Isolation of esophageal stem cells with potential for therapy

Panagiotis Maghsoudlou; Daniel Ditchfield; Dorota H.K. Klepacka; Panicos Shangaris; Luca Urbani; Stavros P. Loukogeorgakis; Simon Eaton; Paolo De Coppi

doi:10.1007/s00383-014-3615-6

Isolation of esophageal stem cells with potential for therapy

Panagiotis Maghsoudlou, Daniel Ditchfield, Dorota H.K. Klepacka, Panicos Shangaris, Luca Urbani, Stavros P. Loukogeorgakis, Simon Eaton, Paolo De Coppi^*

^*Corresponding author for this work

Women & Children's Health

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

6 Citations (Scopus)

Abstract

Purpose

Long-gap esophageal atresia represents a significant challenge for pediatric surgeons and current surgical approaches are associated with significant morbidity. A tissue-engineered esophagus, comprising cells seeded onto a scaffold, represents a therapeutic alternative. In this study, we aimed to determine the optimal techniques for isolation and culture of mouse esophageal epithelial cells and to isolate CD34-positive esophageal epithelial stem cells from cadaveric mouse specimens.

Methods

Primary epithelial cells were isolated from mouse esophagi by enzymatic dissociation from the mucosal layer (Dispase, Trypsin/EDTA) using three different protocols. In protocol A, isolated mucosa was minced and incubated with trypsin once. In protocol B, intact mucosal sheets underwent two trypsin incubations yielding a single-cell suspension. In protocol C, intact mucosa explants were plated epithelial side down. Epithelial cells were cultured on collagen-coated wells.

Results

Initial findings showed that Protocol B gave the best results in terms of yield, viability, and least contamination with different cell types and microbes. Esophageal epithelial cells isolated using Protocol B were stained for CD34 and sorted using fluorescence-activated cell sorting (FACS). Of the total cells sorted, 8.3 % (2–11.3) [%median (range)] were CD34 positive.

Conclusions

Our results demonstrate that mouse esophageal epithelial cells can be successfully isolated from fresh mouse esophagi using two consecutive trypsin incubations of intact mucosal sheets. Furthermore, the cells obtained using this method were successfully stained for CD34, a putative esophageal epithelial stem cell marker. Further research into the factors necessary for the successful proliferation of CD34 positive stem cell lines is needed to progress toward clinical application.

Original language	English
Pages (from-to)	1249-1256
Number of pages	8
Journal	Pediatric Surgery International
Volume	30
Issue number	12
Early online date	30 Oct 2014
DOIs	https://doi.org/10.1007/s00383-014-3615-6
Publication status	Published - Dec 2014

Keywords

Esophageal stem cells
Esophagus
Tissue engineering

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10.1007/s00383-014-3615-6Licence: CC BY

Cite this

@article{efed08ef7b524b688d2bb7715afb1166,

title = "Isolation of esophageal stem cells with potential for therapy",

abstract = "PurposeLong-gap esophageal atresia represents a significant challenge for pediatric surgeons and current surgical approaches are associated with significant morbidity. A tissue-engineered esophagus, comprising cells seeded onto a scaffold, represents a therapeutic alternative. In this study, we aimed to determine the optimal techniques for isolation and culture of mouse esophageal epithelial cells and to isolate CD34-positive esophageal epithelial stem cells from cadaveric mouse specimens.MethodsPrimary epithelial cells were isolated from mouse esophagi by enzymatic dissociation from the mucosal layer (Dispase, Trypsin/EDTA) using three different protocols. In protocol A, isolated mucosa was minced and incubated with trypsin once. In protocol B, intact mucosal sheets underwent two trypsin incubations yielding a single-cell suspension. In protocol C, intact mucosa explants were plated epithelial side down. Epithelial cells were cultured on collagen-coated wells.ResultsInitial findings showed that Protocol B gave the best results in terms of yield, viability, and least contamination with different cell types and microbes. Esophageal epithelial cells isolated using Protocol B were stained for CD34 and sorted using fluorescence-activated cell sorting (FACS). Of the total cells sorted, 8.3 % (2–11.3) [%median (range)] were CD34 positive.ConclusionsOur results demonstrate that mouse esophageal epithelial cells can be successfully isolated from fresh mouse esophagi using two consecutive trypsin incubations of intact mucosal sheets. Furthermore, the cells obtained using this method were successfully stained for CD34, a putative esophageal epithelial stem cell marker. Further research into the factors necessary for the successful proliferation of CD34 positive stem cell lines is needed to progress toward clinical application.",

keywords = "Esophageal stem cells, Esophagus, Tissue engineering",

author = "Panagiotis Maghsoudlou and Daniel Ditchfield and Klepacka, {Dorota H.K.} and Panicos Shangaris and Luca Urbani and Loukogeorgakis, {Stavros P.} and Simon Eaton and {De Coppi}, Paolo",

