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Keywords:

  • neural stem cells;
  • enteric nervous system;
  • enteric neural precursors;
  • enteric neuromuscular disorders;
  • transplantation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENTERIC NEUROMUSCULAR DISORDERS
  5. AN OVERVIEW OF STEM CELLS
  6. WHAT IS THE IDEAL SOURCE OF STEM CELLS FOR USE IN THE ENS?
  7. HOW DO WE OPTIMIZE SURVIVAL AFTER TRANSPLANTATION?
  8. CHALLENGES AND FUTURE DIRECTIONS
  9. REFERENCES

The main goal of this review is to summarize the status of the research in the field of stem cells transplantation, as it is applicable to the treatment of gastrointestinal motility. This field of research has advanced tremendously in the past 10 years, and recent data produced in our laboratories as well as others is contributing to the excitement on the use of neural stem cells (NSC) as a valuable therapeutic approach for disorders of the enteric nervous system characterized by a loss of critical neuronal subpopulations. There are several sources of NSC, and here we describe therapeutic strategies for NSC transplantation in the gut. These include using NSC as a relatively nonspecific cellular replacement strategy in conditions where large populations of neurons or their subsets are missing or destroyed. As with many other recent “breakthroughs” stem cell therapy may eventually prove to be overrated. However, at the present time, it does appear to provide the hope for a true cure for many currently intractable diseases of both the central and the peripheral nervous system. Certainly more extensive research is needed in this field. We hope that our review will encourage new investigators in entering this field of research ad contribute to our knowledge of the potentials of NSC and other cells for the treatment of gastrointestinal dysmotility. Developmental Dynamics 236:33–43, 2007. © 2006 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENTERIC NEUROMUSCULAR DISORDERS
  5. AN OVERVIEW OF STEM CELLS
  6. WHAT IS THE IDEAL SOURCE OF STEM CELLS FOR USE IN THE ENS?
  7. HOW DO WE OPTIMIZE SURVIVAL AFTER TRANSPLANTATION?
  8. CHALLENGES AND FUTURE DIRECTIONS
  9. REFERENCES

The enteric nervous system (ENS) is a relatively autonomous collection of neurons and supportive cells that is present in the gut wall of all vertebrates and regulates the activity of the gastrointestinal (GI) tract, including secretion, absorption, and motility. The neural elements of the ENS are arranged into ganglionated plexuses located within the muscle wall of the gut in two relatively distinct collections found either submucosally (submucosal plexus) or within the muscle layer (myenteric plexus). Immediate neural control of muscle activity is controlled by the enteric neurons located in the myenteric plexus, which express and release either excitatory (acetylcholine, substance P) or inhibitory (nitric oxide, vasoactive intestinal peptide) neurotransmitters. Enteric motor reflexes and programmed activity requires precisely timed sequential activation of inhibitory and excitatory neurons (Wood et al.,1999; Kunze and Furness,1999). Such a system allows the generation of a coordinated motor response such as peristalsis with relaxation of the segment aboral to a bolus, in association with contraction of the segment orad to it (Grundy and Schemann,2006; Grundy et al.,2006). In addition, inhibitory nerves are critical for the normal function of gastrointestinal sphincters, where they are normally quiescent and switched on as needed physiologically. The inhibitory neurotransmitter that has received the most attention in the past decade has been nitric oxide (NO), which is produced by the enzyme neuronal nitric oxide synthase (nNOS; Guslandi,1994; Boeckxstaens et al.,1994; Kim et al.,1999; Mashimo and Goyal,1999; Takahashi,2003). All of the neurons and glia that comprise the ENS are derived from the neural crest cells that are instructed to differentiate under the influence of both intrinsic and extrinsic cues, which are only just beginning to be understood (Fig 1).

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Figure 1. Analyses in vitro and in vivo have identified a series of growth factors and transcriptional regulators (markers) affecting different stages of neurogenesis of neural crest-derived progenitor cells. This schematic diagram summarizes work from many laboratories and includes some information not explicitly described in the body of the text; the compiled data provide a roadmap for following important hallmark events in the development of autonomic, enteric, and sensory neurons. Neural crest cells segregated from the neuroepithelium can be identified by the expression of FoxD3 and SOX10 (among other markers); cells expressing FoxD3 give rise to neurons and not melanocytes. SOX10 maintains multipotency in neural crest-derived cells as well as neurogenic potential. In the enteric nervous system, SOX10 and Pax3 together regulate RET, which is required for normal development of these neurons. Progenitor cells differentiate into sympathetic, parasympathetic, enteric, or sensory neurons in part dependent upon instructive signals encountered early at or near the time of egress from the neural tube. Additionally, extrinsic cues encountered during migration or at sites where neural crest-derived cells differentiate influence patterns of gene expression. In autonomic ganglia, expression of HAND2 appears to select cells as noradrenergic sympathetic ganglion neurons as well as functioning in cell type-specific gene expression. In the sensory neuron lineage, the POU domain transcription factor Brn3a, expressed downstream of Ngn, regulates a large array of genes influencing cell death, neurotransmitter expression, and axon guidance. The signaling molecule sonic hedgehog (SHH) is necessary for the expression of neurogenin. In the enteric nervous system, HAND2 is expressed downstream of Phox2b (Christos Goridis, personal communication) in all segments of the developing gut; the function of HAND2 in development of enteric neurons is unknown. The neurotrophin receptor TrkC is expressed early by neural crest-derived cells whose potential is restricted to neuronal or glial lineages as well as a subset of enteric neurons. (Reprinted from Howard,2005)

