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CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2013;11:354 –358
ADVANCES IN TRANSLATIONAL SCIENCE
Joseph H. Sellin, Section Editor
Tissue Engineering the Small Intestine
RYAN G. SPURRIER and TRACY C. GRIKSCHEIT
Division of Pediatric Surgery, Children’s Hospital Los Angeles, Keck School of Medicine of the University of Southern California, Los Angeles, California
Short bowel syndrome (SBS) results from the loss of a
highly specialized organ, the small intestine. SBS and its
current treatments are associated with high morbidity and
mortality. Production of tissue-engineered small intestine
(TESI) from the patient’s own cells could restore normal
intestinal function via autologous transplantation. Improved understanding of intestinal stem cells and their
niche have been coupled with advances in tissue engineering techniques. Originally described by Vacanti et al of
Massachusetts General Hospital, TESI has been produced
by in vivo implantation of organoid units. Organoid units
are multicellular clusters of epithelium and mesenchyme
that may be harvested from native intestine. These clusters
are loaded onto a scaffold and implanted into the host
omentum. The scaffold provides physical support that permits angiogenesis and vasculogenesis of the developing tissue. After a period of 4 weeks, histologic analyses confirm
the similarity of TESI to native intestine. TESI contains a
differentiated epithelium, mesenchyme, blood vessels, muscle, and nerve components. To date, similar experiments
have proved successful in rat, mouse, and pig models. Additional experiments have shown clinical improvement and
rescue of SBS rats after implantation of TESI. In comparison with the group that underwent massive enterectomy
alone, rats that had surgical anastomosis of TESI to their
shortened intestine showed improvement in postoperative
weight gain and serum B12 values. Recently, organoid units
have been harvested from human intestinal samples and
successfully grown into TESI by using an immunodeficient
mouse host. Current TESI production yields approximately
3 times the number of cells initially implanted, but improvements in the scaffold and blood supply are being
developed in efforts to increase TESI size. Exciting new
techniques in stem cell biology and directed cellular differentiation may generate additional sources of autologous
intestinal tissue for direct translation to human therapy.
Keywords: Tissue Engineering; Small Intestine; Short Bowel Syndrome; Organoid Unit; Stem Cell; Regeneration.
Technological Primer
S
hort bowel syndrome (SBS) describes the constellation of
metabolic, nutritional, and physiological derangements resultant from the loss of 70%–75% of small bowel length. It is a
highly morbid condition, often resulting in failure to thrive and
multiorgan system dysfunction.1,2 Disease processes in the
adult population resulting in SBS include ischemia, inflamma-
tory bowel disease, and trauma, whereas intestinal atresias,
necrotizing enterocolitis, Hirschsprung’s disease, volvulus, and
gastroschisis predominate among pediatric patients.3 To date,
no reliable, curative therapy has been developed, and most
patients are initially managed with lifestyle modification and
total parenteral nutrition (TPN). Long-term TPN carries a high
risk of central line sepsis and parenteral nutrition–associated
liver disease.4 Although evolving understanding of parenteral
nutrition–associated liver disease and the use of fish oil– based
lipid emulsions composed of omega-3 fatty acids show promise
in reducing such complications, morbidity remains high.5,6
Novel therapies such as teduglutide, a glucagon-like peptide 2
analogue, may restore intestinal structural and functional integrity by promoting repair and growth of the mucosa. Multiple
randomized placebo-controlled trials among SBS patients have
shown intestinotrophic and proabsorptive effects, with resultant reduction in need for parenteral support.7–9
Despite these advances in medical therapy, many SBS patients will require surgical intervention such as a bowel lengthening procedure or intestinal transplant. Surgical procedures
for bowel lengthening have been refined during the past few
decades and include longitudinal intestinal lengthening and
tailoring and serial transverse enteroplasty among others.10 A
recent review of autologous intestinal reconstruction surgery
showed successful transition to enteral feeding among TPNdependent patients at a rate of 67% after longitudinal intestinal
lengthening and tailoring and 34% after serial transverse enteroplasty.11 Other patients, such as those with near-total loss
of small bowel length, require intestinal transplant if they fail
more conservative treatments. As the largest immune organ in
the body, the intestine is highly immunogenic and fails engraftment at much higher rates than other transplanted organs
despite lifelong immunosuppression, which itself is fraught
with numerous consequences.12–14 Other limitations also exist
including the low numbers of available donor organs, particularly in the pediatric population.15 Despite all of the above
interventions, the overall 5-year mortality for patients with SBS
is 30% and as high as 45% among pediatric patients requiring
small bowel transplant.1,16,17 Therefore, alternative solutions to
this complex problem are being investigated.
