Author's Accepted Manuscript

Stem cells and biopharmaceuticals: vital rolesin the growth of tissue-engineered smallintestine

GG Belchior, MC Sogayar, TC Grikscheit

PII: S1055-8586(14)00038-9DOI: http://dx.doi.org/10.1053/j.sempedsurg.2014.06.011Reference: YSPSU50491

To appear in: Seminars in Pediatric Surgery

Cite this article as: GG Belchior, MC Sogayar, TC Grikscheit, Stem cells andbiopharmaceuticals: vital roles in the growth of tissue-engineered smallintestine, Seminars in Pediatric Surgery, http://dx.doi.org/10.1053/j.semped-surg.2014.06.011

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Stem cells and biopharmaceuticals: vital roles in the growth of tissue-engineered small

intestine

Belchior GG1, Sogayar MC1, Grikscheit TC2 1Cell and Molecular Therapy Center – NUCEL, Department of Biochemistry, Chemistry Institute,

University of São Paulo, São Paulo, Brazil. 2Developmental Biology and Regenerative Medicine Program, Saban Research Institute,

Children’s Hospital Los Angeles, California, United States of America.

Keywords: tissue-engineered small intestine, stem cells, biopharmaceuticals

Abstract

Tissue engineering currently constitutes a complex, multidisciplinary field exploring ideal sources

of cells in combination with scaffolds or delivery systems in order to form a new, functional organ

to replace native organ lack or loss. Short bowel syndrome (SBS) is a life threatening condition

with high morbidity and mortality rates in children. Current therapeutic strategies consist of costly

and risky allotransplants, which demand lifelong immunosupression. A promising alternative is the

implantation of autologous organoid units (OU) to create a tissue-engineered small intestine

(TESI). This strategy is proven to be stem cell- and mesenchyme-dependent. Intestinal stem cells

(ISCs) are located at the base of the crypt, and are responsible for repopulating the cycling mucosa

up to the villus tip. The stem cell niche governs the biology of ISCs and, together with the rest of

the epithelium, communicates with the underlying mesenchyme to sustain intestinal homeostasis.

Biopharmaceuticals are broadly used in the clinic to activate or enhance known signaling

pathways, and may greatly contribute to the development of a full-thickness intestine by increasing

mucosal surface area, improving blood supply and determining stem cell fate. This review will

focus on tissue engineering as a means of building the new small intestine, highlighting the

importance of stem cells and recombinant peptide growth factors as biopharmaceuticals.

Introduction

Organ failure may arise from congenital or acquired conditions and with enhanced care and

increasing survival of premature infants may be an early life event. Tissue engineering has

emerged within the field of regenerative medicine as a field focused on developing tissue and

organ surrogates to restore, maintain, or improve biological functions [1]. The oldest example of

tissue engineering is a passage from the book of Genesis in which woman is created from a rib

taken from man [2]. The first report of an organ being actually transplanted dates back to the

1950’s, consisting of a kidney transplant between identical twins [3]. Since then, this field has

greatly matured, evolving from employing pre-existing organs to creating de novo, complex ones.

Due to its importance in providing the cellular and molecular building blocks for tissue

growth and maintenance, the small intestine is a vital organ, especially in infants born with GI

abnormalities or who undergo intestinal resection for various neonatal medical conditions. Loss of

portions or the entirety of segments of the small intestine may lead to variable degrees of

absorption debilities, such as short bowel syndrome (SBS), and ultimately to death. One of the

main strategies to overcome loss of intestinal tissue is to increase the surface area in order to

restore the absorption level to that of the healthy organ. An ideal therapeutic approach would

eliminate drawbacks of current treatments, such as the need for immunosuppressive drugs and total

parenteral nutrition (TPN), allowing a better quality of life, and a higher survival rate for patients.

Intestinal stem cells (ISCs) maintain the pool of precursor and differentiated cells of the

dynamically proliferating intestinal epithelium. Differentiated cells are shed from the tip of the villi

after a 7-10 day journey from their production at the base of the crypt [4]. Preserving the intestinal

stem cell niche with cells that produce important signaling proteins, along with the supporting

mesenchyme, appears to be crucial for therapeutic approaches [5].

Biopharmaceuticals are engineered biological molecules produced for therapy and/or

diagnosis. They encompass hundreds of currently marketed products, ranging from nucleic acid

sequences to complex proteins with post-translational modifications, such as growth factors, which

control numerous cell and tissue processes. Several epithelial growth factors have proven to be

important players in intestinal growth [6]. Every engineered organ requires vascular ingrowth in

order to receive adequate nutrition and oxygenation, but sufficient vascularization is not frequently

obtained. One of the main causes rests on the shortage of growth factors where they are most

required [7]. Thus, local administration of growth factors that induce angiogenesis and other

beneficial processes is logical and should be promising for the correct development and growth of

organs in the context of regenerative medicine.