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ENTERIC NEUROMUSCULAR DISORDERS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENTERIC NEUROMUSCULAR DISORDERS
  5. AN OVERVIEW OF STEM CELLS
  6. WHAT IS THE IDEAL SOURCE OF STEM CELLS FOR USE IN THE ENS?
  7. HOW DO WE OPTIMIZE SURVIVAL AFTER TRANSPLANTATION?
  8. CHALLENGES AND FUTURE DIRECTIONS
  9. REFERENCES

Enteric neuromuscular disorders are a diverse group of diseases whose pathobiology in general is poorly understood. They can be congenital or acquired, predominantly affect a subpopulation of cells (e.g., muscle, nerve, or interstitial cells of Cajal), and be regional (limited to a segment of the gut) or more diffuse in their expression (Mertz,2003; De Giorgio et al.,2004; DeGiorgio and Camilleri,2004). Thus inhibitory neuronal dysfunction is an important cause of gastrointestinal dysmotility and may be part of a global loss of neurons such as in Hirschsprung's disease, a more selective loss as in achalasia or congenital hypertrophic pyloric stenosis or due to relatively isolated loss of expression of nNOS as has been reported in diabetic gastropathy (Mearin et al.,1993; McCallum and Brown,1998; Hirano,1999; Mashimo et al.,2000; Sivarao et al.,2001; Camilleri,2003; Belknap,2003; Chelimsky and Chelimsky,2003).

The treatment of the disorders discussed above is far from satisfactory and remains palliative at best. Simple pharmacological replacement of enteric neurotransmitters is a crude and ineffective method of treatment for most of these disorders because of inability to mimic temporally and spatially specific patterns of neuronal activation in health. Theoretically, a real cure will restore or replace missing or dysfunctional neurons with healthy ones. Hopes for such a prospect have been raised by recent advances in the field of neural stem cell biology and experimental evidence of the efficacy of neural stem cell transplantation to treat neurodegenerative conditions of the central (CNS) and peripheral (PNS) nervous systems (Ostenfeld and Svendsen,2003; Burns et al.,2004; Tai and Svendsen,2004).

In the following sections, we briefly review the current knowledge of neural stem cells with particular emphasis on their ability to generate neurons and glia of the ENS. It should be realized at the onset that such approaches are in their infancy, and there is not enough information to formulate a definite “road map.” Indeed, even in the CNS, where work began more than a decade ago, many fundamental issues remain unsolved, particularly regarding the ideal stem cell source for transplantation and the best approach to achieve an appropriate, functional, and long-lasting integration of transplanted stem cells into the host tissue (Pluchino et al.,2005a,b; Martino and Pluchino,2006). In this review, we will discuss our working strategy to approach these issues and the underlying rationale.

AN OVERVIEW OF STEM CELLS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENTERIC NEUROMUSCULAR DISORDERS
  5. AN OVERVIEW OF STEM CELLS
  6. WHAT IS THE IDEAL SOURCE OF STEM CELLS FOR USE IN THE ENS?
  7. HOW DO WE OPTIMIZE SURVIVAL AFTER TRANSPLANTATION?
  8. CHALLENGES AND FUTURE DIRECTIONS
  9. REFERENCES

Our definition of stem cells has remained unchanged since the demonstration of the existence of bone marrow precursors that had the property, at the single cell level, of (1) self-renewal and (2) multilineage differentiation (Weissman,2000). Since then, the existence of putative progenitor cells has been reported in virtually every organ and with the successful growth of human embryonic stem cells in 1998 by Thomson et al. (1998), stem cell biology, its therapeutic impact, and ethical concerns have come to occupy a more or less permanent place on the front page. A brief review of stem cell nomenclature and biology is, therefore, important to understand this rapidly growing field.

The fertilized mammalian egg is totipotent, that is, can give rise to the entire organism. Shortly thereafter, it divides to form the blastocyst, the inner cell mass which consists of pluripotent stem cells that can give rise to virtually every type of cell except the placenta and, hence, cannot form an organism by themselves in utero. Three types of mammalian pluripotent stem cell lines have been isolated for potential clinical use—embryonal carcinoma (EC) cells, the stem cells of testicular tumors; embryonic stem (ES) cells derived from preimplantation embryos; and embryonic germ (EG) cells derived from primordial germ cells (PGC) of the postimplantation embryo. In nature, the pluripotent stem cells give rise to more specialized cells that are committed to specific lineages such as blood, skin, nervous system, etc., and are called multipotent stem cells. Such stem cells are present in many tissues of adult animals and are important in tissue repair and homeostasis. Examples include hematopoeitic stem cells (HSC) and neural stem cells (NSC). Finally, stem cells can be unipotent, sometimes also termed progenitor cells, such as the spermatogonial stem cell that produces only one type of cell, the spermatozoon.

