title: In Vivo Transplantation of Enteric Neural Crest Cells into Mouse Gut; Engraftment, Functional Integration and Long-Term Safety.
abstract: OBJECTIVE
  Enteric neuropathies are severe gastrointestinal disorders with unsatisfactory outcomes. We aimed to investigate the potential of enteric neural stem cell therapy approaches for such disorders by transplanting mouse enteric neural crest cells (ENCCs) into ganglionic and aganglionic mouse gut in vivo and analysing functional integration and long-term safety.
  
  
  METHODS
  Neurospheres generated from yellow fluorescent protein (YFP) expressing ENCCs selected from postnatal Wnt1-cre;R26R-YFP/YFP murine gut were transplanted into ganglionic hindgut of wild-type littermates or aganglionic hindgut of Ednrbtm1Ywa mice (lacking functional endothelin receptor type-B). Intestines were then assessed for ENCC integration and differentiation using immunohistochemistry, cell function using calcium imaging, and long-term safety using PCR to detect off-target YFP expression.
  
  
  RESULTS
  YFP+ ENCCs engrafted, proliferated and differentiated into enteric neurons and glia within recipient ganglionic gut. Transplanted cells and their projections spread along the endogenous myenteric plexus to form branching networks. Electrical point stimulation of endogenous nerve fibres resulted in calcium transients (F/F0 = 1.16 ± 0.01;43 cells, n = 6) in YFP+ transplanted ENCCs (abolished with TTX). Long-term follow-up (24 months) showed transplanted ENCCs did not give rise to tumours or spread to other organs (PCR negative in extraintestinal sites). In aganglionic gut ENCCs similarly spread and differentiated to form neuronal and glial networks with projections closely associated with endogenous neural networks of the transition zone.
  
