Regulatory Role of miRNAs in Human Induced Pluripotent Stem Cells Differentiation

• Other Authors: Li, Lu (author.)
• Format: Others
• Language: English; Chinese
• Published: 2016
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292182

人类诱导性多功能干细胞（hiPSCs）具有分化成所有体细胞的潜能。研究发现小分子RNA（miRNA）与hiPSCs的分化具有极大相关性。然而，miRNAs是否是胚层形成和细胞系分化的关键尚不明确。因此，为了阐明miRNAs在hiPSCs分化中的作用，我将hiPSCs诱导成三个胚层及其代表细胞系，分别是内胚层以及肝细胞，中胚层以及肾干细胞，外胚层以及神经干细胞。通过采集分化过程中不同时间点样本的RNA，我们对三个胚层及其代表细胞系进行了RNA表达谱分析和比较。我们利用生物信息学方法成功预测了细胞系特异性miRNAs的下游靶基因和发育相关基因的上游调控miRNAs。其中，肝细胞特异性miR-192-3p/5p以及miR-193-3p,神经干细胞特异性miR-362-5p，肾发育相关miR-105-5p对各自细胞系分化的影响均被实验证实。本研究表明miR-105-5p可以通过调节多囊蛋白-1（PC1）和多囊蛋白-2（PC2）诱导正常的胚胎肾发育。然而miR-105-5p的异常表达会干扰PC1和PC2之间的表达平衡，并影响与常染色体显性遗传性多囊肾病（ADPKD）相关的信号通路。本研究不仅阐明了hiPSCs分化过程中的miRNA表达谱，为研究人类胚胎发育提供资料，也进一步证实诱导不同细胞系之间转换的可行性。 === Human induced pluripotent stem cells (hiPSCs) have the potential to differentiate into all somatic cell types. Previous studies suggested that the correlation of lineage specification of hiPSCs and miRNAs is high. However, whether miRNAs are key regulators in germ layer formation and lineage specification remains obscure. To investigate the roles of miRNAs in differentiation, we have established hiPSCs differentiation platform for three germ layers, namely, hepatocytes for endoderm, nephron progenitors for mesoderm, and neural progenitors for ectoderm, respectively. Profiling of miRNA expression in samples collected from different time-points during differentiation with microarrays allowed vertical and horizontal comparisons among the three representative lineages of the three germ layers. With either identifying the downstream genes of lineage-critical miRNAs or the upstream miRNAs of developmental regulator genes bioinformatically, we successfully predicted the functions of certain miRNAs in lineage differentiation. The predicted effects of hepatocyte-specific miR-192-3p/5p and miR-193, neuron progenitor-specific miR-362-5p, and renal differentiation-associated miR-105-5p on lineage specification, respectively, were further determined experimentally. The study showed that the normal expression of miR-105-5p contributed to embryonic kidney development regulated by Polycystin-1 (PC1) and Polycystin-2 (PC2). Dysregulation of miR-105-5p potentially perturbed the balance between PC1 and PC2, and consequently affected the pathway involved in the development of autosomal polycystic kidney disease (ADPKD). My studies not only provided a comprehensive view of the miRNA expression dynamics during hiPSCs differentiation, but also possibilities of interpreting the physiologic process in human embryonic development and manipulating cell type specification. === Submitted by Li Lu === For partial fulfilment of the Degree of Doctor of Philosophy in Biomedical Sciences at the Chinese University of Hong Kong in (August 2016) === Li, Lu. === Thesis Ph.D. Chinese University of Hong Kong 2016. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only.