.82)%,GFAP阳性率为(5.56 ± 0.93)%,Oligo2阳性率为(3.21 ± 1.00)%(图4I)。以结果提示hiPSC-NPCs与DON具有良好的生物相容性。在体外培养14 d后,构建的类神经网络组织处于一个相对成熟的状态,大部分的hiPSC-NPCs向成熟神经元方向分化。

    transl

    fig

    图4  DON支架及类神经网络组织的形态学观察和各标志物的表达情况

    Fig. 4  Morphological observation of DON scaffold and neural network-like tissue and expression of the representative markers of neural network-like tissues

    A: Diameter of DON; B: Morphology of DON scaffold observed under scanning electron microscope; C: Morphology of neural network-like tissue observed under scanning electron microscopy; D: Tunnel staining of neural network-like tissue; E-H: NF, Map2, GFAP, Oligo2 expressed by neural network-like tissue; I: Quantitative analysis of Map2, GFAP and Oligo2. D-J: Scale bars in D-H = 50 μm.

    icon Download:  Full-size image | High-res image | Low-res image

    2.5 hiPSC源性类神经网络组织表达的递质类型分析

    hiPSC源性类神经网络组织递质类型的表达如图5所示。体外培养28 d后,行免疫荧光技术对构建的类神经组织上的hiPSC-NPCs进行Map2、ChAT、glutamate和GAD67的染色,分别观察Map2和ChAT、Map2和glutamate、Map2和GAD67的共标情况,以统计Map2阳性神经元表达的神经递质类型。据结果分析,发现种植于DON上的Map2阳性神经元中有(56.65 ± 3.27)%的ChAT阳性率(图5A-C,J)、(74.68 ± 2.60)% Glutamate阳性率(图5D-F,J),(9.05 ± 3.62)%的GAD67阳性率(图5G-I,J)。以上结果表明种植于DON材料上的hiPSC-NPCs大多数分化为能够表达ChAT和glutamate的兴奋性神经元,只有少数分化为表达GAD67的抑制性神经元,提示构建的类神经网络组织是兴奋性的。

    transl

    fig

    图5  hiPSC源性类神经网络组织递质类型的表达

    Fig. 5  Neurotransmitter types expressed by hiPSC-derived neural network-like tissue

    A-I: Co-labeling immunostaining of Map2 and ChAT, Glutamate or GAD67 of hiPSC-derived neural network-like tissue; J: Quantitative analysis of ChAT, Glutamate and GAD67. Scale bars in A-I = 50 μm.

    icon Download:  Full-size image | High-res image | Low-res image

    2.6 hiPSC源性类神经网络组织的功能性分析

    hiPSC源性类神经网络组织的功能性分析结果如图6所示。体外培养28 d后,利用免疫组织化学染色对种植于DON上的hiPSC-NPCs进行Map2、SYP、PSD95的染色,观察到Map2阳性的神经元能够分别与突触前膜蛋白SYP和突触后膜蛋白PSD95的共标(图6A-F)。透射电镜下观察到种植于DON上的细胞状态良好,细胞胞体与长突起、细胞与细胞之间具有一定的接触,并具有突触结构的形成(图6G)。在高倍镜下能观察到细胞与细胞之间膜结构的增厚(图6H)。前面的结果表明种植于DON材料上的hiPSC-NPCs大多数分化为能够表达ChAT和glutamate的兴奋性神经元,只有少数分化为表达GAD67的抑制性神经元,提示构建的类神经网络组织是兴奋性的(图5),为了进一步明确表达ChAT和glutamate的兴奋性神经元的功能,我们进行全细胞膜片钳电流钳记录这些神经元的电生理学特性,发现hiPSC源性神经元在注入正电流(500 ms, 20~50 pA)可以记录到成串的动作电位(AP)(图6I)。全细胞膜片钳电压钳下,也记录到hiPSC源性神经元的自发兴奋性微小突触后电流(mEPSC)(图6J),但没有记录到自发抑制性微小突触后电流(mIPSC)(图6K),电生理结果说明hiPSC 细胞经诱导确实分化成神经元,且这些神经元之间形成了兴奋性的突触连接。

    transl

    fig

    图6  hiPSC源性类神经网络组织的功能性分析

    Fig. 6  Functional analysis of hiPSC-derived neural network-like tissue

    A-F: Co-labeling immunostaining of Map2 and SYP or PSD95 of hiPSC-derived neural network-like tissue; G: Cell bodies and neurites observed under transmission electron microscopy; H: Synaptic structure observed under transmission electron microscopy; I: Action potential detected by whole-cell recording; J: Spontaneous miniature excitatory postsynaptic current was detected; K: No spontaneous miniature inhibitory postsynaptic current was detected. Scale bars in A-F = 50 μm; Scale bars in G = 1 μm; Scale bars in H = 200 nm.

