Method Article

Single-cell RNA Sequencing for Profiling Ganglionic and Aganglionic Colonic Segments from Patients with Hirschsprung Disease

DOI:

10.3791/69825

⸱

May 22nd, 2026

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Gastrointestinal Surgery, The Second Affiliated Hospital of Zunyi Medical University

In This Article

Summary

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This protocol describes a single-cell RNA sequencing workflow to analyze immune-cell infiltration and cell-type-specific transcriptional differences between ganglionic and aganglionic colonic segments in Hirschsprung disease.

Abstract

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Hirschsprung disease (HSCR) is a congenital intestinal motility disorder characterized by the absence of enteric neurons in the distal bowel. Compared with bulk RNA sequencing, single-cell RNA sequencing (scRNA-seq) enables gene expression profiling at the single-cell level, resolving cellular heterogeneity that is masked in population-averaged analyses. A standardized scRNA-seq workflow was established to characterize the transcriptional landscape of ganglionic and aganglionic segments from patients with HSCR. Colon tissues were collected, and hematoxylin and eosin staining was performed to identify ganglionic and aganglionic regions. Tissues were enzymatically and mechanically dissociated into single-cell suspensions, followed by filtration, viability assessment, and construction of single-cell transcriptome libraries. Libraries that passed quality control were sequenced, and raw data were processed for cell quantification and quality filtering to remove low-quality cells and technical artifacts. After normalization and dimensionality reduction, cells were clustered and annotated based on established marker genes. Downstream analyses compared cell-type composition between ganglionic and aganglionic tissues and identified differentially expressed genes within key immune cell subsets. The results reveal substantial differences in the immune microenvironment between ganglionic and aganglionic segments in HSCR.

Introduction

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Hirschsprung disease (HSCR) is one of the most common congenital intestinal motility disorders, with an incidence of approximately 1 in 5,000 live births worldwide1. The disease arises from the failure of enteric neural crest cells (ENCCs) to migrate into the distal intestine during embryonic weeks 5–12, resulting in the absence of enteric ganglia in the affected segments2. The aganglionic segment consequently exhibits persistent spastic contraction and impaired peristalsis, leading to proximal intestinal dilation. Clinically, this manifests as functional obstruction, causing constipation, abdominal distension, and....

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Protocol

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This study was approved by the Institutional Ethics Committee of the Affiliated Hospital of Zunyi Medical University (Approval No.: KYLL-2024-030). The study complies with the Declaration of Helsinki. Informed consent (oral and written) was obtained from all participants and their guardians. Table 1 presents the information on HSCR patients.

Collect colonic tissues, including ganglionic and aganglionic segments, from HSCR patients diagnosed and undergoing surgery in the Department of Pediatric Surgery at the Affiliated Hospital of Zunyi Medical University. Include ganglionic and aganglionic segments from two male HSCR patie....

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Results

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Hematoxylin and eosin (HE) staining (Figure 1) revealed distinct histological differences between ganglionic and aganglionic segments. Compared with ganglionic segments, aganglionic segments exhibited increased mucosal exfoliation, a marked reduction in goblet cells and submucosal neurons, and blurred boundaries between the mucosal and muscular layers. In a subset of sections, tissue preservation was suboptimal, with partial epithelial detachment and disruption of tissue architecture, limiti.......

