sRNA-seq Analysis of Human Embryonic Stem Cells and Definitive Endoderm Reveals Differentially Expressed MicroRNAs and Novel IsomiRs with Distinct Targets

Abstract

MicroRNAs (miRNAs) are noncoding, regulatory RNAs expressed dynamically during differentiation of human embryonic stem cells (hESCs) into defined lineages. Mapping developmental expression of miRNAs during transition from pluripotency to definitive endoderm (DE) should help to elucidate the mechanisms underlying lineage specification and ultimately enhance differentiation protocols. In this report, next generation sequencing was used to build upon our previous analysis of miRNA expression in human hESCs and DE. From millions of sequencing reads, 747 and 734 annotated miRNAs were identified in pluripotent and DE cells, respectively, including 77 differentially expressed miRNAs. Among these, four of the top five upregulated miRNAs were previously undetected in DE. Furthermore, the stem-loop for miR-302a, an important miRNA for both hESCs self-renewal and endoderm specification, produced several highly expressed miRNA species (isomiRs). Overall, isomiRs represented >10% of sequencing reads in >40% of all detected stem-loop arms, suggesting that the impact of these abundant miRNA species may have been overlooked in previous studies. Because of their relative abundance, the role of differential isomiR targeting was studied using the miR-302 cluster as a model system. A miRNA mimetic for miR-302a-5p, but not miR-302a-5p(13), decreased expression of orthodenticle homeobox 2 (OTX2). Conversely, isomiR 302a-5p(13) selectively decreased expression of tuberous sclerosis protein 1, but not OTX2, indicating nonoverlapping specificity of miRNA processing variants. Taken together, our characterization of miRNA expression, which includes novel miRNAs and isomiRs, helps establish a foundation for understanding the role of miRNAs in DE formation and selective targeting by isomiRs.

MicroRNA dynamics during human embryonic stem cell differentiation to pancreatic endoderm

“…For example, during days 1-2, miR-375, miR-1263, miR-132, miR-708, miR-489, and miR-9 were all highly expressed, confirming results from our previous work on miRNAs associated with the transition from pluripotency to definitive endoderm [20]. However, our data sheds new light on other miRNAs.…”

“…an extended protocol such as was used in our previous study [20]. Here we observed both a blunted expression of miR-375 and a sharper decline in expression (Supplementary Table 2) when compared with our previous work [20,48].…”

“…Of the 747 and 734 annotated miRNAs that were identified in pluripotent and DE cells, respectively, 77 were differentially expressed [20]. These experiments demonstrated that DE can be distinguished from pluripotency through a unique miRNA profile.…”

“…Because a single miRNA can have multiple RNA targets within a given cell, these small non-coding RNAs can simultaneously disrupt multiple post-transcriptional networks [8,9,10]. Recent studies have highlighted the importance of miRNAs in hESC differentiation [11,12,13,14,15,16,17,18,19,20] and pancreatic development [21] through knockout studies of genes essential for miRNA biogenesis. These knockout experiments resulted in decreased miRNA expression, impaired ability of the hESCs to differentiate, and decreased formation of endocrine tissue [21].…”

“…Previously, we used microarray analysis and Next Generation Sequencing (NGS) to explore differences in miRNA expression profiles between pluripotent hESCs and definitive endoderm (DE) [18,20]. Of the 747 and 734 annotated miRNAs that were identified in pluripotent and DE cells, respectively, 77 were differentially expressed [20].…”