REVIEW ARTICLE

Neural stem cell transplantation in the enteric nervous

system: roadmaps and roadblocks

K.-H. SCHAFER,* M.-A. MICCI� & P. J. PASRICHA�

*Department of Biotechnology, University of Applied Sciences, Kaiserslautern, Germany

�Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of Texas Medical Branch, Galveston,

TX, USA

�Division of Gastroenterology and Hepatology, Stanford University, Stanford, CA, USA

Abstract The enteric nervous system (ENS) is vulner-

able to a variety of genetic, metabolic or environ-

mental threats, resulting in clinical disorders

characterized by loss or malfunction of neuronal

elements. These disorders have been difficult to treat

and there is much enthusiasm for novel therapies such

as neural stem cell (NSC) transplantation to restore

ENS function in diseased segments of the gut. Recent

research has indicated the potential for a variety of

innovative approaches to this effect using NSC

obtained from the central nervous system (CNS) as

well as gut derived enteric neuronal progenitors. The

main goal of this review is to summarize the current

status of NSC research as it applies to the ENS,

delineate a roadmap for effective therapeutic strate-

gies using NSC transplantation and point out the

numerous challenges that lie ahead.

Keywords enteric nervous system, motility disorders,

neural stem cells, transplantation.

INTRODUCTION

Like other nervous systems in the body, the enteric

nervous system (ENS) is markedly restricted in its

ability to replace dead or damaged neurons. This

has left the ENS vulnerable to a variety of genetic,

metabolic or environmental threats, resulting in clin-

ical disorders such as achalasia, gastroparesis, neuro-

pathic forms of pseudo-obstruction, enteric neuronal

dysplasia, colonic inertia and Hirschsprung�s disease.

There is a paucity of effective therapies for these and

other disorders of motility. Further, there are intrinsic

limits to a pharmacological approach to modulate a

system that is as delicately and intricately inter-

dependent as the ENS.1 This is best illustrated by the

lack of clinical efficacy of exogenous cholinomimetics

such as bethanechol, which though capable of aug-

menting muscle tone, do not result in effective

propulsion. Moreover, even drugs that are capable of

producing coordinated activity require an intact neu-

ronal circuitry, and in its absence, will fail. The

promise of replacing dysfunctional or dead neurons

by neural stem cell (NSC) transplantation is therefore

tremendously appealing.

NEURAL STEM CELLTRANSPLANTATION: STATE-OF-THE-ART

The replacement of the ENS by adequate neuronal and

glial cells was, until recently, considered more of a

futuristic vision than a near term reality. Early

attempts to reconstitute the ENS in vivo were under-

taken by several groups, using isolated mature enteric

ganglia with some limited success in forming neuronal

networks (Fig. 1).2,3 More recently, the explosion of

research on stem cells in this decade has stimulated

scientists to explore their therapeutic potential also for

the ENS. Stem cells are uncommitted cells that are

Address for correspondence

Pankaj Jay Pasricha, Division of Gastroenterology and Hepa-tology, Stanford University School of Medicine, 300 PasteurDrive, Alway Building, Rm M211, Stanford, CA 94305-5187,USA.Tel: +1 650 725 3362; fax: +1 650 723 5488;e-mail: pasricha@stanford.eduReceived: 7 December 2008Accepted for publication: 12 December 2008

Neurogastroenterol Motil (2009) 21, 103–112 doi: 10.1111/j.1365-2982.2008.01257.x

� 2009 The AuthorsJournal compilation � 2009 Blackwell Publishing Ltd 103

capable of self-renewal (the ability to give rise to more

uncommitted stem cells) and are also able to generate a

progeny of specialized cells, in response to the appro-

priate developmental cues. During development, NSC

give rise to more committed progenitor cells that in

turn generate the fully differentiated neuronal and glial

elements of the nervous system.4–6 A common neuro-

ectodermal stem cell is believed to be parent to NSC

that give rise to the central nervous system (CNS-

NSC), and to neural crest stem cells (NCSC) that

migrate into the gut and form the ENS. Both CNS-

NSC, NCSC and the more committed enteric neuronal

progenitor (ENP) cells isolated from the fetal or post-

natal gut have been studied for their ability to re-pop-

ulate the ENS.7–14 A list of the experimental

approaches for re-populating the mammalian ENS

and the models used are summarized in Table 1. This

list clearly shows that, with few exceptions, the

majority of the work conducted so far relies on the

generation of neurospheres (a surrogate for stem cells,

as will be discussed more in details later) and is limited

to extremely short-term, and generally ex vivo testing

of their ability to integrate into the ENS. However,

many fundamental questions remain unanswered, as

summarized in Table 2.