Tissue engineering could provide syngeneic intestinal tissue
for autologous transplantation via in vitro manipulation of
Abbreviations used in this paper: SBS, short bowel syndrome; TESI,
tissue-engineered small intestine; TPN, total parenteral nutrition.
© 2013 by the AGA Institute
1542-3565/$36.00
http://dx.doi.org/10.1016/j.cgh.2013.01.028
April 2013
stem cells. This field uses a combination of principles from
engineering and developmental biology. The goal of current
intestinal engineering efforts is to treat patients with SBS by
providing replacement tissue with structure and function indistinguishable from native intestine.
Findings
The microstructure of the intestinal epithelium is composed of crypts and villi. There is a division of labor among the
differentiated cell types of the villi to provide efficient transport
across this large surface area. Goblet cells secrete mucus that
contains important peptides such as Muc2. Enteroendocrine
cells regulate multiple hormone-dependent processes.18 Enterocytes are responsible for the critical role of nutrient absorption.
Within the crypts are Paneth cells interspersed between the
rapidly cycling Lgr5⫹ intestinal stem cells. Paneth cells have
long been known to contribute to the gut barrier via production of defensins and antimicrobial peptides, whereas their role
in maintaining the microcellular stem cell niche through signaling via WNT and Notch continues to be studied.19 –22 The
intestinal stem cell has 2 roles that facilitate the rapid turnover
of the epithelium: self-replication into identical daughter cells
and differentiation into the various differentiated epithelial cell
types.18 The specific location of the intestinal stem cell has long
been debated but was initially described by Bjerknes and
Cheng.23 They defined crypt-based columnar cells that populate
the base of the crypts between Paneth cells. Through lineage
tracing, these crypt-based columnar cells were confirmed as the
origin of all 4 differentiated cell types of the small intestine.24,25
Although characterized as within the crypt base, the intestinal stem cell is not readily identifiable by microscopy, so investigators sought a definitive cell marker. The first such marker,
Lgr5, was found to be expressed by crypt base cells adjacent to
Paneth cells. Hans Clevers of the Hubrecht Institute showed
these Lgr5 cells to be multipotent, thus confirming the identity
of intestinal epithelial stem cells.26 Another marker, Bmi1, was
found in cells located predominantly 4 cells above the crypt
base (or ⫹4). These Bmi1 cells are also multipotent and required
for crypt maintenance.27 These 2 groups of intestinal stem cells
are now understood to serve complementary functions, with a
quiescent Bmi1 population at ⫹4 and Lgr5 for rapid turnover at
the crypt base.28 Numerous additional markers of putative
intestinal stem cells have also been proposed including Mushashi-1 (Msi-1), doublecortin-like kinase 1 (DCLK1), mouse telomerase reverse transcriptase (mTert), and prominin 1
(Prom1).29 –33
These advances in stem cell biology occurred in parallel with
the development of the field of tissue engineering. In 1988,
Vacanti et al of Massachusetts General Hospital initiated in vivo
implantation of organoid units, multicellular clusters of epithelium and mesenchyme harvested from rat intestine, in a
variation from an original protocol by Evans et al.34,35 These
clusters were loaded onto a scaffold that provided physical
support and allowed for angiogenesis and vasculogenesis of the
developing tissue. After a period of weeks, the tissue-engineered
intestine was harvested and examined.34,35
Subsequent investigations have resulted in the growth of
every region of the gastrointestinal tract including tissue-engineered esophagus, stomach, small intestine, and colon in rodent models.36 –39 Tissue-engineered colon, esophagus, and
small intestine have had their rudimentary function confirmed
TISSUE ENGINEERING THE SMALL INTESTINE
355
in replacement models.38,40,41 In the case of tissue-engineered
small intestine (TESI), histologic analyses demonstrated the
presence of all 4 differentiated intestinal cell types, which confirmed the similarity of TESI to native intestine (Figure 1).37,42
Beyond the epithelium, TESI contains the same key components as native tissue necessary for function: blood vessels,
muscle, and nerve components. TESI mesenchyme contains
intestinal subepithelial myofibroblasts to contribute to wound
healing and immunity. All of these tissues have been confirmed
to originate from the TESI donor rather than the host.37 The
many transgenic tools of the murine model may facilitate further investigation into the mechanism of tissue formation.
