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Cell Stem Cell
Article
Transplantation of Expanded Fetal
Intestinal Progenitors Contributes
to Colon Regeneration after Injury
Robert P. Fordham,
1,2,6
Shiro Yui,
3,4,6
Nicholas R.F. Hannan,
1,2
Christoffer Soendergaard,
5
Alison Madgwick,
1
Pawel J. Schweiger,
3
Ole H. Nielsen,
5
Ludovic Vallier,
1,2
Roger A. Pedersen,
1,2
Tetsuya Nakamura,
4
Mamoru Watanabe,
4
and Kim B. Jensen
1,3,
*
1
Wellcome Trust & Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, CB2 1QR, UK
2
Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, University of Cambridge, Cambridge, CB2 0SZ, UK
3
BRIC: Biotech Research and Innovation Centre, University of Copenhagen, DK-2200 Copenhagen N, Denmark
4
Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, 113-8519, Japan
5
Department of Gastroenterology, Medical Section, Herlev Hospital, Faculty of Health and Medical Sciences, University of Copenhagen,
DK-2730 Herlev, Denmark
6
These authors contributed equally to this work
*Correspondence: kim.jensen@bric.ku.dk
http://dx.doi.org/10.1016/j.stem.2013.09.015
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.
SUMMARY
Regeneration and homeostasis in the adult intestinal
epithelium is driven by proliferative resident stem
cells, whose functional properties during organismal
development are largely unknown. Here, we show
that human and mouse fetal intestine contains pro-
liferative, immature progenitors, which can be
expanded in vitro as Fetal Enterospheres (FEnS). A
highly similar progenitor population can be estab-
lished during intestinal differentiation of human
induced pluripotent stem cells. Established cultures
of mouse fetal intestinal progenitors express lower
levels of Lgr5 than mature progenitors and propa-
gate in the presence of the Wnt antagonist Dkk1,
and new cultures can be induced to form mature in-
testinal organoids by exposure to Wnt3a. Following
transplantation in a colonic injury model, FEnS
contribute to regeneration of colonic epithelium by
forming epithelial crypt-like structures expressing
region-specific differentiation markers. This work
provides insight into mechanisms underlying devel-
opment of the mammalian intestine and points to
future opportunities for patient-specific regeneration
of the digestive tract.
INTRODUCTION
Fertilization of the oocyte initiates a series of events that,
following gastrulation, leads to organ formation in the developing
fetus. During this process, pluripotent stem cells progressively
lose potential as the early embryo is patterned along its axes
and organ structures are specified. Tissue-specific programs
subsequently direct the formation and maturation of adult or-
gans, which are maintained throughout life by stem cells with
tissue-restricted lineage potential. It remains unclear whether
transitory stem cell states exist in the embryo, responsible for
tissue maturation, or whether maturation is achieved via adult
tissue-specific stem cells in the fetal tissue. Understanding the
process of tissue maturation in vivo has implications for the
directed differentiation of pluripotent cells into functionally
mature tissue types (Zorn and Wells, 2009).
The intestinal epithelium is continuously replenished by resi-
dent stem cells. The mature mammalian small intestine is a
tube-like structure with an inner epithelial lining facing the lumen.
This layer is organized into differentiated villi protruding into the
lumen and proliferative crypt compartments invaginated into the
underlying mesenchyme. Intestinal Stem Cells (ISCs) reside at
the crypt base and give rise to all the differentiated cell types
(Barker et al., 2007, 2012). Development of the small intestine
follows a specific pattern. Villus formation in humans begins
around the ninth week of gestation and embryonic day 15
(E15) in mouse. In the human, crypt formation occurs before
birth, whereas in the mouse this happens during the first 2
postnatal weeks (Montgomery et al., 1999; Spence et al.,
2011a). Beyond these morphological rearrangements, the
mechanisms of initial intestinal lineage differentiation and func-
tional maturation are less well characterized. Despite temporal
differences in the ontogeny of the small intestine between human
and mouse, the overall process of development is identical,
making the mouse an accessible model to interrogate the pro-
cess of human intestinal maturation.
Our understanding of the mature intestine has been acceler-
ated by the establishment of culture conditions for long-term
maintenance of adult mouse and human intestinal epithelium
in vitro (Jung et al., 2011; Sato et al., 2009, 2011a). In this system,
single ISCs or dissociated crypt fragments are embedded in
Matrigel where they exhibit self-organization into ‘‘mini-guts.’’
Here we describe the identification of proliferative progenitors
captured in the human fetal intestine and during intestinal differ-
entiation of human induced pluripotent stem cells (hiPSCs). This
Cell Stem Cell 13, 1–11, December 5, 2013 ª2013 The Authors 1
Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
Injury, Cell Stem Cell (2013), http://dx.doi.org/10.1016/j.stem.2013.09.015
is recapitulated in murine tissues, where fetal progenitors can
transition spontaneously and by Wnt induction into an adult
state. Finally, we present evidence that fetal progenitors can
contribute to the regeneration of adult colonic epithelium in vivo,
as proof of principle that developmentally immature cells have
clinical potential.
RESULTS
Fetal Human Intestinal Epithelium Can Be Propagated
Long-Term In Vitro as Fetal Enterospheres
Previous studies have described the establishment of organoid
cultures from mature human gut epithelium (Jung et al., 2011;
Sato et al., 2011a). To investigate the in vitro potential of imma-
ture gut epithelium, we analyzed human fetal intestinal tissue
around gestational week 10. At this stage, crypts have not
formed and the human intestine consists of a series of undulating
Figure 1. Derivation of Immature Intestinal
Progenitors from Human Fetal and Pluripo-
tent Cells
(A) Whole mount of human gestational week 10
small intestine.