WHAT IS THE IDEAL SOURCE OF STEM CELLS FOR USE IN THE ENS?

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENTERIC NEUROMUSCULAR DISORDERS
  5. AN OVERVIEW OF STEM CELLS
  6. WHAT IS THE IDEAL SOURCE OF STEM CELLS FOR USE IN THE ENS?
  7. HOW DO WE OPTIMIZE SURVIVAL AFTER TRANSPLANTATION?
  8. CHALLENGES AND FUTURE DIRECTIONS
  9. REFERENCES

Many recent investigations have generated excitement about the use of either embryonic pluripotent stem cell lines or multipotent stem cell lines derived from fetal or adult tissue to replace cells or rebuild tissue lost or destroyed by disease. There is considerable debate in the literature on the relative merits of the two broad cell types; however, there appears to be a growing consensus that research needs to be done along both lines for the next several years before the true therapeutic potential, if any, of either approach can be established (Gage,2000).

ES Cells and ES Cell-Derived Neural Precursors

Pluripotent ES cell lines possess several distinguishing attributes that may represent potential advantages, depending upon the proposed application. To begin with, they are capable of giving rise to more diverse offspring than multipotent cells. The second important difference relates to the potentials of ES cells to be expanded in culture indefinitely without losing differentiation potential. By contrast, it has been difficult if not impossible to expand adult multipotent stem cells in culture without losing their undifferentiated state. However, although attractive because of their pluripotentiality, there are some obstacles and risks associated with the use of ES cells for therapy, even setting aside the so-called ethical controversies. Spontaneously, only 0.2% of ES cells generate neurospheres (Tropepe et al.,2001), and the number and complexity of signals and transformations required to achieve a neuronal subtype may be formidable (Temple,2001). Recent studies do show, however, that such differentiation can be achieved in vitro (Zhang et al.,2001); whether the neuronal precursors derived from ES cells behave differently than those derived from the CNS remains to be investigated. In addition, there exists at least one identifiable risk that appears to be particularly associated with ES cells: human ES cells injected into mice can produce teratoma-like growths (Reubinoff et al.,2000,2001).

Our preliminary data suggest that grafted ES cells do not differentiate into mature neurons or glia (Sasselli et al.,2006) and that, for an ES-based transplant strategy to successfully repopulate the ENS, the cells will either need pretransplantation “nudging” or further manipulation of the post-transplant environment to acquire an enteric neuronal phenotype, a strategy that we are currently pursuing. The idea that ES cells can be used as a reservoir of cells from which differentiated cells can be isolated has been validated in both rodent and human models (Gorba and Allsopp,2003; Bibel et al.,2004). The advantage of using neurons, glia, or precursors derived from ES cells is that they may not be regionally specified (by contrast with local tissue-specific neural progenitors) and, thus, may be more capable of site-specific integration. Several investigators have shown that cell surface markers, in vitro manipulation of culture conditions, or using tissue-specific promoters can be used to isolate neural stem cells or more restricted precursors. When transplanted in the rodent CNS or spinal cord, ES-derived neural precursors implant and give rise to neurons and glia and are functionally active. The generation of peripheral neurons and neural crest derivatives from ES cells has also been reported (Mizuseki et al.,2003; Pomp et al.,2005). Moreover, and of relevance to this review, our preliminary results suggest that enteric neurons can be generated from ES cells in vitro (Sasselli et al.,2006).

Neural stem cell-like cells have also been observed or induced to arise from a diverse population of other stem cell populations, including those resident in bone marrow (Jiang et al.,2003), and adipose tissue and skin (Safford et al.,2002,2004; Joannides et al.,2004). Whether these derived precursors can truly give rise to sustained and functional neuronal cells after transplantation remains to be established.

CNS-Derived NSC

NSC have been identified in both the developing and adult CNS and PNS of rodents and humans. These cells can be grown in culture and display the ability to self-renew, although they are more restricted than ES cells and give rise predominantly to neurons and glia (astrocytes and oligodendrocytes). In the CNS, NSC have been identified and isolated from various sites within the rodent brain, including the cerebellum, cerebral cortex, hippocampus, and subventricular zone (the richest source along with the hippocampus), and spinal cord. However, NSC from different sites are not necessarily identical, displaying different growth characteristics, trophic factor requirements, and specific patterns of differentiation (Mayer-Proschel,1997; Rao,1999; Morrison,2001).

