<b>Endothelial cells differentiated from hiPSCs form aligned network structures in engineered neural tissue</b>

The data that support the findings of <i>Endothelial cells differentiated from hiPSCs form aligned network structures in engineered neural tissue</i><br>hiPSCs were differentiated to endothelial cells following the protocol developed by Hildebrandt et al., 2021 (https://pubmed.ncbi.nlm.nih.gov/33410098/) with the modification of culturing the hiPSC-derived cells on Geltrex-coated plates. Immunofluorescence data for differentiation monitoring and the resulting cells characterisation were collected using Opera Phenix confocal spinning disc microscope and analysed with Columbus Image Analysis Software v2.9.1. RT-PCR data from differentiation monitoring and resulting cell characterisation were collected using GoScript Reverse Transcriptase Kit (Promega) and Power SYBR Green PCR Master Mix using SimpliAmp Thermal Cycler and QuantStudio3 instrument, analysed by QuantStudio Design and Analysis Software v1.5.1 with hypoxanthine guanine phosphoribosyl transferase (HPRT1), ribosomal protein lateral stalk subunit P0 (RPLP0) and ribosomal protein S18 (RPS18) reference genes. Viability data of hiPSC-derived endothelial cells within engineered neural tissue (EngNT-EC) covers local viability, using ReadyProbes Cell Viability Imaging Kit Blue/Red, and whole construct viability, using Lactase Dehydrogenase (LDH) Assay Kit (Cytotoxicity) collected using a Zeiss LSM710 confocal microscope and a SpectraMAX M5 Multi-Mode Microplate Reader, respectively. Alignment of cells within EngNT-EC was captured by Volocity^TM v6.5.1 analysis of 20 µm z-stacks taken using Zeiss LSM710 confocal microscope at predetermined positions along the construct stained with Rhodamine 110 Phalloidin and Hoechst 33342. Viability and alignment assays were performed 24 h after EngNT-EC construct formation. EngNT-EC were cultured for 2 or 4 days to allow for endothelial network formation. These samples were stained with Rhodamine 110 Phalloidin and Hoechst 33342, imaged on Zeiss LSM710 confocal microscope with 40 µm z-stacks taken at predetermined positions and analysed for angiogenic features using ImageJ plugin Angiogenesis Analyzer. EngNT-EC constructs were further analysed for lumen number from 10 µm sections stained with Rhodamine 110 Phalloidin and Hoechst 33342, imaged on Zeiss AxioLab A1 fluorescent microscope. Neurite length and alignment data were collected using ImageJ and Volocity^TM v6.5.1, respectively, analysing the co-culture of neurons on the EngNT-EC, cultured for 1 or 4 days, and acellular constructs imaged using Zeiss LSM710 confocal microscope of samples stained with anti-betaIII tubulin and Hoechst 33342.Data collected between October 2022 and May 2024Supplement Video 1 - Representative phase contrast video of hiPSC-ECs at Passage 4 during the matrix gel tubulogenesis assay. The imaging frame was consistent over time, with 1 h between each frame. Scale bar 400 µm.本数据集支持《hiPSCs来源的内皮细胞在工程化神经组织中形成对齐网络结构》的研究发现。研究按照Hildebrandt等于2021年开发的方案（https://pubmed.ncbi.nlm.nih.gov/33410098/），将人诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）诱导分化为内皮细胞，并对方案进行修改：将hiPSC来源的细胞接种于包被Geltrex的培养板中进行培养。使用Opera Phenix共聚焦转盘显微镜收集用于分化监测及细胞特性鉴定的免疫荧光数据，并采用Columbus图像分析软件v2.9.1完成数据分析。采用GoScript逆转录酶试剂盒（Promega公司）、Power SYBR Green PCR预混液，结合SimpliAmp热循环仪与QuantStudio3实时荧光定量PCR仪，收集用于分化监测及细胞特性鉴定的逆转录聚合酶链式反应（reverse transcription-polymerase chain reaction, RT-PCR）数据；以次黄嘌呤鸟嘌呤磷酸核糖转移酶1（HPRT1）、核糖体蛋白侧柄亚基P0（RPLP0）及核糖体蛋白S18（RPS18）作为内参基因，采用QuantStudio设计与分析软件v1.5.1进行数据分析。工程化神经组织-内皮细胞（EngNT-EC）中hiPSC来源内皮细胞的活力数据包含局部活力与整体构建体活力两部分：前者采用ReadyProbes细胞活力成像试剂盒Blue/Red检测，后者采用乳糖脱氢酶（Lactase Dehydrogenase, LDH）细胞毒性检测试剂盒检测，分别使用Zeiss LSM710共聚焦显微镜与SpectraMAX M5多功能酶标仪完成数据采集。采用Zeiss LSM710共聚焦显微镜，在经罗丹明110鬼笔环肽与Hoechst 33342染色的构建体预设位置采集20 μm z轴堆叠图像，通过Volocity™ v6.5.1软件分析以获取EngNT-EC内细胞的对齐情况。在EngNT-EC构建体形成24小时后开展活力与对齐检测实验。将EngNT-EC培养2天或4天以促进内皮网络形成，随后采用罗丹明110鬼笔环肽与Hoechst 33342对样本进行染色，使用Zeiss LSM710共聚焦显微镜采集预设位置的40 μm z轴堆叠图像，并通过ImageJ插件Angiogenesis Analyzer分析血管生成相关特征。对经罗丹明110鬼笔环肽与Hoechst 33342染色的10 μm切片样本，采用Zeiss AxioLab A1荧光显微镜成像，进一步分析EngNT-EC构建体的管腔数量。收集神经突长度与对齐数据：分别采用ImageJ与Volocity™ v6.5.1软件，对培养1天或4天的EngNT-EC共培养神经元样本以及无细胞构建体进行分析；样本经抗βIII微管蛋白与Hoechst 33342染色后，使用Zeiss LSM710共聚焦显微镜完成成像。本数据集的采集时间为2022年10月至2024年5月。补充视频1：第4代hiPSC来源内皮细胞进行基质胶管形成实验的代表性相差显微镜视频，成像帧率为每小时1帧，全程保持成像视野一致，比例尺为400 μm。