1 Division of Molecular Neurobiology, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Division of Immunoregulation, MRC National Institute for Medical Research, The
Ridgeway, Mill Hill, London NW7 1AA, UK
Author for correspondence (e-mail:
vpachni{at}nimr.mrc.ac.uk)
Accepted 4 September 2003
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SUMMARY |
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Key words: Enteric nervous system, Neural crest progenitors, Hirschsprung's disease
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Introduction |
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HSCR is the most common neurocristopathy in humans affecting 1:4500
newborns (Chakravarti, 2001;
Swenson, 2002
). It appears
either sporadically or has a familial basis often associated with other
developmental defects. The main form of treatment of HSCR is surgical
resection of the aganglionic bowel and anastomosis of the remaining gut
segments, a procedure aimed at relieving the life threatening consequences of
obstruction, and restoring bowel movements
(Swenson, 2002
). However, the
outcome of this approach is often unsatisfactory as it is frequently
associated with short-term postoperative complications or failure to restore
bowel function in the long-term (Tsuji et
al., 1999
). The better molecular and cellular understanding of
HSCR pathogenesis and the identification of genes that play an important role
in enteric neurogenesis in mammals offers new opportunities for the
development of therapies based on gene or cell replacement strategies.
Among the genes that have been identified as critical players in ENS
development is Ret which encodes the receptor tyrosine kinase (RTK)
RET, the main signalling component of cell surface multisubunit receptors for
glial cell line-derived neurotrophic factor (GDNF) and other members of the
GDNF family of ligands (Baloh et al.,
2000; Saarma,
2000
). Mutations in RET account for approximately 50% of
familial HSCR cases (Chakravarti,
2001
) and mice with null mutations in either Ret
(Retk-) or Gdnf (Gdnf-) have
complete intestinal aganglionosis (Durbec
et al., 1996
; Enomoto et al.,
2001
; Moore et al.,
1996
; Pichel et al.,
1996
; Sanchez et al.,
1996
; Schuchardt et al.,
1994
). Ret encodes two isoforms, RET9 and RET51, which
differ in their carboxy-terminal sequences
(Tahira et al., 1990
).
Analysis of the effects of monoisoformic alleles of Ret, which encode
either RET9 (miRet9) or RET51
(miRet51), demonstrated that RET9 is sufficient to support
normal development of the ENS and that expression of RET51 in the absence of
RET9 is associated with failure of enteric ganglia formation in the distal two
thirds of the colon (de Graaff et al.,
2001
).
Several transcription factors have also been identified as important
regulators of ENS development. Among them is SOX10, a member of the high
mobility group (HMG) family of proteins, which include the SRY
testis-determining factor and other members of the SOX subfamily
(Kuhlbrodt et al., 1998;
Wegner, 1999
). The critical
role of SOX10 in PNS development is highlighted by the severe defects of
sensory, autonomic and enteric ganglia in mice lacking SOX10
(Britsch et al., 2001
;
Herbarth et al., 1998
;
Kapur, 1999
;
Paratore et al., 2001
;
Southard-Smith et al., 1998
).
In addition, humans and mice heterozygous for mutations in this locus have
distal colonic aganglionosis (Herbarth et
al., 1998
; Parisi and Kapur,
2000
; Pingault et al.,
1998
; Southard-Smith et al.,
1998
). Defects in ENS development have also been identified in
mice homozygous for a null mutation of Mash1 (Ascl1-Mouse
Genome Informatics) a locus encoding a basic helix-loop-helix (bHLH) proneural
factor (Johnson et al., 1990
).
MASH1-deficient embryos have a defect in gut colonisation by neural crest
cells, while mutant newborn animals lack specific subsets of enteric neurons
(Blaugrund et al., 1996
).
Enteric neurogenesis is associated with a highly regulated programme of
gene expression that defines distinct stages of cell commitment and
differentiation. Thus, during the early stages of migration PENCCs express
Sox10, which is important for the maintenance of the undifferentiated
multipotential state of ENS progenitors, but are negative for MASH1 or RET
(SOX10+/MASH1-/RET-) (Durbec et al.,
1996; Kim et al.,
2003
; Paratore et al.,
2002
). However, as PENCCs arrive in the vicinity of the foregut,
they begin to express Mash1 and Ret (SOX10+/MASH1+/RET+)
(Blaugrund et al., 1996
;
Durbec et al., 1996
).