Moreover, expanding on the theme of CNS research-

ers,15 many important issues need to be addressed in

order for the promise of cell replacement therapy to

become a reality. These can be summarized as follows:

(i) what is the ideal stem cell source for transplanta-

tion? (ii) what is the most appropriate route and

method of stem cell administration? (iii) does func-

tional restoration require faithful recreation of myen-

teric ganglia and related structures? (iv) what is the

best approach (including in vitro preparation and

post-transplantation manipulation) to achieve an

appropriate, functional, and long-lasting integration of

transplanted stem cells into the host tissue? and (v)

what should the first clinical targets be? This review

will suggest possible pathways to clinical trials as well

as the many gaps in our knowledge.

Potential sources of stem cells for ENS therapy

When it comes to the issue of transplanting cells for

the restoration of the neuronal network in the gut, the

central question is: what is the source of the cells to be

used? There are legions of approaches for all kinds of

diseases using various types of stem cells, and the right

choice is not clear. Some of these sources, such as

embryonic or haematopoietic stem cells not only have

great potential but also may have severe drawbacks.

Thus, there are several theoretical advantages of

embryonic stem cell lines for ENS restoration. Firstly,

they can be maintained and expanded in culture

without losing their �stemness�. Secondly, they can

potentially give rise to many more cell types (e.g.

interstitial cells of Cajal) than just neurons or glia, an

attribute that could be particularly useful in certain

diseases states where multiple lineages are affected. In

an appropriate environment, embryonic stem-derived

neural precursors have been shown to generate suc-

cessfully both central and peripheral neurons, glia,

enteric neurons and other neural crest derivatives.16–19

However, the use of embryonic stem cells is ethically

restricted, can produce teratoma-like growths,20,21 and

further, as with cells from other lineages (bone mar-

row, skin, adipose tissue, etc.), may require additional

and perhaps intense reprogramming by as yet poorly

defined protocols to produce an enteric neuronal

phenotype. A more feasible and perhaps suitable type

of cell therefore may be one that is already pro-

grammed for a neuronal fate, i.e. a NSC. These can

be derived from the CNS, neural crest or postmigratory

enteric neural progenitor population. However, before

discussing the merits of each of these sources, it is

A B

Figure 1 Transplantation of isolated myenteric plexus into the submucosal layer of adult rats cecum (A). The dissociated myentericplexus cells were labelled with Hoechst stain prior to transplantation. (B) PGP9.5 immunoreactivity in grafted cells 7 days aftertransplantation. Secondary neuronal network is visible in the submucous layer. (From Schaefer3). LM, circular muscle; CM,longitudinal muscle; Muc, mucosa; MP, myenteric plexus; T: transplanted cells.

K.-H. Schafer et al. Neurogastroenterology and Motility

� 2009 The AuthorsJournal compilation � 2009 Blackwell Publishing Ltd104

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26

Volume 21, Number 2, February 2009 NSC transplantation in the ENS

� 2009 The AuthorsJournal compilation � 2009 Blackwell Publishing Ltd 105

important to first clarify that most of the literature

consists of studies on neurospheres and not a pure stem

cell population.

Neurospheres vs stem cells When putative NSC are

isolated in culture from their source organs, they

characteristically grow and proliferate in floating

spheroid colonies called neurospheres. Several investi-

gators have successfully isolated neurospheres from

both the rodent and human gut which appear to be

similar to their CNS-derived counterparts.14,22,23 As in

the CNS, neurospheres continue to form the starting

material for most studies published to date in the ENS

field. However, although this feature has been used as a

surrogate marker for �stemness�,24 the neurosphere is

actually a very heterogeneous entity, consisting of

progenitor cells at various levels of commitment as

well as neurons, glia and other terminally differentiated

cell types. Only 3–4% of the cells within neurospheres

are actually true stem cells in that they are self-

renewing and can give rise to all three neural lineages.25

Although it is clearly desirable to start with a relatively

homogenous population of NSC, this goal has been

difficult to attain because of the lack of a suitable

marker. Amongst the studies shown in Table 1, only

two have started with a relatively homogenous popu-

lation based on cell sorting by expression of either the

Ret receptor9 or the neurotrophin receptor p75,26 and

only one of these performed clonal analysis to prove the

true stem cell nature of these cells.26 It is not clear

whether these receptors represent universal markers

for ENS-NSC that can be applied to different species or

at different stages of development, as most investiga-

tors appear to have limited themselves to using neur-

ospheres. It may therefore be necessary to identify

additional and perhaps more specific markers in the

self-renewing stem cell population that will allow us to

rigorously prove their stemness and consistently and

reproducibly isolate them from the gut.

