An optogenetic approach for assessing formation of neuronal connections in a co-culture system

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripot...

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Main Authors: Su, Colin T. E., Yoon, Su-In, Marcy, Guillaume, Chin, Eunice W. M., Augustine, George James, Goh, Eyleen Lay Keow
Other Authors: Lee Kong Chian School of Medicine (LKCMedicine)
Format: Article
Language:English
Published: 2015
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Online Access:https://hdl.handle.net/10356/93695
http://hdl.handle.net/10220/38336
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-936952022-06-08T05:44:04Z An optogenetic approach for assessing formation of neuronal connections in a co-culture system Su, Colin T. E. Yoon, Su-In Marcy, Guillaume Chin, Eunice W. M. Augustine, George James Goh, Eyleen Lay Keow Lee Kong Chian School of Medicine (LKCMedicine) DRNTU::Science::Biological sciences::Human anatomy and physiology::Neurobiology Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations. Published version 2015-07-15T08:00:42Z 2019-12-06T18:43:49Z 2015-07-15T08:00:42Z 2019-12-06T18:43:49Z 2015 2015 Journal Article Su, C. T. E., Yoon, S.-I., Marcy, G., Chin, E. W. M., Augustine, G. J., & Goh, E. L. K. (2015). An optogenetic approach for assessing formation of neuronal connections in a co-culture system. Journal of Visualized Experiments, 96, e52408. 1940-087X https://hdl.handle.net/10356/93695 http://hdl.handle.net/10220/38336 10.3791/52408 25742527 en Journal of visualized experiments © 2015 Creative Commons Attribution-NonCommercial License. This paper was published in Journal of Visualized Experiments and is made available as an electronic reprint (preprint) with permission of Creative Commons Attribution-NonCommercial License. The published version is available at: [http://dx.doi.org/10.3791/52408]. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Science::Biological sciences::Human anatomy and physiology::Neurobiology
spellingShingle DRNTU::Science::Biological sciences::Human anatomy and physiology::Neurobiology
Su, Colin T. E.
Yoon, Su-In
Marcy, Guillaume
Chin, Eunice W. M.
Augustine, George James
Goh, Eyleen Lay Keow
An optogenetic approach for assessing formation of neuronal connections in a co-culture system
description Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.
author2 Lee Kong Chian School of Medicine (LKCMedicine)
author_facet Lee Kong Chian School of Medicine (LKCMedicine)
Su, Colin T. E.
Yoon, Su-In
Marcy, Guillaume
Chin, Eunice W. M.
Augustine, George James
Goh, Eyleen Lay Keow
format Article
author Su, Colin T. E.
Yoon, Su-In
Marcy, Guillaume
Chin, Eunice W. M.
Augustine, George James
Goh, Eyleen Lay Keow
author_sort Su, Colin T. E.
title An optogenetic approach for assessing formation of neuronal connections in a co-culture system
title_short An optogenetic approach for assessing formation of neuronal connections in a co-culture system
title_full An optogenetic approach for assessing formation of neuronal connections in a co-culture system
title_fullStr An optogenetic approach for assessing formation of neuronal connections in a co-culture system
title_full_unstemmed An optogenetic approach for assessing formation of neuronal connections in a co-culture system
title_sort optogenetic approach for assessing formation of neuronal connections in a co-culture system
publishDate 2015
url https://hdl.handle.net/10356/93695
http://hdl.handle.net/10220/38336
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