This review provides an overview on the advancements of tissue-engineered small intestine

(TESI) research and the potentially vital combination of stem cells and biopharmaceuticals to

improve the quality of life and life span of pediatric patients suffering from SBS.

Small Intestine and Short Bowel Syndrome

The small intestinal epithelium is composed of villi and crypts, which are a confluent sheet of

specialized cells. Villi are luminal protrusions that increase surface area thousands of times for

better absorption of carbohydrates, peptides and amino acids, lipids, vitamins, ions and water. The

absorptive capacity of the small intestine relies on enterocytes, the most abundant specialized cell

type of the small intestine epithelium, bearing microvilli on their apical region and significantly

increasing the intestinal surface. Goblet cells are distributed between enterocytes and secrete

mucin, a family of O-glycosylated proteins, which are essential to secrete mucous and protect the

luminal surface. Enteroendocrine cells release several peptide hormones that regulate satiety,

energy and lipid metabolism, glucose homeostasis and several other food ingestion-related

metabolic functions. M (membranous or microfold) cells are present in close proximity to

lymphoid follicles and participate in antigen-sampling [8-11]. Crypts are depressions found

between the villi, and are also found in the colon. The base of the crypts is occupied by Paneth

cells, which secrete lysozyme and antimicrobial defensins and support the crypt base cell. Paneth

cells are key to maintenance of the intestinal barrier that separates the lumen content from the

sterile underlying mucosa in addition to supporting the Lgr5+ intestinal stem cells (ISC) through an

intimate association with these cells [12]. ISCs and progenitor cells are also found in the crypt, and

maintain the cell numbers of the dynamically renewing epithelium [11, 13]. The surrounding

mesenchyme ensures full function of the small intestine by signaling the epithelial stem cells and

contributing vascular supply and an innervated muscularis for peristalsis [14].

The onset of intestinal failure (IF) results from obstruction, dysmotility, surgical resection,

congenital defect or disease-associated loss of absorption, characterized by the inability to maintain

protein-energy, fluid, electrolyte or micronutrient balance [1], which may ultimately lead to death.

Short bowel syndrome (SBS) is the most common type of IF which was first reported in the late

1800's [15]. It is caused by intestinal loss or resection, which in the term infant is considered to

occur if more than 50-75% of the small bowel is removed, with higher severity being associated

with simultaneous resection of the ileocecal valve or, in addition, colon [11]. The resulting

shortened intestinal remnant leads to an inability to sustain homeostasis on a normal diet due to

inadequate surface area for absorption, demanding additional nutrition supplementation via the

parenteral route (PN) [15]. The length of the remaining small intestine is highly associated with

neonatal PN dependence even though longer bowel lengths sometimes do not correlate with

weaning from PN [16].

The multifactorial causes of pediatric SBS are not unique to a specific clinical setting or age

group. Nonetheless, the main etiologies usually are necrotizing enterocolitis (NEC), intestinal

atresia, inflammatory bowel disease, gastroschisis, and malrotation with volvulus. Postoperative

SBS and malignancies, together with trauma and motility disorders, also gain importance in older

children [15, 16]. Accurate numbers of SBS cases in children are difficult to obtain, even though

the incidence and prevalence of SBS in adults are estimated to be three and four per million,

respectively [16, 17]. Byrne et al. estimated that approximately ten to twenty thousand patients

received home-delivered total PN for SBS in the United States [18]. A study in Canada involving

175 neonates admitted from 1997 to 1999 and followed until 2001 estimated the incidence of SBS

to be 24.5 per 100,000 live births, with a much higher incidence in infants born at less than 37

weeks [19]. A more recent study with infants born from January 1st, 2002 through June 30th, 2005

comprised of a cohort of hospitalized neonates encompassing 16 tertiary care centers in the USA

demonstrated an incidence of SBS between 0.7% and 1.1%, which correlated with birth weight

[20].

The mortality associated with SBS is seen not only in the early postoperative period, from

complications associated with the underlying disease process and attendant surgery, but, also, in

the long term, when patients succumb from the delayed complications of intestinal failure-

associated liver disease (IFALD) and sepsis [21]. Sepsis in SBS is mainly caused by catheter-

associated bloodstream infection (CABSI) and bacterial overgrowth can also affect up to 60% of

children with SBS [22], apart from the many complications which may arise from PN [15]. A large

intestinal transplant center in the USA has reported a five-year survival of 95% in SBS patients

weaned from PN as opposed to 52% in patients remaining on PN [16, 23].