In the CNS, the NSC phenotype has been distin-

guished by the expression of nestin,27,28 and the low to

absent expression of both peanut agglutinin and heat

stable antigen (nestin+, PNAlo,HASlo).29 Nestin is an

intermediate filament protein whose expression is

widely used to identify mammalian neuronal precursor

cells or stem cells. The stem cell is initially positive for

nestin, but secondary progenitor cells lose this prop-

erty. However, it is not completely specific: in the

adult brain, nestin is expressed not only in NSC in the

subependymal zone but also in reactive astrocytes, and

in other organs, nestin also stains endothelial or

glandular cells.30 A further problem with nestin is that

it is not expressed on the surface, making it difficult to

isolate these cells based on cell sorting techniques.

Despite these limitations, nestin positivity, along with

neurosphere generation, has become almost synony-

mous with stemness in the hands of most investiga-

tors. In rodents, neuronal precursors have also been

isolated from the embryonic and postnatal guts using

antibodies to specific markers known to be expressed

by enteric neural crest-derived cells: Ret28 and p75 (the

low-affinity receptor for nerve growth factor).26,31

However, it is not known that an approach using these

techniques to isolate a more uniform population of

precursor cells actually leads to better engraftment and

functional restoration as compared with neurospheres

alone.

There is therefore obviously a need for investigators

to go �beyond the neurosphere� and put greater efforts

towards the identification of markers with both high

specificity and selectivity, that will allow to harvest

stem cells in larger quantities, using minimally inva-

sive techniques, and without other �contaminating� cell

types. Such strategies will allow the delivery of a pure

source of neuronal tissue, eliminating several con-

founding variables affecting the post-transplantation

outcome.

Heterologous vs autologous sources Although neuro-

spheres can be obtained relatively easily from a variety

of sources, there are both ethical and immunological

problems associated with their origin. Heterologous

transplantation of stem cells into the ENS works rel-

atively well in animal models without immuno-

suppression,32 but it is not clear whether this will also

be true for long-term survival and functional benefit in

clinical situations. Further, ethical issues, while sur-

mountable, will continue to present challenges for the

use of stem cells from tissues obtained from dead or

aborted donors. Clearly, therefore, the best source to be

used will be cells isolated from the patient itself,

preferably from the same organ as the intended target.

This may be feasible even in disorders such as

Hirschsprung�s disease, in which the failure to develop

Table 2 Important questions on the functional potential ofharvested neural progenitors

How does the developmental age of cells effect migrationand differentiation properties and engraftment potential?

How does in vitro culturing prior to transplantation effectthe above?

Can stem cells be nudged towards a better transplantationoutcome by in vitro manipulation of culture conditions?

If the gut contains an abundance of neural progenitors, whydoes the ENS not appear to be capable of repairing itself?

K.-H. Schafer et al. Neurogastroenterology and Motility

� 2009 The AuthorsJournal compilation � 2009 Blackwell Publishing Ltd106

ganglia may be caused by defects either in the NCSC or

in the environment they need to inhabit [e.g. with

endothelin receptor or glial cell line-derived neuro-

trophic factor (GDNF) mutations]. It is presumed that

in the latter group of patients, the autologous source of

stem cells will be the ganglionic segment but it

remains to be seen whether these cells are actually

effective in repairing the ENS. In this regard, one of the

authors (Karl-Herbert Schafer) has been successful in

isolating neurospheres from the ganglionic or the

transitional zone of Hirschsprung�s disease patients

(Fig. 2, Karl-Herbert Schafer, unpublished data).

