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Real-time monitoring of neural differentiation of human mesenchymal stem cells by electric cell-substrate impedance sensing.

Park HE, Kim D, Koh HS, Cho S, Sung JS, Kim JY - J. Biomed. Biotechnol. (2011)

Bottom Line: The resistance value of cells cultured in NDM was automatically measured in real-time and found to increase much more slowly over time compared to cells cultured in non-differentiation media.Overall results suggest that the relatively slow change in resistance values measured by ECIS method can be used as a parameter for slowly growing neural-differentiating cells.However, to enhance the competence of ECIS for in vitro real-time monitoring of neural differentiation of MSCs, more elaborate studies are needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Gachon University of Medicine and Science, Incheon 406-799, Republic of Korea.

ABSTRACT
Stem cells are useful for cell replacement therapy. Stem cell differentiation must be monitored thoroughly and precisely prior to transplantation. In this study we evaluated the usefulness of electric cell-substrate impedance sensing (ECIS) for in vitro real-time monitoring of neural differentiation of human mesenchymal stem cells (hMSCs). We cultured hMSCs in neural differentiation media (NDM) for 6 days and examined the time-course of impedance changes with an ECIS array. We also monitored the expression of markers for neural differentiation, total cell count, and cell cycle profiles. Cellular expression of neuron and oligodendrocyte markers increased. The resistance value of cells cultured in NDM was automatically measured in real-time and found to increase much more slowly over time compared to cells cultured in non-differentiation media. The relatively slow resistance changes observed in differentiating MSCs were determined to be due to their lower growth capacity achieved by induction of cell cycle arrest in G0/G1. Overall results suggest that the relatively slow change in resistance values measured by ECIS method can be used as a parameter for slowly growing neural-differentiating cells. However, to enhance the competence of ECIS for in vitro real-time monitoring of neural differentiation of MSCs, more elaborate studies are needed.

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Cell count and cell cycle transition of hMSCs during neural differentiation. (a) Cell counts were determined 36 hr after changing to a NDM (corresponding to 76 hr after culturing in NGM and to 60 hr after changing to a NIM). Data represent means ± SD (n = 4). *P < .01. (b) The numbers of death cells were determined by counting the trypan blue stained cells in a hemocytometer. (c) Flow cytometric cell cycle analysis of hMSCs cultured under 3 different culture conditions. DNA content-related cell cycle distribution of hMSCs was determined 24 hr after changing to a NDM. Data represent means ± SD (n = 6–9). *P < .01. NGM: normal growth media; NIM: neural induction media; NDM: neural differentiation media.
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fig5: Cell count and cell cycle transition of hMSCs during neural differentiation. (a) Cell counts were determined 36 hr after changing to a NDM (corresponding to 76 hr after culturing in NGM and to 60 hr after changing to a NIM). Data represent means ± SD (n = 4). *P < .01. (b) The numbers of death cells were determined by counting the trypan blue stained cells in a hemocytometer. (c) Flow cytometric cell cycle analysis of hMSCs cultured under 3 different culture conditions. DNA content-related cell cycle distribution of hMSCs was determined 24 hr after changing to a NDM. Data represent means ± SD (n = 6–9). *P < .01. NGM: normal growth media; NIM: neural induction media; NDM: neural differentiation media.

Mentions: In principle, impedance values of the cell layer in ECIS can increase as the number of cells and/or adhesion capacity of cells increases. To determine whether relatively lower impedance values observed in the cell layer in NDM compared to those in the other groups were due to lower cell numbers, we examined cell counts in the 3 different media. As shown in Figure 5(a), the number of cells in NDM was 2.4 times lower than that of the other groups (P < .05). However, cell death rates were not significantly different between the 3 groups (Figure 5(b)). We performed cell cycle analysis by FACS to determine whether such cell count variations were due to differences in the cell cycle process. As shown in Figure 5(c), G0/G1 cell ratio was higher in cells in NDM than in the other groups (P < .01). These findings indicate that cells in NDM have a delayed cell cycle and are less proliferative, which explains why lower cell counts were obtained in NDM compared to the other media.