(B) Higher magnification of villi (arrow) and inter-
villus regions (arrowhead) in (A).
(C–E) Immunohistochemistry analysis for Ki67 (C),
PAS staining (D), and Lysozyme (E) in week 10
human small intestine.
(F and G) Spheroid cultures from week 10 human
small intestinal epithelium, grown with (G) and
without (F) prostaglandin E2 (PGE2) (2.5 mM).
(H and I) Intestinal tissue derived from directed
differentiation of human induced pluripotent stem
cells (hiPSCs), cultured with (I) and without (H)
PGE2.
(J) Relative expression levels of intestinal lineage
markers in material from undifferentiated human
induced pluripotent stem cells (hiPSC), iPSC-
derived intestine (Int. diff.), human primary fetal
enterospheres (hFEnS), human adult organoids
(hOrgs), primary fetal human small intestine (FhSI),
and primary adult human small intestine (AhSI).
Red and green colors reflect increased and
decreased deviation from the mean, respectively.
(K) Detection of VILLIN (green) and CHGA (red) in
hiPSC-FEnS.
The scale bars represent 2 mm in (A) and 100 mmin
(C)–(E) and (K). See also Figure S1 and Table S1.
villi, with proliferation localized primarily
to the intervillus regions (Figures 1A–
1C). Here a subset of cells is weakly pos-
itive for Periodic Acid Schiff’s (PAS),
though they do not have the mature
morphology of goblet cells and there are
no detectable Lysozyme
+ve
Paneth cells
(Figures 1D and 1E). The reduced level
of secretory differentiation was confirmed
at the transcriptional level (Figure 1J).
Fetal human intestinal tissue at around
gestational week 10 was dissected and
dissociated epithelial fragments were
seeded in Matrigel. The conditions used for propagation of adult
murine organoids (EGF, Noggin, and R-spondin1 [ENR]) caused
the growth of small granular spheres that could not be main-
tained long-term without the addition of prostaglandin-E2
(PGE2) (Figures 1F and 1G). We term these human Fetal Entero-
spheres (hFEnS). hFEnS are highly proliferative and can be
passaged repeatedly by mechanical dissociation for over
2 months with no spontaneous transition into budding organoids
during this time.
Intestinal Tissue from Human Pluripotent Cells Has
Fetal Characteristics
Human induced pluripotent stem cells (hiPSCs) can be differen-
tiated into intestinal epithelium (Spence et al., 2011b). We set out
to determine whether hiPSC-derived intestinal tissue transitions
through a fetal state. Using a chemically defined protocol, PSCs
were directed toward definitive endoderm (DE) and further
Cell Stem Cell
Fetal Intestinal Progenitors and Tissue Maturation
2Cell Stem Cell 13, 1–11, December 5, 2013 ª2013 The Authors
Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
Injury, Cell Stem Cell (2013), http://dx.doi.org/10.1016/j.stem.2013.09.015
patterned into posterior DE (Hannan et al., 2013). Raised aggre-
gates of cells forming from the sheet of posteriorized endoderm
were transferred as small clumps to Matrigel (Figure S1A avail-
able online). Again PGE2 facilitated the formation of larger cystic
epithelial spheroids, morphologically analogous to primary
hFEnS (Figures 1H and 1I). These structures were maintained
for over 2 months, through repeated passaging. In both cases
PGE2 provides a pro-proliferative signal that drives the growth
of spherical structures. hiPSC-FEnS also require low levels of
Wnt3a to support growth, suggesting that although morpholog-
ically alike, they possess slightly different properties. Expression
analysis verifies the immature nature of human FEnS and hiPSC-
FEnS when compared to human adult organoids as well as fetal
and adult intestine (Figures 1J and S1B). iPSC-derived FEnS had
Villin present at the apical cell membrane in the spherical struc-
ture, and its immature nature is further supported by the lack of
secretory Chromogranin-A
+ve
cells (Figure 1K).
Establishment of FEnS from Immature Mouse Intestine
We reasoned that development of the mouse intestine would
provide an accessible model system to interrogate intestinal
maturation more closely. The mouse intestine at embryonic
day 16 (E16) resembles the human intestine at around 10 gesta-
tional weeks with high proliferation in the intervillus regions and
scattered immature goblet cells (Figures 2A, S2A, S2B, S2E,
and S2F). By postnatal week 2, mature crypts are forming (Fig-
ures 2B, S2C, and S2D) and mature Lysozyme
+ve
Paneth cells
can now be detected in the proliferative zones (Figures S2G
and S2H). The appearance of secretory cells is also evident by
expression analysis during the course of intestinal development
(Figure 2C).