Source tissues Central nervous system-derived NSC

remain the most well characterized and studied of all

NSC and appear to be closest to clinical reality, at least

for CNS diseases. Interest in the use of fetal NSC as a

therapeutic tool has also been fuelled in part by a report

of the isolation of a human fetal NSC that can be

expanded in vitro for years and that can be readily

differentiated into neurons and glia, thus holding the

promise of a renewable, plentiful and standardized

source of human neural cells;33,34 recent studies show

that such an approach is useful for adult subventricular

zone cells as well.35 Indeed, CNS-NSC provided the

earliest in vivo proof-of-principle for successful func-

tional engraftment in the gut.7 However, long-term

survival is still an issue as more than 90% of the grafted

neurons usually die upon grafting, both in animal and

in human studies.36,37 A large portion of this cell death

occurs as programmed cell death, or apoptosis, and

occurs within the first week after transplantation.38,39

Therefore, CNS-NSC treatment for gastrointestinal

neuromuscular disorders will require strategies to

circumvent or attenuate this phenomenon.

Although NCSC and differentiated CNS cells share a

common progenitor,40,41 NCSC are potentially more

attractive because in nature, they give rise to both the

peripheral nervous system and the ENS, in addition to

smooth muscle cells, pigment cells, bone and cartilage

in other regions of the body.42,43 However, it can

logically be argued that the most appropriate cell type

for ENS therapy is the postmigratory ENP. These cells

are downstream of the NCSC and appear to be more

committed than other neural crest derivatives (such as

sciatic nerve stem cells) in terms of their commitment

to a neuronal fate.44 Recent developments have added

to the enthusiasm for this approach. These include the

discovery of endogenous NSC within the immature as

well as adult ENS.5,26,44–46 NSC isolated from the

small intestine of lactating and adult mice express

nestin, vimentin, and the pro-neural transcription

factors neurogenin-2 (ngn-2), Sox-10 and Mash-1.45

These cells can differentiate into various cell types,

particularly neurons, smooth muscle, and glia with the

neurons expressing several characteristic neurotrans-

mitters and receptors including calcitonin gene-related

peptide, neuropeptide Y, peptide YY, substance P,

vasoactive intestinal polypeptide, galanin and c-KIT.

However, in keeping with the previous discussion, it

should be pointed out that these experiments used

unfractionated primary cell cultures of whole gut and

therefore it is difficult to ascribe any of these attributes

to those emanating from pure NSC.

It is not known whether CNS-NSC can also express

such a profile but these results do suggest that ENP are

more likely to respond to gut-specific environmental

cues. Indeed, isolation and expansion of precursor cells

from the developing and postnatal human ENS have

recently been reported using bowel samples from

human fetuses and children (from the ninth week of

gestation to 5 years postnatal). Such cells can be

differentiated and also be transplanted after dissocia-

tion into aganglionic bowel in vitro.11–13 Another

advantage of ENP, particularly for autologous

approaches, lies in the accessibility of the gut by

minimally invasive means (unlike the CNS). This has

been highlighted by the recent dramatic discovery of

potential neuronal progenitors in the mucosa and

submucosal region which can be accessed by perform-

ing a simple mucosal biopsy. These cells can give rise

to neurospheres in vitro which can be differentiated

into neurons.47 Although it has still to be proven

A B

Figure 2 Neurospheres derived from either the ganglionic(A) or transitional (B) areas of children undergoing surgery forHirschsprung�s disease showing that the transitional zonegives rise to smaller spheres, which is consistent with thefinding of Bondurand et al.22 in an animal model. The neur-ospheres were generated by isolating myenteric plexus orsingle ganglia from both areas using a technique based onenzymatical (collagenase) digestion and mechanical agita-tion.60 Briefly, the resected areas were stored on ice and pro-cessed within hours from surgery. Muscle and submucouslayer were separated and the muscle tissue from the mostproximal, ganglionic, as well as from the transitional zone wasincubated in a collagenase solution (1 mg mL)1) for up to 5 h.After vortexing, muscle cells and myenteric plexus could beidentified in both samples. The plexus tissue was dissociatedand plated in a stem cell medium as previously reported14

(Karl-Herbert Schafer, unpublished data).

Volume 21, Number 2, February 2009 NSC transplantation in the ENS

� 2009 The AuthorsJournal compilation � 2009 Blackwell Publishing Ltd 107

whether this method can generate all the neurons

required to restore fully gastrointestinal function, this

is a very promising and exciting development for the

field.

Another attractive source for ENP is the appendix

which harbours a fully developed ENS48 and can easily

be removed by minimal invasive surgery. Thus, enteric

nervous tissue can be isolated from surgically removed

appendices and neurospheres generated and neuronal

and glial cells cultivated (Karl-Herbert Schafer, unpub-

lished data).

