Culture conditions govern mouse embryonic stem cell behaviour: dependence on heparan sulfate and optimisation of synthetic polymer substrates

Williams, Chloe Culture conditions govern mouse embryonic stem cell behaviour: dependence on heparan sulfate and optimisation of synthetic polymer substrates. Doctor of Philosophy thesis, University of Liverpool.

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Abstract

Human embryonic stem cell (hESC)-based therapies will only become viable once we eliminate the use of animal-derived material during ESC scale-up. Some groups have demonstrated the expansion of hESCs in xeno-free systems but the effect on downstream self-renewal and differentiation is poorly understood. Heparan sulfate (HS) is a master regulator of cellular behavior but the role of HS during ESC expansion is unclear, as is the exogenous source of HS in cultures. It has been shown that mESCs synthesise low levels of low-sulfated HS, but it is unclear if culture condition has any impact. In the studies here, three discrete culture conditions were employed for E14 mESC expansion along with immunostaining and RT-qPCR to study marker expression for differentiation to the three lineages and corresponding BM synthesis. SAX-HPLC was used to characterise soluble HS from cells/medium/serum. A varierty of polymers were tested as synthetic alternatives for ESC expansion. It was found that HS-deficient embryoid bodies (EBs) (derived from EXT1-/- mESCs in normal culture conditions) remained in a pluripotent state and lacked a typical differentiation pattern. Furthermore, HS-deficient mESCs could not be maintained in the absence of serum, highlighting a link between serum and HS. EBs derived from E14 mESCs cultured in the absence of serum displayed unusual differentiation patterns, which were rescued by exogenous porcine mucosal heparin (PMH). Feeder cells displayed cell-surface HS but feeder-cell conditioned medium (CM) was predominantly an unsulfated structure. An array of low and highly sulfated HS structures were identified in serum-alone. 10-fold more HS was purified from serum-free feeder-free (-F –FBS) CM compared to the other mESC CM (with/without feeders but in the presence of serum; +/-F +FBS). Furthermore, unlike +/-F +FBS conditions, highly sulfated HS disaccharide UA2S–GlcNS6S was the major constituent in –F-FBS and Sulf2 levels were significantly reduced. Poly-Ɛ-lysine macroporous substrates supported mESC and kidney-derived stem cells (KSCs-GFP) adherence and proliferation, further enhanced by adsorbing RGD or per-sulfated HS structures to the surface of the poly-Ɛ-lysine. The key conclusions from these studies were that serum is a source of HS, without which, mESCs behave uncharacteristically; that synthetic HS-mimetic structures could represent an alternative to serum; and poly-Ɛ-lysine shows great promise to replace current animal-derived coating materials for ESC expansion.

Item Type:	Thesis (Doctor of Philosophy)
Additional Information:	Date: 2013-05 (completed)
Subjects:	?? QH301 ??
Divisions:	Faculty of Health and Life Sciences
Depositing User:	Symplectic Admin
Date Deposited:	06 Aug 2013 09:34
Last Modified:	16 Dec 2022 04:39
DOI:	10.17638/00012147
Supervisors:	Turnbull, Jerry ORCID: 0000-0002-1791-754X Murray, Patricia Edgar, David
URI:	https://livrepository.liverpool.ac.uk/id/eprint/12147