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Discussion

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Single-cell RNA sequencing was applied to profile transcriptional features of surgically resected colonic tissues (ganglionic and aganglionic segments) from patients with Hirschsprung diseases4,5. Particular attention was given to the tissue dissociation strategy, as dissociation parameters influence cell recovery, cell-type representation, and RNA integrity. Enzymatic digestion conditions were optimized to balance sufficient tissue dissociation with preservation.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported by Guizhou Science and Technology Project (Guizhou Science and Technology Cooperation - Foundation - ZK - [2024] General 344) (to X.F. Yang) and Zunyi Science and Technology Project Contract (Science and Technology Cooperation) (Zunyi Science and Technology Cooperation HZ (2023) No.) (to Y. Qu).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
BeeswaxSinopharm Chemical Reagent CO., Ltd.8012-89-3 https://www.reagent.com.cn/ProductSearch/8012-89-3
BeeswaxSinopharm Chemical Reagent CO., Ltd.8012-89-3 https://www.reagent.com.cn/ProductSearch/8012-89-3
Bovine serum albuminThermofisher30066575 https://www.thermofisher.cn/order/catalog/product/30066575?SID=srch-srp-30066575
Bovine serum albuminThermofisher30066575 https://www.thermofisher.cn/order/catalog/product/30066575?SID=srch-srp-30066575
CellTypistN/ANAhttp://celltypist.org/tutorials/onlineguide
CellTypistN/ANAhttp://celltypist.org/tutorials/onlineguide
Cluster profiler packageBioconductorv4.8.1https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html
Cluster profiler packageBioconductorv4.8.1https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html
Collagenase IIThermofisher17101015 https://www.thermofisher.cn/order/catalog/product/17101015?SID=srch-srp-17101015
Collagenase IIThermofisher17101015 https://www.thermofisher.cn/order/catalog/product/17101015?SID=srch-srp-17101015
Cover glassCITOTEST80340-0130 https://cn.citotest.com/pd40971476.html
Cover glassCITOTEST80340-0130 https://cn.citotest.com/pd40971476.html
Disposable microtome bladeFeatherS-35 https://www.feather.co.jp/en/m_Products/pathology01.html
Disposable microtome bladeFeatherS-35 https://www.feather.co.jp/en/m_Products/pathology01.html
DNase IThermofisher18047019 https://www.thermofisher.cn/order/catalog/product/18047019?SID=srch-srp-18047019
DNase IThermofisher18047019 https://www.thermofisher.cn/order/catalog/product/18047019?SID=srch-srp-18047019
Drying ovenBORUKDHG-9140A http://www.shboruike.com/sys-pd/84.html
Drying ovenBORUKDHG-9140A http://www.shboruike.com/sys-pd/84.html
Dulbecco’s Modified Eagle MediumThermofisher12491015 https://www.thermofisher.cn/order/catalog/product/12491015
Dulbecco’s Modified Eagle MediumThermofisher12491015 https://www.thermofisher.cn/order/catalog/product/12491015
Ethyl alcoholSinopharm Chemical Reagent CO., Ltd.10009218 https://www.reagent.com.cn/ProductSearch/10009218
Ethyl alcoholSinopharm Chemical Reagent CO., Ltd.10009218 https://www.reagent.com.cn/ProductSearch/10009218
Fetal bovine serumThermofisherA5669801https://www.thermofisher.cn/order/catalog/product/A5669801
Fetal bovine serumThermofisherA5669801https://www.thermofisher.cn/order/catalog/product/A5669801
Hematoxylin and eosin staining kitBeyotimeC0105S https://m.beyotime.com/mobilegoods.do?method=code&code=C0105S
Hematoxylin and eosin staining kitBeyotimeC0105S https://m.beyotime.com/mobilegoods.do?method=code&code=C0105S
Hydrochloric acidSinopharm Chemical Reagent CO., Ltd.10011028 https://www.reagent.com.cn/ProductSearch/10011028
