Investigation of human embryonic stem cell differentiation towards endothelial lineages

Cardiovascular disease represents a significant socio-economic burden and minimally invasive therapies that address the needs of patients suffering with peripheral arterial disease and critical limb ischemia are needed. Cell therapies have been proposed as an alternative to pharmacological and surgical treatments, yet, have demonstrated somewhat limited efficacy. However, the unlimited capacity for self-renewal, and the ability to differentiate into cell types from all three germ layers, including endothelial cell (EC) forming mesoderm, make human embryonic stem cells (hESC) and human induced pluripotency stem cells a promising source for well-defined differentiated cell populations with high angiogenic capacity. Numerous endothelial differentiation protocols have been published with high differentiation efficiencies achieved recently. However, most of these approaches are not optimised for clinical purposes due to the use of poorly defined, non cGMP compatible reagents, or require additional processing steps, such as cell sorting, complicating the clinical approval of these therapies. Therefore, here it was aimed to develop and optimise a clinically compatible hESC-EC differentiation protocol that avoids using poorly defined reagents, and yields high percentages of cells expressing EC markers without the use of cell sorting. A novel, serum free hESC-EC differentiation protocol was developed in the lab. The use of Pluronic F-127 well coating was demonstrated as a low cost alternative to low adherence wells. Furthermore, inhibition of TGFB signalling during hESC-EC differentiation to increase the differentiation efficiency was evaluated and did not reveal any additional benefits and thus was not included in the optimised protocol. The optimised protocol consists of embryoid body based mesodermal induction phase, followed by plating and monolayer culture for vascular specification. By day 7, approximately 30% of cells express endothelial markers CD31 and CD144. In addition, transient induction of mesodermal gene, followed by induction of endothelial progenitor and endothelial gene expression was demonstrated, following the expected gene expression patterns. It was proposed that high throughput screening using hESC lines carrying fluorescent reporter constructs could be used to optimise the differentiation protocol for increased efficiency, thus, avoiding the need of cell sorting prior to therapeutic use. Here, reporter constructs where ETV2, ROBO4 and CDH5 promoter sequence fragments were cloned upstream from florescent reporter sequences were generated and preliminary validation was attempted in NCI60, HUVEC and HSVEC cell lines, and during the hESC-EC differentiation. Reporter gene expression was not observed in any of the validation experiments, suggesting that these constructs were not functional. Similarly, previously published CDH5 and commercially sourced ETV2 reporter constructs were validated during the hESC-EC differentiation. Preliminary testing of these reporter constructs showed non-specific reporter gene expression, therefore, the work with reporter constructs work was not pursued further. Next, rational targeting of novel signalling pathways that may contribute to the hESC-EC differentiation was employed as an alternative approach for the optimisation of hESC-EC differentiation. Firstly, it was hypothesised that intracellular cAMP levels could be targeted pharmacologically to increase the differentiation efficiency, and to induce expression of arterial and arterial phenotype associated genes, which could reduce the need for cell sorting and deliver arterial cell populations with a superior angiogenic profile. Forskolin treatments induced increased intracellular cAMP levels during the hESC-EC differentiation, yet, this did not result in increased arterial or arterial phenotype associated gene expression. However, an increase in the percentage of cells expressing EC markers was observed in Forskolin treated differentiations, mainly mediated via an increase in the CD144low CD31+ cell population. Additionally, it was hypothesised that angiotensin II (Ang II) signalling may play a role in hESC-EC differentiation and may be exploited to increase the endothelial differentiation efficiency. Indeed, differential renin angiotensin system receptor expression was demonstrated during hESC-EC differentiation, supporting a role for Ang II signalling in endothelial development. However, no significant differences in the differentiation efficiency and total cell numbers were observed when Ang II and AT1R antagonist Losartan treatments, in combination or alone, were applied during the hESC-EC differentiation. In contrast, a significant reduction in total cell numbers and a trend of reduced differentiation efficiency was observed when AT2R antagonist PD-123319 was used in combination with Ang II. These observations highlight the negative effects of AT1R signalling during hESC-EC differentiation and show that signalling via AT2R counterbalances these effects. In summary, a novel endothelial differentiation protocol was developed and rational selection of signalling pathways for the optimisation of the hESC-EC differentiation was employed. Here, the role of cAMP and Ang II signalling during hESC-EC was demonstrated, highlighting the contribution of various signalling systems to endothelial differentiation. Both of these signalling systems can be easily manipulated in a clinically compliant manner, and therefore represent an attractive target during clinically compatible hESC-EC differentiation. Further research is needed to investigate the underlying mechanisms of the observed effects and to evaluate other, novel signalling pathways that may be targeted to enhance endothelial differentiation. The work described has highlighted the difficulties of establishing efficient, clinically compatible hESC-EC differentiation methods, which are needed to provide highly defined cell populations for future cell therapies and tissue engineering.