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doi = "10.1007/s00383-014-3615-6",

language = "English",

volume = "30",

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journal = "Pediatric Surgery International",

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T1 - Isolation of esophageal stem cells with potential for therapy

AU - Maghsoudlou, Panagiotis

AU - Ditchfield, Daniel

AU - Klepacka, Dorota H.K.

AU - Shangaris, Panicos

AU - Urbani, Luca

AU - Loukogeorgakis, Stavros P.

AU - Eaton, Simon

AU - De Coppi, Paolo

PY - 2014/12

Y1 - 2014/12

N2 - PurposeLong-gap esophageal atresia represents a significant challenge for pediatric surgeons and current surgical approaches are associated with significant morbidity. A tissue-engineered esophagus, comprising cells seeded onto a scaffold, represents a therapeutic alternative. In this study, we aimed to determine the optimal techniques for isolation and culture of mouse esophageal epithelial cells and to isolate CD34-positive esophageal epithelial stem cells from cadaveric mouse specimens.MethodsPrimary epithelial cells were isolated from mouse esophagi by enzymatic dissociation from the mucosal layer (Dispase, Trypsin/EDTA) using three different protocols. In protocol A, isolated mucosa was minced and incubated with trypsin once. In protocol B, intact mucosal sheets underwent two trypsin incubations yielding a single-cell suspension. In protocol C, intact mucosa explants were plated epithelial side down. Epithelial cells were cultured on collagen-coated wells.ResultsInitial findings showed that Protocol B gave the best results in terms of yield, viability, and least contamination with different cell types and microbes. Esophageal epithelial cells isolated using Protocol B were stained for CD34 and sorted using fluorescence-activated cell sorting (FACS). Of the total cells sorted, 8.3 % (2–11.3) [%median (range)] were CD34 positive.ConclusionsOur results demonstrate that mouse esophageal epithelial cells can be successfully isolated from fresh mouse esophagi using two consecutive trypsin incubations of intact mucosal sheets. Furthermore, the cells obtained using this method were successfully stained for CD34, a putative esophageal epithelial stem cell marker. Further research into the factors necessary for the successful proliferation of CD34 positive stem cell lines is needed to progress toward clinical application.

AB - PurposeLong-gap esophageal atresia represents a significant challenge for pediatric surgeons and current surgical approaches are associated with significant morbidity. A tissue-engineered esophagus, comprising cells seeded onto a scaffold, represents a therapeutic alternative. In this study, we aimed to determine the optimal techniques for isolation and culture of mouse esophageal epithelial cells and to isolate CD34-positive esophageal epithelial stem cells from cadaveric mouse specimens.MethodsPrimary epithelial cells were isolated from mouse esophagi by enzymatic dissociation from the mucosal layer (Dispase, Trypsin/EDTA) using three different protocols. In protocol A, isolated mucosa was minced and incubated with trypsin once. In protocol B, intact mucosal sheets underwent two trypsin incubations yielding a single-cell suspension. In protocol C, intact mucosa explants were plated epithelial side down. Epithelial cells were cultured on collagen-coated wells.ResultsInitial findings showed that Protocol B gave the best results in terms of yield, viability, and least contamination with different cell types and microbes. Esophageal epithelial cells isolated using Protocol B were stained for CD34 and sorted using fluorescence-activated cell sorting (FACS). Of the total cells sorted, 8.3 % (2–11.3) [%median (range)] were CD34 positive.ConclusionsOur results demonstrate that mouse esophageal epithelial cells can be successfully isolated from fresh mouse esophagi using two consecutive trypsin incubations of intact mucosal sheets. Furthermore, the cells obtained using this method were successfully stained for CD34, a putative esophageal epithelial stem cell marker. Further research into the factors necessary for the successful proliferation of CD34 positive stem cell lines is needed to progress toward clinical application.

KW - Esophageal stem cells

KW - Esophagus

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SN - 0179-0358

VL - 30

SP - 1249

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JO - Pediatric Surgery International

JF - Pediatric Surgery International

IS - 12

ER -

Isolation of esophageal stem cells with potential for therapy

Abstract

Keywords

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