Enteric Neurogenesis

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