Halim, Ahmad Sukari
(2017)
Involvement of MAPK/ERK and Akt signaling
pathways in the proliferation of keratinocytes
cocultured with adipose-derived stem cells (ACSS)-Porous
Chitosan Scaffold (PSC).
Project Report.
Pusat Pengajian Sains Perubatan, Universiti Sains Malaysia.
(Submitted)
Abstract
Secretory factors of adipose-derived stem cells have paracrine effects that may have potential in
accelerating proliferation of keratinocytes by coculturing both of the cells. This study was conducted to
evaluate the indirect effects of ASCs-PCS on the growth of keratinocytes by paracrine activities. Human
adipose-derived stem cells and human epidermal keratinocytes were isolated and verified with their specific
markers. Distribution and proliferation of ASCs on porous chitosan scaffold were assessed by scanning
electron microscope, live/dead assay and Alamar blue assay. Coculture groups were divided into two groups;
culture ASCs and culture of ASCs-PCS. Proliferation of keratinocytes in the coculture wells were analysed by
Alamar blue assay at 24 hours and 72 hours of coculture while the concentration human epidermal growth
factor in culture supernatant were quantified at 72 hours of coculture. Adipose-derived stem cells culture
have been established and displayed high expression of CD29, CD73, CD90, CD105 and low expression of
CD34. Keratinocytes culture expressed involucrin and cytokeratin 6 as demonstrated by
immunocytochemistry. Percentage of proliferation of ASCs within PCS was more than 80% accompanied
with spindle-shaped features and good viability. After 72 hours of coculture, the percentage of nHEK in the
coculture group was increased due to growth medium. ELISA assay revealed no secretion of EGF from ASCs.
This study demonstrated via dual -chamber coculture model, human -derived ASCs and PCS showed no
growth stimulatory effect towards the growth of keratinocytes via the tested growth factor, EGF. The
proliferation of nHEK in the coculture wells possibly due to other growth factor or other mechanism.
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