The use of multipotent NSC for treatment has several advantages with respect to ES cells. First, cells of a given lineage (e.g., NSC to produce neurons) are “naturally poised” to generate a particular tissue (National Academy Press,2001) and may require little or no manipulation either in vitro or in vivo. Second, such stem cells are able to migrate to sites of injury, or other “homing” sites within an organ (Aboody et al.,2000). Third, they can produce biological factors that may have additional beneficial effects for their “native” tissue (Noble,2000). Thus 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; Ourednik and Ourednik,2004,2005; Martino and Pluchino,2006). A potential problem with NSC is that, by contrast with pluripotent ES cells, multipotent human adult stem cells undergo replicative senescence and crisis after approximately 50 cell divisions, related to the lack of telomerase activity and progressive shortening of the telomeres (Cheng et al.,2005). Fetal multipotent cells may have an advantage in this respect, but recent studies also indicate that the challenge of growing even adult stem cells in large numbers may be overcome in the near future (Gottlieb,2002).

Most investigators isolate NSC by dissecting out a region of the fetal or adult brain, typically the subventricular zone (SVZ). The tissue is then disaggregated, and the dissociated cells are grown in culture medium containing a high concentration of fibroblast growth factor-2 (FGF-2) and/or epidermal growth factor (EGF) and allowed to proliferate. It appears that FGF is mitogenic at the beginning of neurogenesis and EGF-responsive cells emerge later (Santa-Olalla and Covarrubias,1999; Martens et al.,2000). In vitro, FGF but not EGF, promotes self-renewal of NSC, whereas together the two growth factors permit the initial commitment of NSC into neuronal and glial phenotypes (Maric et al.,2003).

In culture, NSC can be characteristically made to grow and proliferate in colonies called neurospheres, which are floating spheroid structures containing a mixture of NSC and more differentiated (progenitor) cells suspended together in an extracellular matrix. These structures have served as convenient assays to test both the multipotentiality of NSC (by characterizing the types of cells present within) as well as their ability for self-renewal (with the assumption that isolated NSC should themselves give rise to secondary neurospheres; Campos,2004). Only 3% to 4% of the cells within neurospheres are actually true stem cells, in that they can give rise to all three neural lineages either in vivo or in vitro when induced to differentiate upon withdrawal of growth factors (EGF and FGF) or by specific inductive signals (trophic factors such as glia-derived growth factor [GDNF], brain-derived neurotrophic factor, or nerve growth factor [NGF]; Gritti et al.,1996).

In the past, it has been difficult, if not impossible, to distinguish tissue-specific stem cells from progenitor cells, which can also be found in the same tissue and represent more differentiated cells that divide and give rise to fully differentiated cell types (National Institutes of Health,2001). Rietze et al. have recently provided a potential means to distinguish the NSC phenotype by virtue of nestin positivity, and low to absent expression of both peanut agglutinin and heat stable antigen (nestin+, PNAlo, HASlo; Rietze et al.,2001). Nestin is an intermediate filament protein whose expression is widely used to identify mammalian neuronal precursor cells or stem cells (Lendahl et al.,1990; Zimmerman et al.,1994). The stem cell is initially positive for nestin, but secondary progenitor cells become negative for nestin. 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, similar to what has been described for another putative stem cell marker, Msi-1 (Musashi-1; Sakakibara and Okano,1997; Sakakibara et al.,2001). Furthermore, Uchida et al. have shown that CD133, a marker used to enrich for hematopoietic stem cells, can also be used to isolate human NSC (Uchida et al.,2000). If these results are validated, we may be able to purify NSC to a much greater degree. For the present, however, most investigators rely on neurosphere generation and nestin positivity as surrogate markers for NSC. Traditionally, it has been easier to work with fetal NSC because of their relative abundance; however, it should be noted that it is not yet established with complete certainty that they behave in the same way as their adult counterparts. Interest in the use of fetal NSC as a therapeutic tool has also been fueled 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 (Kuhn and Svendsen,1999; Svendsen et al.,1999).

CNS-Derived NSC Can Improve Function in a Mouse Model of Gastroparesis

Until very recently, CNS-derived NSC (CNS-NSC) were the most well-characterized and experimentally studied neuronal precursor population. Hence, we used these cells for our initial experiments a few years ago, even though we were well aware of the fact that enteric neurons may represent a different lineage. The potentials of CNS-NSC for the treatment of disorders of the ENS have recently been explored by us. NSC isolated from the SVZ of fetal rodent brain express RET and proliferate in response to GDNF (RET is the tyrosine-kinase receptor for GDNF, a trophic factor that plays a critical role during the development of the ENS) in vitro (Micci et al.,2001). Moreover, they express the enzyme responsible for the production of NO (nNOS), the major neurotransmitter in the ENS. When co-cultured with colonic smooth muscle cells CNS-NSC differentiate into neurons and form functional contact with smooth muscle cells (Fig. 2).