  
  CONCLUSIONS
  Transplanted ENCCs successfully engrafted into recipient ganglionic and aganglionic gut showing appropriate spread, localisation and, importantly, functional integration without any long-term safety issues. This study provides key support for the development and use of enteric neural stem cell therapies.
authors.affiliation: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, 30 Guilford Street, London, United Kingdom.
authors.forename: Julie E
authors.initials: JE
authors.lastname: Cooper
authors.affiliation: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, 30 Guilford Street, London, United Kingdom.
authors.forename: Conor J
authors.initials: CJ
authors.lastname: McCann
authors.affiliation: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, 30 Guilford Street, London, United Kingdom.
authors.forename: Dipa
authors.initials: D
authors.lastname: Natarajan
authors.affiliation: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, 30 Guilford Street, London, United Kingdom.
authors.forename: Shanas
authors.initials: S
authors.lastname: Choudhury
authors.affiliation: Laboratory for Enteric NeuroScience, Translational Research Center for GastroIntestinal Disorders, Department of Clinical and Experimental Medicine, University of Leuven, Leuven, Belgium.
authors.forename: Werend
authors.initials: W
authors.lastname: Boesmans
authors.affiliation: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, 30 Guilford Street, London, United Kingdom.|Centre for Digestive Diseases, Blizard Building, Barts & The London School of Medicine & Dentistry, Queen Mary, University of London, London, United Kingdom.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Delalande
authors.affiliation: Laboratory for Enteric NeuroScience, Translational Research Center for GastroIntestinal Disorders, Department of Clinical and Experimental Medicine, University of Leuven, Leuven, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Vanden Berghe
authors.affiliation: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, 30 Guilford Street, London, United Kingdom.|Department of Clinical Genetics, Erasmus MC University Medical Centre, Rotterdam, The Netherlands.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Burns
authors.affiliation: Stem Cells and Regenerative Medicine, UCL Institute of Child Health, 30 Guilford Street, London, United Kingdom.|Department of Gastroenterology, Great Ormond Street Hospital NHS Foundation Trust, Great Ormond Street, London, United Kingdom.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Thapar
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D060846:Cell Engineering; D064987:Cell- and Tissue-Based Therapy / Q000379:methods*; D004558:Electric Stimulation; D015870:Gene Expression; D006085:Graft Survival; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology* / Q000294:innervation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009412:Nerve Fibers / Q000378:metabolism; D009432:Neural Crest / Q000166:cytology* / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D009457:Neuroglia / Q000166:cytology* / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D044023:Receptor, Endothelin B / Q000235:genetics / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D019076:Transgenes
chemical_list: D001426:Bacterial Proteins; D015415:Biomarkers; C000602969:EDNRB protein, mouse; D008164:Luminescent Proteins; D044023:Receptor, Endothelin B; C054562:yellow fluorescent protein, Bacteria; D002118:Calcium
references.citation: Development. 1992 Sep;116(1):167-75
references.pmid: 1483385
references.citation: Int J Colorectal Dis. 2006 Sep;21(6):554-9
references.pmid: 16267668
references.citation: Am J Physiol Gastrointest Liver Physiol. 2014 Oct 1;307(7):G741-8
references.pmid: 25125684
references.citation: J Pediatr Surg. 2014 Dec;49(12):1809-14
references.pmid: 25487489
references.citation: IEEE Trans Image Process. 1998;7(1):27-41
references.pmid: 18267377
references.citation: Neurogastroenterol Motil. 2004 Oct;16(5):515-31
references.pmid: 15500508
references.citation: Neurogastroenterol Motil. 2013 Jan;25(1):61-9.e7
references.pmid: 22998406
references.citation: J Clin Invest. 2011 Sep;121(9):3412-24
references.pmid: 21865647
references.citation: PLoS One. 2015;10(3):e0119467
references.pmid: 25799576
references.citation: Neurogastroenterol Motil. 2013 Jul;25(7):601-e464
references.pmid: 23534441
references.citation: Gut. 2007 Apr;56(4):489-96
references.pmid: 16973717
references.citation: Adv Parasitol. 2011;76:195-233
references.pmid: 21884893
references.citation: Curr Biol. 1998 Dec 3;8(24):1323-6
references.pmid: 9843687
references.citation: Am J Physiol Gastrointest Liver Physiol. 2010 Feb;298(2):G222-32
references.pmid: 19959818
references.citation: J Pediatr Gastroenterol Nutr. 2011 Dec;53 Suppl 2:S43-5
references.pmid: 22235473
references.citation: Gastroenterology. 2009 Dec;137(6):2063-2073.e4
references.pmid: 19549531
references.citation: Development. 2006 May;133(10):2075-86
references.pmid: 16624853
references.citation: Neurogastroenterol Motil. 2010 Mar;22(3):331-e86
references.pmid: 19775251
references.citation: Dev Biol. 2012 Jun 1;366(1):64-73
references.pmid: 22290331
references.citation: Front Cell Neurosci. 2013 Oct 21;7:183
references.pmid: 24155689
references.citation: PLoS One. 2014;9(5):e97792
references.pmid: 24871092
references.citation: J Neurosci. 2011 Oct 26;31(43):15352-61
references.pmid: 22031881
references.citation: Neurogastroenterol Motil. 2013 Jan;25(1):70-8.e8-9
references.pmid: 23030503
references.citation: Nat Methods. 2012 Jul;9(7):671-5
references.pmid: 22930834
references.citation: Hepatogastroenterology. 2009 Nov-Dec;56(96):1670-4
references.pmid: 20214215
references.citation: BMC Dev Biol. 2001;1:4
references.pmid: 11299042
references.citation: Gastroenterology. 2009 Jun;136(7):2214-25.e1-3
references.pmid: 19505425
references.citation: Nat Methods. 2012 Jul;9(7):676-82
references.pmid: 22743772
references.citation: Br J Surg. 2001 Apr;88(4):545-52
references.pmid: 11298623
references.citation: J Pharmacol Exp Ther. 1983 Nov;227(2):538-44
references.pmid: 6195330
references.citation: Neurogastroenterol Motil. 2006 Oct;18(10):876-87
references.pmid: 16961690
references.citation: World J Gastroenterol. 2012 Jun 28;18(24):3050-7
references.pmid: 22791940
references.citation: J Physiol Sci. 2016 Jan;66(1):67-76
references.pmid: 26335766
references.citation: J Neurogastroenterol Motil. 2015 Oct 1;21(4):552-9
references.pmid: 26424040
references.citation: Int J Dev Biol. 2005;49(2-3):143-50
references.pmid: 15906227
references.citation: J Clin Invest. 2013 Mar;123(3):1182-91
references.pmid: 23454768
references.citation: Gastroenterology. 2002 Nov;123(5):1459-67
references.pmid: 12404220
references.citation: J Pediatr Surg. 2014 Aug;49(8):1319-24; discussion 1324-5
references.pmid: 25092099
references.citation: Curr Opin Pharmacol. 2011 Dec;11(6):617-23
references.pmid: 22056114
references.citation: J Pediatr Surg. 2009 Jun;44(6):1249-55; discussion 1255-6
references.pmid: 19524749
references.citation: Nat Rev Gastroenterol Hepatol. 2014 May;11(5):317-28
references.pmid: 24322895
references.citation: Auton Neurosci. 2006 Apr 30;125(1-2):81-5
references.pmid: 16476573
references.citation: Gastroenterology. 2008 Jul;135(1):205-216.e6
references.pmid: 18515088
references.citation: Cytotechnology. 2015 Aug;67(4):661-70
references.pmid: 25230796
references.citation: Development. 2003 Dec;130(25):6387-400
references.pmid: 14623827
references.citation: Wiley Interdiscip Rev Dev Biol. 2013 Jan-Feb;2(1):113-29
references.pmid: 23799632
references.citation: J Med Genet. 2008 Jan;45(1):1-14
references.pmid: 17965226
references.citation: J Pediatr Surg. 2013 Mar;48(3):619-28
references.pmid: 23480922
references.citation: J Gastroenterol. 2003;38(5):421-30
references.pmid: 12768383
references.citation: PLoS One. 2014;9(4):e93605
references.pmid: 24699866
references.citation: Dev Biol. 2005 Nov 1;287(1):125-33
references.pmid: 16197939