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Title: Characterisation of encapsulated embryonic stem cells using silac-based proteomics
Author: Alsobaie, Sarah
ISNI:       0000 0004 7233 0930
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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The embryonic stem cells have two hallmarks characters, the ability to reproduce self-renewal and generate other cell lineages. Despite the excessive advance in stem cell research, their clinical applications delay owing lack of an optimal culture condition in vitro. Combining biometrical scaffold with stem cells provides a promising way to cellular delivery and tissue transplant. In vitro 3D culture offers both a model to understand self-renewal and stem cell behaviour in vivo as a route to industrial production. We have used a variety of analytical tools including proteomics and quantitative RT-PCR to compare 3D (both static & dynamic) with 2D cultures. We further show that 3D dynamic culture increases expression of the master gene regulators (Oct4, Nanog and Sox2). Consistent with this Rex1, an inner-cell mass (ICM) associated marker, is over-expressed in 3D dynamic culture whereas Fgf5, an epiblast transition marker is down-regulated. Using SILAC-based proteomics to compare 2D vs. 3D dynamic culture, we show that encapsulated stem cell are characterized by glycolytic pathways down-regulation and increased mitochondrial respiratory proteins. Additionally, ECM proteins expression (laminin, fibronectin, heparan sulfate (HS) proteoglycans, agrin, and nidogen-2) were up-regulated. Cells shortly after encapsulation in 3D constructs (both static &dynamic) showed high oxygen uptake rates compared to 2D culture. After 9 days of encapsulation glucose uptake increased in both static & dynamic 3D cultures and was combined with a pronounced increase in cell density and up-regulation of hypoxia induce-factors (HIf-1α and Hif-2α). This shift in metabolism toward anaerobic glycolysis is associated with high expression of hexokinas2 (Hk2) and increased lactate production. Despite extensive cell proliferation at day 18, where cell numbers reached up to 80k/ bead, cells in dynamic culture switched back from anaerobic to aerobic metabolism. This alteration in energy consumption was not associated with nutrient depletion since both glucose and glutamate were not depleted in the culture medium. By monitoring the oxygen consumption, hypoxia-induced factors expression as well as Oct4 levels at different time points, we demonstrate that the stem cells self-renewal status in 3D condition is regulated despite the metabolism transitions. We postulate that the 3D culture environment provides the niche required sensing and responding to external and internal stimuli different from recognised in vitro embryonic stem cell behaviour.
Supervisor: Cass, Tony Sponsor: Saudi Arabia. Safārah (UK)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral