12.1.1

Preface

The enteric nervous system (ENS) is susceptible to various genetic, metabolic, and environmental threats, resulting in clinical disorders characterized by loss or malfunction of neuronal components. These disorders are difficult to treat, highlighting the need for novel therapies, such as the transplantation of NSCs to restore the function of ENS in diseased segments of the gut.

Common neuroectodermal stem cells are precursors to NSC, which are components of the central nervous system (CNS-NSC), and to neural crest stem cells (NCSC), which migrate into the gut and form the ENS. CNS-NSC, NCSC and the more committed enteric neuronal progenitor (ENP) cells isolated from the fetal or postnatal gut, may be able to repopulate the ENS.

12.1.2

Potential Sources of Stem Cells for ENS Therapy

Potential sources of stem cells, such as embryonic or hematopoietic stem cells have great advantages but also severe drawbacks. Several theoretical advantages of using embryonic stem (ES) cell lines for restoration of the ENS are as follows: (i) ES cells can be maintained and expanded in culture without losing their stemness; (ii) because of their pluripotency, ES cells can potentially induce various types of cells (e.g., interstitial cells of Cajal; ICC) in addition to neurons and glia; (iii) in an appropriate environment, ES-derived neural precursors can successfully induce central and peripheral neurons, glia, enteric neurons, and other neural crest derivatives.

However, the use of ES cells is ethically restricted and can produce teratoma-like growths. The latter is common in induced pluripotent stem (iPS) cells, although numerous trials have attempted to overcome this drawback.

Neurospheres, generated from mouse ES cells and cocultured with organotypic preparations of gut tissue consisting of the longitudinal muscle layers with the adherent myenteric plexus (LMMP) led to an upregulation in the expression of pan-neuronal markers βIII-tubulin and protein gene protein 9.5 (PGP 9.5) and specialized markers peripherin and neuronal nitric oxide synthase (nNOS) in neurospheres generated from ES cells at the transcriptional and protein levels. However, after in vivo transplantation into the mouse pylorus, grafted neurospheres generated from ES cells failed to acquire a distinct phenotype at least 1 week following transplantation. These results suggest that localized inhibition may influence the differentiation of neurospheres generated from ES cells under in vivo conditions.

Cells from other lineages (adipose tissue, bone marrow, and skin) may require additional and intensive reprogramming to produce an enteric neuronal phenotype.

NSC may be more feasible and applicable compared with cells from other lineages (adipose tissue, bone marrow, and skin) because they are already programmed for a neuronal fate. NSC can be derived from the CNS, the neural crest, or postmigratory ENP populations.

12.1.3

Neurospheres Versus Stem Cells

Putative NSC, isolated in culture from their source organs characteristically grow and proliferate in floating spheroid colonies called neurospheres. Neurospheres have been successfully isolated from the rodent and human gut; these cells appear to be similar to their CNS-derived counterparts. Only 3%–4% of the cells within neurospheres are true stem cells; those are self-renewable and inducible for all three neural lineages. Cell sorting using the expression of either the Ret or the low-affinity receptor for nerve growth factor p75, enable the isolation of a relatively homogenous population, although additional and more specific markers for the self-renewing population of stem cell would be beneficial.

In the CNS, the NSC phenotype is distinguished by the expression of nestin (nestin +), and the low or absent expression of both peanut agglutinin (PNA ^lo ) and heat stable antigen (HAS ^lo ). Nestin is an intermediate filament; the expression of nestin, although not completely specific, is widely used to identify mammalian neuronal precursor cells or stem cells. Positivity for nestin indicates stemness with some limitations along with neurosphere generation. The PNA ^lo HAS ^lo population accounts for 63.2% of the NSC activity present in the unsorted population, suggesting that most of the NSC in the periventricular region possess this phenotype.

In rodents, neuronal precursors have also been isolated from the embryonic and postnatal gut using antibodies to Ret and p75, which are specific markers expressed by the enteric neural crest (ENC)-derived cells. However, it is unknown whether this isolated and more uniform population of precursor cells actually can deliver better engraftment and restoration of function compared with those delivered by neurospheres alone.

12.1.4

Heterologous and Autologous Sources of Neurospheres

Although neurospheres are easily obtained from various sources, there are ethical and immunological concerns associated with their origin. Heterologous transplantation of stem cells into the ENS works relatively well in animal models without using immunosuppression ; however, long-term survival and functional benefits in clinical situations remain to be clarified.

The best possible source would be cells isolated from the patient, preferably from the same organ as the intended target. This would be useful for treating disorders, such as Hirschsprung’s disease (HSCR), in which failure to develop ganglia is caused by defects in the NCSC, or for cases of dysfunction arising from mutations in the endothelin receptor or glial cell line-derived neurotrophic factor (GDNF).

The autologous source of stem cells in HSCR patients is the ganglionic segment; however, whether these cells are effective in repairing the ENS remains unknown.

12.1.5

Source Tissues for NSC and ENP

CNS-NSC are the most well characterized of all NSC and may be more feasible for clinical applications, such as for treating diseases of the CNS. CNS–NSC also provided the earliest in vivo proof of principle for successful functional transplantation into the gut. However, long-term survival is a concern; studies, conducted in animal and in humans, indicate that > 90% of the transplanted neurons apoptose within the first week after transplantation. Therefore, therapies using CNS-NSC for gastrointestinal neuromuscular disorders will need to circumvent or attenuate apoptosis.

Endogenous NSC are found within the immature and adult ENS. NSC isolated from the small intestine of lactating and adult mice express nestin, vimentin, and the proneural transcription factors, neurogenin-2 (NGN-2), Sox-10, and Mash-1. These cells can differentiate into various cell types, particularly neurons, smooth muscle, and glia. The neurons expressed several sensory and motor neurotransmitters in the CNS and ENS.

The most appropriate cell type for ENS therapy is the postmigratory ENP. These ENP cells are downstream of the NCSC and appear to be more committed than other neural crest derivatives.

ENP are more likely to respond to gut-specific environmental conditions. Isolation and expansion of precursor cells from the developing and postnatal human ENS have been achieved using bowel samples from human fetuses and children (9th week of gestation to 5th year postnatal). After dissociation, such cells can be differentiated and transplanted into an aganglionic bowel in vitro.

Unlike the CNS, the gut is accessible by minimally invasive procedures, which is another reason for using ENP for autologous approaches. ENP are found in the mucosal and submucosal regions that can be accessed with a simple mucosal biopsy. Another attractive source for ENP is the appendix, which can be removed using minimally invasive surgery. The neurospheres, induced by these cells in vitro, can be differentiated into neurons, although it is still unknown whether this promising method can generate all the neurons required to restore gastrointestinal function.

Endoscopic techniques provide access to the muscular layer and associated ganglia in a less invasive manner. These layers may provide an alternative source of stem cells.

12.1.6

Potential Routes of Delivering NSC (or ENP) to Target Areas

Because the gut can be accessed by less invasive methods, NSC (or ENP) can be delivered to target areas in the gut using numerous potential routes.

When the defect is limited to a relatively small and well-defined region of the gut, cells can be delivered using the direct image-guided injection in which visualization or ultrasound allows precise placement of the injected cells. This technique can be used in patients with achalasia or HSCR. Cell suspensions or single neurospheres can be directly injected into the affected sphincteric region after pull-through surgery.

Therapy is challenging for more widespread aganglionosis because of the required number of cells and method of delivery. Administering multiple injections along the length of the affected gut may result in cells migrating after transplantation and eventually filling the gaps in aganglionic regions of the gut.

Another approach is to deliver cells via selected arterial cannulation or intravenous injection. Surprisingly, NSC injected intravenously have been shown to cross the blood-brain-barrier and induce recovery in various disease models including multiple sclerosis; the mechanism appears to involve hijacking of the endothelial transport mechanism and utilizing the cell adhesion molecule CD44. When the serotonin 4 (5-HT ₄ ) receptor agonist is orally coadministered, NSC, injected intravenously, can cross the blood-gut barrier, and induce neurogenesis in the affected region.

Finally, serosally directed transplantation via intraperitoneal injection may result in the cells homing into the aganglionic areas of the gut, presumably following guidance cues.

12.1.7

Endoscopic Delivery of Enteric NSC

Endoscopy allows the targeted delivery of numerous cells in a minimally invasive manner. Enteric NSC are surgically transplanted using colonoscopic injection. Enteric neurospheres are prepared as follows: the LMMP is dissected from the intestines of C57BL/6 J mice, in which all the cells express Discosoma sp. red fluorescent protein (DsRed); LMMP is then dissociated and filtered to obtain a single-cell suspension. The suspension is cultured ex vivo to generate enteric neurospheres. These neurospheres are immunoreactive for the neural crest cell marker, p75. For transplantation, neurospheres are resuspended in phosphate buffered saline at the density of 1000 cells/μL. In vivo neuronal differentiation is confirmed 1 week after transplantation by colocalization of DsRed with the neuronal marker PGP 9.5. Differentiation into glia is confirmed using immunoreactivity to the glial marker, S100.

Compared to laparotomy, this approach is less invasive and allows for the controlled delivery of cells into the desired location under direct visual control; multiple endoscopic microinjections can be used to target larger areas. Overall, endoscopic delivery of enteric NSC appears to be safe and effective.

Conversely, intraperitoneal injection of cells into aganglionic mice and intravascular delivery for CNS disease, shows that cells can distribute spontaneously to the gut wall rather than to specific sites. Ectopic spreading of enteric NSC to undesired locations may cause potential problems, such as risk for malignant transformation, altered host organ function, and decreased transplant efficiency.

12.1.8

Promising Scaffold for NSC Delivery

Pluronic F-127 (poloxamer 407) is an injectable synthetic hydrogel that has a reversible mechanism for gelation and is nontoxic, biocompatible, and biodegradable. Pluronic F-127 is thermosensitive, which enables it to hold encapsulated cells in its structure and favors initial cell adhesion inside the defective site. Additionally, it enhances cell attachment and collagen formation, leading to improved levels of angiogenesis, and making this type of hydrogel biomaterial a promising candidate for encapsulating NSC.

Encapsulation in an extracellular matrix or gel, such as pluronic F-127, allows NSC a more gradual contact with their immediate environment while maintaining trophic support by incorporation of nutrients, growth factors, or antiapoptotic agents into the protective material.

12.1.9

Potential Targets for NSC Therapy

Potential targets for NSC therapy should have the following features: (i) easy accessibility, (ii) relatively restricted target area for innervation, (iii) a requirement for a single physiological effect, (iv) absence of satisfactory alternatives, and (v) lack of clinical urgency to enable the harvesting and in vitro preparation of autologous NSC. The success of the transplantation should be easily demonstrable by objective physiological assays, which is especially critical for human patients.

12.3.1

Brain-Derived Neurotrophic Factor (BDNF) Facilitates ENS Regeneration In Vivo

In vivo enteric neurogenesis from ENC-derived ENS progenitors in the enteric NSC, occurs after lower gut surgery and localized coadministration of BDNF as a regenerative agent for the impaired ENS.

Defecation plays an important physiological role in the lower gut. The defecation reflex in guinea pigs is composed of a rectal contraction (R-R reflex) induced by rectal distension and a simultaneous relaxation of the internal anal sphincter (R-IAS reflex). The R-IAS reflex is suppressed after rectal transection and end-to-end anastomosis, which disrupts the continuity of ENS between the rectum and IAS. This surgery simulates a lower anterior resection for rectal cancer. Eight weeks after the surgery, the R-IAS reflex is restored to the levels similar to those of the control animals, and is accompanied by the regeneration of reflex pathways.