Induction of Mash1 is likely to be associated with neuronal
specification (Lo and Anderson,
1995
; Lo et al.,
1997
) while RET at this stage is required for the survival,
proliferation, differentiation and migration of ENCCs
(Gianino et al., 2003
;
Taraviras et al., 1999
).
Terminal differentiation of subsets of committed neurogenic progenitors
(indicated by expression of neuronspecific tubulin-TuJ1 and PGP9.5) takes
place from the earliest stages of gut colonisation and continues for at least
two weeks after birth.
Progenitors of the ENS have been isolated from rodent embryos and partially
characterised in culture. Using antibodies against RET and
fluorescence-activated cell sorting (FACS), Anderson and colleagues isolated
from the gut of rat embryos, a population of cells which in clonogenic assays
generated mostly neurons (Lo and Anderson,
1995). However, the equivalent murine cell population when grafted
into foetal gut maintained in organotypic culture generated both neuronal and
glial progeny (Natarajan et al.,
1999
). More recently, a self-renewing population of neural crest
stem cells (NCSCs) has been isolated from the gut of foetal and postnatal rats
using antibodies against cell surface markers and flow cytometry
(Bixby et al., 2002
;
Kruger et al., 2002
). In vitro
clonogenic assays and in vivo engraftment of such gut-derived NCSCs showed
that they are capable of generating both neuronal and glial cells. However,
the ability of these cells to re-colonise gut and differentiate within its
wall has not been established.
Isolation of ENS progenitors from fresh tissue so far has relied on the use of cell surface markers, an approach that generally requires relatively large amounts of starting material. Here, we have used cultures of dissociated gut and retrovirus-mediated gene transfer to isolate multipotential progenitors of enteric neurons and glia from both foetal and postnatal gut at least up to 2 weeks after birth. We describe the phenotypic characteristics of these cells, their progressive differentiation in vitro and their ability to generate both neuronal and glial progeny upon transplantation into foetal gut maintained in organ culture. Finally, we show that similar progenitors can be isolated from the normoganglionic gut segments of mice with colonic aganglionosis. We discuss the implications of our findings for the treatment of HSCR by cell replacement strategies.
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Materials and methods |
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Isolation of enteric nervous system progenitor cells (EPCs) from
foetal and postnatal gut
To generate neurosphere-like bodies (NLBs) from foetal gut tissue, whole
gut was dissected from E11.5 embryos (wild-type or from
Retk- intercrosses) in L15 medium (Invitrogen, UK), washed
with Ca2+- and Mg2+-free PBS (Invitrogen, UK) and
digested for 6 minutes with a mixture of 1 mg/ml dispase/collagenase (Roche,
UK) at room temperature (RT). Dissociated tissue was washed sequentially with
1x PBS and NCSC culture medium
(Morrison et al., 1999), which
included 15% chicken embryo extract (CEE) and basic fibroblast growth factor
(bFGF; 20 ng/ml; R&D Systems), and plated onto tissue culture dishes
coated with 20 µg/ml fibronectin (Sigma). Cultures were re-fed every 2
days. Once NLBs appeared, human recombinant epidermal growth factor (hrEGF)
(20 ng/ml; Calbiochem) was added to the medium.
To generate postnatal NLBs, small intestines from P2-P14 wild-type (Parkes) or miRet51 animals were removed and cleaned. Using pairs of forceps, the outer smooth muscle layers along with the myenteric plexus were peeled off from the underlying tissue. These strips were washed in 1x PBS and treated with 1 mg/ml collagenase (Sigma) for 45 minutes at 37°C. The resulting cell suspension was washed in NCSC medium and plated on fibronectin-coated six-well dishes (NUNC). A similar protocol was used to generate NLBs from newborn Retk- homozygous animals. Foetal or postnatal NLBs were either cultured for an additional 5-10 days to isolate enteric nervous system progenitor cells (EPCs) or dissociated and re-plated at low density to generate secondary or tertiary NLBs.
