Novel genetic features of human and mouse Purkinje cell differentiation defined by comparative transcriptomics
Novel genetic features of human and mouse Purkinje cell differentiation defined by comparative transcriptomics
复制标题
通过比较转录组学定义人和小鼠浦肯野细胞分化的新遗传特征
DOI：
10.1101/2020.01.07.897371
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
M. Hatten
中科院分区：
文献类型：
--
作者：
David E. Buchholz;T. Carroll;A. Kocabas;Xiaodong Zhu;Hourinaz Behesti;P. Faust;Lauren Stalbow;Yin Fang;M. Hatten
为了比较分化的人多能干细胞衍生的浦肯野细胞（hPSC-PC）和发育中的小鼠浦肯野细胞（PC）的整体基因表达特征，我们衍生了hPSC-PC并比较了来自人和小鼠PC的基因表达数据集。我们优化了一种分化方案，产生了与小鼠P21 PC基因表达最相似的hPSC-PC。小鼠PC基因在出生后发育过程中表达的元基因预测分析确定了经典的PC标记基因以及新的线粒体和自噬基因途径。这些关键基因表达模式在分化hPSC-PC中是保守的。我们进一步确定了小鼠和hPSC-PC之间关键基因组的时间和表达差异，并证实了一种新的人PC标记物CD 40 LG在人小脑组织中的表达。分化中的人类多能干细胞（hPSC）和发育中的小鼠神经元之间的比较转录组学提供了一种强有力的方法来比较人类和小鼠神经元中的遗传和表观遗传途径。为了分析人浦肯野细胞（PC）的分化，我们优化了一种方案，以产生人多能干细胞衍生的浦肯野细胞（hPSC-PC），当与小鼠小脑胶质细胞和颗粒细胞培养时形成突触，并发射大的钙电流，用遗传编码的钙指示剂jRGECO 1a测量。为了直接比较hPSC-PC与发育中的小鼠PC的整体基因表达，我们使用翻译核糖体亲和纯化（TRAP）。作为第一步，我们使用Tg（Pcp 2-L10 a-Egfp）TRAP小鼠在发育中的出生后小鼠PC中分析活跃转录的基因，并使用元基因投射来确定PC基因表达随时间变化的最显著模式。然后，我们创建了转基因Pcp 2-L10 a-Egfp TRAP hPSC系以分析分化中的hPSC-PC中的基因表达，发现分化的hPSC-PC的关键基因表达途径与晚期幼年小鼠PC（P21）的关键基因表达途径最接近匹配。比较生物信息学在小鼠和人类PC的分化过程中识别了经典的PC基因特征以及新型线粒体和自噬基因途径。此外，我们确定了在hPSC-PC中表达的基因，但不是小鼠PC，并证实了一种新的人PC基因，CD 40 LG，在hPSC-PC和天然人小脑组织中表达的蛋白质表达。因此，本研究提供了hPSC-PC和小鼠PC基因表达的直接比较，以及用于产生具有人类特异性基因表达的分化的hPSC-PC以用于建模发育和退行性小脑疾病的稳健方法。
Significance To compare global gene expression features of differentiating human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) and developing mouse Purkinje cells (PCs), we derived hPSC-PCs and compared gene expression datasets from human and mouse PCs. We optimized a differentiation protocol that generated hPSC-PCs most similar in gene expression to mouse P21 PCs. Metagene projection analysis of mouse PC gene expression over postnatal development identified both classical PC marker genes as well as novel mitochondrial and autophagy gene pathways. These key gene expression patterns were conserved in differentiating hPSC-PCs. We further identified differences in timing and expression of key gene sets between mouse and hPSC-PCs and confirmed expression of a novel human PC marker, CD40LG, in human cerebellar tissue. Comparative transcriptomics between differentiating human pluripotent stem cells (hPSCs) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate human pluripotent stem cell-derived Purkinje cells (hPSC-PCs) that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2-L10a-Egfp TRAP hPSC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides a direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders.
登录
查看更多内容
影响因子：
56.9
作者：
OBERDICK, J;SMEYNE, RJ;MORGAN, JI
通讯作者：
MORGAN, JI
影响因子：
23.9
作者：
Yu P;Pan G;Yu J;Thomson JA
通讯作者：
Thomson JA
影响因子：
8.3
作者：
Korshunov, Vyacheslav A.;Daul, Matthew;Berk, Bradford C.
通讯作者：
Berk, Bradford C.
结节性硬化症复合物神经元模型中的线粒体动力学和线粒体受损。
影响因子：
8.8
作者：
Ebrahimi-Fakhari D;Saffari A;Wahlster L;Di Nardo A;Turner D;Lewis TL Jr;Conrad C;Rothberg JM;Lipton JO;Kölker S;Hoffmann GF;Han MJ;Polleux F;Sahin M
通讯作者：
Sahin M
影响因子：
3.5
作者：
Hahn, Y;Bera, TK;Lee, B
通讯作者：
Lee, B