Derivation of vascular endothelium from human embryonic stem cells: the roles of microRNAs in endothelial differentiation and commitment

With their unique ability to differentiate into any cell of the three germ layers (endoderm, ectoderm and mesoderm) and their capacity for unlimited self-renewal, pluripotent stem cells (PSC), including human embryonic (hESC) and induced pluripotent stem cells (hiPSCs), are thought to hold great potential as an unlimited source of functional, transplantable cells for a diverse range of scientific and clinical applications. Specifically, in the context of cardiovascular and ischemic diseases, it is believed that hESC-derived endothelial cells (hESC-ECs) may be used to stimulate angio- and vasculogenesis in ischemic tissues, therefore restoring blood supply to the affected area. Despite the publication of numerous methods for the derivation of hESC-ECs, differentiation efficiency is often low, or protocols involve the use of cumbersome isolation techniques. Currently, mechanisms governing the commitment of pluripotent cells to this specific lineage remain poorly understood, although numerous studies have highlighted a role for microRNAs (miRNA; miR). miRNAs are short, non-coding RNAs, ~22 nucleotides in length, which act post-transcriptionally to control the expression of their specific mRNA targets. It was, therefore, hypothesised that specific miRNAs play crucial roles during hESC-EC differentiation and commitment. The aim of this study was to identify novel miRNAs with roles in early mesodermal and endothelial commitment, and their potential mechanisms of action, in two newly developed hESC-EC differentiation protocols. Identified miRNAs could then be modulated to drive hESCs toward an endothelial lineage, therefore, allowing for increased differentiation efficiencies. In order to study the role of miRNAs during commitment of pluripotent cells to the endothelial lineage, two distinct hESC-EC differentiation systems were developed. The first was a direct system, whereby pluripotent cells were taken at d0 and differentiated directly to hESC-ECs. By d7 of direct differentiation, ~40% of cells were CD31+CD144+ hESC-ECs, and this was coupled with a significant downregulation of pluripotent-associated genes and surface markers at this time point. Furthermore, it was demonstrated that CD31+CD144+ hESC-ECs could be isolated and cultured for a further 7 days. Resultant d14 hESC-ECs were ~100% CD31+CD144+ and were functional, demonstrated by their ability to form tubules on a Matrigel matrix. The second method for hESC-EC generation was indirect, and was developed using a pre-existing hematopoietic differentiation system. In vivo, development of the hematopoietic and vascular systems are closely linked, with a number of publications demonstrating the existence of a bipotent progenitor population with the ability to generate both endothelial and hematopoietic lineages, known as hemogenic endothelium (HE). In vitro studies using hPSCs have also identified and characterised HE populations. Using the cell surface marker profiles defined during these studies, a CD31+CD144+CD235a-CD43-CD73- HE population was demonstrated to exist on d7 of hematopoietic differentiation. Optimisation was then performed, to drive HE cells toward an EC phenotype, and generate a second, indirect hESC-EC differentiation protocol. By d10 of indirect hESC-EC differentiation, cells formed a confluent monolayer, expressed endothelial markers CD144, CD31 and CD73, and were negative for hematopoietic and pluripotent-associated markers. Profiling miRNAs involved in early stages of mesodermal and endothelial specification required identification of a progenitor population, indicating the beginning of lineage commitment. Using time course analysis, a CD326lowCD56+ mesoderm progenitor (MP) population was identified on d3 during direct hESC-EC differentiation, before the appearance of endothelial-associated markers, and coinciding with the peak in the expression of mesoderm-associated genes. Further characterisation was performed using fluorescence activated cell sorting (FACS), to isolate a pure CD326lowCD56+ MP samples, and TLDA card analysis, to examine the expression of 48 different genes. The existence of this population was also demonstrated in the indirect hESC-EC system, where CD326lowCD56+ MP cells were also present on d3 of differentiation, before the expression of hematopoietic- or endothelial-associated markers were detected. Global analysis of changes in miRNA expression during direct hESC-EC was then performed using a miRNA microarray screen, using the previously characterised CD326lowCD56+ MP, as well as d0 pluripotent cells, the d3 negative cell fraction (NCF) and d7 hESC-ECs. Overall, it was observed that pluripotency-associated miRNAs, such as the miR-302 family, were significantly downregulated during hESC-EC differentiation, with miRNAs associated with endothelial function and angiogenesis significantly upregulated as cells moved toward and endothelial phenotype. These findings were also validated in samples taken during indirect hESC-EC differentiation. Direct comparisons between d3 CD326lowCD56+ MP and NCF samples were performed to identify novel miRNAs with potential roles in early mesoderm and endothelial commitment, and led to the identification of 56 differentially expressed miRNAs. Most interestingly, -3p and -5p strands of a number of miRNA stem loops, including miR-145 and miR-483, were found to be regulated in a similar manner during hESC-EC differentiation. Upon additional analysis, it was discovered that both strands of the miR-143 stem loop, transcribed in a cluster with miR-145, were also regulated in the same fashion. Therefore, the miR-143/145 cluster and miR-483 were chosen as miRNA candidates for further investigation. Modulation of the miR-143/145 cluster and miR-483 was performed during direct hESC-EC differentiation. It was hypothesised that overexpression of these miRNAs would lead to increased differentiation efficiency, assessed via the percentage of CD31+CD144+ hESC-ECs present on d7. Therefore, lentiviral vectors, for overexpression of these specific miRNAs, were produced and cells infected on d0 of differentiation. Although no significant differences were recorded when miRNAs were overexpressed in the system, there are a large number of studies which could still be performed in order to fully interrogate the roles of miR-143/145 and miR-483 in the differentiation and commitment of pluripotent cells to both mesodermal and endothelial lineages. In summary, vast changes in global miRNA expression profiles during hESC-EC differentiation indicate an important role for these regulatory RNAs. Investigation was performed using two newly developed methods for hESC-EC generation and miRNAs were screened using microarray technology. Although the initial miRNA modulation studies were unsuccessful in increasing the efficiency of hESC-EC differentiation, further work must be completed in order to fully interrogate the roles of these specific miRNAs