To isolate EPCs, NLBs were trypsinised at RT, washed with NCSC medium and passed through a mesh (pore size 50 µm) to produce a near single cell suspension, which was plated as before. Cells were infected using a mixture of NCSC medium and a GFP-expressing retrovirus suspension (1:1) in the presence of polybrene (5 µg/ml). GFP-expressing cells were isolated 24 hours later by fluorescence activated cell sorting (FACS) and plated in complete NCSC medium at a density of 50-100 cells per SonicsealTM well and cultured for up to 15 days. Human recombinant (hr) GDNF protein (Peprotech) was used at 10 ng/ml either in complete medium or in NCSC medium lacking CEE, EGF or FGF.
Manually cut pieces of NLBs and isolated EPCs were grafted into
organotypically cultured E11.5 mouse embryo gut as described previously
(Natarajan et al., 1999).
Detailed protocols of all the procedures described here are available upon
request.
Retrovirus production
BOSC cells were transfected with the GFP-expressing retroviral vector pMX
(Kitamura, 1998), using
CaCl2 and 25 µM chloroquine for 10 hours. Cells were washed,
allowed to recover and viral particles collected after 36 hours in DMEM medium
plus 10% foetal calf serum, 100 U/ml antibiotic mixture, 1 mM L-glutamine (2.5
ml/10 cm plate). Viral stocks were filtered (0.45 µ) and stored at
-80°C.
Immunostaining
Cultures were fixed in 4% PFA (in 1x PBS) for 10 minutes at RT. After
washing twice in PBS + 0.1% Triton X-100 (PBT), they were incubated with
blocking solution (PBT + 1% BSA + 0.15% glycine) at 4°C (overnight) or at
RT (for 2-3 hours). Primary antibodies were diluted in blocking solution as
follows: TuJ1(mouse; Babco, UK) 1:1000, PGP9.5 (rabbit; Biogenesis, UK) 1:400,
vasointestinal peptide, VIP (rabbit; Biogenesis, UK) 1:500, neuropeptide Y,
NPY (rabbit; Biogenesis, UK) 1:100, CGRP (rabbit; Biogenesis, UK) 1:100,
tyrosine hydroxylase, TH (rabbit; Chemicon, UK) 1:1000, RET (rabbit;
Immuno-Biological Labs, Japan) 1:50, GFAP (rabbit; DAKO, USA) 1:400,
phospho-histone-3 (PH3) (rabbit; Upstate) 1:500, SOX10 (mouse; kindly provided
by Dr David Anderson) (Lo et al.,
2002) 1:10, Mash1 (mouse; from D. Anderson)
(Lo et al., 1991
) 1:1, GFP
antibody (mouse or rabbit; Molecular Probes) both 1:1000. Cultures were
incubated with primary antibodies at RT (for 5-6 hours) or at 4°C
(overnight). After several washes with PBT, secondary antibodies were added in
blocking solution for 2-4 hours at RT at the following dilutions: anti-mouse
FITC-conjugated (Jackson labs) 1:500, anti-rabbit FITC-conjugated (Jackson
labs) 1:500, anti-mouse AlexaFlour (Molecular Probes) 1:500, anti rabbit Alexa
Flour (Molecular Probes) 1:500. Preparations were counter stained with
TO-PRO-3-iodide (Molecular Probes; 1:3000 in PBS) and mounted using
VectashieldTM (Vector Laboratories) or mounted directly in Vectashield
containing DAPI. Immunostained cultures were examined with a Bio-Rad confocal
microscope or a Zeiss epifluorescence microscope (Axiophot). Images were
analysed using Metaphor software package (Universal Imaging) and figures were
compiled using Adobe Photoshop 7.
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Results |
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During embryogenesis, all enteric neurons and glia originate from neural
crest-derived progenitors (Le Douarin, 1999;
Yntema and Hammond, 1954). To
confirm that NLBs were also derived from cells of neural crest origin, we
examined gut cultures from RET-deficient animals in which the gastrointestinal
tract fails to be colonised by ENCCs
(Durbec et al., 1996
;
Schuchardt et al., 1994
).