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Figure 2. Top: β-Tubulin immunoreactivity in rat central nervous system-derived neural stem cells (CNS-NSC) in co-culture with human colonic smooth muscle cells (visible in the background) indicating that CNS-NSC differentiate into neurons. Bottom: Intracellular calcium measurements in human colonic smooth muscle cells in co-culture with rat CNS-NSC in response to the nicotinic agonist DMPP. Cells were loaded with the calcium-sensitive fluorescent indicator Fura-2 and changes in intracellular calcium were measured in colonic smooth muscle cells in response to bath application of the nicotinic agonist DMPP (20 μM, indicated by the white arrows). A: DMPP induces an increase in intracellular calcium in colonic smooth muscle cells when in co-culture with rat CNS-NSC. B: This effect is not observed when colonic smooth muscle cells are cultured without rat CNS-NSC. C: The DMPP-induced response in colonic smooth muscle cells in co-culture with rat CNS-NSC was blocked by the nicotinic receptor antagonist hexamethonium, demonstrating that DMPP is acting on nicotinic receptor. D: Moreover, the response was blocked by the neurokinin A receptor antagonist (CP-099994-01), suggesting that it is mediated by substance P. These data show that rat CNS-NSC stimulated with DMPP induce an increase in smooth muscle calcium, demonstrating that they make functional contact with smooth muscle cells in vitro.

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When CNS-NSC are transplanted in the stomach of a transgenic mouse model of gastroparesis (the nNOS−/− mouse, characterized by delayed gastric emptying and the inability of the pyloric sphincter to relax in response to nerve stimulation), CNS-NSC graft and differentiate into neurons and glia, with the majority of neurons expressing nNOS. Moreover, 1 week after implantation of CNS-NSC, significant improvement of gastric function (known to be impaired in nNOS−/− mice) is observed, thus providing the first functional evidence that transplantation of CNS-NSC in the gut might be beneficial in the treatment of motility disorders (Fig. 3; Micci et al.,2005a). Although promising, these data are limited in their short-term nature and further investigation are necessary to assess long-term survival of CNS-NSC in the gut and their functional effect on motility.

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Figure 3. Left: Transplanted neural stem cells (NSC) survive in the pyloric wall and differentiate. A,B: NSC grafts in cross-sections of nNOS−/− mouse pylorus, 1 week post-transplantation showing immunoreactivity for the neuronal marker PGP9.5 and the glial marker GFAP. LM, longitudinal muscle; CM, circular muscle. Scale bar = 20 μm. Center: NSC transplantation results in increased relaxation of the pyloric muscle. A: Electrical field stimulation of pyloric circular muscle under NANC conditions in nNOS−/− mice. B: Quantification of NANC-induced relaxations in response to EFS in nNOS−/− mice as described in A. C: L-NAME (100 μM) blocks relaxations, indicating that the relaxations are NO mediated. Data are means ± SEM. *P < 0.05 and **P = 0.001. Right: Transplanted NSC improve gastric emptying in nNOS−/− mice. Gastric emptying of liquids in nNOS−/− mice 1 week after NSC transplantation or vehicle. *P < 0.01; n = 6 mice for vehicle and 8 mice for NSC. (Reprinted from Micci et al.,2005a.)

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Neural Crest Stem Cells and ENP

Within the developing CNS, a group of cells arise from the neural crest, the neural crest stem cells (NCSC). NCSC have been isolated and characterized in multiple species from the neural crest. NCSC generate the PNS and also migrate throughout the gut to form the ENS (Gershon,1997; Heuckeroth and Pachnis,2006). Like CNS-NSC, NCSC are multipotent and generate differentiated progeny by means of the production of more restricted progenitor cells, but in addition to neurons and glia they can also generate non-neuronal cells (i.e., smooth muscle cells, pigment cells, bone and cartilage of the face and skull). NCSC, therefore, differ from CNS-NSC because of their extensive ability to migrate, their epithelial to mesenchymal transition, and the phenotypes that can arise from them. Evidence from chick embryo experiments suggest that NCSC and differentiated CNS cells share a common progenitor (Bronner-Fraser and Fraser,1989; Mujtaba et al.,1998). Spinal cord neuroepithelial cells can generate p75+/nestin+ cells that are morphologically and antigenically similar to previously characterized NCSC (Mujtaba et al.,1998). CNS-NSC–derived p75+ cells differentiate into peripheral neurons, smooth muscle, and Schwann cells in both mass and clonal culture (p75 is the low-affinity receptor for NGF, whose function during development is not completely understood). Moreover, clonal analysis indicated that individual CNS-NSC could generate both CNS and PNS derivatives, providing evidence for the first time of a direct lineage relationship between these two distinct cell types. However, it is unclear whether NCSC can generate CNS derivatives. When NCSC were transplanted into the spinal cord, they gave rise to Schwann cells but not to CNS derivatives, suggesting that the NCSC is a more restricted precursor. These concepts are illustrated in Figure 4.

