Ye, Yee May
(2015)
Expansion, characterisation and
differentiation of human neural stem
cells.
Project Report.
Universiti Sains Malaysia.
(Submitted)
Abstract
Stroke irreversibly damages affected brain part, leading to permanent neuronal
impairment. Neural stem cell (NSC)-based therapy is a potential stroke treatment due to
its ability to self-renew and to differentiate into various viable neuronal cells for damaged brain tissue regeneration. Here, human NSCs were expanded from GIBCO® human NSC line and stroke patients’ brain subventricular zone (SVZ) tissue biopsy with ethical approval. NSCs obtained from GIBCO® cell line were used for pilot testing of NSC characterisation and differentiation ability in vitro. GIBCO® NSCs were cultured in complete StemPro®NSC SFM while human brain SVZ tissues were cultured in an adherent layer with serum-free medium containing bFGF and EGF.
Characterisation of NSC was performed using qRT-PCR and Western blot; while NSC
differentiation was performed using Neuron Differentiation Medium and Astrocytes
Differentiation Medium. Both assays used human normal brain cell line (SVG-pl2) as
negative control. qRT-PCR data illustrated that NSCs showed over 4000-fold difference
in Nestin and 6000-fold difference in CD133 expression compared to SVG-pl2,
indicated that Nestin and CD 133 were specific to NSC. These results were consistent
with the Western blot data in which Nestin protein with approximately 200 kDa was
identified in NSCs but was absent in SVG-pl2. Upon four days in culture with
differentiation media, NSCs did not show morphological changes towards neurons and
astrocytes respectively, compared to non-treated cells. This is due to the limited time
available to perform this assay in present study. Longer culture duration which is more
than one week will be performed in future to obtain more accurate result. Meanwhile,
two clonal neurospheres were obtained from SVZ tissue culture, indicated that the
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