Intestine was dissected from individual progeny of intercrossed
Retk- heterozygotes and cultured as described above. As
expected, intestine from wild-type (Ret+/+) or
heterozygous (Ret+/k-) embryos or neonates formed NLBs
with many TuJ1+ and GFAP+ cells (Fig.
2A,B and data not shown). In contrast, neither NCSC-like colonies
nor NLBs were present in gut cultures generated from
Retk-/k- animals (Fig.
2C). Furthermore, staining of mutant cultures for TuJ1 and GFAP,
failed to identify any neurons or glial cells
(Fig. 2D). In summary, our
experiments suggest that the culture conditions we have employed here support
the expansion, propagation and differentiation of neurogenic and gliogenic
progenitors from foetal and postnatal gut.
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EPCs recapitulate the ontogenetic profile of ENS progenitors
To further characterise the EPC colonies, GFP+ cells were isolated from
NLBs, plated at clonal densities and analysed 1, 3 and 10 days after plating
for expression of several molecular markers that define various stages of
cellular differentiation in the ENS. On day 1, approximately 25% of foetal
EPCs expressed Sox10 (Fig.
5A1-A2). This percentage matches the clonogenic efficiency of
these cells and suggests that only Sox10-expressing GFP+ cells have
the potential to form multilineage colonies. Immunostaining for MASH1 and RET
identified no positive cells at this stage
(Fig. 5B1-C2) which, together
with the virtual absence of TuJ1 and GFAP staining
(Fig. 4D,J) suggested that,
when isolated from NLBs, EPCs expressed only markers of undifferentiated
progenitors but lacked markers of neuronal commitment and neuronal or glial
differentiation.
|
On day 10, 25% of cells in all colonies were positive for the neuronal markers TuJ1 (Fig. 4) and PGP9.5 (Fig. 5H1). Double labelling experiments showed that all PGP9.5-expressing cells were negative for SOX10 (Fig. 5H1-H3). Also, cells expressing relatively low levels of PGP9.5 (PGP9.5low) co-expressed Mash1 but the vast majority of PGP9.5high cells were negative for MASH1 (Fig. 5J1-J3). Finally, cells expressing TuJ1, also expressed the highest levels of Ret (Fig. 5L1-L3). In addition to neuronal markers, 87% of day-10 foetal EPC colonies included cells expressing GFAP. Double staining showed that GFAP+ cells were generally SOX10+, but a large fraction of SOX10+ cells were negative for GFAP (Fig. 5I1-I3). As no mature neurons expressed SOX10, these data are consistent with the idea that this transcriptional regulator is expressed in undifferentiated EPCs and their gliogenic progeny. Also, GFAP was absent from cells expressing high levels of Mash1 or Ret (Fig. 5K1-K3 and M1-M3).
The molecular mechanisms underlying differentiation of postnatal EPCs were likely to be similar to those operating in their foetal counterparts. Thus, on day 1, 25% of GFP+ cells isolated from postnatal NLBs were expressing Sox10 but all cells at this stage were negative for MASH1 and RET (Fig. 6A1-C2). On day 3, all colony-associated cells maintained expression of Sox10, a subset of them induced Mash1 and Ret but no TuJ1 was detected (Fig. 6D1-G2). By day 10, a large number of PGP9.5+/SOX10-neurons were present in colonies (Fig. 6H1-H3), most of which also contained GFAP+/SOX10+ cells (Fig. 6I1-I3). Also, the relative expression of PGP9.5, TuJ1, MASH1, RET and GFAP at this stage was similar to that of foetal EPC colonies (Fig. 6J1-M3). Taken together, our findings indicate that clonogenic EPCs isolated from foetal and postnatal NLBs probably represent undifferentiated bipotential ENS progenitors. As is the case for the in vivo progenitors, cultured EPCs initially acquire markers of neurogenic and gliogenic commitment and eventually differentiate into mature neurons and glia.