Real-time monitoring of neural differentiation of human mesenchymal stem cells by electric cell-substrate impedance sensing.

Park HE, Kim D, Koh HS, Cho S, Sung JS, Kim JY - J. Biomed. Biotechnol. (2011)

Cell count and cell cycle transition of hMSCs during neural differentiation. (a) Cell counts were determined 36 hr after changing to a NDM (corresponding to 76 hr after culturing in NGM and to 60 hr after changing to a NIM). Data represent means ± SD (n = 4). *P < .01. (b) The numbers of death cells were determined by counting the trypan blue stained cells in a hemocytometer. (c) Flow cytometric cell cycle analysis of hMSCs cultured under 3 different culture conditions. DNA content-related cell cycle distribution of hMSCs was determined 24 hr after changing to a NDM. Data represent means ± SD (n = 6–9). *P < .01. NGM: normal growth media; NIM: neural induction media; NDM: neural differentiation media.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Cell count and cell cycle transition of hMSCs during neural differentiation. (a) Cell counts were determined 36 hr after changing to a NDM (corresponding to 76 hr after culturing in NGM and to 60 hr after changing to a NIM). Data represent means ± SD (n = 4). *P < .01. (b) The numbers of death cells were determined by counting the trypan blue stained cells in a hemocytometer. (c) Flow cytometric cell cycle analysis of hMSCs cultured under 3 different culture conditions. DNA content-related cell cycle distribution of hMSCs was determined 24 hr after changing to a NDM. Data represent means ± SD (n = 6–9). *P < .01. NGM: normal growth media; NIM: neural induction media; NDM: neural differentiation media.
Mentions: In principle, impedance values of the cell layer in ECIS can increase as the number of cells and/or adhesion capacity of cells increases. To determine whether relatively lower impedance values observed in the cell layer in NDM compared to those in the other groups were due to lower cell numbers, we examined cell counts in the 3 different media. As shown in Figure 5(a), the number of cells in NDM was 2.4 times lower than that of the other groups (P < .05). However, cell death rates were not significantly different between the 3 groups (Figure 5(b)). We performed cell cycle analysis by FACS to determine whether such cell count variations were due to differences in the cell cycle process. As shown in Figure 5(c), G0/G1 cell ratio was higher in cells in NDM than in the other groups (P < .01). These findings indicate that cells in NDM have a delayed cell cycle and are less proliferative, which explains why lower cell counts were obtained in NDM compared to the other media.

Bottom Line: The resistance value of cells cultured in NDM was automatically measured in real-time and found to increase much more slowly over time compared to cells cultured in non-differentiation media.Overall results suggest that the relatively slow change in resistance values measured by ECIS method can be used as a parameter for slowly growing neural-differentiating cells.However, to enhance the competence of ECIS for in vitro real-time monitoring of neural differentiation of MSCs, more elaborate studies are needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Gachon University of Medicine and Science, Incheon 406-799, Republic of Korea.

ABSTRACT
Stem cells are useful for cell replacement therapy. Stem cell differentiation must be monitored thoroughly and precisely prior to transplantation. In this study we evaluated the usefulness of electric cell-substrate impedance sensing (ECIS) for in vitro real-time monitoring of neural differentiation of human mesenchymal stem cells (hMSCs). We cultured hMSCs in neural differentiation media (NDM) for 6 days and examined the time-course of impedance changes with an ECIS array. We also monitored the expression of markers for neural differentiation, total cell count, and cell cycle profiles. Cellular expression of neuron and oligodendrocyte markers increased. The resistance value of cells cultured in NDM was automatically measured in real-time and found to increase much more slowly over time compared to cells cultured in non-differentiation media. The relatively slow resistance changes observed in differentiating MSCs were determined to be due to their lower growth capacity achieved by induction of cell cycle arrest in G0/G1. Overall results suggest that the relatively slow change in resistance values measured by ECIS method can be used as a parameter for slowly growing neural-differentiating cells. However, to enhance the competence of ECIS for in vitro real-time monitoring of neural differentiation of MSCs, more elaborate studies are needed.

Show MeSH
Related in: MedlinePlus