Finally, endoscopic techniques are being developed

that can provide access to the muscular layer

and associated ganglia in a relatively non-invasive

manner.49,50 It is quite possible that these layers may

provide an alternative source for stem cells in the future.

Although these locations are very promising auto-

logous sources for enteric NSC, further studies and

more effort have to be directed at characterizing the

amount and quality of neurospheres that can be

obtained from individual biopsies or appendices.

Potential routes of delivery

The accessibility of the gut by minimally invasive

means allows one to envision a number of potential

routes of delivery of NSC or ENP to target areas in the

gut. The cells can be injected directly under visual or

ultrasound control into the affected tissue. This

procedure will allow for a precise injection of cells

within a layer of the gut where they would have an

optimal chance for engraftment and regeneration of the

neuronal circuitry. This approach would work best

when the defect is limited to a relatively small and

well-defined region of the gut. For example, this

procedure can be used in achalasia or Hirschsprung�sdisease patients that have undergone pull-through

surgery, where cell suspensions or single neurospheres

can be directly injected into the affected sphincteric

region. For more widespread aganglionosis, therapy

will be more challenging both in terms of the cell

numbers required and the method of delivery. One

approach is to administer multiple injections along the

length of the affected gut and hope that post-trans-

plantation migration will eventually cover the �gaps�.Another approach is to attempt delivery via selected

arterial canulation or perhaps even intravenously.

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 utilizing

CD44.51,52 Finally, serosally directed transplantation

via intraperitoneal injections with the hope that the

cells will home into the aganglionic areas of the gut,

presumably following guidance cues, as has been

shown recently.11

EFFECTS OF NSC ON THE ENS:RECONSTRUCTION OR BYSTANDER?

Even if the progenitor cells reach the desired segment

of the gut, there is still the question of finding the

appropriate intramural zone – myenteric plexus or

muscularis propria. This can be achieved by sophisti-

cated imaging techniques including endoscopic ultra-

sound or by direct visualization at surgery. However, it

is not clear that this is necessary. We have shown that

a significant therapeutic effect can be accomplished

without necessarily achieving an anatomically and

physiologically correct recreation of myenteric gan-

glia.7 While the latter goal is ideal, it may not be

essential. Indeed, it is becoming increasingly clear

from the CNS literature that replacing affected cell

populations (or structural components like myelin) and

their connections is not the only mechanism by which

stem cells can promote functional recovery. As in our

studies, they can deliver missing neurotransmitters,

provide a tissue scaffold for host axonal regeneration,

and provide neurotrophic and other active molecules

that promote endogenous growth and repair (so-called

�bystander� effects).53,54 Thus, anatomic precision may

not be a critical factor for a successful functional

outcome after NSC transplantation.

OPTIMIZING POST-TRANSPLANTATIONFATE

This is perhaps the most formidable challenge to

overcome. As mentioned previously, in most experi-

mental studies, the majority, or all of the transplanted

cells die after a relatively short period after transplan-

tation. Cell sources can be optimized, progenitors can

be �primed� and appropriate and adequate delivery

ensured; however, after transplantation the fate of the

NSC becomes largely under the influence of factors

that can affect both survival and differentiation. How-

ever, these factors are poorly understood and difficult

to manipulate adequately.

One can conceptualize the various phases that

progenitor neurons have to undergo after transplanta-

tion; although somewhat overlapping, each has its own

set of perils (Table 3). Thus, the transfer from the

controlled media of the culture dish to an active

organic environment can be expected to be very

stressful physiologically. This may be further

K.-H. Schafer et al. Neurogastroenterology and Motility

� 2009 The AuthorsJournal compilation � 2009 Blackwell Publishing Ltd108

compounded if the method of administration involves

direct injection into the wall of the gut. The target

layers of the muscularis propria are tightly packed and

trauma to both host and transplanted tissue is inevi-

table, made worse by the ensuing tissue injury

response. It may therefore be helpful to provide some

protection to the stem cells while they establish a

�beach-head� during this phase. This could be in the

form of encapsulation in an extracellular matrix or gel

suspension which facilitates a more gradual contact of

the stem cells with their immediate environment

while maintaining trophic support (such as by the

incorporation of nutrients, growth factors or anti-

apoptotic agents into the protective material) (Fig. 3,

Karl-Herbert Schafer, unpublished data).

