If cell therapy is to be used to treat gastrointestinal motility disorders, it is crucial to determine whether neural stem/progenitors can migrate and differentiate into neurons with the appropriate neurochemical and electrophysiological properties after transplantation into the postnatal gut in vivo. In the present study, we showed that NSs generated from neural crest–derived cells from both the fetal and the postnatal gut survived, proliferated, migrated, and differentiated into glial cells and a range of neuron subtypes that exhibited neurochemical, morphological, and electrophysiological characteristics similar to those of resident enteric neurons.
The wall of the embryonic gut consists largely of undifferentiated mesenchyme at the time of migration of neural crest–derived cells. The present study showed that both fNS-derived cells and more clinically relevant pNS-derived cells were capable of migrating substantial distances in the gut wall of postnatal mice, consisting of concentric layers of differentiated cells. 4 weeks after transplantation of NSs about 0.25 mm in diameter, fNS- and pNS-derived cells occupied an average of about 9 mm2
and 7 mm2
, respectively, and graft-derived neurites occupied an area around twice the size of the graft-derived cell bodies. Previous studies reported very limited migration of CNS stem cells when transplanted into the stomach of adult mice (4
), or when enteric progenitor cells were cocultured with the hindgut of chick or mouse embryos after the gut had been fully colonized with neural crest–derived cells (24
). The differences in the extent of migration between the current study and earlier studies might reflect differences between the migratory abilities of CNS- and ENS-derived progenitor cells within the postnatal gut, differences between gut regions in their ability to permit migration of stem/progenitor cells, differences between the environment of the late embryonic gut versus the postnatal gut, and/or differences of the environment of the gut in vivo and in culture. Although we showed that the ENS progenitor cells migrated extensively in the postnatal mouse gut, stem/progenitor cells will need to be introduced in greater numbers and at multiple sites to treat humans with enteric neuropathies due to the larger size.
Previous studies have shown that ENS NSs, like their CNS counterparts, are composed of a mixture of progenitors and differentiated progeny (neurons and glial cells) (55
). To examine whether some of the graft-derived neurons we detected were the progeny of transplanted cells, we injected EdU into the recipient mice immediately after transplantation of the NSs. 4 weeks later, Hu+
neurons that had incorporated EdU were readily encountered, which showed that at least some neurons were generated in vivo from transplanted proliferating progenitors.
The neural circuits controlling motility consist of a variety of subtypes of enteric neurons, including intrinsic sensory neurons, interneurons, cholinergic motor neurons that mediate contraction of the gut wall, and inhibitory motor neurons that mediate relaxation and express NOS (56
). For cell therapy for motility disorders, all neuron subtypes will need to be generated from transplanted progenitors and form appropriate connections with each other and with muscle cells. In the present study, grafted ENS progenitor cells gave rise to neurons possessing neurochemical and morphological features similar to those of normal enteric neurons. For example, graft-derived neurons expressed a number of markers characteristic of subtypes of enteric neurons in mice, humans, and other species, including ChAT, VAChT, NOS, calbindin, and calretinin (43
). These markers are not, however, expressed exclusively by neurons in the ENS, but are also expressed by some classes of neurons elsewhere in the nervous system. Importantly, most of the graft-derived NOS neurons possessed lamellar dendrites, which is notable because enteric NOS neurons possess lamellar dendrites that are the sites of many of their synaptic inputs (61
). The proportions of graft-derived neurons expressing NOS and ChAT were similar to those of myenteric neurons in the neighboring region of distal colon. This is reassuring for the generation of an ENS in the aganglionic region of patients with Hirschsprung disease, in which all neuron subtypes will need to be generated (63
); however, for enteric neuropathies such as achalasia, in which there is degeneration of specific classes of enteric neurons (3
), manipulation of the cells prior to transplantation is likely to be required to bias the differentiation of cells to particular neuron subtypes.
Our study showed that transplanted ENS progenitor-derived cells migrate and settle in locations similar to those occupied by neural crest–derived cells during normal development. Furthermore, varicose, graft-derived neurites were present in the muscle layers and formed close associations with myenteric neurons of the recipient and with other graft-derived neurons. Thus, cues must exist in the postnatal gut that graft-derived neurites can use to navigate to specific targets. However, graft-derived fibers were rarely observed in the mucosa, and graft-derived neurons were not observed in the submucosa. This may be because the NSs were transplanted into the outer part of the external muscle. During normal development, the secondary migration of cells from the myenteric region to the submucosa to form submucosa ganglia requires netrin/DCC (deleted in colonic cancer) signaling (64
). It is also possible that components of this signaling pathway, or the extracellular matrix molecules involved in netrin/DCC-mediated migration such as laminin (65
), are not expressed at sufficient levels in the colon of 2- to 3-week-old mice to promote the migration of stem/progenitor cells into the submucosa. Although most of the neurons involved in the regulation of motility in small mammals are found in the myenteric plexus, some of the intrinsic sensory neurons involved in the circuits controlling motility are located in the submucosal plexus (66
). It is therefore highly likely that submucosal ganglia are also essential for a fully functioning bowel, not only for the control of motility, but also for water and electrolyte transport. Hence, techniques will need to be developed to generate submucosal ganglia that consist of appropriate functional types of neurons from transplanted cells.