BDNF potentiates the formation of enteric neural networks in the gut-like organ differentiated from mouse ES cells. Two weeks after localized treatment with BDNF using a gelatin sponge saturated with BDNF solution (10 ^{− 6} g/mL) and applied at the site of rectal anastomosis, the R-IAS reflex recovers, and bundles of fine nerve fibers interconnect the oral and anal ends of the myenteric plexus ( Figs. 12.1 and 12.2 ).

Effects of treatment with BDNF on changes in the mean rectal distension-induced rectal (R-R) and internal anal sphincter (R-IAS) reflex indices in the same guinea pigs after 2 weeks following surgery. Control: without a gelatin sponge (GS) saturated with brain-derived neurotrophic factor (BDNF) ( n = 4). BDNF: with a GS saturated with BDNF (10 ^{− 6} g/mL) at the site of rectal anastomosis ( n = 3). *, P < 0.01 vs. control.

Representative images of immunostaining for neurofilaments at the site of anastomosis following treatment without (A) with BDNF (B) for 2 weeks after surgery. (C) Bundles of fine nerve fibers interconnect the oral and anal ends of the myenteric plexus (see arrow heads ). Calibration bar, 200 μm.

(Reproduced and modified from Katsui R, Kuniyasu H, Matsuyoshi H, Fujii H, Nakajima Y, Takaki M. The plasticity of the defecation reflex pathway in the enteric nervous system of guinea pigs. J Smooth Muscle Res 2009; 45 :1–13.)

Neurons, found in the granulation tissue of the anastomotic site treated with BDNF, express NSC markers, such as p75 in the ENS, distal less homeobox 2 (DLX2) in the CNS, and cell proliferating markers, proliferating cell nuclear antigen (PCNA), and 5-bromo-2′-deoxyuridine (BrdU) used for the detection of proliferating cells in living tissues. Cells concurrently labeled with BrdU and neurofilament (NF) have been found in the granulation tissue of the anastomotic site treated with BDNF.

These results indicate that BDNF may induce the formation of neurons from the enteric NSC in the newly formed granulation tissue at anastomotic sites and/or promote the migration of neurons into the sites, thereby facilitating recovery of ENS physiological function. Therefore, it may be possible to repair anal dysfunction by promoting the regeneration of reflex pathways in the ENS using localized application of BDNF without the transplantation of exogenous cells.

12.3.2

Drawbacks of Using BDNF for Regeneration of ENS In Vivo

BDNF upregulates its receptor, the receptor tropomyosin-related kinase B (TrkB), at the rectal anastomosis. Upregulation of TrkB is related to an increased risk of metastasis in patients with rectal cancer. Furthermore, BDNF can exacerbate the inflammation generated at the site of rectal anastomosis after surgery, as observed in guinea pigs; this indicates that the safety of using BDNF as a therapeutic drug for the regeneration of ENS after rectal surgery needs to be further evaluated.

12.3.3

Genetic Factors Impact the Development of ENS

ENS forms from ENCDC that originate primarily in the vagal region of the neural tube. The processes of migration, proliferation, differentiation into neurons or glia, clustering into ganglia, and formation of an extensively interconnected network are controlled by numerous genetic factors. Several key molecules involved in these processes are RET , GDNF , EDNRB , EDN3 (ET-3) , PHOX2B , and SOX10 ( Table 12.1 ).