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Figure 4. During development, early neuro-ectodermal stem cells generate two subpopulations of multipotent neural stem cells: Central nervous system-derived neural stem cells (CNS-NSC) that give rise to neurons and glia of the CNS and neural crest stem cells that generate the neurons and glia of the peripheral nervous system (PNS), including the enteric nervous system (ENS), as well as other non-neural lineages. Recent reports suggest that individual CNS-NSC could generate both CNS and PNS derivatives, providing evidence for the first time of a direct lineage relationship between these two distinct cell types. Specific markers expressed by each population are indicated in red.

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NSC have also been isolated from the immature as well as adult ENS (Kruger et al.,2002; Sidebotham et al.,2002a,b; Bixby et al.,2002; Suarez-Rodriguez and Belkind-Gerson,2004; Burns,2005), a discovery that raises the possibility of an intrinsic mechanism for enteric neuronal replacement within the gut, even though we are as yet unaware of what it takes to harness this potential. Whether these cells have the same potential of NCSC is not clear but are probably more restricted to a gut phenotype and more appropriately termed enteric neuronal precursors, or ENP. Such precursor cells have been isolated from the embryonic and postnatal gut of rats using antibodies to specific markers known to be expressed by enteric neural crest-derived cells: RET (the tyrosine-kinase receptor for GDNF; Lo and Anderson,1995) and p75 (Kruger et al.,2002).

ENP have also been isolated from the gut without using these markers. When the embryonic or postnatal gut is dissociated and the cells cultured in high concentrations of EGF and/or FGF, neurospheres are generated that contain nestin+ proliferating progenitor cells, neurons, and glia (Bondurand et al.,2003; Schafer et al.,2003; Suarez-Rodriguez and Belkind-Gerson,2004). These cells partially recapitulate the genetic program of ENS development from NCSC in that they express Sox10 but lack detectable levels of RET or lineage specification markers, such as MASH1 (the transcription factor Sox10 plays a critical role in development of the PNS including sensory, autonomic, and enteric ganglia [Britsch et al.,2001] while Mash1 encodes a basic helix–loop–helix pro-neural factor that is required for specific subsets of enteric neurons; Blaugrund et al.,1996).

ENP isolated from the small intestine of lactating and adult mice can differentiate into various cell types, particularly neurons, smooth muscle, and glia with the neurons expressing several sensory and motor neurotransmitters present in the CNS and ENS, including calcitonin gene-related peptide, neuropeptideY, peptideYY, substance P, vasoactive intestinal polypeptide, and galanin; along with glia, these neurons form elaborate intercellular connections. Both endothelin 3 and SOX10 are important factors in maintaining these cells in an uncommitted state (Bondurand et al.,2006). GDNF promotes ENS precursor survival through PI-3 kinase (Srinivasan et al.,2005), but its effects on proliferation are opposed by Sonic hedgehog (Shh), which also prevents their differentiation (Fu et al.,2004).

ENP differ from their neural crest counterparts in the sciatic nerve—the cells from the gut are more responsive to neurogenic factors, while sciatic nerve cells are more responsive to gliogenic factors (Bixby et al.,2002). When transplantated into peripheral nerves in vivo, sciatic NSC generate only glia, while gut NSC give rise primarily to neurons. Thus ENP, by virtue of their genetic lineage, may have quite different potentials than their CNS counterparts and may be more suitable for therapeutic purposes as they are more likely to respond to gut-specific environmental cues.

There are several compelling reasons to think that ENP may become the preferred route for clinical use. First, it has been shown by Dr. Pachnis' laboratory that multipotential progenitors can be derived from biopsy-sized gut segments, allowing minimally invasive harvesting of tissue (Bondurand et al.,2003). The gut is readily accessible endoscopically in humans, thus rendering this an attractive feature. The same team has also shown that ENP can also be isolated from unaffected segments of the gut of Ret mutant animals. Furthermore, isolation and expansion of precursor cells from the developing and postnatal human ENS has recently been reported using bowel samples from human fetuses and children (from the ninth week of gestation to 5 years postnatal). Neurospheres obtained by such methods could be differentiated and also be transplanted after dissociation into aganglionic bowel in vitro (Rauch et al.,2005), thus confirming earlier experimental results with murine ENP (Bondurand et al.,2003). These are exciting results and highlight the potential for the use of ENP in postnatal motility disorders.

HOW DO WE OPTIMIZE SURVIVAL AFTER TRANSPLANTATION?

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENTERIC NEUROMUSCULAR DISORDERS
  5. AN OVERVIEW OF STEM CELLS
  6. WHAT IS THE IDEAL SOURCE OF STEM CELLS FOR USE IN THE ENS?
  7. HOW DO WE OPTIMIZE SURVIVAL AFTER TRANSPLANTATION?
  8. CHALLENGES AND FUTURE DIRECTIONS
  9. REFERENCES

