A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons

Frontiers in Neuroscience

Neuroscience

Erik Bélanger¹  Nizar Chetoui¹  François Paquet-Mercier¹  Marie-Eve Paquet²  Niraj Patel³  Vincent Ouellet³  Antoine G. Godin⁴  Pierre Marquet⁴ 

[1] CERVO Brain Research Centre, Laval University, Quebec, QC, Canada;CERVO Brain Research Centre, Laval University, Quebec, QC, Canada;Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Quebec, QC, Canada;Department of Psychiatry and Neuroscience, Laval University, Quebec, QC, Canada;CERVO Brain Research Centre, Laval University, Quebec, QC, Canada;Department of Psychiatry and Neuroscience, Laval University, Quebec, QC, Canada;CERVO Brain Research Centre, Laval University, Quebec, QC, Canada;Centre for Optics, Photonics and Lasers (COPL), Laval University, Quebec, QC, Canada;

关键词: human iPSCs (induced pluripotent stem cells);    neurodifferentiation;    GECI (genetically encoded Ca indicator);    human synapsin1 (hSyn) promoter;    CAG promoter;    calcium imaging;    disease modeling;    neuronal network;   

DOI  :  10.3389/fnins.2023.1247397

 received in 2023-06-26, accepted in 2023-08-16,  发布年份 2023

来源: Frontiers

【 摘 要 】

IntroductionHuman induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process.MethodsIn this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter.ResultsThe retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types.DiscussionOur results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures.

【 授权许可】

Unknown   
Copyright © 2023 Patel, Ouellet, Paquet-Mercier, Chetoui, Bélanger, Paquet, Godin and Marquet.