TY - JOUR
T1 - miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro
AU - Ishikawa, Mitsuru
AU - Aoyama, Takeshi
AU - Shibata, Shoichiro
AU - Sone, Takefumi
AU - Miyoshi, Hiroyuki
AU - Watanabe, Hirotaka
AU - Nakamura, Mari
AU - Morota, Saori
AU - Uchino, Hiroyuki
AU - Yoo, Andrew S.
AU - Okano, Hideyuki
PY - 2020/2/25
Y1 - 2020/2/25
N2 - Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer's disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid β42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.
AB - Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer's disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid β42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.
KW - Alzheimer’s disease
KW - excitatory neuron
KW - human pluripotent stem cell
KW - microRNA-124
KW - microRNA-9/9
KW - neurogenin2
KW - presenilin1
KW - presenilin2
UR - http://www.scopus.com/inward/record.url?scp=85092493416&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85092493416&partnerID=8YFLogxK
U2 - 10.3390/cells9030532
DO - 10.3390/cells9030532
M3 - Article
C2 - 32106535
AN - SCOPUS:85092493416
SN - 2073-4409
VL - 9
JO - Cells
JF - Cells
IS - 3
M1 - 136
ER -
