Mesenchymal stem cells (MSCs) have been considered an alternative source of neuronal lineage cells, which are difficult to isolate from brain and expand in vitro. Previous studies have reported that MSCs expressing Nestin ($Nestin^+$ MSCs), a neuronal stem/progenitor cell marker, exhibit ...
Mesenchymal stem cells (MSCs) have been considered an alternative source of neuronal lineage cells, which are difficult to isolate from brain and expand in vitro. Previous studies have reported that MSCs expressing Nestin ($Nestin^+$ MSCs), a neuronal stem/progenitor cell marker, exhibit increased transcriptional levels of neural development-related genes, indicating that $Nestin^+$ MSCs may exert potential with neurogenic differentiation. Accordingly, we investigated the effects of the presence of $Nestin^+$ MSCs in bone-marrow-derived primary cells (BMPCs) on enhanced neurogenic differentiation of BMPCs by identifying the presence of $Nestin^+$ MSCs in uncultured and cultured BMPCs. The percentage of $Nestin^+$ MSCs in BMPCs was measured per passage by double staining with Nestin and CD90, an MSC marker. The efficiency of neurogenic differentiation was compared among passages, revealing the highest and lowest yields of $Nestin^+$ MSCs. The presence of $Nestin^+$ MSCs was identified in BMPCs before in vitro culture, and the highest and lowest percentages of $Nestin^+$ MSCs in BMPCs was observed at the third (P3) and fifth passages (P5). Moreover, significantly the higher efficiency of differentiation into neurons, oligodendrocyte precursor cells and astrocytes was detected in BMPCs at P3, compared with P5. In conclusion, these results demonstrate that neurogenic differentiation can be enhanced by increasing the proportion of $Nestin^+$ MSCs in cultured BMPCs.
Mesenchymal stem cells (MSCs) have been considered an alternative source of neuronal lineage cells, which are difficult to isolate from brain and expand in vitro. Previous studies have reported that MSCs expressing Nestin ($Nestin^+$ MSCs), a neuronal stem/progenitor cell marker, exhibit increased transcriptional levels of neural development-related genes, indicating that $Nestin^+$ MSCs may exert potential with neurogenic differentiation. Accordingly, we investigated the effects of the presence of $Nestin^+$ MSCs in bone-marrow-derived primary cells (BMPCs) on enhanced neurogenic differentiation of BMPCs by identifying the presence of $Nestin^+$ MSCs in uncultured and cultured BMPCs. The percentage of $Nestin^+$ MSCs in BMPCs was measured per passage by double staining with Nestin and CD90, an MSC marker. The efficiency of neurogenic differentiation was compared among passages, revealing the highest and lowest yields of $Nestin^+$ MSCs. The presence of $Nestin^+$ MSCs was identified in BMPCs before in vitro culture, and the highest and lowest percentages of $Nestin^+$ MSCs in BMPCs was observed at the third (P3) and fifth passages (P5). Moreover, significantly the higher efficiency of differentiation into neurons, oligodendrocyte precursor cells and astrocytes was detected in BMPCs at P3, compared with P5. In conclusion, these results demonstrate that neurogenic differentiation can be enhanced by increasing the proportion of $Nestin^+$ MSCs in cultured BMPCs.
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제안 방법
These results emphasize the importance of Nestin+ MSCs in the mass production of neuronal-lineage cells from MSCs. Accordingly, in this study, we aimed to identify the presence of MSCs with NSPC characteristics in uncultured and cultured bone-marrow-derived primary cells (BMPCs) and investigate their effects on BMPC differentiation into neuronal-lineage cells.
5 mM isobutylmethylxanthine (IBMX; Sigma-Aldrich), 1 mM dibutyryl cyclic AMP (dbcAMP; Sigma-Aldrich), 10 μg/L human basic fibroblast growth factor (bFGF; Peprotech, Rocky Hill, NJ), and 10 μg/L human epidermal growth factor (hEGF; Peprotech) was conducted for 7 days. Cells of neural lineage were rinsed with DPBS, retrieved using 0.25% trypsin-EDTA, and adjusted for flow cytometric analysis.