Hydrochloric acidSinopharm Chemical Reagent CO., Ltd.10011028 https://www.reagent.com.cn/ProductSearch/10011028
Microscope slideCITOTEST80302-3111 https://cn.citotest.com/pd530614548.html
Microscope slideCITOTEST80302-3111 https://cn.citotest.com/pd530614548.html
Mounting mediumBeyotimeC0173-100ml https://m.beyotime.com/mobilegoods.do?method=code&code=C0173-100ml
Mounting mediumBeyotimeC0173-100ml https://m.beyotime.com/mobilegoods.do?method=code&code=C0173-100ml
Optical microscopeNikonECLIPSE SI https://www.microscope.healthcare.nikon.com/ja_JP/products/upright-microscopes/eclipse-si
Optical microscopeNikonECLIPSE SI https://www.microscope.healthcare.nikon.com/ja_JP/products/upright-microscopes/eclipse-si
Paraffin wax Sinopharm Chemical Reagent CO., Ltd.8002-74-2 https://www.reagent.com.cn/ProductSearch/8002-74-2
Paraffin wax Sinopharm Chemical Reagent CO., Ltd.8002-74-2 https://www.reagent.com.cn/ProductSearch/8002-74-2
Paraformaldehyde Fix SolutionBeyotimeP0099-3L https://m.beyotime.com/mobilegoods.do?method=code&code=P0099-3L
Paraformaldehyde Fix SolutionBeyotimeP0099-3L https://m.beyotime.com/mobilegoods.do?method=code&code=P0099-3L
Penicillin–streptomycinThermofisher15140122 https://www.thermofisher.cn/order/catalog/product/15140122?SID=srch-srp-15140122
Penicillin–streptomycinThermofisher15140122 https://www.thermofisher.cn/order/catalog/product/15140122?SID=srch-srp-15140122
Phosphate-buffered salineThermofisher10010023 https://www.thermofisher.cn/order/catalog/product/10010023
Phosphate-buffered salineThermofisher10010023 https://www.thermofisher.cn/order/catalog/product/10010023
Qualitative filter paperSolarbioYA0166https://www.solarbio.com
Qualitative filter paperSolarbioYA0166https://www.solarbio.com
R software packageR Foundation for Statistical Computingv4.2.2https://www.r-project.org/
R software packageR Foundation for Statistical Computingv4.2.2https://www.r-project.org/
Rotary MicrotomeLeicaLeica RM2125 RTShttps://www.leicabiosystems.com/en-jp/histology-equipment/microtomes/histocore-biocut/
Rotary MicrotomeLeicaLeica RM2125 RTShttps://www.leicabiosystems.com/en-jp/histology-equipment/microtomes/histocore-biocut/
ScanpyCommunity-developedv1.9.3https://scanpy.readthedocs.io/en/latest/?utm_source=chatgpt.com
ScanpyCommunity-developedv1.9.3https://scanpy.readthedocs.io/en/latest/?utm_source=chatgpt.com
SeuratCRANv4.3.0https://cran.r-project.org/web/packages/Seurat/index.html
SeuratCRANv4.3.0https://cran.r-project.org/web/packages/Seurat/index.html
Tissue dehydratorWuhan Junjie Electronics Co., LTDJT-12H http://www.whjjbl.com/front/page/126495/product_id/46166.html
Tissue dehydratorWuhan Junjie Electronics Co., LTDJT-12H http://www.whjjbl.com/front/page/126495/product_id/46166.html
Tissue embedding machineWuhan Junjie Electronics Co., LTDJB-L8 http://www.whjjbl.com/front/page/126495/product_id/46064.html
Tissue embedding machineWuhan Junjie Electronics Co., LTDJB-L8 http://www.whjjbl.com/front/page/126495/product_id/46064.html
Trypan blue SolarbioC0040https://www.solarbio.com/haveRegion?id=48&all=0&key=%20Trypan%20Blue
Trypan blue SolarbioC0040https://www.solarbio.com/haveRegion?id=48&all=0&key=%20Trypan%20Blue
XyleneSinopharm Chemical Reagent CO., Ltd.10023418 https://www.reagent.com.cn/ProductSearch/10023418
XyleneSinopharm Chemical Reagent CO., Ltd.10023418 https://www.reagent.com.cn/ProductSearch/10023418

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Tags

Single Cell RNA SequencingHirschsprung DiseaseGanglionic ColonAganglionic ColonGene Expression ProfilingEnteric NeuronsCell Type CompositionImmune MicroenvironmentTissue DissociationDifferential Gene Expression

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