The remaining organ has an intrinsic adaptation in which the small intestinal epithelium

partially compensates for the loss in absorptive surface through enhanced epithelial growth to

maintain nutritional status [15]. Adaptation is a complex physiological response with several

morphological and cellular changes to the intestinal mucosa, requiring exogenous assistance for

improving patients’ quality of life and survival rate. Several pharmacological and surgical

approaches have been developed for the treatment of SBS [24]. One of the main therapeutic

strategies consists of increasing the absorptive area, for which intestinal transplantation is a viable

option, but one that may lead to graft rejection and lifelong use of immunosuppressive drugs, in

addition to the problems of donor scarcity, high cost and morbidity from surgery [25-27].

Conversely, techniques based on tissue engineering with intestinal stem cells and the regenerative

capability of the intestine represent a great opportunity as potential alternative therapies for this

syndrome.

Tissue-Engineered Small Intestine

Early attempts based on serosal patching to increase the intestinal absorptive surface led to

undesired physiological effects including decreases in intestinal remnant growth, villus height, body

weight, and mucosal protein content. These changes resulted in overall impaired intestinal

adaptation and absorption following massive enterectomy [28, 29]. Seeding of conduits with

isolated epithelial cells was unsuccessful due to limited growth [30]. Recent tactics to engineer the

small intestine in vitro were successful but the surface area generated is not sufficient for human

therapy and most in vitro approaches generate only intestinal epithelium without surrounding

mesenchymal structures.

Salerno-Gonçalvez and collaborators created a system for generating organotypic human

intestinal mucosa from fibroblasts, lymphocytes, epithelial cells and endothelial cells in a 3D

bioreactor under microgravity and in the presence of gelled collagen-1 mimicking extracellular

matrix (ECM). Differentiated cell types and villus-like structures were obtained and function was

confirmed by pathogen response. Salmonella enterica serovar Typhi was presented to epithelial

cells and alterations in morphology and cytokine production were verified. Cell membrane

alterations (rearrangement and ruffling) were detected 1 hour after infection and increased over

time. Infection also resulted in production of cytokines (IL-1β, IL-6, IL-8, IL-11, IL-12p70, IL-17a,

IL-21 and TNF-α). Even though these results demonstrated satisfactory functionality, no

mesenchyme was formed. [31].

In a different laboratory, by means of a bioreactor-assisted system, 3D co-cultures of Caco-2

cells (from human epithelial colorectal adenocarcinoma) and human microvascular endothelial cells

(hMECs) generated multilayers of prismatic, enterocyte-resembling cells more efficiently than 2D

cultures. Expression of occludin, villin and E-cadherin, and the efflux transporter p-glycoprotein

were similar to the native jejunum, and staining for CD31 (PECAM) and von Willebrand factor

(vWF) indicated hMEC were present during the whole 14-day culture period [32]. The tumor origin

of Caco-2 cells, and absent mesenchymal structures such as nerves, are future challenges for this

model.

Human pluripotent stem cells (both embryonic (ESCs) and induced pluripotent stem cells

(iPSCs)) have been directly differentiated in vitro to form a tissue resembling that of the fetal

intestine, presenting secretory and absorptive functions [33]. Even though mesenchymal markers

FOXF1 (forkhead box F1) and vimentin detected through immunofluorescence and anatomical

microscopic analysis indicated the presence of a mesenchymal layer in the formed tissue, it lacked

blood vessels and nerves. Therefore, these strategies also contain impediments for correct growth of

a full-thickness intestine in patients in the future. For the reasons above, approaches that preserve

the mesenchyme along with the epithelium are possibly better suited for a pediatric therapy aiming

at small intestine replacement.

TESI originated in a 1988 work by the Vacanti Lab at Massachusetts General Hospital.

Before these experiments, cell transplantation techniques involved the injection of cell suspensions

directly into tissues or the vascular system. By seeding mouse and rat cells and cell clusters from

different origins onto biodegradable, polymeric scaffolds and implanting them into host animals

they obtained satisfactory cell viability, proliferation and engraftment rates, especially for

hepatocytes and fetal small intestinal cells [34]. The development of isolated organoid units (OUs)

from the intestine of the suckling rat to produce all of the epithelial cell lineages represented an

important step forward. In an adaptation from a method elaborated by Evans et al. in the early

1990’s, the Vacanti group reported the generation of TESI in vivo. Cells were isolated in the form

of organoid units (OU), which consisted of multicellular clusters of epithelium and mesenchyme.