Despite encouraging initial results reported by many investigators (including ourselves), there are still significant limitations that prevent the clinical development of NSC replacement as a therapy for GI neurodegenerative diseases. Specifically, posttransplant survival of NSC represents a critical limiting factor for successful anatomical and functional repopulation of the host tissue. Upon grafting, more than 90% of neurons usually die, both in animal and human studies (Freeman et al.,1995; Rosenstein,1995). Transplanted NSC can die from a variety of causes: physical injury, immune attack by the host, lack of trophic factors, or toxic environmental factors (free radicals, cytokines, etc.). Such death can either be due to necrosis or apoptosis. A large portion of this cell death occurs as apoptosis, and occurs within the first week after transplantation (Mahalik et al.,1994; Zawada et al.,1998). Apoptotic cell death is commonly observed in neural precursor cells even when they are grown in vitro in free-floating neurospheres, with approximately 25–30% of these cells showing caspase-3 activation, the majority of which localizes with nestin positivity; much of this appears to be driven by receptor-independent intrinsic pathways under the control of the Bcl-2 family of proteins (Milosevic et al.,2004). Therefore, until an effective approach to prevent the loss of NSC after transplantation becomes available, it will not be possible to develop a clinically relevant NSC-based treatment for GI neuromuscular disorders.

Intrinsic Apoptotic Programming May Be More Important Than Host Immune Responses in Determining Survival of Transplanted NSC

Our previously published results have shown that inhibition of apoptosis using a selective inhibitor of caspase-1 (Ac-YVAD-cmk) at the time of CNS-NSC transplantation, results in a significant improvement in graft survival 1 week posttransplantation (Micci et al.,2005b). Moreover, immunosuppression with cyclosporine A, a commonly used drug for organ transplantation, does not improve cell survival of CNS-NSC transplanted in the gut. In particular, we have shown that the effect of the caspase-1 inhibitor on graft survival is associated with reduced apoptosis and increased proliferation of grafted CNS-NSC.

Caspase-1 is also known as ICE (interleukin converting enzyme), because it converts pro-interleukin (IL) -1β to the active form; in addition, it also activates another pro-inflammatory cytokine, IL-18 (interferon-γ–inducing factor; Friedlander et al.,1997a,b; Friedlander and Yuan,1998; Friedlander,2003). Thus caspase-1 activation in appropriate cells (possibly induced by cytokines such as TNF) will result in the amplification of the inflammatory response by means of the production of IL-1β and interferon (IFN) -γ. A second important role for caspase-1 is induction of neuronal apoptosis, at least in pathological conditions. The treatment of neuronal precursors with a pharmacological inhibitor of caspase-1 (Ac-YVAD-cmk) before transplantation has been shown to prevent apoptosis from trophic deprivation in vitro and improve short-term survival and functional outcomes after transplantation of embryonic nigral cells in vivo (Schierle et al.,1999; Hansson et al.,2000; Karlsson et al.,2005).

Despite the encouraging results that we reported at 1 week using this approach, the number of grafted cells was drastically reduced at 2 and 4 weeks post-transplantation. This finding might suggest that the pharmacological treatment with a caspase inhibitor might not be sufficient in producing a long-lasting effect on graft survival, possibly due to the fact that the caspase inhibitor is only present at the time of transplantation. Long-term survival of grafted cells, therefore, is a problem of critical importance that will need to be addressed in future studies. A possible approach would be to down-regulate specific caspases using siRNA or antisense oligonucleotides in the NSC before grafting. An alternative approach involves the overexpression of an anti-apoptotic protein such as Bcl-2, which may result in a longer and more specific inhibition of apoptosis in the NSC after transplantation in the host tissue.

Role of Trophic Factors in Determining Survival

During development, postmigratory neural progenitors from NCSC colonize the gastrointestinal tract and form most of the ENS. Interaction of the receptor tyrosine kinase (RET) expressed by these cells with gut-derived neurotrophic growth factors GDNF and NTN is critical in this process. Similar to our report on rat NSC (Micci et al.,2001), our preliminary data have shown that mouse CNS-NSC share a critical feature with NCSC precursors in that they also express the receptor complex for GDNF (RET and GFRα1) and respond to GDNF in vitro. This finding could suggest that a subset of CNS-NSC share some phenotypic features with NCSC and, hence, could potentially be more likely to successfully colonize the gut. Studies from adult mice suggest that GDNF is expressed in the gut, but at relatively low levels.

In preliminary experiments we have tested the hypothesis that GDNF can further improve survival of grafted CNS-NSC in the mouse GI tract (unpublished). RET+ and RET fractions were prepared from CNS-NSC and transplanted into the pyloric wall of recipient mice. To test the effect of GDNF, mice received daily injections of human recombinant GDNF or vehicle. However, neither RET sorting nor GDNF supplementation in any group was able to improve survival. Thus, as yet, we do not have a specific strategy to improve survival of grafted CNS-NSC using trophic factors.

How Does the Environment Participate in Determining the Outcome After Transplantation?

Enteric dysmotility is seldom due to the isolated loss of a single neurotransmitter as in nNOS−/− mice. Instead, it typically occurs either due to a congenital absence of neural crest derivatives (as in Hirschsprung's disease) or due to an inflammatory process targeting the myenteric ganglia (as in achalasia). Understanding the effects of such an environment on transplanted NSC is very important in making decisions about the use of cell replacement therapies in neonatal or very young patients (such as those with Hirschsprung's disease) or in patients with a damaged or inflamed environment (such as those with achalasia or auto-immune forms of neuronal destruction).

