TY - JOUR
T1 - Precise nanoinjection delivery of plasmid DNA into a single fibroblast for direct conversion of astrocyte
AU - Park, Hang Soo
AU - Kwon, Hyosung
AU - Yu, Jewon
AU - Bae, Yeonju
AU - Park, Jae Yong
AU - Choi, Kyung Ah
AU - Choi, Yeonho
AU - Hong, Sunghoi
N1 - Funding Information:
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning [2012M3A9C6050124, 2012M3A9C6050131, 2017M3A9C6026996 and 2017M3A9C027000].
PY - 2018/10/31
Y1 - 2018/10/31
N2 - Direct conversion is a powerful approach to safely generate mature neural lineages with potential for treatment of neurological disorders. Astrocytes play a crucial role in neuronal homeostasis and their dysfunctions contribute to several neurodegenerative diseases. Using a single-cell approach for precision, we describe here a robust method using optimized DNA amounts for the direct conversion of mouse fibroblasts to astrocytes. Controlled amount of the reprogramming factors Oct4, Sox2, Klf4 and cMyc was directly delivered into a single fibroblast cell. Consequently, 2500 DNA molecules, no more or less, were found to be the optimal amount that dramatically increased the expression levels of the astrocyte-specific markers GFAP and S100b and the demethylation gene TET1, the expression of which was sustained to maintain astrocyte functionality. The converted astrocytes showed glutamate uptake ability and electrophysiological activity. Furthermore, we demonstrated a potential mechanism whereby fibroblast was directly converted into astrocyte at a single-cell level; this was achieved by activating BMP2 pathway through direct binding of Sox2 protein to BMP2 gene. This study suggests that nanotechnology for directly injecting plasmid DNAs into cell nuclei may help understand such a conversion at single-cell level.
AB - Direct conversion is a powerful approach to safely generate mature neural lineages with potential for treatment of neurological disorders. Astrocytes play a crucial role in neuronal homeostasis and their dysfunctions contribute to several neurodegenerative diseases. Using a single-cell approach for precision, we describe here a robust method using optimized DNA amounts for the direct conversion of mouse fibroblasts to astrocytes. Controlled amount of the reprogramming factors Oct4, Sox2, Klf4 and cMyc was directly delivered into a single fibroblast cell. Consequently, 2500 DNA molecules, no more or less, were found to be the optimal amount that dramatically increased the expression levels of the astrocyte-specific markers GFAP and S100b and the demethylation gene TET1, the expression of which was sustained to maintain astrocyte functionality. The converted astrocytes showed glutamate uptake ability and electrophysiological activity. Furthermore, we demonstrated a potential mechanism whereby fibroblast was directly converted into astrocyte at a single-cell level; this was achieved by activating BMP2 pathway through direct binding of Sox2 protein to BMP2 gene. This study suggests that nanotechnology for directly injecting plasmid DNAs into cell nuclei may help understand such a conversion at single-cell level.
KW - Quantitative and controllable injection
KW - a single cell
KW - astrocytes
KW - direct conversion
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U2 - 10.1080/21691401.2018.1446019
DO - 10.1080/21691401.2018.1446019
M3 - Article
C2 - 29506416
AN - SCOPUS:85042905572
VL - 46
SP - 1114
EP - 1122
JO - Artificial Cells, Nanomedicine and Biotechnology
JF - Artificial Cells, Nanomedicine and Biotechnology
SN - 2169-1401
IS - sup1
ER -