In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function [version 1; peer review: 2 approved]
The development of the human brain involves multiple cellular processes including cell division, migration, and dendritic growth. These processes are triggered by developmental cues and lead to interactions of neurons and glial cells to derive the final complex organization of the brain. Development...
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| Format: | Article |
| Language: | English |
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Wellcome
2020-02-01
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| Series: | Wellcome Open Research |
| Online Access: | https://wellcomeopenresearch.org/articles/5-16/v1 |
| _version_ | 1811259127533404160 |
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| author | Yojet Sharma Sankhanil Saha Annu Joseph Harini Krishnan Padinjat Raghu |
| author_facet | Yojet Sharma Sankhanil Saha Annu Joseph Harini Krishnan Padinjat Raghu |
| author_sort | Yojet Sharma |
| collection | DOAJ |
| description | The development of the human brain involves multiple cellular processes including cell division, migration, and dendritic growth. These processes are triggered by developmental cues and lead to interactions of neurons and glial cells to derive the final complex organization of the brain. Developmental cues are transduced into cellular processes through the action of multiple intracellular second messengers including calcium. Calcium signals in cells are shaped by large number of proteins and mutations in several of these have been reported in human patients with brain disorders. However, the manner in which such mutations impact human brain development in vivo remains poorly understood. A key limitation in this regard is the need for a model system in which calcium signaling can be studied in neurons of patients with specific brain disorders. Here we describe a protocol to differentiate human neural stem cells into cortical neuronal networks that can be maintained as live cultures up to 120 days in a dish. Our protocol generates a 2D in vitro culture that exhibits molecular features of several layers of the human cerebral cortex. Using fluorescence imaging of intracellular calcium levels, we describe the development of neuronal activity as measured by intracellular calcium transients during development in vitro. These transients were dependent on the activity of voltage gated calcium channels and were abolished by blocking sodium channel activity. Using transcriptome analysis, we describe the full molecular composition of such cultures following differentiation in vitro thus offering an insight into the molecular basis of activity. Our approach will facilitate the understanding of calcium signaling defects during cortical neuron development in patients with specific brain disorders and a mechanistic analysis of these defects using genetic manipulations coupled with cell biological and physiological analysis. |
| first_indexed | 2024-04-12T18:25:47Z |
| format | Article |
| id | doaj.art-ea341c0468934ddf9af8edc00e63443e |
| institution | Directory Open Access Journal |
| issn | 2398-502X |
| language | English |
| last_indexed | 2024-04-12T18:25:47Z |
| publishDate | 2020-02-01 |
| publisher | Wellcome |
| record_format | Article |
| series | Wellcome Open Research |
| spelling | doaj.art-ea341c0468934ddf9af8edc00e63443e2022-12-22T03:21:15ZengWellcomeWellcome Open Research2398-502X2020-02-01510.12688/wellcomeopenres.15626.117119In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function [version 1; peer review: 2 approved]Yojet Sharma0Sankhanil Saha1Annu Joseph2Harini Krishnan3Padinjat Raghu4Cellular Organization and Signalling, National Centre for Biological Sciences - TIFR, Bangalore, Karnataka, 560065, IndiaCellular Organization and Signalling, National Centre for Biological Sciences - TIFR, Bangalore, Karnataka, 560065, IndiaCellular Organization and Signalling, National Centre for Biological Sciences - TIFR, Bangalore, Karnataka, 560065, IndiaCellular Organization and Signalling, National Centre for Biological Sciences - TIFR, Bangalore, Karnataka, 560065, IndiaCellular Organization and Signalling, National Centre for Biological Sciences - TIFR, Bangalore, Karnataka, 560065, IndiaThe development of the human brain involves multiple cellular processes including cell division, migration, and dendritic growth. These processes are triggered by developmental cues and lead to interactions of neurons and glial cells to derive the final complex organization of the brain. Developmental cues are transduced into cellular processes through the action of multiple intracellular second messengers including calcium. Calcium signals in cells are shaped by large number of proteins and mutations in several of these have been reported in human patients with brain disorders. However, the manner in which such mutations impact human brain development in vivo remains poorly understood. A key limitation in this regard is the need for a model system in which calcium signaling can be studied in neurons of patients with specific brain disorders. Here we describe a protocol to differentiate human neural stem cells into cortical neuronal networks that can be maintained as live cultures up to 120 days in a dish. Our protocol generates a 2D in vitro culture that exhibits molecular features of several layers of the human cerebral cortex. Using fluorescence imaging of intracellular calcium levels, we describe the development of neuronal activity as measured by intracellular calcium transients during development in vitro. These transients were dependent on the activity of voltage gated calcium channels and were abolished by blocking sodium channel activity. Using transcriptome analysis, we describe the full molecular composition of such cultures following differentiation in vitro thus offering an insight into the molecular basis of activity. Our approach will facilitate the understanding of calcium signaling defects during cortical neuron development in patients with specific brain disorders and a mechanistic analysis of these defects using genetic manipulations coupled with cell biological and physiological analysis.https://wellcomeopenresearch.org/articles/5-16/v1 |
| spellingShingle | Yojet Sharma Sankhanil Saha Annu Joseph Harini Krishnan Padinjat Raghu In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function [version 1; peer review: 2 approved] Wellcome Open Research |
| title | In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function [version 1; peer review: 2 approved] |
| title_full | In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function [version 1; peer review: 2 approved] |
| title_fullStr | In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function [version 1; peer review: 2 approved] |
| title_full_unstemmed | In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function [version 1; peer review: 2 approved] |
| title_short | In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function [version 1; peer review: 2 approved] |
| title_sort | in vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function version 1 peer review 2 approved |
| url | https://wellcomeopenresearch.org/articles/5-16/v1 |
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