This work also suggested that the growth of ex-vivo gut epithelium was dependent on the

surrounding tissue and the growth factors produced by it, shedding light on the importance of

maintaining cellular relationships within the explant [35]. Another article published by the same

group in 1998 described the use of tubular polyglycolic acid (PGA) scaffolds sprayed with 5%

poly-L-lactic acid (PLLA) to heterotopically transplant intestinal epithelial OUs containing a

mesenchymal core. In this approach, organoid units are isolated through enzymatic digestion and

sedimentation, after which they are seeded onto the scaffolds and implanted in the omentum or

other vascularized space.

Consistent results showed that OU survived and formed a complex composite that resembled

a small intestine, indicating that morphogenesis, cytodifferentiation and phenotypic maturation of

the organ had occurred [36, 37]. Lewis rats subjected to 75% small bowel resection had increased

body weight by 36 weeks after anastomosis of TESI to the native jejunum in a side-to-side fashion.

These TESI had increased length and diameter, as well as a well-developed mucosa, at 10 weeks

[38]. Lewis rats subjected to TESI anastomosis after massive resection gained weight faster and

more efficaciously, presented normal serum B12 levels and longer transit times when compared to

controls (resection only)[39]. Later studies would show that tissue-engineered intestinal tissues

were capable of generating a mature immune system with macrophages, T, B and NK cells, and of

presenting SGLT1 transporter expression [40, 41]. Adaptations of the OU isolation protocol for the

small intestine resulted in a portfolio of engineered gut portions that extended to esophagus,

stomach and large intestine [42-44], proving both flexibility and efficiency of the method as a

candidate for treating diseases of GI organs that demand regeneration.

An alternate approach has been xenograft models of transplanted human fetal intestine into

host animals. These models allowed survival and mostly epithelial proliferation of the intestinal

tissue [45, 46]. Several reports clarified aspects of human intestinal immunity, development, ability

to survive in the host, expression of regulatory genes and ability to form a fully differentiated

epithelium [47-52]. The xenotransplantation studies provided insights into the role of neutrophils

and cytokine production in human intestine infection, the importance of genes, such as p53, for

regeneration of the newly formed intestine and the potential of a xenograft approach being

transferred to the clinic, but they are analogs to the imperfect current allotransplantation therapies.

The mesenchymal elements, which are critical to long-term function including peristalsis, may not

actively regenerate, and a fetal donor source has ethical difficulties for actual human therapy.

Postnatal human intestinal tissue growth has not been explored in these models.

In two recently published papers, Barthel et al. and Levin et al. were the first to document that

seeding of organoid units from postnatal human large and small intestinal tissue onto a

biodegradable PGA/PLLA scaffold formed full-thickness human engineered large and small

intestine in an irradiated NOD/SCID gamma-chain deficient mouse model. The human TESI

developed similarly to reported murine TESI, forming crypts and villi with all four specialized

epithelial cell types, mucosa and a mesenchyme from human origin with cells expressing muscular

and neural markers [53, 54]. Similarly, human tissue-engineered colon recapitulated native

architecture. Previous studies had not been able to demonstrate growth of intestinal xenografts

from human tissue other than of fetal origin, (Figure 1).

Epithelial-mesenchymal interactions are critical in the governance of proliferation and

differentiation of the intestinal epithelium, and have been investigated for decades [55]. Perhaps one

of the greatest contributions of the mesenchyme to the juxtaposed epithelium is to the maintenance

of the ISC niche located at the base of the crypt. The intestinal epithelium is the fastest self-

renewing tissue in the adult, with the epithelium totally replaced every 3-7 days [11, 56]. The niche

is a restricted tissue microenvironment composed of proliferating and differentiating epithelial cells,

where the Paneth cell has proven to be pivotal [12]. It is also composed of surrounding

mesenchymal cells, including enteric neurons, intraepithelial lymphocytes and (myo)fibroblasts,

and blood vessels [57]. It is responsible for controlling the balance between stem cells self-renewal

and differentiation, hence controlling progeny number to prevent both complete depletion of the

stem cell pool and uncontrolled proliferation that could lead to tumor formation [58]. It is through a

tightly regulated crosstalk with the mesenchyme that the stem cell niche-dependent cellular

dynamics of the epithelium is sustained. A work by Cheng and Leblond (1974), provided evidence

that intestinal stem cells located in the crypts, also known as crypt-base columnar cells (CBCCs),

were pluripotent, responsible for generating the four differentiated cell types of the intestinal

epithelium [59]. Many ISC markers have been proposed, namely Bmi1, Lgr5, CD133, DcamKL-1

and Musashi [60-64]. In the intestine, Lgr5 (Leucine-rich repeat-containing G-protein-coupled

receptor) is exclusively expressed in CBCCs, which are truly capable of forming “crypt-like

structures” and all of the four differentiated cell types of the intestine in vitro. In addition, Lgr5- and

