Enteric Neurospheres Are Not Specific to Neural Crest Cultures: Implications for Neural Stem Cell Therapies

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Abstract

Objectives

Enteric neural stem cells provide hope of curative treatment for enteric neuropathies. Current protocols for their harvesting from humans focus on the generation of ‘neurospheres’ from cultures of dissociated gut tissue. The study aims to better understand the derivation, generation and composition of enteric neurospheres.

Design

Gut tissue was obtained from Wnt1-Cre;Rosa26 ^Yfp/Yfp transgenic mice (constitutively labeled neural crest cells) and paediatric patients. Gut cells were cultured either unsorted (mixed neural crest/non-neural crest), or following FACS selection into neural crest (murine-YFP+ve/human-p75+ve) or non-neural crest (YFP-ve/p75-ve) populations. Cultures and resultant neurospheres were characterized using immunolabelling in vitro and following transplantation in vivo.

Results

Cultures of (i) unsorted, (ii) neural crest, and (iii) non-neural crest cell populations generated neurospheres similar in numbers, size and morphology. Unsorted neurospheres were highly heterogeneous for neural crest content. Neural crest-derived (YFP+ve/p75+ve) neurospheres contained only neural derivatives (neurons and glia) and were devoid of non-neural cells (i.e. negative for SMA, c-Kit), with the converse true for non-neural crest-derived (YFP-ve/p75-ve) ‘neurospheres’. Under differentiation conditions only YFP+ve cells gave rise to neural derivatives. Both YFP+ve and YFP-ve cells displayed proliferation and spread upon transplantation in vivo, but YFP-ve cells did not locate or integrate within the host ENS.

Conclusions

Spherical accumulations of cells, so-called ‘neurospheres’ forming in cultures of dissociated gut contain variable proportions of neural crest-derived cells. If they are to be used for ENS cell replacement therapy then improved protocols for their generation, including cell selection, should be sought in order to avoid inadvertent transplantation of non-therapeutic, non-ENS cells.

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Most cited references 33

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Fate of the mammalian cardiac neural crest.

X Jiang, D Rowitch, P Soriano … (2000)

A subpopulation of neural crest termed the cardiac neural crest is required in avian embryos to initiate reorganization of the outflow tract of the developing cardiovascular system. In mammalian embryos, it has not been previously experimentally possible to study the long-term fate of this population, although there is strong inference that a similar population exists and is perturbed in a number of genetic and teratogenic contexts. We have employed a two-component genetic system based on Cre/lox recombination to label indelibly the entire mouse neural crest population at the time of its formation, and to detect it at any time thereafter. Labeled cells are detected throughout gestation and in postnatal stages in major tissues that are known or predicted to be derived from neural crest. Labeling is highly specific and highly efficient. In the region of the heart, neural-crest-derived cells surround the pharyngeal arch arteries from the time of their formation and undergo an altered distribution coincident with the reorganization of these vessels. Labeled cells populate the aorticopulmonary septum and conotruncal cushions prior to and during overt septation of the outflow tract, and surround the thymus and thyroid as these organs form. Neural-crest-derived mesenchymal cells are abundantly distributed in midgestation (E9.5-12.5), and adult derivatives of the third, fourth and sixth pharyngeal arch arteries retain a substantial contribution of labeled cells. However, the population of neural-crest-derived cells that infiltrates the conotruncus and which surrounds the noncardiac pharyngeal organs is either overgrown or selectively eliminated as development proceeds, resulting for these tissues in a modest to marginal contribution in late fetal and postnatal life.

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Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors.

R Zufferey, John Donello, D Trono … (1999)

The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy. Here, we explored the possibility that the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) might help resolve this problem. Insertion of the WPRE in the 3' untranslated region of coding sequences carried by either oncoretroviral or lentiviral vectors substantially increased their levels of expression in a transgene-, promoter- and vector-independent manner. The WPRE thus increased either luciferase or green fluorescent protein production five- to eightfold, and effects of a comparable magnitude were observed with either the immediate-early cytomegalovirus or the herpesvirus thymidine kinase promoter and with both human immunodeficiency virus- and murine leukemia virus-based vectors. The WPRE exerted this influence only when placed in the sense orientation, consistent with its predicted posttranscriptional mechanism of action. These results demonstrate that the WPRE significantly improves the performance of retroviral vectors and emphasize that posttranscriptional regulation of gene expression should be taken into account in the design of gene delivery systems.

