A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine

Jonathan D. Windster; Andrea Sacchetti; Gerben J. Schaaf; Eric M.J. Bindels; Robert M.W. Hofstra; Rene M.H. Wijnen; Cornelius E.J. Sloots; Maria M. Alves

doi:10.15252/embr.202255789

A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine

Jonathan D. Windster, Andrea Sacchetti^*, Gerben J. Schaaf, Eric M.J. Bindels, Robert M.W. Hofstra, Rene M.H. Wijnen, Cornelius E.J. Sloots, Maria M. Alves^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

Original language	English
Article number	e55789
Journal	EMBO Reports
Volume	24
Issue number	4
DOIs	https://doi.org/10.15252/embr.202255789
Publication status	Published - 5 Apr 2023

Bibliographical note

Funding Information:
We would like to thank R.M. Hoogenboezem (Department of Hematology, Erasmus Medical Center) for processing our raw single‐cell data and M.P. Verhagen (Department of Pathology, Erasmus Medical Center) for fruitful discussions and bio‐informatic assistance. This work was supported by a grant from the Stichting Sophia Kinderziekenhuis Fonds to MMA and RMWH (S17‐18).

Funding Information:
We would like to thank R.M. Hoogenboezem (Department of Hematology, Erasmus Medical Center) for processing our raw single-cell data and M.P. Verhagen (Department of Pathology, Erasmus Medical Center) for fruitful discussions and bio-informatic assistance. This work was supported by a grant from the Stichting Sophia Kinderziekenhuis Fonds to MMA and RMWH (S17-18).

Publisher Copyright:
© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

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EMBO Reports - 2023 - Windster - A combinatorial panel for flow cytometry‐based isolation of enteric nervous system cellsFinal published version, 2.15 MBLicence: CC BY-NC-ND

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@article{d49624cb1b89450386c6e88ec2c99f5b,

title = "A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine",

abstract = "Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.",

author = "Windster, {Jonathan D.} and Andrea Sacchetti and Schaaf, {Gerben J.} and Bindels, {Eric M.J.} and Hofstra, {Robert M.W.} and Wijnen, {Rene M.H.} and Sloots, {Cornelius E.J.} and Alves, {Maria M.}",

note = "Funding Information: We would like to thank R.M. Hoogenboezem (Department of Hematology, Erasmus Medical Center) for processing our raw single‐cell data and M.P. Verhagen (Department of Pathology, Erasmus Medical Center) for fruitful discussions and bio‐informatic assistance. This work was supported by a grant from the Stichting Sophia Kinderziekenhuis Fonds to MMA and RMWH (S17‐18). Funding Information: We would like to thank R.M. Hoogenboezem (Department of Hematology, Erasmus Medical Center) for processing our raw single-cell data and M.P. Verhagen (Department of Pathology, Erasmus Medical Center) for fruitful discussions and bio-informatic assistance. This work was supported by a grant from the Stichting Sophia Kinderziekenhuis Fonds to MMA and RMWH (S17-18). Publisher Copyright: {\textcopyright} 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.",

year = "2023",

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doi = "10.15252/embr.202255789",

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AU - Windster, Jonathan D.

AU - Sacchetti, Andrea

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AU - Bindels, Eric M.J.

AU - Hofstra, Robert M.W.

AU - Wijnen, Rene M.H.

AU - Sloots, Cornelius E.J.

AU - Alves, Maria M.

N1 - Funding Information: We would like to thank R.M. Hoogenboezem (Department of Hematology, Erasmus Medical Center) for processing our raw single‐cell data and M.P. Verhagen (Department of Pathology, Erasmus Medical Center) for fruitful discussions and bio‐informatic assistance. This work was supported by a grant from the Stichting Sophia Kinderziekenhuis Fonds to MMA and RMWH (S17‐18). Funding Information: We would like to thank R.M. Hoogenboezem (Department of Hematology, Erasmus Medical Center) for processing our raw single-cell data and M.P. Verhagen (Department of Pathology, Erasmus Medical Center) for fruitful discussions and bio-informatic assistance. This work was supported by a grant from the Stichting Sophia Kinderziekenhuis Fonds to MMA and RMWH (S17-18). Publisher Copyright: © 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

PY - 2023/4/5

Y1 - 2023/4/5

N2 - Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

AB - Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

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ER -

A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine

Abstract

Bibliographical note

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