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EPCs differentiate into enteric neurons and glia upon grafting into
foetal gut in organ culture
We next tested the ability of NLB cells to colonise foetal gut. For this,
manually cut pieces of foetal or postnatal NLBs were labelled with DiI and
grafted into the wall of guts dissected from E11.5 wild-type mouse embryos and
cultured organotypically for 4 days, as described previously
(Natarajan et al., 1999).
Emigration of cells from both foetal or postnatal NLBs was initiated within 24
hours of grafting and continued until an extensive halo of DiI-positive cells
formed around the site of engraftment (data not shown). Extensive migration of
DiI-labelled cells was also observed from foetal or postnatal NLBs grafted
into guts isolated from Retk- mutant embryos, which fail
to develop intrinsic enteric neurons and glia. In addition, immunostaining of
grafted mutant guts for TuJ1 or GFAP revealed the presence of large numbers of
neurons and glia in the domains that had been colonised by DiI-labelled cells
(Fig. 9B and data not shown).
As expected, such differentiated cells were absent from non-grafted mutant
guts (Fig. 9A). These
experiments indicated that neuronal and glial cells originating in foetal and
postnatal NLBs can colonise wild-type and aganglionic gut in organ
culture.
|
To examine the ability of postnatal EPCs to integrate into the intrinsic ENS and differentiate into mature neurons and glia, similar transplantation experiments were carried out using GFP+ cells isolated from postnatal NLBs. These experiments established that, similarly to foetal EPCs, postnatal EPCs were capable of colonising the gut (67%, n=34) and migrating along the anteroposterior and radial axis of the gut wall (Fig. 9J). In addition, in 73% and 61% of the successfully transplanted guts, we observed GFP+ cells differentiating into neurons (GFP+/TuJ1+) and glia (GFP+/GFAP+), respectively (Fig. 9K,L).
We also tested the ability of EPCs to colonise aganglionic gut in organotypic culture. For this, the gut of Retk-/k- embryos was transplanted with foetal EPCs or postnatal EPCs and analysed by double immunostaining as above. We found that similar to the transplantations into wild-type gut, both the foetal and postnatal EPCs were capable of recolonising aganglionic gut mesenchyme with an efficiency comparable to those observed for wild-type gut (83% n=6 and 60% n=5, respectively; Fig. 9H,M). Furthermore, both foetal and postnatal EPCs were capable of differentiating into neurons (Fig. 9I,N). However, we were unable to detect double positive GFP+/GFAP+ cells (not shown). In summary, our experiments show that both foetal and postnatal EPCs have the potential to colonise the wall of wild-type and aganglionic gut. In addition, both types of progenitors can generate neuronal and glial progeny upon transplantation into wild-type gut wall. However, under our present experimental conditions, grafting into aganglionic gut resulted in the generation of neurons only.
EPCs can be generated from partially aganglionic postnatal gut
Mice homozygous for the hypomorphic allele of Ret
(miRet51) constitute a model for HSCR as they are
characterised by colonic aganglionosis (de
Graaff et al., 2001). To examine whether the apparently
normoganglionic small intestine of miRet51 neonates can
give rise to multilineage ENS progenitors upon dissociation and culture, the
outer muscle layers of the small intestine from P2-8
miRet51 homozygotes were dissociated and cultured under
standard conditions. Wild-type littermates were used as controls. As shown in
Fig. 10A,B, gut cultures from
miRet51 homozygous mutants generated large numbers of NLBs
with a time course similar to that of control cultures. In addition,
GFP-retrovirus transduction of miRet51 NLBs resulted in
the efficient isolation of GFP-expressing cells which in clonogenic assays
were capable of generating neurons and glia
(Fig. 10C). These data suggest
that the normoganglionic intestine of miRet51 homozygous
animals is capable of generating EPCs with an efficiency similar to that of
wild-type postnatal gut.
