Lincon A. Stamp

Transplanted progenitors generate functional enteric neurons in the postnatal colon

Cell therapy has the potential to treat gastrointestinal motility disorders caused by diseases of the enteric nervous system. Many studies have demonstrated that various stem/progenitor cells can give rise to functional neurons in the embryonic gut; however, it is not yet known whether transplanted neural progenitor cells can migrate, proliferate, and generate functional neurons in the postnatal bowel in vivo. We transplanted neurospheres generated from fetal and postnatal intestinal neural crest-derived cells into the colon of postnatal mice. The neurosphere-derived cells migrated, proliferated, and generated neurons and glial cells that formed ganglion-like clusters within the recipient colon. Graft-derived neurons exhibited morphological, neurochemical, and electrophysiological characteristics similar to those of enteric neurons; they received synaptic inputs; and their neurites projected to muscle layers and the enteric ganglia of the recipient mice. These findings show that transplanted enteric neural progenitor cells can generate functional enteric neurons in the postnatal bowel and advances the notion that cell therapy is a promising strategy for enteric neuropathies.

Hirschsprung disease: a developmental disorder of the enteric nervous system.

Hirschsprung disease (HSCR), which is also called congenital megacolon or intestinal aganglionosis, is characterized by an absence of enteric (intrinsic) neurons from variable lengths of the most distal bowel. Because enteric neurons are essential for propulsive intestinal motility, infants with HSCR suffer from severe constipation and have a distended abdomen. Currently the only treatment is surgical removal of the affected bowel. HSCR has an incidence of around 1:5,000 live births, with a 4:1 male:female gender bias. Most enteric neurons arise from neural crest cells that emigrate from the caudal hindbrain and then migrate caudally along the entire gut. The absence of enteric neurons from variable lengths of the bowel in HSCR results from a failure of neural crest‐derived cells to colonize the affected gut regions. HSCR is therefore regarded as a neurocristopathy. HSCR is a multigenic disorder and has become a paradigm for understanding complex factorial disorders. The major HSCR susceptibility gene is RET. The penetrance of several mutations in HSCR susceptibility genes is sex‐dependent. HSCR can occur as an isolated disorder or as part of syndromes; for example, Type IV Waardenburg syndrome is characterized by deafness and pigmentation defects as well as intestinal aganglionosis. Studies using animal models have shown that HSCR genes regulate multiple processes including survival, proliferation, differentiation, and migration. Research into HSCR and the development of enteric neurons is an excellent example of the cross fertilization of ideas that can occur between human molecular geneticists and researchers using animal models. WIREs Dev Biol 2013, 2:113–129. doi: 10.1002/wdev.57

Birthdating of myenteric neuron subtypes in the small intestine of the mouse.

There are many different types of enteric neurons. Previous studies have identified the time at which some enteric neuron subtypes are born (exit the cell cycle) in the mouse, but the birthdates of some major enteric neuron subtypes are still incompletely characterized or unknown. We combined 5‐ethynynl‐2′‐deoxyuridine (EdU) labeling with antibody markers that identify myenteric neuron subtypes to determine when neuron subtypes are born in the mouse small intestine. We found that different neurochemical classes of enteric neuron differed in their birthdates; serotonin neurons were born first with peak cell cycle exit at E11.5, followed by neurofilament‐M neurons, calcitonin gene‐related peptide neurons (peak cell cycle exit for both at embryonic day [E]12.5–E13.5), tyrosine hydroxylase neurons (E15.5), nitric oxide synthase 1 (NOS1) neurons (E15.5), and calretinin neurons (postnatal day [P]0). The vast majority of myenteric neurons had exited the cell cycle by P10. We did not observe any EdU+/NOS1+ myenteric neurons in the small intestine of adult mice following EdU injection at E10.5 or E11.5, which was unexpected, as previous studies have shown that NOS1 neurons are present in E11.5 mice. Studies using the proliferation marker Ki67 revealed that very few NOS1 neurons in the E11.5 and E12.5 gut were proliferating. However, Cre‐lox‐based genetic fate‐mapping revealed a small subpopulation of myenteric neurons that appears to express NOS1 only transiently. Together, our results confirm a relationship between enteric neuron subtype and birthdate, and suggest that some enteric neurons exhibit neurochemical phenotypes during development that are different from their mature phenotype. J. Comp. Neurol. 522:514–527, 2014.