During the second phase, as transplanted cells start

engaging with their neighbours, they face other threats

to their survival that include host immunity (in case of

heterologous transplants) as well as the lack of target-

derived trophic factors. The former is relatively easy to

overcome, if necessary, with pharmacological methods

but the latter is still problematic, in part because the

exact nature and mix of environmental factors are

complicated and not fully understood. Thus, a non-

permissive environment may involve an over-expres-

sion of factors such as laminin that push the cells

towards (premature) differentiation or a lack of neuro-

trophic factors such as GDNF. For instance, both

laminin55 and GDNF56–58 vary in their expression in

the colon in Hirschsprung�s disease, depending on the

colonic region as well as with the nature of the genetic

defect. In the future, one could envision customizing

and administering a �cocktail� of factors that are

designed for the target genetic background and that

could be administered locally either with the trans-

planted cells or in adjacent areas to facilitate this phase.

In the �final� phase, transplanted NSC have ideally

assumed a long-lasting mutually beneficial equilib-

rium with their environment and achieved pheno-

typic stability that is maintained by the appropriate

signals from their targets as well as adaptive gene

plasticity. Perhaps the biggest threat at this stage is

persistence of the original insult that led to neuronal

loss in the first place (e.g. an auto-immune disorder);

this will need to be addressed by either treatment

directed at the underlying disease or careful selection

of the stem cells from sources that may not express

the vulnerable antigen or its presenting molecule. It

is also not clear whether some capacity for

self-renewal will be required to maintain a stable

population in the long-term.

Theoretically, many of the therapeutic manipula-

tions discussed above can be addressed by genetic

modification of the stem cells prior to transplantation.

However, it is likely that safety concerns will prevent

these from being implemented at least for the first

generation or two of this therapy.

A B

Figure 3 (A) Mouse ENS-derived neurospheres cultured in a 3-dimensional gel (extracellular matrix, ECM from Sigma Aldrich,St Louis, MO, USA), demonstrating the innervation potential of freshly explanted neural stem cells. Briefly, enteric neurosphereswere harvested, centrifuged and the supernatant completely removed. The pellet was topped with 50 lL of ice-cold extracellularmatrix gel (ECM; Sigma), resuspended and plated on coverslips in drops of 10 lL. The ECM solution was allowed to gel in anincubator for 15 min before adding media. The sphere started to develop neurite outgrowth within hours. (B) This picture representsa magnified view of (A) showing increased density of fibres between the single embedded spheres (Karl-Herbert Schafer,unpublished data).

Table 3 Post-transplantation phases and challenges

Phase I: ExpeditionarySpace constraintsEarly inflammation and injury response

Phase II: EngagementTarget homing and contactTrophic deprivation and adaptationHost immune response

Phase III: IntegrationFunctional interaction with neighboursPhenotypic stabilityLong-term survival and/or capacity for self-renewal

Volume 21, Number 2, February 2009 NSC transplantation in the ENS

� 2009 The AuthorsJournal compilation � 2009 Blackwell Publishing Ltd 109

First clinical targets

There are some intuitively obvious features that

characterize a relatively easy disease target for NSC

therapy in the gut. These features include ease of

access, a relatively limited target area for innervation, a

requirement for a single physiological effect (and hence

single neurotransmitter production) and the lack of

satisfactory alternatives. Further, there should be no

clinical urgency so that harvesting and in vitro prep-

aration of autologous NSC can be performed carefully.

Finally, the success of the transplantation should be

easily demonstrable by objective physiological assays

(e.g. manometric, radiological or electromyographical

studies). Good candidate diseases therefore include

achalasia or residual anal sphincter dysfunction after

surgical pull through for Hirschsprung�s disease, both

of which require only a neural relaxatory component.

CONCLUSIONS

The use of NSC for the restoration of function in the

aganglionic gut is feasible. This review has suggested

possible pathways to clinical trials as well as the many

gaps in our knowledge. A cautious approach would

suggest that it is too early to take this therapy to

patients as the biological basis for a successful out-

come remains largely unknown. Clearly, much more

fundamental research is needed before we can make

significant progress. However, it is perhaps also impor-

tant to consider, in parallel, pilot human studies that

can provide early signals on the direction for which

this research should take. In this regard, one can draw

from the experience (and early success) of myoblast and

fibroblast injections for relief of urinary incontinence,

coupled with the relative safety of using autologous

stem cells.59 These early trials will supplement and

provide direction for the more fundamental research

that is also clearly required as we go down this new and

very exciting therapeutic road.

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