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Discussion |
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Our strategy for the isolation of EPCs was not dependent on expression of
cell surface or intracellular markers on ENS progenitors; instead, it was
based exclusively on the ability of neural crest-derived cells in the gut to
maintain their proliferative capacity and differentiation potential, leading
to formation of NLBs. However, gene expression analysis of EPCs and their
progeny in clonogenic assays showed that they undergo changes in patterns of
gene expression which reproduce those observed in endogenous ENS progenitors
(Young et al., 2003;
Young et al., 1999
). Thus,
similarly to the early migratory vagal neural crest cells, EPCs express
Sox10 but lack detectable levels of RET or lineage specification
markers, such as MASH1 (SOX10+/RET-/MASH1-). However, shortly after plating, a
subset of EPC progeny express RET and MASH1 and differentiate into mature
neurons. SOX10+/RET-progenitor cells had not been identified previously in
mouse embryonic or postnatal gut. In a recent study, Young and colleagues
showed that the majority of ENS progenitors express Sox10 and
Ret at all embryonic stages examined
[(Young et al., 2003
;
Young et al., 1999
) and
personal communication]. This raises the question of the cellular origin of
EPCs in the cell culture system described here. One possibility is that our
protocol preferentially expands a relatively small subpopulation of
pre-existing SOX10+/RET-/MASH1-cells which represent early migrating vagal
neural crest cells that persist in the mouse gut throughout enteric
neurogenesis. An alternative hypothesis would be that our culture conditions
reprogram a relatively late ENS progenitor to acquire properties
characteristic of an earlier precursor cell type. Such cell reprogramming has
been observed in vitro before. For example, under certain culture conditions,
oligodendrocyte precursor cells (OPCs) revert to self-renewable multipotential
neural stem cells, which can generate neurons, astrocytes and oligodendrocytes
(Kondo and Raff, 2000
). It is
therefore possible that signals in our cultures reprogram neural crest-derived
cells in mouse gut to acquire characteristics of NCSCs.
A characteristic property of NCSCs isolated from neural tube explants or
prospectively identified from peripheral tissues at later stages of
embryogenesis, is their ability to self-renew and generate similar
multipotential progeny (Morrison et al.,
1999; Stemple and Anderson,
1992
). The ability of EPCs to generate multiple lineages (in
addition to the neuronal and glial ones) is currently unclear. A small
percentage of SMA+/GFP+ cells have been isolated from NLBs but the ability of
EPCs to generate myofibroblasts [as is the case for other NCSCs
(Morrison et al., 1999
)]
requires further experimentation. The isolation of bipotential progenitors
from primary NLBs at relatively late stages of gut cultures and from secondary
and tertiary NLBs, suggests the presence of self-renewing progenitors in these
structures. Such progenitors could divide asymmetrically to generate committed
neurogenic and gliogenic precursors as well as bipotential progenitors
isolated as EPCs. The presence of self-renewing progenitors in NLB cultures is
further supported by our recent data indicating that dissociation and
replating of primary EPC colonies at clonal densities gives rise to neuron-
and glia-containing secondary colonies. Although the formation of such
secondary colonies was inefficient (due primarily to failure of cells to
survive upon replating) we were able to reproducibly generate four to five
multipotential secondary colonies per experiment, thus establishing in
principle the ability of EPCs to self renew in vitro. These studies, together
with recent experiments indicating that EPCs express Phox2B (N.B. and
V.P., unpublished data), a locus encoding a homeodomain-containing
transcription factor necessary for the specification of autonomic and enteric
neural crest cells lineages (Pattyn et
al., 1997
; Pattyn et al.,
1999
), suggest that EPCs are likely to represent self-renewing
progenitors of the sympathoenteric lineage
(Durbec et al., 1996
).
In addition to reproducing stages of cell differentiation observed for
endogenous ENS progenitors, EPCs and their progeny appear to respond to
signals that play a critical role in mammalian ENS development. A series of
studies have shown that GDNF controls several aspects of mammalian ENS
development by activating RET signalling in ENCCs and their progeny
(Taraviras and Pachnis, 1999).
Among the known effects of RET activation are the proliferation of ENS
progenitors, a response likely to determine the number of enteric neurons in
the mature ENS (Gianino et al.,
2003
) and the morphological differentiation of ENCCs
(Taraviras et al., 1999
).