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Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury.

Catia Laranjeira, Katarina Sandgren, Nicoletta Kessaris … (2011)

The enteric nervous system (ENS) in mammals forms from neural crest cells during embryogenesis and early postnatal life. Nevertheless, multipotent progenitors of the ENS can be identified in the adult intestine using clonal cultures and in vivo transplantation assays. The identity of these neurogenic precursors in the adult gut and their relationship to the embryonic progenitors of the ENS are currently unknown. Using genetic fate mapping, we here demonstrate that mouse neural crest cells marked by SRY box-containing gene 10 (Sox10) generate the neuronal and glial lineages of enteric ganglia. Most neurons originated from progenitors residing in the gut during mid-gestation. Afterward, enteric neurogenesis was reduced, and it ceased between 1 and 3 months of postnatal life. Sox10-expressing cells present in the myenteric plexus of adult mice expressed glial markers, and we found no evidence that these cells participated in neurogenesis under steady-state conditions. However, they retained neurogenic potential, as they were capable of generating neurons with characteristics of enteric neurons in culture. Furthermore, enteric glia gave rise to neurons in vivo in response to chemical injury to the enteric ganglia. Our results indicate that despite the absence of constitutive neurogenesis in the adult gut, enteric glia maintain limited neurogenic potential, which can be activated by tissue dissociation or injury.

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Author and article information

Contributors

Mirella Dottori: Role: Academic Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 23 March 2015

Publication date Collection: 2015

Volume: 10

Issue: 3

Electronic Location Identifier: e0119467

Affiliations

[1 ]Stem Cells and Regenerative Medicine, UCL Institute of Child Health, London, United Kingdom

[2 ]Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands

[3 ]Department of Gastroenterology, Great Ormond Street Hospital for Children, London, United Kingdom

University Of Melbourne, AUSTRALIA

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: NT AJB EB DN. Performed the experiments: EB DN JC RK MC. Analyzed the data: EB DN JC RK MC AJB NT. Contributed reagents/materials/analysis tools: EB DN JC RK MC AJB NT. Wrote the paper: EB DN JC RK MC JMD CM AJB NT.

[¤a]

Current address: Royal Hospital for Sick Children, Glasgow, United Kingdom

[¤b]

Current address: Unit of Pediatric Gastroenterology, Digestive Endoscopy, Hepatology and Care of the Child with Liver Transplantation, Department for Woman and Child Health, University Hospital of Padova, Padova, Italy

[¤c]

Current address: Centre for Digestive Diseases, Barts & The London School of Medicine & Dentistry, Queen Mary, University of London, London, United Kingdom

* E-mail: n.thapar@ 123456ucl.ac.uk (NT); alan.burns@ 123456ucl.ac.uk (AJB)

Article

Publisher ID: PONE-D-14-37730

DOI: 10.1371/journal.pone.0119467

PMC ID: 4370605

PubMed ID: 25799576

SO-VID: a4edf051-bd0d-4f5a-9cc5-f5ae7060775a

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

History

Date received : 29 August 2014

Date accepted : 27 January 2015

Page count

Figures: 7, Tables: 1, Pages: 18

Funding

This work was supported by Great Ormond Street Hospital Children's Charity, W1018C (NT and AJB); Great Ormond Street Hospital Children's Charity, V1222 (NT); and Medical Research Council, G0800973 (NT and AJB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Data Availability All relevant data are within the paper and its Supporting Information files.

Enteric Neurospheres Are Not Specific to Neural Crest Cultures: Implications for Neural Stem Cell Therapies

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Most cited references 33

Fate of the mammalian cardiac neural crest.

Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors.

Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury.

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