    icon Download:  Full-size image | High-res image | Low-res image

    3 讨 论

    在本研究中,我们选择了易获取、不具道德和伦理争议的外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)来源的hiPSCs。HiPSCs具有高度全能性和高度增殖能力,直接将其移植到体内具有致畸胎瘤的风险

    9-10。为了降低这种致瘤性,我们将hiPSCs诱导成为人神经前体细胞(human neural precursor cells, hNPCs)。实验结果表明,我们的方案能够诱导出高于95 %比例的hNPCs,诱导后的细胞具有一定的增殖能力,但是不具有hiPSCs的残留。hNPCs在体外可以分化为神经元、星形胶质细胞和少突胶质细胞,具有良好的生物安全性,但是由于SCI发生后机体内恶劣的微环境,往往会导致移植的hNPCs向星形胶质细胞分化,较少分化为神经元,引起损伤区胶质瘢痕的增生,不利于损伤后的恢复11-13。此外,神经干细胞的原位移植具有异位迁移的风险,移植细胞会在迁移部位大量增殖,影响迁移部位组织的正常生理功能或引起该部位的疼痛14。故我们选择在体外将hiPSC-NPCs向神经元方向诱导分化。
    transl

    hiPSCs向神经元的诱导分化可通过转染逆转录病毒或慢病毒实现。但这种方法是将重编程因子整合于细胞基因启动子附近,会改变细胞内源性基因的表达,具有潜在毒性,存在插入突变的风险

    15。为获取更安全的hiPSC源性神经元,研究者开发了mRNAs诱导转染技术16。转染mRNAs虽能快速实现神经元高分化率,但仍然存在转基因蛋白异质性表达的风险。近年来,使用药物诱导hiPSCs向神经元方向分化,消除了使用病毒、mRNAs转染可能引起的潜在风险,具有较高的安全性17-19。本研究中,在hiPSCs培养过程中使用STEMdiff SMADi Neural induction medium阻断TGF-β/BMP依赖的SMAD信号通路可诱导hiPSCs向中枢神经系统hNPCs分化20-22。Xu等23使用含N2、B27、BDNF、db-cAMP等营养因子的神经元诱导培养基成功构建hiPSC源性神经网络,我们在此方法的基础上对诱导方案进行改良,实现了较高的神经元分化率。
    transl

    SCI发生后,由于内环境的复杂性,干细胞在迁移的过程中容易受其他因素影响,发生转化甚至失活,存活率较低,因此可以通过给予一些外源性的支持更好的地达到干细胞治疗的效果

    24。生物材料可以模拟细胞外基质,局部改善微环境,减少胶质瘢痕的形成和炎症反应的发生,改善细胞滞留,抑制细胞的坏死或凋亡,有利于损伤脊髓组织重新连接,重建神经传导回路25。因此,将干细胞和具有药物或生物活性分子的生物材料联合应用,利用组织工程的技术手段,为干细胞提供合适的生态位,促进轴突再生、突触和神经环路的重建,可以在一定程度上修复或取代受损组织的功能和解剖结构,是治疗SCI的有效手段26。课题组的前期研究工作表明,DON去除了原始猪视神经细胞外基质成分中的髓磷脂相关糖蛋白和软骨素硫酸蛋白多糖等轴突抑制分子,保存了Ⅳ型胶原蛋白和层粘连蛋白等轴突促细胞外基质蛋白,为改善SCI后抑制性微环境提供了可行途径8。因此,我们创新性地提出将hiPSC-NPCs种植于DON上,在体外构建一种具有较高神经元分化率的类神经网络组织。
    transl

    SCI会导致神经元及其轴突传导束的丢失,引起部分神经环路的沉默。因此,通过移植能够与宿主神经环路进行突触整合的兴奋性神经网络组织作为神经元中继器(neuronal relay),将来自大脑的下行兴奋性信息穿过损伤区传递到脊髓尾端,重新激活沉默的神经环路,有助于SCI后运动功能的恢复