Importance of Findings
Beginning in the late 1980s with intestinal cell cultures
in vitro, Evans et al34 demonstrated the ability to grow rat
intestinal epithelial cells in culture by modifying conditions
such as culture medium and growth factors. Further modifications to in vitro conditions of cell culture, including creation of
a microgravity environment with a rotating bioreactor, have
facilitated the growth of villus-like structures from a monolayer
of fibroblasts and endothelial cells.43 Recently, single Lgr5⫹
stem cells have been cultured in vitro without mesenchymal
cells and successfully developed into crypt and villus structures.
However, these single cells yielded organoids in only 6% of
cultures and required the support of basement membrane preparation (Matrigel; BD Biosciences, San Jose, CA) and supplementation of multiple exogenous signals usually produced by
supporting niche cells.44 Although cell culture methods are
capable of producing differentiated intestinal epithelium, such
in vitro techniques have been unable to produce vasculature or
supporting mesenchyme, which has proved crucial for maintaining the stem cell niche.22 In this regard and others, the in
vivo tissue engineering model is unique and more suited to
human therapy.
Translation of Findings Into Routine
Clinical Diagnosis and Treatment
Beyond replicating the structure of native intestine and
defining the underlying molecular mechanisms, the ultimate
goal of tissue engineering is treatment of SBS. To that end, a
number of experiments have been undertaken in animals that
demonstrate feasibility for translation of TESI therapy to humans.
Grikscheit et al were able to demonstrate clinical improvement after rescue with TESI by using a Lewis rat model of SBS.
As compared with the group that underwent massive enterectomy alone, the TESI group showed improvement in postoperative weight gain and serum B12 levels.40 With evidence that
TESI therapy experimentally improves SBS outcomes, the next
logical step was to engineer small intestine in a large animal
model that was representative of human scale. Therefore, Sala
et al42 harvested small intestine for organoid unit preparation
from Yorkshire swine. With the donor animal still under anesthesia, autologous organoid units were loaded onto a biodegradable scaffold and implanted into the omentum of the
animal during the same operation. The results demonstrated
that successful growth of TESI was a promising pilot for future
investigational new drug—enabling large animal studies.
356
SPURRIER AND GRIKSCHEIT
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 11, No. 4
Figure 1. TESI in the mouse model demonstrates normal epithelial differentiation. (A) Alcian blue staining of the goblet cells in the native and the
tissue-engineered small intestine, respectively. (B) Lysozyme staining demonstrates presence of Paneth cells in their crypt location in tissueengineered intestine and native intestine. (C) Chromogranin A⫹B staining identifies enteroendocrine cells in both native and tissue-engineered
intestine. (D) The brush border of the enterocytes is stained by using villin specific antibody in both tissues. Scale bar, 40.0 ␮m. From Sala FG,
Matthews JA, Speer AL, et al. A multicellular approach forms a significant amount of tissue-engineered small intestine in the mouse. Tissue Eng Part
A 2011;17:1841–1850. Reprinted with permission from AAAS.
The ability to engineer small intestine is not a phenomenon
limited to animal tissues. Human organoid units have been
implanted in immunodeficient mouse hosts and successfully
grown into tissue-engineered small intestine and colon.45,46 On
transition to human therapy, the patient will serve as his or her
own bioreactor for autologous TESI growth, thus avoiding the
need for immunosuppression. The TESI could then be moved
on the vascular pedicle into position for surgical anastomosis.
