miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro
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 tra...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-02-01
|
| Series: | Cells |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4409/9/3/532 |
| _version_ | 1797715422713217024 |
|---|---|
| author | Mitsuru Ishikawa Takeshi Aoyama Shoichiro Shibata Takefumi Sone Hiroyuki Miyoshi Hirotaka Watanabe Mari Nakamura Saori Morota Hiroyuki Uchino Andrew S. Yoo Hideyuki Okano |
| author_facet | Mitsuru Ishikawa Takeshi Aoyama Shoichiro Shibata Takefumi Sone Hiroyuki Miyoshi Hirotaka Watanabe Mari Nakamura Saori Morota Hiroyuki Uchino Andrew S. Yoo Hideyuki Okano |
| author_sort | Mitsuru Ishikawa |
| collection | DOAJ |
| description | 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 (<i>Ngn2</i>), a proneural factor. While it has been established that <i>Ngn2</i> 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 <i>Ngn2</i> 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 <i>presenilin-1</i> (<i>PS1</i>) or <i>presenilin-2</i> (<i>PS2</i>) 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 <i>Ngn2</i> and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery. |
| first_indexed | 2024-03-12T08:06:33Z |
| format | Article |
| id | doaj.art-1c96d6775aaa44be9c35851c261e3bb7 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-12T08:06:33Z |
| publishDate | 2020-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-1c96d6775aaa44be9c35851c261e3bb72023-09-02T19:27:57ZengMDPI AGCells2073-44092020-02-019353210.3390/cells9030532cells9030532miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In VitroMitsuru Ishikawa0Takeshi Aoyama1Shoichiro Shibata2Takefumi Sone3Hiroyuki Miyoshi4Hirotaka Watanabe5Mari Nakamura6Saori Morota7Hiroyuki Uchino8Andrew S. Yoo9Hideyuki Okano10Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, JapanDepartment of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, JapanDepartment of Anesthesiology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, JapanDepartment of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, JapanDepartment of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, JapanDepartment of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, JapanDepartment of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, JapanDepartment of Anesthesiology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, JapanDepartment of Anesthesiology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, JapanDepartment of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USADepartment of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, JapanObtaining 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 (<i>Ngn2</i>), a proneural factor. While it has been established that <i>Ngn2</i> 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 <i>Ngn2</i> 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 <i>presenilin-1</i> (<i>PS1</i>) or <i>presenilin-2</i> (<i>PS2</i>) 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 <i>Ngn2</i> and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.https://www.mdpi.com/2073-4409/9/3/532human pluripotent stem cellexcitatory neuronneurogenin2microrna-9/9*microrna-124alzheimer’s diseasepresenilin1presenilin2 |
| spellingShingle | Mitsuru Ishikawa Takeshi Aoyama Shoichiro Shibata Takefumi Sone Hiroyuki Miyoshi Hirotaka Watanabe Mari Nakamura Saori Morota Hiroyuki Uchino Andrew S. Yoo Hideyuki Okano miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro Cells human pluripotent stem cell excitatory neuron neurogenin2 microrna-9/9* microrna-124 alzheimer’s disease presenilin1 presenilin2 |
| title | miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro |
| title_full | miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro |
| title_fullStr | miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro |
| title_full_unstemmed | miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro |
| title_short | miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro |
| title_sort | mirna based rapid differentiation of purified neurons from hpscs advancestowards quick screening for neuronal disease phenotypes in vitro |
| topic | human pluripotent stem cell excitatory neuron neurogenin2 microrna-9/9* microrna-124 alzheimer’s disease presenilin1 presenilin2 |
| url | https://www.mdpi.com/2073-4409/9/3/532 |
| work_keys_str_mv | AT mitsuruishikawa mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT takeshiaoyama mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT shoichiroshibata mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT takefumisone mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT hiroyukimiyoshi mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT hirotakawatanabe mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT marinakamura mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT saorimorota mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT hiroyukiuchino mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT andrewsyoo mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro AT hideyukiokano mirnabasedrapiddifferentiationofpurifiedneuronsfromhpscsadvancestowardsquickscreeningforneuronaldiseasephenotypesinvitro |