CD133-positive cells are located at sites at the base of the crypt classically postulated as belonging

to stem cells [14, 56, 65]. CD45-negative cells have also been proposed to be small intestine

epithelial stem cells isolated as a side population [66]. Bjerknes et al. showed that progenitor cells

were also capable of dividing to generate the large amount of specialized cells the intestine requires

for performing its function properly [67]. In order to keep up with such active rhythm, the niche

stem cells guarantee constant production of progenitor cells that proliferate and follow the dogmatic

crypt-villus axis, subsequently shed at the tip of the villi. The first cell progeny generated by these

stem cells are rapidly cycling daughter cells committed to continue dividing and to move up the

crypt towards the villus. These cells are designated transit-amplifying (TA) cells, which proliferate

four to five times before differentiating [58, 68, 69].

Methodological approaches have been developed that confirmed the postulated migration

pattern of these cells [70]. Both stem cells and TA cells are regulated by the evolutionarily

conserved canonical Wnt/β-catenin signaling pathway, which targets Lgr5 expression and is

thought to be the master activator of intestinal crypt proliferation [58, 71-73]. Wnt blocks β-catenin

degradation, resulting in stabilization and translocation to the nucleus where it forms β-catenin/TCF

complexes [4]. Absence of Wnt signal targets β-catenin for degradation through the

ubiquitination/proteasome pathway, elicited by the destruction complex composed of APC, Axin,

CKI and GSK3 [74-76]. β-catenin-mediated gene expression determines the physical structure of

the ISC niche, in coordination with Ephrins and Eph proteins, which control cell migration patterns.

The nuclear/cytoplasmic β-catenin pattern indicates that Wnt acts forming a gradient along the crypt

axis, with its signaling components being shown to be present in both epithelial cells of the crypt

and mesenchymal cells [77, 78]. Interactions between the intestinal epithelium and the mesenchyme

also occur through the hedgehog, platelet-derived growth factor (PDGF) and bone morphogenetic

protein (BMP) pathways. The Sonic hedgehog (Shh) and Indian hedgehog (Ihh) ligands are

expressed in the small intestine epithelium of the mouse, becoming concentrated to intervillus

regions as villus formation occurs. Conversely, receptors (Ptch1 and Ptch2) and effectors (Gli1,

Gli2 and Gli3) are constrained to the mesenchyme lying beneath [79]. Hedgehog is proposed to be

essential for villus establishment, but negatively regulates crypt formation. PDGFA is also produced

in the epithelium, with its receptors located in the mesenchyme. This signaling pathway is important

for villus shaping and control of mesenchyme behavior [80]. For BMP signaling, on the other hand,

opposite expression patters are found: BMP2 and BMP4 ligands are produced in the mesenchyme

and their receptor (BMPR1A) is present in the endothelium. Indeed, BMP pathway activation is

higher in the villus epithelium and BMP acts as a Hedgehog mediator, and a blocker of ectopic

crypt formation [68, 81, 82] (Figure 2).

Vacanti et al. reported that “mesenchymal elements from the host” were present in the

isolated cell clusters and suggested that the final three-dimensional tissue would be a chimera from

both the donor and the host [34]. Indeed, lineage tracing in the mouse showed that all the

differentiated cell types of TESI formed by implantation of actinGFP organoid units came from cells

that belonged to the donor, which was also the case for ganglion cells. However, CD31-stained

blood vessels were both GFP-positive and GFP-negative, suggesting vascular anastomosis between

donor and host [14], a communication critical for TESI growth. The growing TESI and its

proliferating neomucosa display high metabolic demands. Nutrient and oxygen intake and drainage

of cellular waste are provided by the blood supply. The vascular architecture around the engineered

intestine is similar to that found in the native intestine. However, the concentration of antigenic

growth factors such as VEGF (Vascular Endothelial Growth Factor) and bFGF (basic Fibroblast

Growth Factor) in the engineered intestine are significantly lower than that found in the juvenile

bowel [83]. This indicated that supplementing the implanted composite with factors that induce

blood vessel growth could accelerate growth and maturation, resulting in a functional organ in a

shorter period of time. Several other controllable events happen during the development of the

neointestine. Therefore, strong evidence indicates that administration of specific biopharmaceuticals

such as recombinant peptide factors may greatly contribute to tissue-engineered organs.