To investigate whether fetal murine intestine contains equiva-
lent FEnS progenitors, we seeded epithelial cells from the prox-
imal half of the small intestine. During a developmental time
course, we observed that FEnS form exclusively up to P2,
whereas organoids are formed from P15 and onward (Figures
2D–2I). Interestingly, analysis of material from P2 to P15 illus-
trates the formation of both FEnS and organoids with an
increasing fraction of the latter (Figures 2G2I). Murine FEnS
(mFEnS) are morphologically indistinguishable from hFEnS and
can be expanded through fortnightly passaging for at least 2
years (Passage n z100). During their serial passaging we
observe no spontaneous maturation or morphological and
karyotypic alterations (Figure 2J). Although PGE2 is not required
for maintenance of mFEnS, it does provide a pro-proliferative
effect independent of Wnt signaling (Figure S2I). As has been
reported for the adult colonic cultures, this is most likely via
cAMP-mediated block of anoikis and stimulation of MAP kinase
signaling (Jung et al., 2011). Established mFEnS can grow
without R-spondin1 and in the presence of the natural Wnt
antagonist DKK1, Porcupine inhibitor (which inhibits Wnt secre-
tion), and tankyrase inhibitor (which stabilizes the Axin2/APC
complex responsible for degradation of b-catenin), hereby
demonstrating that FEnS can be maintained independently of
Wnt signaling (Figures S2J and S2K). This distinguishes them
from adult organoids.
Characterization of mFEnS revealed that they consist of a
polarized epithelium with Villin localized to the apical surface,
similar to the small intestine (Figures 2K and 2L). Moreover,
FEnS phenocopy the differentiation patterns of the immature
epithelium as there are no detectable secretory cell markers at
both the protein and RNA level and reduced expression of adult
stem cell markers (Figures 2M–2P and S3A). BrdU incorporation
analysis showed that proliferative cells in mFEnS are scattered
across the whole surface, whereas proliferative zones in organo-
ids are restricted to the crypt domains (Figures 2Q and 2R). The
overall morphology and growth of FEnS as spheres are reminis-
cent of that reported for organoids that form as a result of
augmented Wnt signaling following loss of APC (Sato et al.,
2011b). However, expression analysis demonstrates distinct
expression patterns between FEnS and APCnull organoids (Fig-
ure S3B). In particular, it is clear that loss of APC causes
increased levels of adult stem cell markers, whereas these are
generally reduced in the fetal state (Figure S3B). In summary,
this demonstrates that progenitors within the fetal small intestine
have a unique behavior that sets them aside from both normal
and cancerous adult stem cells.
In Vitro Maturation of Fetal Enteric Progenitors
Intestinal maturation in vivo has been proposed to follow a wave
from proximal to distal sites (Spence et al., 2011a). To assess the
positional effect along the length of the small intestine, we
analyzed the regional differences in in vitro growth potential at
postnatal day 2 (Figure 3A). Contrary to expectations, FEnS
formed from proximal tissue, whereas more distal tissues formed
organoids (Figures 3A and 3B). Gene expression analysis
showed that the ability to form organoids correlates with
increased levels of Lgr5 and Axin2 (Figure 3C). Analysis of the
cultured material from the proximal and mid regions of the small
intestine shows variable but comparable expression of Wnt
target genes, suggesting that FEnS can respond to Wnt stimula-
tion and that this represents a transitory and dynamic cellular
state (Figure 3D). In line with the observed adult stem cell
behavior, the distal part of the small intestine expresses higher
levels of secretory lineage markers, which are characteristic of
the adult small intestine, and contains a greater number of
Ulex europaeus agglutinin I (UEA-I) reactive secretory cells (Fig-
ures 3C, 3E–3E00, and 3F). This further supports a distal to prox-
imal wave of tissue maturation.
In the mature intestine, Lgr5 marks ISCs, and single sorted
Lgr5
+ve
cells give rise to adult organoids (Barker et al., 2007;
Sato et al., 2009). In the immature intestine Lgr5 is expressed
by cells in the intervillus regions (Figure 4A). We hypothesized
that Lgr5 expression defines progenitors permissive for transi-
tioning into the adult state. In line with this, Lgr5-EGFP
+ve
cells
sorted from neonatal intestinal epithelium form organoids
in vitro, whereas FEnS are formed from cells in the Lgr5-EGFP
ve
population (Figures 4B–4E). It is impossible to assess whether
organoids form exclusively from Lgr5
+ve
cells, as a large propor-
tion of Lgr5-expressing cells in the Lgr5 knockin model are
EGFP
ve
due to the mosaic nature of the mouse model.
To assess the relationship between organoids and FEnS,
we analyzed samples from P2. Approximately one-half of the
structures grow in a manner indistinguishable from fetal tissues
(Figure S4A, Movie S1), whereas the rest followed a distinct
pattern indicative of spontaneous differentiation (Figure S4B,
Movie S2). All structures grow exponentially for around 7 days.
At this point some structures collapse and start to form budding
Cell Stem Cell
Fetal Intestinal Progenitors and Tissue Maturation
Cell Stem Cell 13, 1–11, December 5, 2013 ª2013 The Authors 3
Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
Injury, Cell Stem Cell (2013), http://dx.doi.org/10.1016/j.stem.2013.09.015
protrusions from the surface (Figure S4B). After passaging, these
P2 organoids become R-spondin1 dependent and identical to
structures obtained from more mature intestinal tissue (Fig-
ure S4C, Movie S3).
Since Lgr5 and Axin2 are both Wnt target genes, and given the
dynamic regional expression correlating with organoid formation
(Figure 3C), we investigated whether Wnt3a can induce intestinal
maturation in vitro. Stimulation of cells from E16 proximal intes-
tine, which normally only form FEnS, promoted the transition into
budding organoids in a proportion of the forming structures (Fig-
ure S4D). This effect is enhanced upon passaging and the form-
ing organoids can subsequently be maintained without exoge-
nous Wnt in an R-spondin1-dependent manner (Figure S4Dix).
