Han-Lin Shuai1, Rui-Ling Yan1, Hong Song1, Dan-Liang Chen1, Xin Luo2. 1. Department of Obstetrics and Gynecology, the First Affiliated Hospital of Jinan University, Guangzhou, 510630, China. 2. Department of Obstetrics and Gynecology, the First Affiliated Hospital of Jinan University, Guangzhou, 510630, China. Electronic address: luoxin121@yeah.net.
Abstract
OBJECTIVE: Three different kinds of transfection reagents were used to mediate the transfection of gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) into human umbilical-cord-derived mesenchymal stem cells (hUCMSCs). The efficacy of different transfection reagents and the feasibility of NMR tracer in vitro of magnetized stem cells were estimated. METHODS: After purification by tissue explants adherent method, the biological characteristics of hUCMSCs in vitro were identified by subculture and amplification. Calcium phosphate, Effectene and liposome2000 were used to transfect Gd-DTPA-labeled hUCMSCs respectively, and cell counting was used to mediate the transfection of Gd-DTPA into hUCMSCs, which were then induced to lipoblast and osteoblast in vitro. The determination of the transfection activities of the transfection reagents was conducted by measuring the magnetic resonance imaging (MRI) signal intensity of the Gd-DTPA-labeled cells and the concentration of gadolinium ion in the cells. Furthermore, the relationship between the signal intensity of Gd-DTPA-labeled hUCMSCsMRI, cell subculture and generations was studied. RESULTS: Primary cells were obtained by tissue explants adherent for two weeks. The cells displayed a long spindle form and grew in swirl. After two passage generations, the cellular morphology became more homogeneous. The result detected by the flow cytometer showed that CD29C, D44, CD90, and CD105 were highly expressed, while no CD45, CD40, and HLA-DR expression was detected in the third generation cells. Directional induction in vitro caused the differentiation into lipoblast and osteoblast. After transfected by calcium phosphate, Effectene and liposome 2000, the signal intensity of stem cells was 2281.2±118.8, 2031.9±59.7 and 1887.4±40.8 measured by MRI. Differences between these three groups were statistically significant (P<0.05). The concentrations of gadolinium ion in three groups of stem cells were 0.178±0.009mg/L, 0.158±0.003mg/L and 0.120±0.002mg/L respectively, examined by inductively coupled plasma atomic emission spectrometry. No significant differences were found among these three groups (P<0.05). The proliferation and differentiation abilities of the Gd-DTPA-labeled stem cells were not affected. A minimum 5×10(4) Gd-DTPA-labeled stem cells could be traced with MRI in vitro and presented in high signal. The trace duration time in vitro was about 12days. CONCLUSIONS: Tissue explants adherent method can be availably applied to purify hUCMSCs. The Effectene method was proved to have the best transfection effect. The proliferation ability and differentiation potency of Gd-DTPA-labeled hUCMSCs were not affected, and the NMR of labeled stem cells in vitro was proved to be feasible.
OBJECTIVE: Three different kinds of transfection reagents were used to mediate the transfection of gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) into human umbilical-cord-derived mesenchymal stem cells (hUCMSCs). The efficacy of different transfection reagents and the feasibility of NMR tracer in vitro of magnetized stem cells were estimated. METHODS: After purification by tissue explants adherent method, the biological characteristics of hUCMSCs in vitro were identified by subculture and amplification. Calcium phosphate, Effectene and liposome2000 were used to transfect Gd-DTPA-labeled hUCMSCs respectively, and cell counting was used to mediate the transfection of Gd-DTPA into hUCMSCs, which were then induced to lipoblast and osteoblast in vitro. The determination of the transfection activities of the transfection reagents was conducted by measuring the magnetic resonance imaging (MRI) signal intensity of the Gd-DTPA-labeled cells and the concentration of gadolinium ion in the cells. Furthermore, the relationship between the signal intensity of Gd-DTPA-labeled hUCMSCsMRI, cell subculture and generations was studied. RESULTS: Primary cells were obtained by tissue explants adherent for two weeks. The cells displayed a long spindle form and grew in swirl. After two passage generations, the cellular morphology became more homogeneous. The result detected by the flow cytometer showed that CD29C, D44, CD90, and CD105 were highly expressed, while no CD45, CD40, and HLA-DR expression was detected in the third generation cells. Directional induction in vitro caused the differentiation into lipoblast and osteoblast. After transfected by calcium phosphate, Effectene and liposome 2000, the signal intensity of stem cells was 2281.2±118.8, 2031.9±59.7 and 1887.4±40.8 measured by MRI. Differences between these three groups were statistically significant (P<0.05). The concentrations of gadolinium ion in three groups of stem cells were 0.178±0.009mg/L, 0.158±0.003mg/L and 0.120±0.002mg/L respectively, examined by inductively coupled plasma atomic emission spectrometry. No significant differences were found among these three groups (P<0.05). The proliferation and differentiation abilities of the Gd-DTPA-labeled stem cells were not affected. A minimum 5×10(4) Gd-DTPA-labeled stem cells could be traced with MRI in vitro and presented in high signal. The trace duration time in vitro was about 12days. CONCLUSIONS: Tissue explants adherent method can be availably applied to purify hUCMSCs. The Effectene method was proved to have the best transfection effect. The proliferation ability and differentiation potency of Gd-DTPA-labeled hUCMSCs were not affected, and the NMR of labeled stem cells in vitro was proved to be feasible.
Authors: Forrest Goodfellow; Gregory A Simchick; Luke J Mortensen; Steven L Stice; Qun Zhao Journal: Adv Funct Mater Date: 2016-02-17 Impact factor: 18.808