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Expansion, characterisation and differentiation of human neural stem cells

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, huma...

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Main Author: Ye, Yee May
Format: Monograph
Language:English
Published: Universiti Sains Malaysia 2015
Subjects:
R Medicine (General)
RA440-440.87 Study and teaching. Research
Online Access:http://eprints.usm.my/60657/1/YEE%20MAY%20YE%20-%20e.pdf
http://eprints.usm.my/60657/
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spelling my.usm.eprints.60657 http://eprints.usm.my/60657/ Expansion, characterisation and differentiation of human neural stem cells Ye, Yee May R Medicine (General) RA440-440.87 Study and teaching. Research 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 culture method used in this study was successful to isolate NSCs. However, neurosphere contains only small amount of true stem cells, thus cell disaggregation and proliferation are suggested in future study to obtain more cells for downstream analysis. Conclusion, NSCs were successfully obtained from both GIBCO® human NSC line and SVZ tissue. However, further expansion, characterisation and differentiation of NSC isolated from SVZ tissue should be performed in future study. Universiti Sains Malaysia 2015 Monograph NonPeerReviewed application/pdf en http://eprints.usm.my/60657/1/YEE%20MAY%20YE%20-%20e.pdf Ye, Yee May (2015) Expansion, characterisation and differentiation of human neural stem cells. Project Report. Universiti Sains Malaysia.
institution Universiti Sains Malaysia
building Hamzah Sendut Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Sains Malaysia
content_source USM Institutional Repository
url_provider http://eprints.usm.my/
language English
topic R Medicine (General)
RA440-440.87 Study and teaching. Research
spellingShingle R Medicine (General)
RA440-440.87 Study and teaching. Research
Ye, Yee May
Expansion, characterisation and differentiation of human neural stem cells
description 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 culture method used in this study was successful to isolate NSCs. However, neurosphere contains only small amount of true stem cells, thus cell disaggregation and proliferation are suggested in future study to obtain more cells for downstream analysis. Conclusion, NSCs were successfully obtained from both GIBCO® human NSC line and SVZ tissue. However, further expansion, characterisation and differentiation of NSC isolated from SVZ tissue should be performed in future study.
format Monograph
author Ye, Yee May
author_facet Ye, Yee May
author_sort Ye, Yee May
title Expansion, characterisation and differentiation of human neural stem cells
title_short Expansion, characterisation and differentiation of human neural stem cells
title_full Expansion, characterisation and differentiation of human neural stem cells
title_fullStr Expansion, characterisation and differentiation of human neural stem cells
title_full_unstemmed Expansion, characterisation and differentiation of human neural stem cells
title_sort expansion, characterisation and differentiation of human neural stem cells
publisher Universiti Sains Malaysia
publishDate 2015
url http://eprints.usm.my/60657/1/YEE%20MAY%20YE%20-%20e.pdf
http://eprints.usm.my/60657/
_version_ 1800715760355508224
score 13.211869

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