Nee, Kang In
(2018)
Therapeutic potentials of hypoxic- and baicalein-enriched fraction-preconditioned human neural stem cells for in vitro ischemic stroke model.
Masters thesis, Universiti Sains Malaysia.
Abstract
Ischemic stroke is the third leading cause of death in Malaysia, closely after
heart disease and cancer. Standard treatments for stroke are not totally efficient to
repair and regenerate the damaged brain tissue and there are possibilities for the
recurrence. Replacement by endogenous adult neural stem cells (NSCs) during
ischemic stroke was insufficient to repair injury site due to low neuronal turnover
that could integrate into functional neuron network. Therefore, it is imperative to
develop alternative therapeutic strategies to improve stroke recovery. Recently,
human NSC grafting has emerged as encouraging approach for treating stroke.
Nonetheless, the therapeutic potential of NSC-based treatment is limited, mainly due
to a large number of implanted cells died after grafting into the injury site. To
circumvent this problem, this study aimed to enhance therapeutic potentials of
human NSCs prior to transplantation through hypoxic and baicalein-enriched fraction
(F5) preconditioning. Hypoxic preconditioning under 2% O2 for 24 h enhanced NSC
self-renewal, survival and multipotency. 60S ribosomal protein large P1 (RPLP1)
and ribosomal protein L13A (RPL13A) were the most reliable reference genes for
qPCR normalization of normoxic- and hypoxic-preconditioned NSCs. Hypoxic
preconditioning induced innate neuroprotective signaling through transcriptional
activation of hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth
factor A (VEGFA), angiopoietin 1 (ANGPT1), neurogenic locus notch homolog
protein 1 (Notch 1), nuclear factor erythroid 2-related factor 2 (Nrf2) and sodium
dismutase 1 (SOD1). Based on the HIF-1α stabilization potential of baicalein at
ambient conditions, F5 was postulated to trigger effects mimic hypoxic
preconditioning under normoxia. Interestingly, preconditioning with 1.56 μg/mL of
F5 for 24 h increased NSC proliferation, viability and lineage specific differentiation.
Hypoxanthine phosphoribosyl transferase 1 (HPRT1) and RPL13A were the most
stably expressed reference genes for qPCR normalization of control (0.1% DMSO)
and F5-preconditioned NSCs. Moreover, F5 preconditioning stimulated hypoxiamimetic
signaling intrinsically via HIF-1α, VEGFA, ANGPT1, Notch 1, Nrf2 and
SOD1 upregulation. Both hypoxic- and F5-preconditioned NSCs were applied to in
vitro ischemic stroke (IVIS) model on wound-healing based culture slide for 72 h of
live imaging. F5-preconditioned NSCs accelerated migration and homing towards
IVIS model over an experimental period of 72 h compared to hypoxicpreconditioned
NSCs. The neuroprotective factors induced by hypoxic
preconditioning are postulated to degrade rapidly when exposed to oxygen.
Contrarily, F5-preconditioned NSCs attained intrinsic neuroprotective mechanisms
without compromising their stability under normoxia. In conclusion, both the
hypoxic and F5 preconditioning had successfully enhanced therapeutic potentials of
NSCs for ischemic stroke. F5-preconditioned NSCs with enhanced therapeutic
efficacy was more likely to be applicable in clinical setting and thus could be a
promising therapeutic tool for ischemic stroke in the future.
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