Statistical analysis

Unless stated, values are presented as mean ± the standard error of the mean (SEM). qRT-PCR results are expressed at relative quantification (RQ) calculated

using 2-ΔΔcT (see section 2.5.3), and error is expressed as RQ max and RQ min, determined by the calculation of RQ using ΔΔCT ± SEM.

Statistical analysis was performed using GraphPad Prism 5 Software (California, USA). Student’s t-test was used to perform comparisons when 2 groups were present, or one-way analysis of variance (ANOVA), followed by a Tukey’s post hoc t-test, in experimental conditions with more than 2 groups. In conditions where multiple readings were taken from the same sample set, paired or repeated measures tests were used. Statistical significance was accepted as p<0.05.

Chapter 3

Development and characterisation of a

direct hESC-EC differentiation

3.1 Introduction

It has been suggested that hPSC-derived ECs, may hold the key to the development of a long term cell-based therapy for the treatment of a wide range of ischemic diseases, such as PAD, CLI, CHD and stroke. It is believed that these cells are able to stimulate angio- and vasculogenesis in vivo, and this has been demonstrated in a number of different publications, with cells generated using a variety of hPSC-EC differentiation protocols (Kane et al., 2010, Orlova et al., 2014a, Patsch et al., 2015).

Thus far, there have been a number of protocols published describing the generation of ECs from hPSCs; either hESCs (hESC-ECs) or hiPSCs (hiPSC-ECs), and these have been reviewed extensively (Descamps et al., 2012). Many early publications used undefined systems, containing serum-supplemented or conditioned medium (Levenberg et al., 2002) or co-culture with stromal cell lines, any of which make the protocol unsuitable for use in a clinical setting. Additionally, undefined culture can lead to introduction of unknown factors, thus making it difficult to study exact mechanisms and pathways involved in specific differentiation and culture systems. Therefore, studies have focused on the development of protocols using more defined conditions. Differentiation efficiency in many of these protocols has, however, been poor, with generation of <10% hPSC-ECs. Recently, a number of publications have demonstrated more efficient generation of these cells (Orlova et al., 2014a, Orlova et al., 2014c, Patsch et al., 2015). Development of efficient differentiation protocols is essential for the translation of any cell-based therapy, due to the large numbers of cells which may be needed for each treatment.

Gaining further understanding of mechanisms of both differentiation and commitment will allow for the development of more efficient protocols. Signalling pathways, molecules or non-coding RNAs involved in differentiation, may be manipulated in order to drive differentiation, and produce higher number of hPSC-ECs for potential transplantation. In order to study mechanisms of mesodermal and early endothelial commitment, and to determine the role of specific factors within this differentiation system, there has also been a focus on the identification of progenitor cell populations, existing within these in vitro hPSC-EC differentiation systems. Early mesodermal progenitor (MP) populations

are, thus far, poorly defined and various studies have suggested an array of cell surface marker profiles, describing these distinct populations (Evseenko et al., 2010, Drukker et al., 2012, Kurian et al., 2015).

One study, published in 2010, suggested that these MP cells can be described as cells negative for the cell surface marker epithelial cell adhesion molecule (EpCAM; CD326) and positive for neural cell adhesion molecule (NCAM; CD56), also known as CD326-CD56+ (Evseenko et al., 2010). During early embryogenesis, epithelial-to-mesenchymal transition (EMT) plays an important role in gastrulation and lineage specification and, specifically, has been shown play a key role in the generation of mesoderm in several experimental organisms (Nakaya et al., 2008). Specifically, FGF, Wnt and TGFβ signalling, have been shown to contribute to mesoderm formation via EMT in vivo (Ciruna et al., 2001, Kemler et al., 2004, Ben-Haim et al., 2006). It was therefore, hypothesised that the earliest stage of mesoderm formation during hESC differentiation in vitro may also be associated with this process. CD56 had previously been shown to be upregulated during EMT in human epithelial breast carcinoma cells, and this upregulation was associated with a loss of E-cadherin and epithelial cell adhesion molecule (Lehembre et al., 2008). Similarly to epithelial cells, hESCs express adhesion molecules such as E-Cadherin and CD326, that may possibly play roles in the maintenance of hESC pluripotency (Lu et al., 2010). In hESC culture, CD326 is localised to Oct4 positive cells, and becomes downregulated during differentiation, with knockdown causing an increase in mesoderm- associated markers in pluripotent cells (Lu et al., 2010, Ng et al., 2010b). Evseenko et al. demonstrated that CD326-CD56+ cells, occurring at day 3.5 of a general hESC-mesoderm differentiation protocol, represent a multipotent mesoderm-committed progenitor population, capable of differentiating towards hematopoietic, endothelial, mesenchymal, smooth muscle and cardiomyocyte lineages, whilst lacking the ability to differentiate to endo- or ectodermal lineages (Evseenko et al., 2010). These cells, herein known as mesoderm progenitors (MP), were described as possible precursors to previously described, more lineage-restricted, mesodermal progenitor cell types.

Experiments within this chapter were designed to develop and characterise an efficient hESC-EC differentiation protocol, generating immature vascular ECs directly from hESCs. Using the marker profile published by Evseenko et al., we

also wanted to probe early MP populations, existing within the direct hESC-EC differentiation system (Evseenko et al., 2010). Identified populations could then be used for investigation and interrogation of factors, mechanisms and pathways involved in hESC-EC commitment, thus allowing for manipulation of the system and the potential to increase differentiation efficiency by driving hESCs toward an EC phenotype.