Continued culture of organoids with high levels of exogenous
Wnt3a produced the cystic morphology previously described
for Wnt overactivity in adult cultures (Sato et al., 2011b;Figures
S4Dviii). In contrast, FEnS could not be induced to transit
to an adult state with Wnt3a (Figures S4Dvi and S4Dvii). The
observed Wnt-stimulated maturation of FEnS to organoids is
associated with the expected upregulation of secretory lineage
markers (Figure S4E). It is clear that FEnS respond to Wnt stim-
ulation, as Lgr5 and Axin2 expression is elevated compared to
Figure 2. Establishment of mFEnS from Immature Mouse Intestine
(A and B) Immunohistochemistry analysis for Phospho-Histone-H3 (pHist) on sections of small intestine from E16 mice (A) and P15 mice (B).
(C) Relative expression levels of intestinal lineage markers in tissue isolated from proximal murine intestine at increasing developmental age from E16 to adult.
Red and green colors reflect increased and decreased deviation from the mean, respectively.
(D–H) Representative images of in vitro structures derived from E14 to P15. The arrow and arrowhead in (G) indicate an FEnS and an organoid, respectively.
(I) Relative proportions of FEnS and organoids present after 2 weeks from E16, P2, and P15 tissues.
(J) Metaphase spread of a cell at day 180 shows a normal karyotype (n = 15).
(K and L) Detection of apical villin expression (green) in adult small intestine (K) and mFEnS (L).
(M–P) Lysozyme expression in adult small intestine (M), cross sections of mFEnS (N), and whole-mount organoids and mFEnS (O and P).
(Q and R) BrdU incorporation analysis in whole mounts of organoids and FEnS (green). b-catenin (red) is used as a counterstain.
The scale bars represent 100 mm. E, embryonic day; P, postnatal day; adult, >3 weeks postnatal. See also Figures S2 and S3.
Cell Stem Cell
Fetal Intestinal Progenitors and Tissue Maturation
4Cell Stem Cell 13, 1–11, December 5, 2013 ª2013 The Authors
Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
Injury, Cell Stem Cell (2013), http://dx.doi.org/10.1016/j.stem.2013.09.015
established cultures and also newly transitory organoids (Fig-
ure S4E); however, the signal is insufficient to induce maturation.
In order to further probe the functional significance of Wnt in
the transition from a fetal to an adult phenotype, epithelial cells
were isolated from P2 proximal small intestine. Because a
proportion of FEnS at this stage naturally transition to the adult
organoids, it is possible to investigate the importance of Wnt
signaling in the establishment of both organoids and FEnS, as
well as the transition between the two states. Epithelial cells
were isolated from the Lgr5-reporter model in order to visualize
Lgr5 expression. Whenever we observe high Lgr5-EGFP expres-
sion, this is in association with structures that are beginning to
transition into the adult state. In medium supplemented with
ENR, EGFP
+ve
cells can be found either in mature crypt domains
(Figures 4F and 4F0) or in regions with columnar morphology (Fig-
ures 4G and 4G0), whereas FEnS structures are seemingly
EGFP
ve
or dim (Figures 4H and 4H0). The addition of Wnt in-
creases the number of formed organoids (Figure 4N) and the re-
gions of Lgr5 expression in the developing structures. This varies
from single positive buds to extensive regions of Lgr5-EGFP
+ve
Figure 3. Adult Stem Cell Behavior Follows a Caudal to Rostral Pattern
(A) Schematic diagram of the Proximal, Mid, and Distal parts of the small intestine and the representative images of cultures derived at P2.
(B) Relative proportion of FEnS and organoids in the different sections of the small intestine.
(C) Expression analysis in material isolated from Proximal, Mid, and Distal regions. Data represent the mean, and the error bars, the SEM (n = 3). Data are ex-
pressed relative to Proximal, on a Log
2
scale.
(D) Expression analysis of cultures from proximal and mid intestine enriched for FEnS and organoids, respectively. Data represent the mean, and the error bars,
the SEM (n = 3), and are normalized to proximal cultures.
(E) Detection of cells of the secretory lineage based on binding of Ulex europaeus agglutinin I (UEA-I) in the proximal, mid, and distal small intestine.
(F) Quantification of UEA-I
+ve
cells. Data represent the mean, and the error bars, the SEM (n = 3).
The scale bars represent 100 mm.
Cell Stem Cell
Fetal Intestinal Progenitors and Tissue Maturation
Cell Stem Cell 13, 1–11, December 5, 2013 ª2013 The Authors 5
Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
Injury, Cell Stem Cell (2013), http://dx.doi.org/10.1016/j.stem.2013.09.015
Figure 4. In Vitro Maturation of Fetal Enteric Progenitors Is Associated with Lgr5 Expression and Wnt Signaling
(A) Detection of Lgr5-EGFP at P2 from Lgr5-EGFP-ires-CreERT2 mice.
(B) Isolation of Lgr5-EGFP
+ve
and Lgr5-EGFP
ve
epithelial cells from P2 small intestine by flow cytometry.
(C) Quantification of proportion of FEnS and organoids formed in vitro from Lgr5-EGFP
ve
and Lgr5-EGFP
+ve
neonatal intestinal epithelial cells.
(D and E) Representative images of structures formed in vitro from Lgr5-EGFP
ve
and Lgr5-EGFP
+ve
neonatal intestinal epithelial cells.
(F–M) Representative images of FEnS and organoids derived from Lgr5-EGFP-ires-CreERT2 mice and cultured in the presence of EGF, Noggin, and R-spondin1
(ENR), ENR and the porcupine inhibitor IWP2 (ENR/IWP2), ENR and Wnt3a (WENR), or WENR in the presence of the tankyrase inhibitor IWR (WENR/IWR). (F0)–(M0)
show grayscale images of EGFP in the derived structures.
