Undifferentiated human embryonic stem cells (hESCs) grow on the extracellular matrix (ECM) substrate fibronectin (FN) in defined feeder-free conditions. The ECM is part of the hESCs pluripotent niche and supports their maintenance, but the contribution to survival remains to be elucidated. Understanding the mechanism of survival is particularly crucial in hESCs, since it affects their expansion in cell culture and ultimately translation of research to the clinic. HESCs bind to FN mainly via alpha5β1- integrin, known to be upstream of important survival cascades in other cell types. However, it is not understood if and how FN/integrin binding supports those molecular pathways in the context of pluripotent hESCs. The aim of this work was to elucidate the survival cascade downstream of the FN/integrin interaction in hESCs. Initially, when hESCs were cultured on a non-integrin activating substrate they initiated an apoptotic response that also occurred when β1-integrin was selectively blocked with antibody, leading the cells to detach from FN. Integrin activation is generally transduced within cells via a complex adhesome of scaffold and kinase proteins, among which the focal adhesion kinase (FAK) plays a key role. Indeed, blocking β1-integrin resulted in dephosphorylation of endogenous FAK in hESCs. When FAK kinase activity was directly inhibited (with small molecule inhibitors), hESCs responded by detaching from FN and activating caspase-3, leading to an increase in apoptosis. Furthermore, flow cytometry analysis showed that the population of hESCs that underwent apoptosis still retained the pluripotency-associated marker NANOG. FAK is a convergent point between growth factor signaling and the PI3K/Akt pathway, with a well-reported role in the maintenance of hESCs. Consistently, FN activated both AKT and its target the ubiquitin ligase MDM2 at the protein levels, while pAkt was reduced after β1-integrin blocking and FAK inhibition. Cell imaging showed that MDM2, which regulates p53 degradation in the nucleus, displayed reduced nuclear localisation after FAK inhibition, opening the possibility for a change in the p53 balance in hESCs. In fact, p53 protein increases after FAK inhibition corresponding also to caspase activation. Further investigation explored if FAK-dependent pathways are also implicated in the maintenance of hESC pluripotency. Inhibition of FAK led the cells that survived apoptosis to lose stem cell morphology, decrease pluripotency-associated markers and change nuclear shape. Moreover, a small pool of FAK was found in the nucleus of hESCs cultured on FN, but decreased after FAK inhibition. FAK was also co- immunoprecipitated with NANOG protein in standard hESC culture while NANOG decreased after sustained FAK inhibition. This data suggests that nuclear roles of FAK could support, together with the cytoplasmic activation of the PI3K cascade, both survival and pluripotency pathways requiring further investigation. In conclusion, the original contribution of this work is to identify in FAK the downstream survival effector of the FN/β1-integrin interaction in hESCs. HESCs survival is maintained by the binding of β1-integrin to FN and activation of FAK kinase and downstream PI3K/Akt, leading to the suppression of p53 and caspase activation. In parallel, promotion of these pathways by FAK is suggested also to support the key pluripotency circuitry, feeding into NANOG. Overall, FAK is proposed here as an important regulator of hESC survival and fate.

There is a huge unmet clinical need to treat damaged articular cartilage such as that caused by osteoarthritis (OA) with an estimated 8.75 million people in the UK having sought treatment for OA (ARUK 2013). Embryonic stem cells (ESCs) offer a promising alternative therapeutic approach, potentially providing an unlimited source of chondrocytes capable of regenerating the damaged cartilage however this is limited by the efficiency of the chondrogenic differentiation protocol. An improved understanding of the posttranscriptional regulation of chondrogenesis by microRNAs (miRNAs) may enable us to improve hESC chondrogenesis. Also the recent discovery that miRNAs are selectively packaged into exosomes which can then be transferred to and be functionally active within neighbouring cells suggests they may have a role in cell-cell communication. This project investigated the regulation of miRNA expression in relation to the transcriptome during hESCs-directed chondrogenesis and the possible role for exosomes during differentiation and in stem cell maintenance of hESCs. Small RNA-seq and whole transcriptome sequencing was performed on distinct stages of hESC-directed chondrogenesis using the Directed Differentiation Protocol (DDP) developed in our lab. Also small RNA-seq was performed on exosomes isolated from hESCs and chondroprogenitors along with the donor cells that the exosomes originated from. This revealed significant changes in the expression of several miRNAs during hESC-directed chondrogenesis including: upregulation of miRNAs transcribed from the four Hox complexes, known cartilage associated miRNAs and the downregulation of pluripotency associated miRNAs. Overall miRome and transcriptome analysis revealed the two hESC lines exhibited slightly different miRome and transcriptome profiles during chondrogenesis, with Man7 displaying larger changes in miRNA and mRNA expression as it progressed through the DDP suggesting it may be more predisposed to undergo chondrogenesis. Integration of miRomes and transcriptomes generated during hESC-directed chondrogenesis identified four key functionally related clusters of co-expressed miRNAs and protein coding genes: pluripotency associated cluster, primitive streak cluster, limb development cluster and an extracellular matrix cluster. Further investigation of these gene/miRNA clusters allowed the identification of several potential novel regulators of hESC-directed chondrogenesis. In accordance with the reported literature the exosomal miRNAs from hESCs and hESC-chondroprogenitors were enriched with a guanine rich motif. Notably, several of these were enriched with targets associated with embryonic skeletal system development suggesting they may play a role in regulating differentiation. Preliminary functional experiments examining pluripotency-associated exosomes suggests they may have a role in regulating hESC stem cell maintenance. However the molecular mechanism by which this is achieved has not been investigated. This research identified main miRome and transcriptome changes during hESC-directed chondrogenesis leading to the identification of several potential novel regulators of chondrogenesis and pluripotency which can be further investigated. This project has also highlighted the potential of exosomal miRNAs to regulate hESC stem cell maintenance and differentiation.