12.3.4

Nongenetic Factors Alter the Structure of ENS

The function of ENS depends on a balance between the specific neuronal subtypes, such as excitatory motor neurons, inhibitory motor neurons, intrinsic primary afferent neurons (IPANs, sensory), and interneurons. These neuronal subtypes differ in morphology, receptors, number of axons, axonal trajectory and function, and the types of neurotransmitters they secrete. Major changes in electrophysiological properties and morphology occur during the postnatal development of the ENS. The factors that guide the identity of neuronal subtypes in the ENS remain poorly understood. However, the ratio of neuronal subtypes is influenced by the timing of cell cycle exit ; numerous factors, such as GDNF, EDN3, sonic hedgehog (Shh), bone morphogenic proteins (BMPs), and retinoic acid (RA), influence the decision to exit the cell cycle.

Serotonin (5-HT), produced by early differentiating enteric neurons, acts as a trophic factor for the surrounding ENCDC; changes in the levels of 5-HT levels influence the ratios of neuronal subtypes. In the postnatal bowel, 5-HT promotes neurogenesis and repair of ENS, partially via the receptors 5-HT ₄ and 5-HT _2B. 5-HT plays offensive and defensive roles in the bowel such as that of regulating intestinal inflammation.

12.3.5

Adult In Vivo Neurogenesis is Induced by 5-HT ₄ Receptor Agonism Without Cell Transplantation

Activation of 5-HT ₄ receptors induces adult enteric neurogenesis in the mouse. The ENS gains neurons throughout the first 4 months of life in wild-type mice; however, this does not occur in mice lacking the 5-HT ₄ receptors.

Production of new neurons, initiated in guinea pigs treated with a 5-HT ₄ agonist, accelerated the return of function (R-IAS reflex) after rectal transection and end-to-end anastomosis that disrupt the continuity of ENS.

A 5-HT ₄ -receptor agonist, mosapride citrate (MOS; 0.1–1.0 mg/kg), applied intravenously and dose-dependently, enhances both the R-R reflex and the R-IAS reflex mediated via ENS. MOS (0.3 and 1 mg/kg), administered intraperitoneally, stimulates the 5-HT ₄ receptor to accelerate the release of acetylcholine (ACh) from cholinergic myenteric neurons; this subsequently activates the alpha7-nicotinic Ach receptor on activated monocytes/macrophages to inhibit the inflammatory reactions in the muscle layer of the rat ileum. MOS (10–100 μM), applied locally at the site of anastomosis or administered orally, promotes the regeneration of the neural circuit in impaired myenteric plexus and recovery of the defecation reflex in the distal gut of rats and guinea pigs. These anti-inflammatory effects and prokinetic effects on both reflex responses may influence MOS-induced enteric neurogenesis.

MOS also generates cells immunopositive for NF, the 5-HT ₄ -receptor, and BrdU, and induces the formation of a neural network in the newly formed granulation tissue at the site of anastomosis 2 weeks after an enteric nerve circuit insult. The numbers of cells immunopositive for the NSC markers DLX2, P75 positive, and NF increase during this time period. In the gut-like organ differentiated from mouse ES cells, MOS produces dense enteric neural networks associated with the upregulated expression of 5-HT ₄ receptor mRNA.

All actions by MOS are inhibited by the specific 5-HT ₄ -receptor antagonist GR113808 (10 µM). Activation of enteric neural 5-HT ₄ -receptors promotes reconstruction of the enteric neural circuit, leading to the recovery of the R-IAS reflex in the distal gut ; this reconstruction possibly involves the enteric NSC.

Similar to 5-HT ₄ agonism, ablation of existing enteric neurons with benzalkonium chloride induces neurogenesis in the gut of adult mice. Additionally, the neurogenic potential of glia can be activated in vivo by tissue dissociation or injury. Although the enteric glia can potentially form both neurons and glia, they are predisposed to form predominantly glia in the adult mice.

Overall, studies conducted currently indicate that neurogenesis can actually occur in the adult mouse ENS; however, it does not occur spontaneously and is difficult to induce.