Non-linear elasticity of core/shell spun PGS/PLLA fibres and their effect on cell proliferation

An efficient delivery system is critical for the success of cell therapy. To deliver cells to a dynamic organ, the biomaterial vehicle should mechanically match with the non-linearly elastic host tissue. In this study, non-linearly elastic biomaterials have been fabricated from a chemically crosslinked elastomeric poly(glycerol sebacate) (PGS) and thermoplastic poly(l-lactic acid) (PLLA) using the core/shell electrospinning technique. The spun fibrous materials containing a PGS core and PLLA shell demonstrate J-shaped stress-strain curves, having ultimate tensile strength (UTS), rupture elongation and stiffness constants of 1 ± 0.2 MPa, 25 ± 3% and 12 ± 2, respectively, which are comparable to skin tissue properties reported previously. Our ex vivo and in vivo trials have shown that the elastomeric mesh supports and fosters the growth of enteric neural crest (ENC) progenitor cells, and that the cell-seeded elastomeric fibrous sheet physically remains in intimate contact with guts after grafting, providing the effective delivery of the progenitor cells to an embryonic and post-natal gut environment.

Enteric Neural Cells From Hirschsprung Disease Patients Form Ganglia in Autologous Aneuronal Colon

Background & Aims Hirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells. Methods Cells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2′-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed. Results ENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia. Conclusions NC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient.

Genetic fate-mapping of tyrosine hydroxylase-expressing cells in the enteric nervous system

During development of the enteric nervous system, a subpopulation of enteric neuron precursors transiently expresses catecholaminergic properties. The progeny of these transiently catecholaminergic (TC) cells have not been fully characterized.

Optogenetic Demonstration of Functional Innervation of Mouse Colon by Neurons Derived From Transplanted Neural Cells

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.

Enteric neural progenitors are more efficient than brain-derived progenitors at generating neurons in the colon

Gut motility disorders can result from an absent, damaged, or dysfunctional enteric nervous system (ENS). Cell therapy is an exciting prospect to treat these enteric neuropathies and restore gut motility. Previous studies have examined a variety of sources of stem/progenitor cells, but the ability of different sources of cells to generate enteric neurons has not been directly compared. It is important to identify the source of stem/progenitor cells that is best at colonizing the bowel and generating neurons following transplantation. The aim of this study was to compare the ability of central nervous system (CNS) progenitors and ENS progenitors to colonize the colon and differentiate into neurons. Genetically labeled CNS- and ENS-derived progenitors were cocultured with aneural explants of embryonic mouse colon for 1 or 2.5 wk to assess their migratory, proliferative, and differentiation capacities, and survival, in the embryonic gut environment. Both progenitor cell populations were transplanted in the postnatal colon of mice in vivo for 4 wk before they were analyzed for migration and differentiation using immunohistochemistry. ENS-derived progenitors migrated further than CNS-derived cells in both embryonic and postnatal gut environments. ENS-derived progenitors also gave rise to more neurons than their CNS-derived counterparts. Furthermore, neurons derived from ENS progenitors clustered together in ganglia, whereas CNS-derived neurons were mostly solitary. We conclude that, within the gut environment, ENS-derived progenitors show superior migration, proliferation, and neuronal differentiation compared with CNS progenitors.

Exposure to GDNF Enhances the Ability of Enteric Neural Progenitors to Generate an Enteric Nervous System

Summary Cell therapy is a promising approach to generate an enteric nervous system (ENS) and treat enteric neuropathies. However, for translation to the clinic, it is highly likely that enteric neural progenitors will require manipulation prior to transplantation to enhance their ability to migrate and generate an ENS. In this study, we examine the effects of exposure to several factors on the ability of ENS progenitors, grown as enteric neurospheres, to migrate and generate an ENS. Exposure to glial-cell-line-derived neurotrophic factor (GDNF) resulted in a 14-fold increase in neurosphere volume and a 12-fold increase in cell number. Following co-culture with embryonic gut or transplantation into the colon of postnatal mice in vivo, cells derived from GDNF-treated neurospheres showed a 2-fold increase in the distance migrated compared with controls. Our data show that the ability of enteric neurospheres to generate an ENS can be enhanced by exposure to appropriate factors.

Different neural crest populations exhibit diverse proliferative behaviors.

The rate of proliferation of cells depends on the proportion of cycling cells and the frequency of cell division. Here, we describe in detail methods for quantifying the proliferative behavior of specific cell types in situ, and use the method to examine cell cycle dynamics in two neural crest derivatives—dorsal root ganglia (DRG) using frozen sections, and the enteric nervous system (ENS) using wholemount preparations. In DRG, our data reveal a significant increase in cell cycle length and a decrease in the number of cycling Sox10+ progenitor cells at E12.5–E13.5, which coincides with the commencement of glial cell generation. In the ENS, the vast majority of Sox10+ cells remain proliferative during embryonic development, with only relatively minor changes in cell cycle parameters. Previous studies have identified proliferating cells expressing neuronal markers in the developing ENS; our data suggest that most cells undergoing neuronal differentiation in the developing gut commence expression of neuronal markers during G2 phase of their last division. Combined with previous studies, our findings show that different populations of neural crest‐derived cells show tissue‐specific patterns of proliferation.