(N) Quantification of proportionof FEnS and organoids formedin the different treatmentgroups (ENR: 18/8; ENR/IWP2: 26/0; WENR:21/30; WENR/IWR: 33/0). Two-
tailedFisher’s exact test showssignificant differencebetween ENR and ENR/IWP2(p = 0.0042), ENR andWENR (p = 0.0297), and WENRand WENR/IWR (p < 0.0001).
(O) Expression analysis of the different treatment groups normalized to the ENR condition. Data represent the mean (n = 2).
(P and Q) Detection of b-catenin (green) in organoids and FEnS. Arrows indicate cells with nuclear localization of b-catenin suggestive of active signaling. P‘-Q’
show b-catenin expression in grayscale.
(legend continued on next page)
Cell Stem Cell
Fetal Intestinal Progenitors and Tissue Maturation
6Cell Stem Cell 13, 1–11, December 5, 2013 ª2013 The Authors
Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
Injury, Cell Stem Cell (2013), http://dx.doi.org/10.1016/j.stem.2013.09.015
cells (Figures 4J–4L0). The transition and Lgr5 expression is
blocked by the addition of porcupine inhibitor to ENR-supple-
mented cultures (Figures 4I, 4I0, and 4N) and by the addition of
the tankyrase inhibitor IWR-1 to cells cultured in the presence
of ENR and Wnt (Figures 4M, 4M0, and 4N). Importantly, these in-
hibitors do not preclude the formation of FEnS. The maturation is
reflected at the RNA level, where Wnt induces a robust increase in
the Paneth cell marker Lysozyme. However, it is also clear that
FEnS in early cultures express endogenous Wnt3a, which drives
both Axin2 and Lgr5 expression within the fetal population of cells
(Figure 4O). In line with the elevated expression of Axin2 and Lgr5,
b-catenin can be observed in the nucleus of cells in FEnS as
well as in the formed organoids (Figures 4P, 4P0, 4Q and 4Q0).
In vivo tissue maturation correlates with the emergence of
secretory Paneth cells, which have been identified as the major
source of epithelial Wnt secretion in the intestinal epithelium
(Sato et al., 2011b; Farin et al., 2012). Although mature
Lysozyme
+ve
Paneth cells cannot be observed until postnatal
week 2 (Figure S2E–S2H), these are preceded by immature
secretory cells, which can be detected based on Cryptdin6
expression (Wong et al., 2012). Assessment of tissues from P2
and P15 demonstrates that Cryptdin6-expressing cells can be
detected as early as P2 (Figures 4R and 4S). This correlates
with the appearance of cells that are weakly positive for the
stem cell marker Olfm4 as well as Wnt3a within the bottom of
the intervillus regions (Figures 4T–4W). This provides an epithe-
lial source of Wnt3a that can drive tissue maturation.
In summary, this demonstrates that exogenous Wnt induces
elevated focal Lgr5 upregulation in the fetal state and that matu-
ration proceeds from these Lgr5 expression domains. Expres-
sion of Wnt3a can be detected in proliferative intervillus regions
as the tissue proceeds into its adult state, suggesting that Wnt
induction in vivo correlates with tissue maturation.
Regeneration of Adult Colonic Epithelium from mFEnS
To assess the differentiation potential of immature intestinal pro-
genitors and whether they represent a transplantable source,
EGFP
+ve
established mFEnS were injected under the renal
capsule of mice (n = 8). In all cases at analysis, EGFP FEnS cells
had either not proliferated or were not detectable. To test a more
physiologically relevant approach, we transplanted EGFP FEnS
into a chemically induced colonic injury model, where the repair
process is associated with endogenous activation of Wnt
signaling (Figure 5A; Yui et al., 2012; Koch et al., 2011). Within
3 hr after the first transplantation, FEnS-derived cells attached
to ulcerated regions in the distal colon and were subsequently
maintained long-term (Figures 5B and S5A–S5H). Initially, cells
engrafted as a single-layered epithelium on top of the denuded
lamina propria (Figures S5I and S5J). Three days following trans-
plantation, grafted regions migrated downward into the underly-
ing mesenchyme. Here they formed epithelial ‘‘pockets’’ with a
central lumen and Ki67
+ve
cells distributed along the length (Fig-
ures 5C, S5K, and S5L). One week after the second transplanta-
tion, engrafted cells formed epithelial crypt-like structures.
These fetal-derived cells, although refractory to maturation
in vitro, adapt to the colonic tissues, with subsets of cells differ-
entiating appropriately into Mucin-2
+ve
and PAS
+ve
goblet cells
and starting to express carbonic anhydrase-II, a specific marker
of colonic tissue. None of this was detected in FEnS (Figures 5C,
S5L, S5N, and S5P–S5R). Importantly, the grafted material did
not express markers normally associated with the small intestine
such as Lysozyme and alkaline-phosphatase (Figures S5S–S5T).
Fetal-derived colonic crypts persisted at 1.5 months after trans-
plantation, with continued evidence for proper differentiation and
proliferation (Figures 5C, S5O, and S5P). Thus, immature enteric
progenitors represent a transplantable source of cells with the
capacity to differentiate in vivo.