Contributions of Biopharmaceuticals to TESI

Biopharmaceuticals are proteins or nucleic acids obtained through recombinant DNA

technology (i.e. by means other than direct extraction from biological sources) for pharmaceutical

use in therapy or in vitro diagnosis [84]. Over 200 biopharmaceuticals are currently available in the

market, illustrating their importance in healthcare. Recombinant therapeutic proteins alone

contributed to 15.6% of the pharmaceutical industry in 2007 and, together with antibodies, totalled

$99 billion for the global biopharmaceutical market in 2009 [85]. Growth factors, mitogens and

other regulating molecules are polypeptides that govern important steps from early stages of

embryonic life until adulthood, regulating multiple functions by altering cell physiology and

behavior. Since building a new organ or tissue usually in some stages recapitulates embryonic

development processes and such molecules are key regulators of developmental events, a

biopharmaceutical-assisted tissue engineering approach could be essential for the future of

regenerative medicine, including the case of complex organs such as the small intestine. A review

of GH (growth hormone) therapy for SBS patients may be found elsewhere [86]. Several other

molecules, namely EGF (epidermal growth factor), IGF (insulin-like growth factor)-I/II, TGF

(transforming growth factor), PDGF, FGF and TNF (tumor necrosis factor)-α have been proven to

exert a positive effect on intestinal epithelial growth in culture and in animal models [86-101]

(Table 1), but only a handful of factors have been used in combination with the TESI approach.

Ramsanahie et al. (2003) provided evidence for the beneficial effects of adding regulatory

molecules to the developing engineered intestine. Glucagon-like peptide-2 (GLP-2) is a

proglucagon derivative produced by enteroendocrine cells that stimulates intestinal growth,

upregulates villus height, increases crypt cell proliferation and decreases enterocyte apoptosis. It

avoids intestinal hypoplasia in patients under TPN and has been reported to control expression and

activity of an intestinal transporter (RefSeq, 2008) [102]. GLP-2 was administrated subcutaneously

to Lewis rats bearing TESI anastomosed to the jejunum. When compared to controls, these animals

had improved intestinal function of the TESI, presenting higher villi, deeper crypts, higher crypt

cells proliferation and reduced apoptosis of epithelial cells, proving that the effects of an exogenous

protein on the tissue-engineered small intestine resembles that of the endogenous protein in the

native organ [103], suggesting that other proteins could also positively affect TESI. Wulkersdorfer

et al. also showed that loading GLP-2 onto PGA discs before seeding rat OUs augmented the

number of tissue-engineered intestinal complexes, especially in the presence of Matrigel®, an ECM

mimic composed mainly of basement membrane proteins. In the same work, holo-transferrin and

Matrigel® resulted in increased surface area of the engineered intestine [6]. Indeed, clinical trials in

humans showed improvement in bone mineral density, weight gain and relative protein absorption

[104, 105].

Insufficient vascularization is a common hurdle for engineered organs, precluding proper

influx of nutrients and oxygen and, consequently, proper growth and function. Initial efforts to pre-

vascularize biodegradable polymers to ameliorate cell attachment and growth had not employed

additional biologicals in the composite [106, 107]. Angiogenesis is the growth of blood vessels

from the pre-existing vasculature. Vascular Endothelial Growth Factor, for instance, comprises a

group of proteins belonging to the PDGF/VEGF growth factor family. As key mediators of

physiological and pathological angiogenesis and lymphangiogenesis, these proteins are therapeutic

targets in several disease contexts [108]. In an effort to improve the vascular architecture of TESI, a

pro-angiogenic VEGF isoform was encapsulated in poly(lactide-co-glycolide) (PLGA)

microspheres and loaded into molded scaffolds containing intestinal OUs. The microspheres

provided a sustained delivery of VEGF culminating in an increased vascular net outside the

developing organs (CD31 staining), which also grew larger and denser than the control

counterparts. Moreover, epithelial cell proliferation increased and no alteration in apoptosis was

detected [7]. Matthews et al also reported the beneficial effects of pro-angiogenic VEGF

overexpression on TESI formation, by means of an inducible, ubiquitous genetic system. Overall,

OUs overexpressing VEGF formed TESI that grew faster and bigger after 4 weeks in vivo when

compared to control, also presenting higher villi and deeper crypts with all differentiated cell types.

CD31/PECAM staining proved capillary density and crypt epithelial cell proliferation was also

increased in the overexpressing TESI [26].

Growing the tissue-engineered small intestine from organoid units on a scaffold requires that

ISCs be co-isolated with cell members of the niche that regulates the stem cells “renew or

differentiate” balance. Wnt is a family of highly-conserved, secreted signaling proteins which play a

crucial role in development and stem cell proliferation control, maintenance and differentiation in

the adult vertebrate, including ISCs [109]. Wnt is palmitoylated in the ER by Porcupine, and

secreted with the assistance of Wntless/Evi in the Golgi and plasma membrane. It then diffuses

through the ECM and acts upon target cells expressing the seven-pass transmembrane receptor

Frizzled and its adjacent Lrp5/6 (Low density lipoprotein receptor-Related Protein) co-receptors