This is the first study to report the electrophysiological properties of neurons generated from neural progenitor cells transplanted into the gut. During ENS development, some cells express pan-neuronal markers, but do not have the electrophysiological properties of neurons (68
). However, graft-derived neurons in the postnatal colon had electrophysiological properties similar to those of mature, functional enteric neurons. Importantly, the presence of fEPSPs showed that the graft-derived neurons had integrated into the neuronal circuitry, although we were unable to determine whether the synaptic inputs arose from other graft-derived neurons, from the recipient’s neurons, or both. There are 2 main electrophysiological classes of myenteric neurons in the ENS, S neurons and AH neurons (53
). S neurons are uniaxonal and show monophasic repolarization after an AP and fEPSPs in response to fiber tract stimulation. In contrast, AH neurons show a biphasic repolarization and a slow afterhyperpolarizing potential after an AP and rarely display fEPSPs (53
). In the current study, only a single electrophysiological class of neuron was observed, possessing properties very similar to S neurons (53
), in the mouse distal colon. Although AH neurons account for about 20% of neurons in the mouse distal colon (53
), we did not encounter graft-derived neurons with AH-type electrophysiology. This might be because AH neurons are not generated from transplanted progenitor cells in the postnatal colon, or because neurons do not develop AH characteristics until 3–4 weeks after transplantation, when all of the electrophysiological experiments were performed.
The neural circuitry controlling motility involves precise connectivity among different functional classes of neurons (26
). Although we showed that graft-derived neurons projected to the correct gut layers, it remains to be determined whether each of the different neurochemical types of graft-derived neurons formed appropriate synaptic connections. Also important is that graft-derived neurons do not make incorrect synaptic connections. However, while no gut motility studies were performed, the recipient animals survived and did not exhibit any obvious signs of motility defects, such as stool retention, which would be suggestive of inappropriate circuitry.
There were no significant differences in the behavior of cells derived from fNSs and pNSs. This is important because patient-derived cells are an accessible source of cells to treat congenital motility disorders such as Hirschsprung disease (13
), and their use will avoid immune responses and the ethical issues associated with some sources of stem/progenitor cells (10
). It remains to be determined whether cells derived from NSs generated from the adult gut have post-transplantation properties similar to those of fNS- and pNS-derived cells.
Our data lay the foundation for studies in which ENS progenitors are transplanted into animal models of enteric neuropathies to determine whether graft-derived neurons ameliorate the motility defects. In preliminary studies, we transplanted genetically labeled (EdnrbKik
) fNSs into the aganglionic region of colon of postnatal sl/sl
mice, a mouse model of Hirschsprung disease (71
). However, very few transplanted cells survived beyond 1 week due to immunological rejection, as the EdnrbKik
mice are on a different genetic background from sl/sl
mice. As a result, detailed studies have had to be postponed until the EdnrbKik
mice are backcrossed onto the same genetic background as the sl/sl
mice. Nonetheless, we performed some preliminary experiments in which NSs were generated from N4 backcrossed mice and implanted into the aganglionic region of sl/sl
mice. After 4 weeks, graft-derived cells were present, some of which had migrated away from the transplant site and formed clusters of Hu+
cells (Supplemental Figure 4, A and B), and graft-derived neurites were abundant in the circular muscle layer. Furthermore, electrophysiological recordings from 2 briefly impaled graft-derived neurons revealed fEPSPs (Supplemental Figure 4C). These preliminary data showed that cells transplanted into the aganglionic region survived and migrated in the absence of endogenous enteric neurons and that graft-derived neurons received synaptic inputs. Our findings of immunological rejection after transplantation of cells between mouse strains strongly suggest that patient-derived cells will be the best source of enteric neurons to transplant into patients with enteric neuropathies. Furthermore, although our data using postnatal donor and recipient mice support the idea that cell therapy might be used to treat pediatric enteric neuropathies, additional studies in which cells isolated from the adult mouse gut are transplanted into adult mice are required to demonstrate proof of principle that cell therapy might also be used to treat adult enteric neuropathies.
In conclusion, the ability of ENS stem/progenitor cells to proliferate, migrate extensively, differentiate into neurons of the appropriate phenotype, associate closely with endogenous enteric neurons, and incorporate into the neuronal circuitry in postnatal colon suggests that cell therapy to replace the diseased ENS in some enteric neuropathies is a distinct possibility.