Browsing by Author "Chua K.H."
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Publication Effects of epidermal growth factor on the proliferation and cell cycle regulation of cultured human amnion epithelial cells(2012) ;Fatimah S.S. ;Tan G.C. ;Chua K.H. ;Tan A.E. ;Hayati A.R. ;Faculty of Medicine and Health Sciences ;Universiti Kebangsaan Malaysia (UKM)Universiti Sains Islam Malaysia (USIM)Human amnion epithelial cells (HAECs) hold great promise in tissue engineering for regenerative medicine. Large numbers of HAECs are required for this purpose. Hence, exogenous growth factor is added to the culture medium to improve epithelial cells proliferation. The aim of the present study was to determine the effects of epidermal growth factor (EGF) on the proliferation and cell cycle regulation of cultured HAECs. HAECs at P1 were cultured for 7 days in medium containing an equal volume mix of HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of EGF (0, 5, 10, 20, 30 and 50. ng/ml EGF) in reduced serum. Morphology, growth kinetics and cell cycle analysis using flow cytometry were assessed. Quantitative gene expression for cell cycle control genes, pluripotent transcription factors, epithelial genes and neuronal genes were also determined. EGF enhanced HAECs proliferation with optimal concentration at 10. ng/ml EGF. EGF significantly increased the proportion of HAECs at S- and G2/M-phase of the cell cycle compared to the control. At the end of culture, HAECs remained as diploid cells under cell cycle analysis. EGF significantly decreased the mRNA expression of p21, pRb, p53 and GADD45 in cultured HAECs. EGF also significantly decreased the pluripotent genes expression: Oct-3/4, Sox2 and Nanog; epithelial genes expression: CK14, p63, CK1 and Involucrin; and neuronal gene expression: NSE, NF-M and MAP 2. The results suggested that EGF is a strong mitogen that promotes the proliferation of HAECs through cell cycle regulation. EGF did not promote HAECs differentiation or pluripotent genes expression. � 2012 The Society for Biotechnology, Japan. - Some of the metrics are blocked by yourconsent settings
Publication Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells(2012) ;Mok P.L. ;Cheong S.K. ;Leong C.F. ;Chua K.H. ;Ainoon O. ;Faculty of Medicine and Health Sciences ;Universiti Kebangsaan Malaysia (UKM) ;Universiti Tunku Abdul Rahman (UTAR)Universiti Sains Islam Malaysia (USIM)Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 � 1.09%) and C-17 (5.62 � 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 � 10.10)% and (21.93 � 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC. � 2011 Springer Science+Business Media B.V. - Some of the metrics are blocked by yourconsent settings
Publication Human mesenchymal stromal cells could deliver erythropoietin and migrate to the basal layer of hair shaft when subcutaneously implanted in a murine model(Elsevier Ltd, 2012) ;Mok P.L. ;Cheong S.K. ;Leong C.F. ;Chua K.H. ;Ainoon O. ;Faculty of Medicine and Health Sciences ;Universiti Kebangsaan Malaysia (UKM) ;Universiti Tunku Abdul Rahman (UTAR)Universiti Sains Islam Malaysia (USIM)Mesenchymal stromal cells (MSC) are an attractive cell-targeting vehicle for gene delivery. MIDGE (an acronym for Minimalistic, Immunologically Defined Gene Expression) construct is relatively safer than the viral or plasmid expression system as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. The objective of this study was to test the ability of the human MSC (hMSC) to deliver the erythropoietin (EPO) gene in a nude mice model following nucleofection using a MIDGE construct. hMSC nucleofected with MIDGE encoding the EPO gene was injected subcutaneously in Matrigel at the dorsal flank of nude mice. Subcutaneous implantation of nucleofected hMSC resulted in increased hemoglobin level with presence of human EPO in the peripheral blood of the injected nude mice in the first two weeks post-implantation compared with the control groups. The basal layer of the hair shaft in the dermal layer was found to be significantly positive for immunohistochemical staining of a human EPO antibody. However, only a few basal layers of the hair shaft were found to be positively stained for CD105. In conclusion, hMSC harboring MIDGE-EPO could deliver and transiently express the EPO gene in the nude mice model. These cells could be localized to the hair follicle and secreted EPO protein might have possible role in hair regeneration. � 2012 Elsevier Ltd.