Analysis of the transcriptome: investigation of human embryonic stem cells during directed differentiation to cardiovascular lineages

Howard, Lynsey (2013) Analysis of the transcriptome: investigation of human embryonic stem cells during directed differentiation to cardiovascular lineages. PhD thesis, University of Glasgow.

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To date, the need for effective treatments to tackle ischaemic diseases such as CHD and PAD remains unmet. As such, there has been a great deal of interest in developing cell therapies in order to address these important pathologies. The main goals of a cell therapy strategy for ischaemic disease remain prompt restoration of blood supply to the affected areas in order to salvage tissue and/or regeneration of tissues previously lost to ischaemia.
Derived from the Inner Cell Mass (ICM) of an embryo at the blastocyst stage, hESC have been proposed as a potential source of functional, transplantable cells for a variety of cell therapy applications. Although successful differentiation of multiple cell types from hESC has been demonstrated, the molecular processes governing the cell commitment process remain poorly understood, and differentiation efficiency often fails to provide the number of cells required to see clinical benefit in the patient. As such, a more thorough transcriptional characterisation of cardiovascular cell types derived from hESC was the goal of this study.
MicroRNAs (miRNA; miR) are small (~22nt), non-coding RNAs which negatively regulate mRNA. MiR-1 and miR-133 were previously shown to play a role in regulating cardiac differentiation with miR-1 potentiating cardiac differentiation and miR-133 having an inhibitory effect. Optimisation of lentiviral vectors showed generation of single pre-miR overexpression lentiviruses for miR-1 and miR-133 in a construct using the SFFV promoter to be possible. Furthermore, it was realised SA461 hESC were unsuitable for cardiac differentiation, however, using a modified version of the LaFlamme protocol in a monolayer system resulted in beating cells with a cardiomyocyte phenotype in H1 hESC. Despite successful overexpression of miR-1 and miR-133, there was very little effect on cardiac differentiation over no virus control.
Previously published methods for the generation of vascular endothelial cells (EC) have reported varying efficiency and target cell population purity (~3 – 30%). This laboratory recently reported the successful generation of functional EC-like cells from hESC in a feeder-free manner. HESC-EC were analysed by LC Sciences miRNA microarray at early time points day 0, day 2, day 4 and day 10 after initiation of differentiation with time-matched pluripotent controls. An induction of miR-99b, -181a and -181b over time was observed, and validated in H1 hESC. In addition, miR-99b, -181a and -181b were also found to be expressed in other mesodermal cell types including adult human saphenous vein endothelial cells (HSVEC). No statistically significant expression of these miRNAs could be found in representative cell types of ectoderm and endoderm germ layers, therefore it was hypothesised that these miRNAs were largely mesoderm specific. Despite initial data showing a significant difference in expression between HSVEC from control patients and patients undergoing coronary artery bypass grafting (CABG), classical pathophysiological stimuli to cause endothelial cell stress did not change the expression of miR-99b, -181a and -181b in vitro.
In order to understand more about gene expression in early lineage commitment, hESC-EC were analysed by Illumina microarray at early timepoints day 0, day 2, day 4 and day 10 after initiation of differentiation with time-matched pluripotent controls. In parallel, primary human saphenous vein endothelial cells (HSVEC) were analysed. Illumina technology permitted whole-genome profiling in a high throughput chip format. Due to overall expression levels being lower intensity than expected, no cut-off of fold-change was applied to the dataset. Analysis of the dataset showed a large number of significantly differentially expressed probes at each time point: Day 2 of endothelial differentiation compared to Day 0 pluripotent control showed 1040 significant differentially expressed probe changes, Day 4 of endothelial differentiation compared to Day 0 pluripotent control showed 2400 significant differentially expressed probe changes and Day 10 of endothelial differentiation compared to Day 0 pluripotent control showed 2157 significant differentially expressed probe changes (all False Discovery Rate <0.05). Although significant downregulation of pluripotency markers were observed, few endothelial associated genes were present at hESC-EC day 10. Analysis of HSVEC compared to hESC-EC Day 10 reveals 6133 significantly differentially expressed probes (FDR <0.05). This suggests that although day 10 hESC-ECs have previously been shown satisfy criteria for endothelial cells in vitro and in vivo, their transcriptional profiling demonstrates that they remain different in comparison to adult ECs.
A transient induction of several transcription factors was observed at hESC-EC day 2, accounting for some 10% of gene changes at this time point. We hypothesised that these transcription factors may play key roles in the early mesoderm/EC commitment process. Of these, FOXA2, a transcription factor not previously associated with mesoderm or EC commitment, was upregulated, and this was further validated by both qRT-PCR and ICC in SA461, H1 and RC10 hESC lines. In addition to gene expression data, an in silico prediction of gene epigenetic status was made using a previously published chromatin immunoprecipitation sequencing (ChIP-SEQ) dataset performed in H9 hESC. Approximately 3000 genes are bivalently marked, meaning they have both H3K4me3 active chromatin and H3K27me3 repressive chromatin at their transcriptional start sites (TSS). This conveys a poised state, with the potential for the gene to be rapidly activated and/or repressed. Of these bivalent genes it was noted that FOXA2 was marked as being potentially bivalent. Upon further investigation using ChIP it was revealed that FOXA2 carried both H3K4me3 and H3K27me3 chromatin modifications in the TSS region, both in H9 and SA461 pluripotent hESC. It was hypothesised that epigenetic modification was responsible for the dynamic expression of FOXA2, although this hypothesis remains to be investigated further. Lastly, several of the miRNA targets for miR-99b, miR-181a and miR-181b were downregulated by hESC-EC day 10 compared to hESC-EC day 0, although whether these targets play a role in refining differentiation to EC warrants further investigation.
In summary, a range of molecular biology techniques were employed to investigate the master control of hESC differentiation. These studies have contributed to existing knowledge on mesodermal and cardiovascular lineage specification. They provide evidence to support the continued in depth investigation of these processes in order to develop a clinically relevant cell therapy for ischaemic diseases.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: human embryonic stem cell, microRNA, epigenetics, cardiovascular differentiation, cardiovascular cell therapy, cloning, molecular biology, microarray
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
Supervisor's Name: Baker, Prof. Andrew H. and Milligan, Prof. Graeme
Date of Award: 2013
Depositing User: Miss Lynsey Howard
Unique ID: glathesis:2013-4068
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 09 Apr 2013 10:38
Last Modified: 09 Apr 2013 10:40

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