Joubert Syndrome-derived induced pluripotent stem cells show altered neuronal differentiation in vitro

Introduction This database includes the script used for the differential gene expression (DGE) analysis linked with the paper "Joubert Syndrome-derived induced pluripotent stem cells show altered neuronal differentiation in vitro." published on "Cell and tissue research". In this paper we analyzed the differentiation steps of JS patient-derived iPSCs towards cerebellar granule cells. We demonstrated that JS patient-derived iPSCs have an impaired expression of the genes of neuronal differentiation, analyzed through real time pcr and immunofluorescence in four different time points from D0, starting day, till D31 end of the differentiation protocol. This data were confirmed by transcriptomic analysis showing an impaired progression along the differentiation time course of the JS-patients derived IPSCs respect to control cells. We also analyzed cilia length and numbers in both patients and control cells, demonstrating notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. Taken together our results shows that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context. Methods Gene expression data from an RNA-seq experiment (Illumina platform) were initially processed with command line interface to align reads on the reference genomes and to count reads falling on coding regions (gene expression counts), with the pipeline outlined in the Materials and Methods section of the paper. The metadata contained sample names, mutated genes defining experimental conditions and time points for each sample. This script, written with the R programming language, performed a comprehensive analysis of the processed next-generation sequencing data (read counts). To ensure the reproducibility of our analysis, we initialized the R environment by setting a specific seed for random number generation. Several R libraries were used in our analysis, including edgeR, RColorBrewer, DESeq2, ggplot2, openxlsx, readxl, and ggrepel. These libraries provided essential functions for statistical analysis, data visualization, and manipulation. The workflow contained in this script consisted of several steps: we generated count-per-million (CPM) values and filtered out genes with low expression. Reads Per Kilobase Million (RPKM) values were then calculated to account for gene length and normalize the data, to ensure compatibility of expression levels across experiments and genes. Differential gene expression was performed using the DESeq2 package, through the creation of a DESeqDataSet object, the estimation of size factors and dispersions, and the fitting of models. Differentially expressed genes between different conditions and time points were visualized with a volcano and heatmap plot. Results Results of the transcriptomic analysis show differences in the expression of genes relating to the central nervous system development already at D8, in particular cerebellum markers (LMX1A, OLIG3) being expressed only in controls cells. At D24, differences in gene expression became more evident, underlining for control samples the expression of genes specific to the terminal phases of cerebellar differentiation (ASTN2, CNTN1, WNT3, PLXNA2, LMX1B) and the expression of genes encoding proteins of neuronal functionality (GRIA4, SYT8, GRIK1, P2RX2, PCP4, GRIK2). In contrast, expression of these genes was significantly lower in JS lines, which showed early differentiation markers still predominantly expressed (ITGB1, CXCL12, HIF1A, EN2, GBX2, BMPR1B, CUL2, RPL37, RPL39, RPL35, PSMD2, and PSMD3). Taken together, these data support the observations, obtained with the other assays in our paper, that JS-iPSC lines show an impaired progression along the differentiation time course and a reduced ability to reach the maturation state seen in controls.引言 本数据库包含与发表于《细胞与组织研究（Cell and Tissue Research）》的论文《Joubert综合征来源诱导多能干细胞体外神经元分化异常》相关的差异基因表达（differential gene expression, DGE）分析脚本。 本研究分析了Joubert综合征（Joubert Syndrome, JS）患者诱导多能干细胞（induced pluripotent stem cells, iPSCs）向小脑颗粒细胞的分化过程。通过实时聚合酶链反应（real-time PCR）与免疫荧光实验，在从起始日D0至分化方案结束日D31的4个不同时间点开展检测，证实JS患者iPSCs的神经元分化相关基因表达存在缺陷。转录组学分析进一步验证了该结果：相较于对照细胞，JS患者来源iPSCs的分化进程存在显著阻滞。此外，本研究还分析了患者与对照细胞的纤毛长度与数量，发现所有分化中的JS患者iPSCs均存在明显的纤毛缺陷。综上，患者来源iPSCs是在神经元背景下分析JS基因突变相关细胞表型的便捷且具有相关性的体外模型。 方法 本研究的RNA测序（RNA-seq）实验数据基于Illumina平台生成，首先通过命令行界面进行处理，将测序reads比对至参考基因组，并统计编码区域的reads计数（基因表达计数），所用流程详见论文的“材料与方法”部分。元数据包含样本名称、定义实验条件的突变基因以及每个样本的时间点信息。本脚本采用R语言编写，用于对处理后的下一代测序数据（read计数）进行全面分析。为确保分析可复现，我们通过设置随机数生成的特定种子来初始化R运行环境。 本分析用到了多个R扩展库，包括edgeR、RColorBrewer、DESeq2、ggplot2、openxlsx、readxl及ggrepel，这些库提供了统计分析、数据可视化与数据操作所需的核心功能。 本脚本包含的分析流程分为多个步骤：首先生成每百万计数（counts per million, CPM）值并过滤低表达基因；随后计算每千碱基每百万reads（reads per kilobase million, RPKM）值，以校正基因长度并对数据进行标准化，确保不同实验与基因间的表达水平具有可比性；使用DESeq2包进行差异基因表达分析，具体步骤包括创建DESeqDataSet对象、估算大小因子与离散度、拟合模型；通过火山图与热图可视化不同条件与时间点间的差异表达基因。 结果 转录组学分析结果显示，早在D8时间点即可观察到与中枢神经系统发育相关基因的表达差异，其中小脑特异性标志物（LMX1A、OLIG3）仅在对照细胞中表达。至D24时间点，基因表达差异更为显著：对照样本可检测到小脑分化终末期特异性基因（ASTN2、CNTN1、WNT3、PLXNA2、LMX1B）以及神经元功能相关蛋白编码基因（GRIA4、SYT8、GRIK1、P2RX2、PCP4、GRIK2）的表达；而JS细胞系中这些基因的表达水平显著降低，且仍主要表达早期分化标志物（ITGB1、CXCL12、HIF1A、EN2、GBX2、BMPR1B、CUL2、RPL37、RPL39、RPL35、PSMD2及PSMD3）。综上，这些数据佐证了本研究中其他实验的观察结果：JS-iPSCs系的分化进程存在阻滞，且相较于对照细胞，其达到成熟状态的能力显著降低。