    27。我们构建的类神经网络组织中,大部分神经元具有兴奋性神经递质的表达,同时具有一定的突触传递功能。我们认为将它移植到体内后,对SCI后沉默的神经环路会具有一定的重新激活作用。除了因部分神经环路沉默导致的肢体功能障碍外,部分颈椎损伤患者还可能会出现痉挛和不自主的肌肉收缩,对患者的心理、生理以及社会生活产生巨大影响28-29。Gong等30通过移植抑制性γ-氨基丁酸(gamma-aminobutyric acid, GABA)能神经元到SCI大鼠中,证实了移植的抑制性神经元能够与内源性运动神经元连接,逆转SCI后运动神经元兴奋性增加导致的痉挛。我们在体外构建的类神经网络组织尚未检测到自发的抑制性突触后电流的形成。为此我们可以考虑,是否可以通过与彭历芝等人构建的hiPSCs定向分化的GABA能神经元联合应用,构建既具有兴奋性突触传递功能,又具有抑制性突触传递功能的类神经网络组织,达到更好的SCI修复效果31。除此之外,我们也可以考虑在维持原有DON结构优势的前提下,通过缓释某些药物或生物活性分子,促进现构建的类神经网络组织抑制性突触传递功能的完善,提高其功能性的协调,实现神经网络组织细胞外基质结构和功能的双重仿生,为体内运用hiPSC源性神经元中继器治疗SCI打下基础。
    transl

    4 结 论

    我们利用天然来源的具有均匀孔道分布的DON生物支架材料和hiPSC-NPCs的有机结合,构建了一种以兴奋性神经元为主的具有突触传递功能的类神经网络组织。

    transl

    参考文献

    1

    de Paulis SArlotta GCalabrese Met al. Postoperative intensive care management of aortic repair[J]. J Pers Med2022128): 1351. [Baidu Scholar] 

    2

    Pieczonka KFehlings MG. Incorporating combinatorial approaches to encourage targeted neural stem/progenitor cell integration following transplantation in spinal cord injury [J]. Stem Cells Transl Med2023124): 207-214. [Baidu Scholar] 

    3

    Biswas AHutchins R. Embryonic stem cells[J]. Stem Cells Dev2007162): 213-222. [Baidu Scholar] 

    4

    Martin-Lopez MFernandez-Muñoz BCanovas S. Pluripotent stem cells for spinal cord injury repair [J]. Cells20211012): 3334. [Baidu Scholar] 

    5

    Takahashi KTanabe KOhnuki Met al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J]. Cell20071315): 861-872. [Baidu Scholar] 

    6

    Zheng YGallegos CMXue Het al. Transplantation of human induced pluripotent stem cell-derived neural progenitor cells promotes forelimb functional recovery after cervical spinal cord injury [J]. Cells20221117): 2765. [Baidu Scholar] 

    7

    Khazaei MAhuja CSNakashima Het al. Gdnf rescues the fate of neural progenitor grafts by attenuating notch signals in the injured spinal cord in rodents [J]. Sci Transl Med202012525): eaau3538. [Baidu Scholar] 

    8

    Sun JHLi GWu TTet al. Decellularization optimizes the inhibitory microenvironment of the optic nerve to support neurite growth[J]. Biomaterials2020258120289. [Baidu Scholar] 

    9

    Abad MMosteiro LPantoja Cet al. Reprogramming in vivo produces teratomas and ips cells with totipotency features[J]. Nature20135027471): 340-345. [Baidu Scholar] 

    10

    Suman SDomingues ARatajczak Jet al. Potential clinical applications of stem cells in regenerative medicine[J]. Adv Exp Med Biol201912011-22. [Baidu Scholar] 

    11

    Kriegstein AAlvarez-Buylla A. The glial nature of embryonic and adult neural stem cells[J]. Annu Rev Neurosci200932149-184. [Baidu Scholar] 

    12

    Cao QLZhang YPHoward RMet al. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage[J]. Exp Neurol20011671): 48-58. [Baidu Scholar] 

    13

    Barnabé-Heider FGöritz CSabelström Het al. Origin of new glial cells in intact and injured adult spinal cord[J]. Cell Stem Cell201074): 470-482. [Baidu Scholar] 

    14

    Steward OSharp KGMatsudaira Yee K. Long-distance migration and colonization of transplanted neural stem cells[J]. Cell20141563): 385-387. [Baidu Scholar] 

    15

    Castillo Bautista CMSterneckert J. Progress and challenges in directing the differentiation of human iPSCs into spinal motor neurons[J]. Front Cell Dev Biol2022101089970. [Baidu Scholar] 

    16

    Goparaju SKKohda KIbata Ket al. Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors[J]. Sci Rep2017742367. [Baidu Scholar] 