Consistent with these studies, we observed that GDNF enhanced cell
proliferation and reduced the percentage of neurons in colonies of foetal and
postnatal EPCs maintained in complete medium. However, GDNF promoted the
morphological differentiation of neurons in the absence of CEE and other
growth factors. These findings highlight the context-dependent outcome of RET
activation (Barlow et al.,
2003
) and suggest that additional signalling molecules (such as
those present in CEE) could modify the effect of GDNF on EPCs. The exact cell
type in EPC colonies that responds to GDNF is presently unclear. Our failure
to detect Ret expression in EPCs shortly after their isolation
suggests that the target of GDNF are committed progenitors, such as the RET+
cells detected after day 3 of culture and their differentiated progeny. This
is consistent with the idea that EPCs represent pre-enteric progenitors
similar to the early vagal neural crest cells that migrate and proliferate
independently of RET activation (Durbec et
al., 1996
; Taraviras et al.,
1999
), but are capable of generating progeny similar to the
RET-dependent ENCCs.
The suggestion that EPCs represent progenitors of the mammalian ENS is
further supported by their ability to generate neuronal subtypes normally
encountered in enteric ganglia. Nearly all colonies of EPCs, generated from
either foetal or postnatal stages, contained neurons that expressed NPY, VIP
or CGRP. The generation of such neuronal subtypes by foetal and postnatal EPCs
is consistent with birthdating studies which indicate that neurons expressing
a wide range of neurotransmitters and neuropeptides, including those described
here, are born between E9.5 and P15 (Pham
et al., 1991). Expression of TH, a characteristic marker of
adrenergic neurons, is normally restricted to a subset of foetal ENS
progenitors and mature neurons between E9.5 and E14.5 and is absent from
postnatal gut (Baetge and Gershon,
1989
; Baetge et al.,
1990
). Our findings that colonies generated from foetal EPCs
produce TH+ neurons further argues that these cells have a developmental
potential similar to that of the endogenous ENS progenitors. However, similar
numbers of TH+ cells were also detected in colonies from postnatal EPCs. The
reasons of this apparent discrepancy are currently unclear. It is possible TH
expression reflects the reprogramming of postnatal cells to become similar to
foetal progenitors. Alternatively, the generation of TH+ neurons from
postnatal EPCs results from the absence of a signal that is normally present
in the late embryonic and postnatal gut, and represses the expression of TH.
Experiments are currently in progress to distinguish between these
possibilities.
Differentiation of foetal and postnatal EPCs upon grafting into
foetal gut in organ culture
Grafting of foetal and postnatal NLBs into the wall of foetal gut
maintained in organ culture resulted in extensive emigration of cells from the
graft and invasion of the surrounding tissue. Such emigration took place in
both wild-type gut (not shown) and in gut from RET-deficient embryos which
lack endogenous neural crest derivatives. Absence of an intrinsic ENS from the
latter allowed us to establish that many of the cells that emigrated from NLBs
were mature neuronal and glial cells. These findings suggest that NLBs
constitute a repository of enteric neurons and glia that can be used to
colonise the wall of aganglionic gut. It will be interesting to determine the
developmental stages during which the gut wall is receptive to colonisation by
EPCs. In addition, it is unclear at this point whether exogenous neurons can
establish functional synapses. Future in vivo transplantation experiments into
aganglionic gut should address this question.
Upon transplantation into wild-type and aganglionic gut, both foetal and postnatal EPCs increased in number and colonised regions of the host gut at relatively long distance from the grafting site, suggesting that EPCs and their progeny have the capacity to respond to migratory and proliferative signals present in the intact gut wall. In addition, foetal and postnatal EPC progeny differentiated into neurons and glial cells upon grafting into wild-type foetal gut. Similarly, neuronal differentiation of EPCs took place within the gut of RET-deficient embryos. However, we were unable to detect EPC-derived GFAP+ cells in grafted aganglionic gut. The reasons for the absence of glial cell differentiation are currently unclear. It is possible that the endogenous neural crest-derived cells present in the gut of wild-type embryos provide critical diffusible or cell-cell contact signals that are necessary for glial cell differentiation. Such signals, which could either be derived from undifferentiated progenitors of enteric neurons or differentiated neurons themselves, are expected to be reduced or absent in RET-deficient embryos which lack all neural crest-derived cells in the gut. Consistent with this hypothesis is the observation that gliogenesis in colonies of EPCs in vitro follows extensive neuronal differentiation.
