Gastroenterology

Gastroenterology

Volume 152, Issue 6, May 2017, Pages 1407-1418
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Optogenetic Demonstration of Functional Innervation of Mouse Colon by Neurons Derived From Transplanted Neural Cells

https://doi.org/10.1053/j.gastro.2017.01.005Get rights and content

Background & Aims

Cell therapy offers the potential to treat gastrointestinal motility disorders caused by diseased or absent enteric neurons. We examined whether neurons generated from transplanted enteric neural cells provide a functional innervation of bowel smooth muscle in mice.

Methods

Enteric neural cells expressing the light-sensitive ion channel, channelrhodopsin, were isolated from the fetal or postnatal mouse bowel and transplanted into the distal colon of 3- to 4-week-old wild-type recipient mice. Intracellular electrophysiological recordings of responses to light stimulation of the transplanted cells were made from colonic smooth muscle cells in recipient mice. Electrical stimulation of endogenous enteric neurons was used as a control.

Results

The axons of graft-derived neurons formed a plexus in the circular muscle layer. Selective stimulation of graft-derived cells by light resulted in excitatory and inhibitory junction potentials, the electrical events underlying contraction and relaxation, respectively, in colonic muscle cells. Graft-derived excitatory and inhibitory motor neurons released the same neurotransmitters as endogenous motor neurons—acetylcholine and a combination of adenosine triphosphate and nitric oxide, respectively. Graft-derived neurons also included interneurons that provided synaptic inputs to motor neurons, but the pharmacologic properties of interneurons varied with the age of the donors from which enteric neural cells were obtained.

Conclusions

Enteric neural cells transplanted into the bowel give rise to multiple functional types of neurons that integrate and provide a functional innervation of the smooth muscle of the bowel wall. Circuits composed of both motor neurons and interneurons were established, but the age at which cells are isolated influences the neurotransmitter phenotype of interneurons that are generated.

Section snippets

Mice

Wnt1::Cre mice26 were time plug−mated to Ai32(RCL-ChR2(H134R)/EYFP) mice (stock no: 012569; The Jackson Laboratory, Bar Harbor, ME), which express ChR2/EYFP fusion protein after exposure to Cre recombinase. Neurospheres were generated from the gut of embryonic (E)14.5 or postnatal (P)3−5 Wnt1::Cre;ChR2-EYFP mice. Wild-type C57BL/6 mice at 3−4 weeks of age were used as recipients.

Isolation and Culture of Enteric Neural Cells as Neurospheres

Enteric neurospheres were generated as described previously21 (see Supplementary Material for details). Neurospheres

Enteric Neural Cells Transplanted Into the Colon Generate Neurons That Project Axons to the Circular Muscle and Myenteric Ganglia of the Recipient

Enteric neural crest-derived cells expressing the light-sensitive ion channel ChR2 were isolated from E14.5 or P3−5 Wnt1::Cre;ChR2EYFP mice and grown in low attachment wells as described previously.21 Seven days after isolation from both the fetal or postnatal bowel, enteric neural cells formed neurospheres around 200 μm in diameter (Figure 1A). Neurospheres generated from E14.5 Wnt1::Cre;ChR2EYFP mice were termed fetal neurospheres and neurospheres generated from P3−5 Wnt1::Cre;ChR2EYFP mice

Discussion

Cell therapy offers the potential to treat diseases of the ENS.4, 9, 11 Significant progress has been made in the isolation of enteric neural cells from the bowel of humans and rodents, and in transplantation of cells into the bowel of rodents.7, 15, 17, 20, 22, 23, 37, 38, 39, 40 However, it had not been possible to determine whether transplanted cells generate different functional classes of enteric neurons, including interneurons and excitatory and inhibitory motor neurons, which are

Acknowledgments

The authors thank Annette Bergner and Jan Morgan for excellent technical assistance, and Vanda Lennon and Piers Emson for generously providing antisera.

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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was supported by National Health and Medical Research Council (NHMRC) project grant APP1079234 to Heather M. Young, Lincon A. Stamp, and Joel C. Bornstein, and NHMRC Senior Fellowship APP1002506 to Heather M. Young. The study sponsor played no role in study design or in the collection, analysis, and interpretation of data.

    Author names in bold designate shared co-first authorship.

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