图1 hiPSCs的形态学观察和各标志物的表达
Published:20 July 2023,
Received:08 February 2023
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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.
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.
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.
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.
hiPSC-NPCs;
DON;
synapse;
excitatory neurons;
neural network-like tissue
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[
DON支架的制备如前期实验方案所述[
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)。
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。
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。
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。
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。
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。
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。
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。
复苏的hiPSCs的形态学观察和各标志物的表达情况如
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.
hiPSC-NPCs的形态学观察和各标志物的表达如
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.
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 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.
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 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.
hiPSC源性类神经网络组织递质类型的表达如
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.
hiPSC源性类神经网络组织的功能性分析结果如
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.
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[
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[
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[
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[
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。
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