Aziz, Nur Syazwani
(2020)
Gene expression analysis of stem cell from exfoliated deciduous teeth in migration and local angiogenesis of tissue repair.
Masters thesis, Universiti Sains Malaysia.
Abstract
Stem cells play essential role during the angiogenesis process of wound
healing. Stem cells from exfoliated deciduous teeth (SHED) possess mesenchymal
stem cell (MSC) characteristics, showing the ability to differentiate into various
lineages, indicating their massive potential to benefit cell-based therapy and tissue
repair. Nevertheless, the behaviour of SHED engaging in angiogenesis in terms of its
migratory capacity and gene expression regulation remains questionable. The present
study aims to analyse the gene expression pattern of SHED undergoing migration and
angiogenic differentiation. Together, the effect of different initial seeding density and
induction medium on the migration of SHED were assessed. SHED were expanded in
vitro and induced for angiogenesis by supplementation of 10 ng/ml of vascular
endothelial growth factor (VEGF) in 2 ml of endothelial growth medium 2 (EGM2).
Scratch test assay was conducted in the transwell chamber to determine the optimum
initial seeding density for SHED by comparing 5,000 cells/cm2 and 10,000 cells/cm2
prior to angiogenic induction. A further comparison was made to assess the effect of
angiogenic induction media on SHED migration rate. Three groups of SHED were
assessed the gene expression analysis: the angiogenic induction group (+A-M),
angiogenic induction with migration group (+A+M), and the control group (-A-M).
RNA was extracted at different time points (day 1, 3, 7, 10 and 14). One-step RT-PCR
was then performed to assess the gene expression level of a series of angiogenic,
migration and MSC gene markers. Overall, the data demonstrated a high capability of
SHED committing to angiogenic lineage. Higher seeding density (10,000 cells/cm2)
increased SHED migration, whereas angiogenic induction suppressed SHED
migratory capacity. SHED positively expressed Ang-1, Il-8, and VE-Cadherin, the
angiogenic markers, as well as the migratory gene markers (CCR1, CXCR4 and
CCL28). SHED also maintains the stemness level by positive expression of CD73,
CD90 and CD105 during the induction protocol. The gene expression pattern of both
angiogenic and migratory gene markers observed within this study indicate the
complexity of understanding these two events during tissue repair. Indeed, a better
understanding of these SHED-chemokine interactions is needed to enable the effective
use of SHED in cell-based therapies.
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