DISCUSSION
In this study, we reveal the existence of a transitory population of
progenitors present during the intestinal growth phase in both
human and murine tissues. Moreover, a population of cells
with similar characteristics can be obtained from pluripotent
stem cells. This population is characterized by distinct prolifera-
tive and differentiation potential and reduced in vitro growth
factor requirements compared to progenitors in the adult intesti-
nal epithelium. Transition of fetal enteric progenitors into an adult
state can be induced in vitro via stimulation with high levels of
Wnt or alternatively by transplantation in vivo into an injury
model. These cells are a valuable asset for understanding tissue
maturation and an attractive source of transplantable progeni-
tors for regenerative therapies.
Studies of human organ development are complicated by the
availability of material. We provide evidence that mouse and
human fetal intestine contain an immature population of epithe-
lial progenitors and that similar immature cells can be obtained
from hPSCs. Here the immature progenitors represent a transi-
tory population of cells. Interestingly, many differentiation proto-
cols from PSCs result in cells with a stable immature phenotype
(Meyer et al., 2009; Nicholas et al., 2013). Based on our results
this is not necessarily a tissue culture artifact but rather a result
of the in vitro stabilization of an otherwise transitory state in vivo.
It is however clear that it is not straightforward to extrapolate
growth factor requirements from mouse to human cells as has
been reported for their adult counterparts (Jung et al., 2011;
Sato et al., 2009, 2011a).
Intestinal maturation has been proposed to follow a rostral-to-
caudal (proximal-to-distal) wave (Spence et al., 2011a). We
observe that FEnS form from the proximal region and organoids
from the distal region, indicating that maturation in actual fact
proceeds in the opposite direction. This is correlated with the
expression pattern of markers of the mature secretory lineage
and correlates with the observation that Lgr5 expression is asso-
ciated with progenitors in a transitory competent state. These
spatial and temporal observations are in agreement with previ-
ous work showing that Lgr5 gene expression is higher in the
ileum than in the duodenum at E18.5 (Garcia et al., 2009). It
(R–W) In situ hybridization for Cryptdin6,Olfm4, and Wnt3a in tissue from P2 and P15. Arrows in (S) and (U) indicate regions of Olfm4 and Wnt3A expression,
respectively.
The scale bars represent 50 mm (F, J, K–L, P–Q, and V–W) or 100 mm (A, D–E, G–I, M, and R–U). Cells are counterstained with DAPI (blue) in (A), (O), (P) and (Q). See
also Figure S4 and Movie S1,Movie S2, and Movie S3.
Cell Stem Cell
Fetal Intestinal Progenitors and Tissue Maturation
Cell Stem Cell 13, 1–11, December 5, 2013 ª2013 The Authors 7
Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
Injury, Cell Stem Cell (2013), http://dx.doi.org/10.1016/j.stem.2013.09.015
(legend on next page)
Cell Stem Cell
Fetal Intestinal Progenitors and Tissue Maturation
8Cell Stem Cell 13, 1–11, December 5, 2013 ª2013 The Authors
Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
Injury, Cell Stem Cell (2013), http://dx.doi.org/10.1016/j.stem.2013.09.015
does remain a possibility that the culture conditions that maintain
adult stem cells in vitro are optimal for the distal intestine at this
developmental time point rather than a reflection of tissue
maturation.
The spatial differences in expression of the Wnt target genes
Lgr5 and Axin2 (Barker et al., 2007; Lustig et al., 2002) prompted
the investigation of Wnt signaling in the developmental transition.
The differing requirements between the mature and immature
states imply that Wnt signaling has a context-dependent role in
development and tissue homeostasis or alternatively that ligands
are dynamically regulated. There are several potential Wnt
ligands in the intestine, where Wnt3a has been shown to play
an autonomous role in epithelial stem cell maintenance (Sato
et al., 2011b; Farin et al., 2012). In line with this, we observe
that the expression of Wnt3A is correlated with the appearance
of adult stem cell markers as well as adult stem cell behavior in
the developing epithelium. Interestingly, this pattern of expres-
sion coincides with the phenotype of the knockout of the major
b-catenin effector, Tcf4, which die shortly after birth with intesti-
nal hypoplasia (Korinek et al., 1998).
In vitro Wnt stimulation and spontaneous maturation can be
blocked by Wnt inhibition. Here, Wnt causes a prominent focal
upregulation of Lgr5 expression in the developing structures.
This is associated with the transition from a thin epithelium to
domains with columnar morphology reminiscent of the cellular
architecture in the small intestine. After the emergence of Paneth
cells, these structures become independent of exogenous Wnt
similar to adult intestinal stem cells. We hypothesize that Lgr5
in this context facilitates the transition by enhancing focal Wnt
stimulation via the Wnt agonist R-spondin1 (de Lau et al.,
2011). This will also explain why established FEnS are resilient
to Wnt stimulation in vitro—they express significantly reduced
levels of Lgr5. Although Wnt signaling mediates the transitioning
of murine FEnS, it might be more complicated for hFEnS, where
a low level of Wnt stimulation is required for their normal
maintenance.
The gold standard for testing the true differentiation potential
of progenitor cells is in vivo transplantation (Lin et al., 2013).
We have previously demonstrated that adult colonic organoids
can engraft into an injury model (Yui et al., 2012). An initial
concern was that due to the striking morphological and growth
similarities between FEnS and APC null adult organoids (Sato
et al., 2011b), transplantation of FEnS in vivo would lead to tumor
formation. However, FEnS cells were shown to attach to
denuded regions of colonic epithelium and subsequently be
incorporated into the colonic epithelium. Furthermore, since
FEnS were unable to survive under the kidney capsule, this sug-
gests that orthotopic transplantation is a more useful readout of
in vivo potential. Our transplantation experiments unequivocally
demonstrate that established FEnS can mature in vivo and
contribute to regeneration of damaged gut epithelium in adult
hosts. Moreover, it is striking that these fetal derivatives from
the small intestine rapidly respond to the new microenvironment
and differentiate appropriately to the regional requirements. This
might reflect their immature behavior although we cannot
exclude the possibility that adult organoids will behave similarly.