    17

    Andersen JRevah OMiura Yet al. Generation of functional human 3d cortico-motor assembloids[J]. Cell20201837): 1913-1929. e26. [Baidu Scholar] 

    18

    Du ZWChen HLiu Het al. Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells[J]. Nat Commun201566626. [Baidu Scholar] 

    19

    Cutarelli AMartínez-Rojas VATata Aet al. A monolayer system for the efficient generation of motor neuron progenitors and functional motor neurons from human pluripotent stem cells[J]. Cells2021105): 1127. [Baidu Scholar] 

    20

    Chambers SMFasano CAPapapetrou EPet al. Highly efficient neural conversion of human es and ips cells by dual inhibition of smad signaling[J]. Nat Biotechnol2009273): 275-280. [Baidu Scholar] 

    21

    Niethamer TKLarson ARO'neill AKet al. Ephrin-b1 mosaicism drives cell segregation in craniofrontonasal syndrome hiPSC-derived neuroepithelial cells[J]. Stem Cell Rep201783): 529-537. [Baidu Scholar] 

    22

    Baarine MKhan MSingh Aet al. Functional characterization of iPSC-derived brain cells as a model for x-linked adrenoleukodystrophy[J]. PLoS One20151011): e0143238. [Baidu Scholar] 

    23

    Xu XTay YSim Bet al. Reversal of phenotypic abnormalities by crispr/cas9-mediated gene correction in huntington disease patient-derived induced pluripotent stem cells[J]. Stem Cell Rep201783): 619-633. [Baidu Scholar] 

    24

    De Becker AⅣRiet. Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?[J]. World J Stem Cells201683): 73-87. [Baidu Scholar] 

    25

    Chen XZhao YLi Xet al. Functional multichannel poly(propylene fumarate)-collagen scaffold with collagen-binding neurotrophic factor 3 promotes neural regeneration after transected spinal cord injury[J]. Adv Healthc Mater2018714): e1800315. [Baidu Scholar] 

    26

    Li GZhang BSun JHet al. An nt-3-releasing bioscaffold supports the formation of trkc-modified neural stem cell-derived neural network tissue with efficacy in repairing spinal cord injury[J]. Bioact Mater2021611): 3766-3781. [Baidu Scholar] 

    27

    Lai BQChe MTFeng Bet al. Tissue-engineered neural network graft relays excitatory signal in the completely transected canine spinal cord[J]. Adv Sci2019622): 1901240. [Baidu Scholar] 

    28

    Sköld CLevi RSeiger A. Spasticity after traumatic spinal cord injury: Nature, severity, and location[J]. Arch Phys Med Rehabil19998012): 1548-1557. [Baidu Scholar] 

    29

    Dragojlovic NRomanoski NLVerduzco-Gutierrez Met al. Prevalence and treatment characteristics of spastic hypertonia on first-time admission to acute inpatient rehabilitation [J]. American journal of physical medicine & rehabilitation20221014): 348-352. [Baidu Scholar] 

    30

    Gong CZheng XGuo Fet al. Human spinal gaba neurons alleviate spasticity and improve locomotion in rats with spinal cord injury[J]. Cell Rep20213412): 108889. [Baidu Scholar] 

    31

    彭历芝位庆帅马瑗锾. 构建具有突触传递潜能的iPSC源性抑制性神经网络组织[J]. 中山大学学报(医学科学版)2023441): 18-25. [Baidu Scholar] 

    Peng LZWei QSMa YHet al. Construction of iPSC-derived inhibitory neural network tissue with synaptic transmission potentials[J]. J Sun Yat-sen Univ(Med Sci)2023441): 18-25. [Baidu Scholar] 

    51

    Views

    196

    Downloads

    0

    CSCD

    Alert me when the article has been cited
    Submit
    Tools
    Download
    Export Citation
    Share
    Add to favorites
    Add to my album

    Related Articles

    No data

    Related Author

    SU Qi-song
    LI Ge
    XU Jin-hai
    JIANG Bin
    WEI Qing-shuai
    ZENG Xiang
    ZENG Yuan-shan
    ZHU Ping

    Related Institution

    School of Biology and Biological Engineering, South China University of Technology
    Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University
    Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital//Guangdong Academy of Medical Sciences, Southern Medical University
    Medical Research Institute, Guangdong Provincial People's Hospital //Guangdong Academy of Medical Sciences, Southern Medical University
    Guangdong Provincial Key Laboratory of Pathogenesis, Targeted Prevention and Treatment of Heart Disease//Guangzhou Key Laboratory of Cardiac Pathogenesis and Prevention
    0