[110]. The third element involved in Wnt recognition by ISCs is the Lgr5 receptor. It is likely that

targeting Wnt isoforms could be beneficial for the crypt intestinal stem cell niche and other Wnt

responsive organs. Indeed, drug candidates that enhance and especially those that block Wnt

signaling have been studied in diseases, such as various types of cancer, Alzheimer’s disease,

osteoporosis, fracture repair and ES/iPS stem cell differentiation. However, this pathway still poses

several challenges as a therapeutic target. Nineteen Wnt ligands and 10 Frizzled receptor isoforms

are known so far, rendering specificity and control of individual downstream events a real challenge

for a biopharmaceutical approach. Additionally, short pulses of Wnt are known to be sufficient for

developmental steps in the embryo, implying that long exposures to Wnt are unnecessary and that a

rigorous control of this signaling protein will be required [111]. Finally, many cell targets are

susceptible to Wnt, increasing possible off-target responses and restraining its use for in vitro and

animal models. Consequently, an approach that enhances the beneficial downstream effects of

endogenous Wnt without generally altering the total Wnt input through a co-mediator might be

better suited for therapy. R-Spondins belong to a thrombospondin type-1 repeat (TSR-1)-containing

proteins superfamily. The R-spondin members (Rspo1-4) also bear positively charged amino acids

at the C-terminal region, and two cysteine-rich, furin-like domains at the N-terminus which are

necessary and sufficient for Wnt signal potentialization. Lgr receptors (Lgr4, -5 and -6), which

physically interact with Lrp5/6 and Frizzleds, have recently been identified as receptors for all four

R-spondins [88, 112, 113]. In particular, intestinal Lgr5+ (stem) cells at the base of the crypt

respond to Rspo1 by becoming highly proliferative. Rspo1 also acts as an in vivo mitogenic growth

factor for the intestinal epithelium in the mouse [88, 93], allowing for expansion of epithelial mass

in culture systems. Thereby, R-Spondins may improve the way TESI develops and is a potential

molecule for clinical use in the future.

The fibroblast growth factor (FGF) family comprises a set of over twenty heparin-binding

proteins in humans. FGFs regulate cell differentiation, proliferation, migration and survival and

display broad angiogenic and mitogenic activity, being required in the development of multiple

organs, including those of the gastrointestinal tract [114-117] (RefSeq, Jul 2008). Among FGF

members, FGF10 has been implicated in dysgenesis of digestive organs when ablated (Fgf10-/-)

[116]. Conversely, Tai et al. were the first to provide evidence that FGF10 overexpression in the

ileal crypt epithelial assists intestinal adaptation following massive small bowel resection [118]. A

2013 report by Torashima et al. has now proven that this protein is also passive of collaborating in

the formation of tissue engineered small intestine. Following the rationale behind transgenic

models, they overexpressed FGF10 in organoid units implanted in host animals under doxycycline

administration, rendering similar results obtained in the VEGF overexpression approach [26]. TESI

size and weight augmented and higher villi and deeper crypts were obtained. Also, the proliferation

of crypt epithelial cells was enhanced and terminal cell differentiation was not compromised [124]

(Torashima et al., 2013; Epub ahead of print).

In view of the crucial role played by peptide growth factors and hormones not only in

embryonic development and several physiological processes, but, also, in tissue engineering, we

have systematically pursued in vitro production of their recombinant human counterparts. Over the

past decade, we achieved heterologous expression in bacteria, mammalian and insect cells of a

number of these recombinant factors/biopharmaceuticals, namely: human prolactin, amylin, FGF,

PDGF, VEGFs, G- and GM-CSFs, TGF-beta 1 and 3, and BMPs 2, 4 and 7. The transgenic cell

lines overproducing some of these peptides have been transferred to local Brazilian biotech and

pharma companies for commercial production and distribution (Sogayar et al., unpublished results).

Conclusion and Future Perspectives

Pediatric patients suffering from intestinal failure, as in short bowel syndrome, are still in

need of a better alternative for their condition to overcome high morbidity and mortality rates.

Tissue engineering techniques have improved considerably over the last two decades and may bring

a gold standard substitute to the currently available therapies, including transplantation. Autologous

tissue-engineered intestine is one promising solution. Preserving the stem cell niche and the

surrounding mesenchyme appears to be of utmost importance and may be critical for the

maintenance of signaling pathways. But additional biological factors may also be necessary.

Indeed, several reports have demonstrated the beneficial effects of adding growth factors, such as

GLP-2, FGF10, and VEGF to the organoid unit-scaffold construct in animal models, but choosing

the best candidates is the next challenge. Also, many promising candidates, such as R-Spondins,

still need to be included in these investigations. Further studies should focus on refinement of

spatio-temporal delivery of peptide factors, which is dependent on dose, stability, number of

biologicals being delivered at once and interaction properties with synthetic and organic scaffolds.

