Towards hES cell-based therapy of Epidermolysis Bullosa

Regenerative medicine offers great hope for therapeutic innovation in many diseases, including those with defective skin, such as the group of inherited blistering skin diseases known as epidermolysis bullosa. The work in this thesis addresses some of the important pre-clinical approaches to generating keratinocytes (the main cell of the outer skin layer, epidermis) from pluripotent stem cells (human embryonic stem cells, hESCs). Undifferentiated hESCs were exposed to a complex microenvironment in organotypic cultures (to mimic conditions for epidermal stem cell development and maintenance); this yielded epidermis-like structures which stained positively for basal keratin 14 and several extracellular matrix molecules, although a stratified epidermis was not produced. To refine the protocol, hESCs were differentiated to an epidermal cell fate in monolayer cultures in vitro prior to subjecting them to organotypic culture. The most efficient technique involved culture of hESCs on native de-cellularized extracellular matrix produced by normal human dermal fibroblasts as a substrate for differentiation. Large epitheliaHike sheets positive for keratin 14 and p63 expression were observed. On subculture, these cells retained their morphological and immunocytochemical characteristics for up to 5 passages and could be successfully cryo-preserved and recovered. Overall, this study explored the potential of pluripotent stem cells as a source of epidermal progenitors, with results that provide proof-of-concept for future cell therapy innovations in defective skin diseases such as epidermolysis bullosa.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2012

Author dc:creator

• Petrova, Anastasia

Advisors dc:contributor.advisor

• McGrath, John Alexander
• Ilic, Dusko

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/79901c47-3f09-41be-b737-fab3127af800

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/79901c47-3f09-41be-b737-fab3127af800