5-HT is endogenous to the mucosa and the ENS; it is a potential initiator of neurogenesis via 5-HT ₄ receptors, although it is not clear whether the enteric glia are precursors for 5-HT ₄ receptor evoked neurogenesis. Newly generated neurons, detected after induction using 5-HT ₄ agonism, first appear in the extraganglionic locations, and then slowly migrate into the myenteric plexus. When first detected, these cells, like the enteric glia, express Sox10. Unlike glia, they are committed to neuronal lineage and lack glial markers, such as glial fibrillary acidic protein (GFAP) and S100β, expressing instead the neuronal markers such as paired-like homeobox 2b (Phox2b), HuC/D, and doublecortin. The predecessors of these cells may have been glia; however, this remains undetermined pending the development of a marker for adult enteric neurogenic stem cells.

The 5-HT ₄ receptor agonist MOS increases the number of c-RET-positive cells and levels of c-RET mRNA in the gel sponges implanted in the necks of rats. The 5-HT ₄ receptor is a G-protein-coupled receptor (GPCR) linked to G protein G _s -cAMP cascades. Activation of the 5-HT ₄ receptor may induce the activation of c-Ret and/or cAMP-dependent protein kinase A (PKA) by elevating the levels of cAMP.

To reveal these pathways, MOS was orally administered to mice and rats for 2 weeks after disruption of ENS continuity, together with the Ret tyrosine kinase inhibitor withaferin A (WA) and 2-indolinone Ret tyrosine kinase inhibitor RPI-1 or the PKA inhibitor N -[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinoline-sulfonamide dihydrochloride (H89). MOS alone significantly ( P < 0.05) increased the number of newly generated neurons in the newly formed granulation tissue at the site of anastomosis, but not when coadministered with WA or RPI-1. Coadministration of H89 failed to alter the MOS-induced increases in neurogenesis. These results indicated that the c-RET signaling pathway, which is vital in ENS development, contributes to enteric neurogenesis facilitated by MOS, although the contribution of PKA activation seems unlikely.

12.3.6

Possible Mechanisms for Enteric Neurogenesis Induced by 5-HT ₄ Receptor Agonism

The expression of particular genes and/or other markers enables the recognition of many putative progenitor stages in enteric neuronal development. Ret encodes a transmembrane receptor kinase, RET which dimerizes when activated by a complex that includes the preferred glycosylphosphatidyl-inositol-anchored coreceptor, GDNF family receptor (GFRα1). A common RET/GDNF/GFRα1-dependent progenitor gives rise to committed lineages both of glia and enteric neurons, which can be distinguished from ENCDC.

The 5-HT ₄ receptor is a GPCR. MOS increases the number of c-RET-positive cells and c-RET mRNA in the implanted GS in rats. c-RET is a receptor tyrosine kinase (RTK) for the GDNF family of ligands. GPCR-mediated signaling pathways include transactivation of RTKs; the differential involvement of RTKs and downstream signaling pathways, activated in response to GPCR-mediated stimulation, elicit a variety of cellular effects during the development, proliferation, differentiation, survival, repair, and synaptic transmission in the CNS. Therefore, it is possible that as a GPCR, 5-HT ₄ receptor cross-communicates with c-RET in the ENS.

Activation of RET results in phosphorylation of several residues, including Y1015 and Y1062. Growth factor receptor-bound protein 2 (GRB2) and phospholipase Cγ (PLCγ) are required for proliferation and/or differentiation of ENS precursors. RAC, RHO, and CDC42 regulate the migration and proliferation of ENCDC. Kinesin-like protein, KIF26A, Sprouty2 (SPRY2), and phosphatase and tensin homolog deleted from chromosome 10 (PTEN) are negative regulators of RET signaling. KIF26A binds to GRB2 and prevents the signaling of RAS-ERK and phosphoinositide 3-kinase (PI3K). SPRY2 binds to RAS and RAF and blocks the activation of the RAS-ERK pathway, whereas PTEN regulates proliferation via the PI3K and Akt signaling cascades ( Fig. 12.3 ).

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