The presence or absence of inflammation in the microenvironment can have a profound effect not only on the survival and differentiation of apoptosis but also on the nature of the therapeutic effect (i.e., differentiation and assumption of a functional target tissue phenotype or modulation of the environment by secretion of growth factors and cytokines, the so-called bystander effect; Martino and Pluchino,2006). Neural stem and progenitor cells express a variety of receptors that enable them to sense and react to signals in their environment such as cytokines produced by blood-borne and resident inflammatory cells (Mueller et al.,2005). These cytokines can have both detrimental and beneficial effects. TNF-α, IL-1β, and IL-6 can profoundly inhibit neurogenesis (Vallieres et al.,2002). TNF, acting by means of TNFR1, can also produce apoptosis in neural precursor cells (Sheng et al.,2005). Microglia can have varying effects on neural precursors, depending upon the nature of the insult: NSC differentiation can be blocked by endotoxin-activated microglia (in association with up-regulation of TNF-α), but induced by microglia exposed to IL-4 or low level of IFN-γ (mediated in part by production of insulin-like growth factor-I). Furthermore, the IL-4–activated microglia show a bias toward oligodendrogenesis, whereas the IFN-gamma–activated microglia show a bias toward neurogenesis (Butovsky et al.,2006). Microglia also release other soluble factors, including chemokines such as CXCL12 and MCP-1, all of which can direct migration and differentiation of neural precursor cells (Aarum et al.,2003; Peng et al.,2004; Belmadani et al.,2005,2006). It is clear, therefore, that an inflamed environment can have profound effects on the fate of transplanted NSC, with the exact outcome varying with the profile of cytokines and other locally produced factors and consequently difficult to predict.

Similarly profound changes occur in the microenvironment of the ENS during early life. It is intuitive that the neonatal environment will be more supportive of transplanted stem cell survival and appropriate differentiation, but this has not been rigorously studied because of the difficulties in performing surgical procedures in animals at this age. When CNS-NSC are transplanted into “ectopic” sites such as the retina, they can survive and differentiate into mature neurons but not necessarily into retinal cells unless the eye is either damaged or from a neonatal animal (Sakaguchi et al.,2003,2004,2005). Successful incorporation of neural precursors into the retina decreases with increasing age of the host eye and is maximal in the first few days after birth in experimental animals (Van Hoffelen et al.,2003). This finding is not surprising since survival and differentiation cues, such as growth or neurotrophic factors are expected to be much more abundant in early life and contribute to better survival, differentiation, and migration of the transplanted cells at younger ages. Thus the expression of GDNF and neurturin, both important factors for the development of a healthy ENS, is highest in embryonic tissue and declines with maturity (Golden et al.,1999).

CHALLENGES AND FUTURE DIRECTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENTERIC NEUROMUSCULAR DISORDERS
  5. AN OVERVIEW OF STEM CELLS
  6. WHAT IS THE IDEAL SOURCE OF STEM CELLS FOR USE IN THE ENS?
  7. HOW DO WE OPTIMIZE SURVIVAL AFTER TRANSPLANTATION?
  8. CHALLENGES AND FUTURE DIRECTIONS
  9. REFERENCES

It is clear, therefore, that many issues need to be addressed and techniques optimized before stem cell therapy can be considered a practical therapeutic approach for disorders of the ENS. There are several potential sources of NSC, from fetal or adult CNS, from fetal or adult gut, from totipotential ES cells, and even from other non-neural stem cell populations (i.e., mesenchymal stem cells), and it is not yet clear which of these will work best. We also need more research on ways to ensure proper geographic location, adequate survival, and neuronal differentiation, particularly in the fetal or inflamed environment. Posttransplant survival appears to be a critical limiting factor for successful anatomical and functional repopulation. This may be determined by both immunological as well as nonimmune mediated mechanisms, including intrinsic programming toward apoptosis. Finally, we have worked so far on a model with a very simple and single molecular deficiency (i.e., lack of nitric oxide); we also need to know more about how to “nudge” these cells to assume the desired phenotype for other therapeutic indications.

Preliminary experimental data are exciting and suggest that it is possible to restore neuronal function to affected segments of the gut. Although, as with many other recent “breakthroughs,” stem cell therapy may eventually prove to be overrated, at the present time it does appear to provide the hope for a true cure for many currently intractable diseases of both the central and the peripheral nervous system.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ENTERIC NEUROMUSCULAR DISORDERS
  5. AN OVERVIEW OF STEM CELLS
  6. WHAT IS THE IDEAL SOURCE OF STEM CELLS FOR USE IN THE ENS?
  7. HOW DO WE OPTIMIZE SURVIVAL AFTER TRANSPLANTATION?
  8. CHALLENGES AND FUTURE DIRECTIONS
  9. REFERENCES