Subsequently, to investigate the effects of in vitro culture of BMPCs on the generation of MSCs with NSPC characteristics, combinatorial expression of CD90 and Nestin was measured at each passage during the culture of BMPCs up to the fifth passage. Finally, to identify the effects of different population ratios of MSCs expressing NSPC marker proteins in cultured BMPCs on differentiation into neuronal lineage cells, BMPCs at passages showing the highest and lowest percentages of MSCs with NSPC characteristics were differentiated into three neuronal lineage cells, and the efficiencies of neurogenic differentiation were compared.
Subsequently, to guide neurogenic differentiation, incubation of pre-induced cells in neural induction medium consisting of HG-DMEM supplemented with 10% (v/v) heat-inactivated FBS, 0.5 mM isobutylmethylxanthine (IBMX; Sigma-Aldrich), 1 mM dibutyryl cyclic AMP (dbcAMP; Sigma-Aldrich), 10 μg/L human basic fibroblast growth factor (bFGF; Peprotech, Rocky Hill, NJ), and 10 μg/L human epidermal growth factor (hEGF; Peprotech) was conducted for 7 days.
Firstly, to determine whether MSCs with NSPC characteristics exist in a heterogeneous cell population derived from bone marrow, combinatorial expression of CD90 (an MSC marker) and Nestin (an NSPC marker) was measured in uncultured BMPCs. Subsequently, to investigate the effects of in vitro culture of BMPCs on the generation of MSCs with NSPC characteristics, combinatorial expression of CD90 and Nestin was measured at each passage during the culture of BMPCs up to the fifth passage. Finally, to identify the effects of different population ratios of MSCs expressing NSPC marker proteins in cultured BMPCs on differentiation into neuronal lineage cells, BMPCs at passages showing the highest and lowest percentages of MSCs with NSPC characteristics were differentiated into three neuronal lineage cells, and the efficiencies of neurogenic differentiation were compared.
대상 데이터
Six three-week-old male Sprague-Dawley (SD) rats were purchased from DBL (Eumseong, Korea) and used as bone marrow cell donors. All animal housing, handling and experimental procedures were performed according to the Animal Care and Use Guidelines of Kangwon National University and were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (IACUC approval no.
이론/모형
Significant differences were determined by analysis of variance (ANOVA), and a least-square difference or Duncan’s method was used for comparisons among groups.
성능/효과
The relative value of each marker protein-positive cell was then calculated using the following equation: Atarget/Areference, where Atarget represents the percentage of cells stained positively with each antibody post-neural differentiation of BMPCs at each passage, and Areference represents the percentage of cells stained positively with each antibody post-neural differentiation of BMPCs at the third passage (B, C and D). In all repeated experiments, BMPCs at the third passage showed a numerically and significantly higher percentage of NeuN-, NG2- and GFAP-positive cells than those at the fifth passage. Data represent the means ± standard deviation (s.
In conclusion, the co-existence of MSCs with NSPC characteristics in an MSC population enhances the neurogenic differentiation of MSCs. Therefore, mass production of neuronal lineage cells from MSCs, the best tool for cell therapy, is possible by enhancing neurogenic differentiation by increasing the proportion of MSCs with NSPC characteristics in an MSC population.
In this study, a small number of Nestin+ MSCs were included in heterogeneous primary cells retrieved from bone marrow, and their proportion in BMPCs increased gradually by in vitro culture, resulting in improved efficiency of neurogenic differentiation. Accordingly, we determined that neurogenic differentiation of MSCs could be enhanced by increasing the number of MSCs with NSPC characteristics in the MSC population.
후속연구
2013). However, the exact mechanism of MSC transformation should be investigated in further studies.
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