The blood supply of neovascularized omentum that supported
its growth would remain intact. Although immediate processing and implantation of organoid units would be ideal, some
patients may come to intestinal resection with clinical conditions that are temporarily unfavorable for immediate tissue
engineering in the SBS patient’s omentum. In cases such as
sepsis due to necrotizing enterocolitis, storage or in vitro expansion protocols may be required.
mm in diameter constructed of polyglycolic acid and poly
(l-lactic acid) coated with type I collagen (Figure 2). This material demonstrates 95% porosity to allow initial imbibition of
nutrients. The type I collagen promotes initial cellular adhesion, and subsequently the scaffold slowly hydrolyzes to allow
the TESI to continue growing without excessive concurrent
foreign body reaction.37 Although TESI has demonstrated nerve
cell growth, specific studies of peristalsis have not been performed. In interposition anastomosis, obstruction did not ensue. Motility will need to be directly measured as the size and
length of TESI increase.37
Roadblocks and/or Limitations
Some simple hollow viscus organs, such as the trachea,
have been engineered on scaffolds without a dedicated blood
supply and grown to scale for whole organ replacement.47 TESI
has been found to contain approximately 3 times the number of
cells initially implanted.37 Current research seeks to improve
this yield and further amplify the implanted stem cell population, with the goal of producing enough tissue for whole organ
replacement. Larger constructs would be more likely to grow
with the support of a definitive blood supply rather than simple
imbibition and neovascularization. However, simple anastomosis of a construct to host vasculature is not sufficient to support
TESI growth, because attempts to implement larger acellular
dermal matrices have failed.48 Although decellularized matrices
have been investigated for their potential biocompatibility with
native tissue and maintenance of architecture, inadequate porosity to allow imbibition of nutrients has limited their application.49 –53 Current TESI scaffolds are a highly porous tube ⬍5
Figure 2. Morphology of TESI in the mouse model. Four weeks after
implantation, the tissue-engineered intestine formed a sphere about twice
the size of the initial implanted polymer (arrow). From Sala FG, Matthews
JA, Speer AL, et al. A multicellular approach forms a significant amount of
tissue-engineered small intestine in the mouse. Tissue Eng Part A 2011;
17:1841–1850. Reprinted with permission from AAAS.
April 2013
Differentiation of intestinal stem cells into each of the mature cell types results from an elegant and high-fidelity cellular
traffic within the crypt that is not entirely defined. As understanding of the intestinal stem cell niche improves, an improved donor cell population may be substituted for the organoid units currently implanted. Alternatively, the Spence lab
has demonstrated the novel approach to induce differentiation
of human pluripotent stem cells into intestinal epithelium. In
this in vitro model the addition of Wnt3a, Activin A, and
fibroblast growth factor to human induced pluripotent stem
cells was adequate for hindgut patterning, specification, and
morphogenesis, with NEUROG3 transcription factor required
for enteroendocrine cell development.54 The factors that contributed to this novel differentiation in vitro are among dozens
of molecules within those families with unknown effects in
vivo. Further effects of any growth factor must be defined
before regulatory approval for transition to human therapy.
Conclusions
SBS results from the loss of a highly specialized organ,
the small intestine. High morbidity and mortality are associated
with both SBS and its current treatments. Tissue engineering is
an emerging field with tremendous potential for novel therapies
to treat these patients. Ideally, restoration of normal intestinal
function would occur via autologous transplantation of TESI.
In vitro and in vivo techniques have evolved such that modest
amounts of small intestine can be reproducibly engineered
from donor stem cells. Continued development of materials
science may provide more efficient scaffolds or integrate composite biomaterials to support larger constructs. New techniques in stem cell biology and directed cellular differentiation
may generate new sources of autologous intestinal tissue. With
continued advancements in each of its disciplines, the future is
promising for the field of tissue engineering in the small intestine.
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Reprint requests
Address requests for reprints to: Tracy C. Grikscheit, MD, 4650
Sunset Boulevard, Mailstop 35, Los Angeles, California 9002.
e-mail: [email protected]; fax: (323) 361-3534.
Conflicts of interest
The authors disclose no conflicts.
Funding
Supported by California Institute for Regenerative Medicine RN2
00946-1 grant.