In summary, we have identified a population of expandable
fetal enteric progenitors from mouse and human that can be
used as a transplantable source. This work has important impli-
cations for understanding the mechanisms underlying intestinal
maturation and demonstrates that immature intestinal progeni-
tors, including fetal-like material derived from human pluripotent
stem cells, have the potential to be used in colonic regenerative
medicine. It will be interesting to see if similar populations of
immature progenitors exist in other endodermal organs.
EXPERIMENTAL PROCEDURES
Mice
Rag2
/
mice were from Taconic Farms and Central Laboratories for Experi-
mental Animals. EGFP transgenic mice and Lgr5-EGFP-ires-CreERT2 mice
are described elsewhere (Barker et al., 2007; Okabe et al., 1997). Experimental
animals were obtained by crossing these with C57BL/6 male or female ani-
mals. All animal experiments in Cambridge were performed under the terms
of a UK Home Office License and transplantation experiments were performed
with the approval of the Institutional Animal Care and Use committee of TMDU.
Transplantation
Transplantation was performed as described on days 7 and 10 following initi-
ation of dextran sulfate sodium-induced colonic injury (Yui et al., 2012). Donor
FEnS were released from the Matrigel and mechanically dissociated into small
sheets of epithelial tissue. Cell fragments from 500–1,000 FEnS were resus-
pended in 200 ml of Matrigel in PBS (1:20), which was instilled into the colonic
lumen using a syringe and a thin flexible catheter. Animals were subsequently
sacrificed at indicated time points.
In Vitro Cultures
Organoids
Primary crypts from proximal adult small intestine were cultured as previously
described with reduced concentration of murine recombinant R-spondin1
(500 ng/ml, R&D Systems; Sato et al., 2009).
FEnS
Fetal small intestines were opened longitudinally and cut into small pieces
prior to dissociation with 2 mM EDTA. Isolated epithelial units were embedded
in Matrigel and maintained in conditions identical to those used for adult orga-
noids. In certain experiments Wnt3a and R-spondin1 from conditioned media
were collected from HEK293 cell lines expressing recombinant Wnt3a and
R-spondin1 (kindly provided by Hans Clevers and Calvin Kuo, respectively).
Relative Wnt/R-spondin1 activity was measured using a TOPflash assay
with a Dual-Luciferase Reporter Assay System (Millipore).
Human Tissue
First-trimester human fetal material was obtained from the John van Geest
Centre for Brain Repair, University of Cambridge, and used with informed con-
sent under an Approved Protocol of Human Tissue Studies. Fetuses were
staged by Crown Rump Length. Fetal intestines were processed for in vitro
epithelial culture, paraffin sections, or RNA extraction, using procedures
Figure 5. Regeneration of Adult Colonic Epithelium from mFEnS
(A) Experimental protocol: gastrointestinal tract dissected from E16 EGFP transgenic mouse fetus (top left). Proximal small intestine was cultured in vitro as FEnS
before mechanical dissociation and intracolonic transplantation into Rag2
/
adult recipients with Dextran Sulfate Sodium (DSS)-induced ulcerative colitis.
(B) Recipient colon at 1 week and 1.5 months posttransplantation. Lower panel shows EGFP
+ve
areas in host colon.
(C) Immunohistological analysis of EGFP
+ve
fetal-derived engraftments for Ki67 (Ki67
+ve
cells marked by arrowheads), carbonic anhydrase II, and PAS, 3 days,
1 week, and 1.5 months after transplantation.
The scale bars represent 1 mm (whole colons) and 200 mm (magnified areas) in (B) and 100 mm in (C). See also Figure S5.
Cell Stem Cell
Fetal Intestinal Progenitors and Tissue Maturation
Cell Stem Cell 13, 1–11, December 5, 2013 ª2013 The Authors 9
Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
Injury, Cell Stem Cell (2013), http://dx.doi.org/10.1016/j.stem.2013.09.015
identical to those described for murine material, with the addition of PGE2
(2.5 mM, Sigma-Aldrich). Adult human intestinal biopsies were obtained from
the Division of Gastroenterology and Hepatology, Department of Medicine,
University of Cambridge, and were used with local ethical permission, under
informed consent.
Adult primary human organoids were derived from biopsies obtained during
routine colonoscopies from the terminal ileum. A single crypt suspension was
obtained through chelation of the washed biopsies in cold chelation buffer
(distilled water with 5.6 mmol/l Na
2
HPO
4
, 8.0 mmol/l KH
2
PO
4
, 96.2 mmol/l
NaCl, 1.6 mmol/l KCl, 43.4 mmol/l sucrose, 54.9 mmol/l D-sorbitol,
0.5 mmol/l DL-dithiothreitol) containing 4 mM EDTA for 45 min followed by
release of the crypts in fresh chelation buffer by vigorous shaking. Isolated
crypts were treated like murine and fetal tissues; however, cultures were
additionally supplemented with 1xN2 and 1xB27 (from invitrogen), 2.5 mM
N-acetylcysteine (Sigma), 40% Wnt3a conditioned medium, 10% R-spondin1
conditioned medium, 10 mM nicotinamide, 10 mM SB202190, and 500 nM
A-83-01. Tissue for primary human cultures was obtained at Herlev Hospital
with local ethical permission and under informed consent.
