Divisions of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology Medicine and Health, Michael Smith Building, Oxford Road, University of Manchester, Manchester Academic Health Science Centre, Manchester, M13 9PT, UK.
Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, United Kingdom.
Sci Rep. 2020 Mar 16;10(1):4744. doi: 10.1038/s41598-020-61734-4.
Human embryonic stem cells (ESCs) offer a promising therapeutic approach for osteoarthritis (OA). The unlimited source of cells capable of differentiating to chondrocytes has potential for repairing damaged cartilage or to generate disease models via gene editing. However their use is limited by the efficiency of chondrogenic differentiation. An improved understanding of the transcriptional and post-transcriptional regulation of chondrogenesis will enable us to improve hESC chondrogenic differentiation protocols. Small RNA-seq and whole transcriptome sequencing was performed on distinct stages of hESC-directed chondrogenesis. This revealed significant changes in the expression of several microRNAs including upregulation of known cartilage associated microRNAs and those transcribed from the Hox complexes, and the downregulation of pluripotency associated microRNAs. Integration of miRomes and transcriptomes generated during hESC-directed chondrogenesis identified key functionally related clusters of co-expressed microRNAs and protein coding genes, associated with pluripotency, primitive streak, limb development and extracellular matrix. Analysis identified regulators of hESC-directed chondrogenesis such as miR-29c-3p with 10 of its established targets identified as co-regulated 'ECM organisation' genes and miR-22-3p which is highly co-expressed with ECM genes and may regulate these genes indirectly by targeting the chondrogenic regulators SP1 and HDAC4. We identified several upregulated transcription factors including HOXA9/A10/D13 involved in limb patterning and RELA, JUN and NFAT5, which have targets enriched with ECM associated genes. We have developed an unbiased approach for integrating transcriptome and miRome using protein-protein interactions, transcription factor regulation and miRNA target interactions and identified key regulatory networks prominent in hESC chondrogenesis.
人胚胎干细胞 (ESC) 为骨关节炎 (OA) 提供了有前途的治疗方法。能够分化为软骨细胞的无限细胞来源具有修复受损软骨的潜力，或通过基因编辑生成疾病模型。然而，它们的使用受到软骨分化效率的限制。对软骨生成的转录和转录后调控的深入了解将使我们能够改进 hESC 软骨分化方案。在 hESC 定向软骨生成的不同阶段进行了小 RNA-seq 和全转录组测序。这揭示了几个 microRNA 的表达发生了显著变化，包括已知的与软骨相关的 microRNA 和来自 Hox 复合物的转录物的上调，以及多能性相关的 microRNA 的下调。miRomes 和 hESC 定向软骨生成过程中产生的转录组的整合，确定了与多能性、原始条纹、肢体发育和细胞外基质相关的关键功能相关的共表达 microRNA 和蛋白编码基因簇。分析确定了 hESC 定向软骨生成的调节剂，例如 miR-29c-3p，其 10 个已建立的靶标被鉴定为共同调节的“ECM 组织”基因，而 miR-22-3p 与 ECM 基因高度共表达，并且可能通过靶向软骨生成调节剂 SP1 和 HDAC4 间接调节这些基因。我们鉴定了几个上调的转录因子，包括参与肢体模式形成的 HOXA9/A10/D13 和 RELA、JUN 和 NFAT5，其靶标富含与 ECM 相关的基因。我们开发了一种使用蛋白质-蛋白质相互作用、转录因子调节和 miRNA 靶标相互作用整合转录组和 miRome 的无偏方法，并确定了在 hESC 软骨生成中突出的关键调节网络。