Implication of EPC isolation for HSCR disease treatment
At present, the only definitive therapy for HSCR is surgical resection of
the aganglionic gut segment and anastomosis of the residual bowel, which is
generally successful in relieving the immediate consequences of intestinal
obstruction (Swenson, 2002;
Tsuji et al., 1999
). However,
this approach is associated with short- and long-term morbidity and mortality
and in a relatively large fraction of patients severe dysmotility persists
postoperatively and can last for years
(Tsuji et al., 1999
). It is
currently unclear whether such peristaltic malfunction is due to the surgical
procedure itself or results from neuronal deficits of the ganglionated gut
associated with the primary cause of aganglionosis. Therefore, we wished to
consider the possibility of restoring peristalsis of aganglionic gut segments
by transplanting ENS progenitors capable of forming a functional enteric
plexus. According to one scenario, such progenitors could be derived from a
small apparently normoganglionic gut segment of an affected individual and
autotransplanted into the aganglionic gut region. Prerequisites for such
procedure would be that (a) multipotential ENS progenitors could be derived
from small gut segments (possibly the size of a biopsy sample), and (b) such
progenitors could also be generated from the normoganglionic bowel segment of
an HSCR patient. We have used wild-type mice and an animal model of HSCR to
address both issues. Our findings show that it is feasible to isolate neural
crest-derived progenitors capable of generating enteric neurons and glia. In
addition, it is likely that such progenitors can be generated from relatively
small gut segments of foetal and postnatal gut. Although in the present study
we did not attempt to define the smallest possible gut segment that could
generate sufficient numbers of EPCs, we have isolated EPCs from whole as well
as segments of E11.5 guts (Fig.
10 and data not shown). This together with the ability of EPCs to
self-renew and proliferate suggests that it is feasible to isolate EPCs form
relative small segments of human gut.
Although the majority of HSCR patients are characterised by regional
aganglionosis which is usually restricted to the distal colon
(Chakravarti, 2001), our recent
analysis of mouse models with a similar phenotype indicated that, in addition
to the colon, neurogenesis in the small intestine is also affected during
embryogenesis. In embryos homozygous for mutations of Ret or
Et-3 (which encode components of the two signalling pathways most
commonly affected in familial cases of HSCR)
(Chakravarti, 2001
) we observed
reduced number and delayed migration of ENCCs prior to the arrival of these
cells in the colon (Natarajan et al.,
2002
) (Barlow et al.,
2003
). Although these cellular deficits were apparently corrected
throughout the small intestine and aganglionosis was eventually restricted to
the colon, the possibility arises that the normoganglionic regions of the gut
harbour subtle deficits. This hypothesis is further supported by a recent
report indicating changes in neurotransmitter expression and the number of
enteric neurons in the ileum of Et-3ls/ls animals
(Sandgren et al., 2002
). Such
defects could provide a potential explanation for the peristaltic
dysregulation observed postoperatively in patients that have undergone removal
of the aganglionic gut segment and anastomosis. In addition, they raise the
question as to whether the genetic deficits associated with aganglionosis
preclude the efficient isolation of multipotential progenitors that could
potentially be used to restore the neuronal plexus and the peristaltic
activity of the affected gut region. Our experiments indicate that EPCs can be
isolated from the apparently normoganglionic segment of the gut of
miRet51/51 embryos and neonates with efficiencies similar
to those observed in wild-type embryos. Furthermore, in clonogenic assays such
mutant EPCs were capable of generating both neuronal and glial cells. However,
it is presently unclear whether miRet51/51 EPCs are
capable of generating the whole range of neuronal and glial subtypes
encountered in similar cultures from wild-type EPCs. Also, it remains to be
seen whether miRet51/51 EPCs are capable of recolonising
wild-type or aganglionic gut upon transplantation into intact gut in vitro or
in vivo. The availability of the EPC clonogenic assay and gut transplantations
will allow us to examine these possibilities and determine the neurogenic and
gliogenic potential of multipotential progenitors from
miRet51/51 animals in vitro and in vivo.
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work
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