Generation, Culture, and Differentiation of hiPSCs
hiPSCs (BBHX8) were derived using retrovirus-mediated reprogramming of
human skin fibroblasts (Rashid et al., 2010). hiPSCs were cultured in a chem-
ically defined, feeder-free culture system (Brown et al., 2011). Cells were
passaged every 7 days using a mixture of collagenase IV or collagenase and
dispase at a ratio of 1:1. hiPSCs were differentiated as outlined in Figure S1
and Table S1 (Hannan et al., 2013). Briefly, iPSCs were differentiated into DE
using Activin-A, BMP4, and LY294002 for 3 days. DE cells were subsequently
cultured with CHIR99021 for 4 days to generate posterior endoderm. Raised
aggregates of posteriorized endoderm were transferred into growth factor-
reduced Matrigel. The cell-Matrigel mix was overlaid with Advanced DMEM/
F12 supplemented with 2 mM GlutaMax (Invitrogen), 10 mM HEPES, and
100 U/ml Penicillin/100 mg/ml Streptomycin containing B27 supplement,
Y-27632 (10 mM), human Noggin (100 ng/ml), human EGF (100 ng/ml), human
R-spondin1 (1 mg/ml), and human Wnt3a (100 ng/ml).
Imaging and Histology
Live imaging of 3D cultures was performed using a Nikon Biostation IM
system. Structures in Matrigel were observed using phase contrast and DIC
microscopy using an Axiovert 200M microscope (Zeiss) equipped with an
AxioCam MRc (Zeiss).
Tissue preparation, staining, and image analysis were carried out as
described previously using antibodies listed in Table S2 (Wong et al., 2012;
Yui et al., 2012). Images of sections were acquired using a DeltaVision system
(Applied Precision) or a Zeiss Imager M.2, equipped with AxioCam MRm and
MRc cameras.
DIG in situ hybridization was carried out essentially as described before us-
ing IMAGE clones (Gregorieff et al., 2005).
RNA Extraction and qRT-PCR
RNA was isolated from intact intestine as described (Wong et al., 2012). Total
RNA was isolated from cultured cells using the Invitrogen PureLink RNA micro
kit. cDNA was synthesized from 100 ng total RNA using the Invitrogen
SuperScript III Reverse transcriptase kit, using random primers. Gene-specific
expression assays (Applied Biosystems) or SYBR Green analysis (Invitrogen)
with optimized primer pairs was used for qPCR on an Applied Biosystems
7500HT RealTime PCR System (Applied Biosystems). Values were normalized
to 18S using the DCt method. Z scores were calculated and used to generate
heatmaps in R.
Isolation of Cells for Flow Cytometry
Cells were isolated essentially as described (Wong et al., 2012). A single-cell
suspension was achieved by subsequent incubation using trypsin. Cell sorting
was carried out using a MoFlo (Beckman Coulter). Ten thousand cells were
seeded into 25 ml Matrigel. Data analysis was performed in FlowJo.
Statistical Analysis
Statistical significance of quantitative data was determined by applying a
two-tailed Student’s t test to raw values or to the average values obtained
from analysis of independent experiments. A two-tailed Fisher’s exact test
was used to analyze the significance of the Wnt and inhibitor culture
experiment.
SUPPLEMENTAL INFORMATION
Supplemental Information for this article includes five figures, two tables, and
three movies and can be found with this article online at http://dx.doi.org/10.
1016/j.stem.2013.09.015.
AUTHOR CONTRIBUTIONS
R.P.F., S.Y., and K.B.J. conceived and designed the study, analyzed the data,
and wrote the manuscript; R.P.F., S.Y., N.R.F.H., C.S., A.M., P.J.S., and K.B.J
performed experimental work; and R.P.F., S.Y., and K.B.J. prepared the fig-
ures. O.H.N., L.V., R.A.P., and M.W. gave conceptual advice. T.N. and K.B.J
supervised the project.
ACKNOWLEDGMENTS
We thank F. Watt for EGFP transgenic mice, SCI Core Facilities for their sup-
port, and R. Barker for facilitating access to human fetal tissue. We thank A.
Martinez-Arias, B. Simons, M. Zilbauer, and the Jensen, Pedersen, and
Watanabe labs for critical discussions. This work was supported by an MRC
PhD Studentship and Centenary Awards (R.P.F.), the Evelyn Trust grant
(N.R.F.H.), EC FP7 ERC starting investigator research grant (L.V.), the Cam-
bridge Hospitals National Institute for Health Research Biomedical Research
Center (L.V.), the Regenerative Medicine Realization Base Network Program
from Japan Science and Technology Agency (JST) (T.N. and M.W.), the
MEXT/JSPS KAKENHI (Grant Number 22229005 to T.N. and M.W.), a Health
and Labor Sciences Research Grant for Research on rare and intractable dis-
eases from the Ministry of Health, Labor and Welfare of Japan (M.W.), Sidney
Sussex College (A.M.), The Danish Cancer Society (K.B.J.), a Wellcome Trust
Career Development Fellowship (K.B.J.), and a Lundbeck Foundation Fellow-
ship (K.B.J.). L.V. is a founder and shareholder of DefiniGEN.
Received: May 22, 2013
Revised: September 4, 2013
Accepted: September 27, 2013
Published: October 17, 2013
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Please cite this article in press as: Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after
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