Azmi, Nur Izyan
(2018)
Effects of ALK-5 inhibitor and transforming growth factor-Beta1 in the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into epithelial-like cells.
Masters thesis, Universiti Sains Malaysia.
Abstract
Stem cells from human exfoliated deciduous teeth (SHED) are capable to
divide, differentiate and mature to the specific types of cells as well as to replenish
themselves to regenerate other living cells. Previous study has showed that SHEDs
could differentiate into epithelial-like cells. Yet, the effects of Transforming Growth
Factor-Beta1 (TGF-β1) or activin like kinase 5 (ALK-5) inhibitor on SHEDs remain
unexplored. Thus, the present study was aimed to investigate the effects of TGF-β1
and ALK-5 inhibitor on SHEDs cultured in Keratinocyte Growth Medium (KGM) on
its potential to differentiate into epithelial-like cells employing MTT [(3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] and alamar blue assays, cell
morphology analysis, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
technique and flow cytometry. A serial dilution of TGF-β1 (0.3125, 0.625, 1.25, 2.5,
5.0, 10.0, 20.0, 40.0, 80.0, and 160.0 ng/ml) and ALK-5 inhibitor (0.156, 0.3125,
0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, and 160.0 μM) concentration was carried
out to determine the cell cytotoxicity for each treatment using MTT assay for 72 hours.
Afterwards, the three selected concentrations for TGF-β1 (0.3125, 0.625, and 1.25
ng/ml) and ALK-5 inhibitor (0.156, 0.3125, and 0.625 μM) were analysed using
alamar blue assay on day 1, 3, 5, 7, and 10 and was done in alpha Minimum Essential
Medium (α-MEM) to determine the population doubling time (PDT). The study wasfurther investigated with observation of the cell morphological changes on SHED
cultured in KGM only, and with selected concentration of TGF-β1 or ALK-5 inhibitor
on day 1, 3, 7, 14, and 21. RNA isolation of SHED culture in three different conditions
were harvested at day 1, 3, 7, 14, and 21. Then, the gene expression analysis of stem
cell, epithelial cell, and specific genes involved in TGF-β signalling were identified
during the differentiation process using Two-step RT-PCR. Apart from that, protein
expression analysis of epithelial markers was also determined using flow cytometer on
day 1 and 21. Based on MTT assay, 0.3125, 0.625, and 1.25 ng/ml TGF-β1 and 0.156,
0.3125, and 0.625 μM ALK-5 inhibitor showed less cytotoxicity effects (more than
50%) and were selected for proliferation assay. The shortest PDT was represented by
1.25 ng/ml TGF-β1 (75 hours) and 0.625 μM ALK-5 inhibitor (68 hours) and these
concentrations including cells in KGM only, showed there were cell morphological
changes compared to control. The gene expression profile showed an absence of
epithelial markers E-cadherin, ΔNp63, and Keratin5 for gene, and E-cadherin and pancytokeratin
for protein expression indicated that the culture conditions unable to
induce the differentiation process into epithelial-like cells. However, the presence of
stem cell markers (NANOG, nestin, Rex1, and vimentin) and specific molecules
involved in TGF-β signalling (TGFβR1, TGFβ1, Smad3, and Smad4) indicated that
the cell culture in three different condition induced epithelial to mesenchymal
transition (EMT) since the presence of TGFβ1 and Smad3 in TGF-β signalling that
have been associated with EMT. Thus, KGM was unable to fully differentiate SHED
into epithelial-like cells and hence, SHED were incapable to undergo mesenchymal to
epithelial transition (MET).
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