These strategies are likely to accelerate the successful development of the regenerating intestine,

making it clinically available to improve the lives of pediatric patients who greatly depend on these

discoveries.

References

1. O'Keefe, S.J., et al., Short bowel syndrome and intestinal failure: consensus definitions and overview. Clin Gastroenterol Hepatol, 2006. 4(1): p. 6-10.

2. Vacanti, C.A., The history of tissue engineering. J Cell Mol Med, 2006. 10(3): p. 569-76.

3. Vacanti, J.P., Beyond transplantation. Third annual Samuel Jason Mixter lecture. Arch Surg, 1988. 123(5): p. 545-9.

4. Schuijers, J. and H. Clevers, Adult mammalian stem cells: the role of Wnt, Lgr5 and R-spondins. Embo Journal, 2012. 31(12): p. 2685-2696.

5. Sala, F.G., et al., Tissue-engineered small intestine and stomach form from autologous tissue in a preclinical large animal model. J Surg Res, 2009. 156(2): p. 205-12.

6. Wulkersdorfer, B., et al., Growth factors adsorbed on polyglycolic acid mesh augment growth of bioengineered intestinal neomucosa. J Surg Res, 2011. 169(2): p. 169-78.

7. Rocha, F.G., et al., The effect of sustained delivery of vascular endothelial growth factor on angiogenesis in tissue-engineered intestine. Biomaterials, 2008. 29(19): p. 2884-90.

8. Moran-Ramos, S., A.R. Tovar, and N. Torres, Diet: friend or foe of enteroendocrine cells--how it interacts with enteroendocrine cells. Adv Nutr, 2012. 3(1): p. 8-20.

9. Bruen, C.M., et al., The effects of food components on hormonal signalling in gastrointestinal enteroendocrine cells. Food Funct, 2012. 3(11): p. 1131-43.

10. Potten, C.S., et al., The stem cells of small intestinal crypts: where are they? Cell Prolif, 2009. 42(6): p. 731-50.

11. Day, R.M., Epithelial stem cells and tissue engineered intestine. Curr Stem Cell Res Ther, 2006. 1(1): p. 113-20.

12. Sato, T., et al., Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature, 2011. 469(7330): p. 415-8.

13. Levin, D.E. and T.C. Grikscheit, Tissue-engineering of the gastrointestinal tract. Curr Opin Pediatr, 2012. 24(3): p. 365-70.

14. Sala, F.G., et al., A multicellular approach forms a significant amount of tissue-engineered small intestine in the mouse. Tissue Eng Part A, 2011. 17(13-14): p. 1841-50.

15. Thompson, J.S., et al., Current management of short bowel syndrome. Curr Probl Surg, 2012. 49(2): p. 52-115.

16. Gutierrez, I.M., K.H. Kang, and T. Jaksic, Neonatal short bowel syndrome. Semin Fetal Neonatal Med, 2011. 16(3): p. 157-63.

17. Bakker, H., et al., Home parenteral nutrition in adults: a european multicentre survey in 1997. ESPEN-Home Artificial Nutrition Working Group. Clin Nutr, 1999. 18(3): p. 135-40.

18. Byrne, T.A., et al., A new treatment for patients with short-bowel syndrome. Growth hormone, glutamine, and a modified diet. Ann Surg, 1995. 222(3): p. 243-54; discussion 254-5.

19. Wales, P.W., et al., Neonatal short bowel syndrome: population-based estimates of incidence and mortality rates. J Pediatr Surg, 2004. 39(5): p. 690-5.

20. Cole, C.R., et al., Very low birth weight preterm infants with surgical short bowel syndrome: incidence, morbidity and mortality, and growth outcomes at 18 to 22 months. Pediatrics, 2008. 122(3): p. e573-82.

21. Peyret, B., et al., Prevalence of liver complications in children receiving long-term parenteral nutrition. Eur J Clin Nutr, 2011. 65(6): p. 743-9.

22. Kaufman, S.S., et al., Influence of bacterial overgrowth and intestinal inflammation on duration of parenteral nutrition in children with short bowel syndrome. J Pediatr, 1997. 131(3): p. 356-61.

23. Nucci, A., et al., Interdisciplinary management of pediatric intestinal failure: a 10-year review of rehabilitation and transplantation. J Gastrointest Surg, 2008. 12(3): p. 429-35; discussion 435-6.

24. Thompson, J.S., et al., Current management of the short bowel syndrome. Surg Clin North Am, 2011. 91(3): p. 493-510.

25. Reyes, J., et al., Current status of intestinal transplantation in children. J Pediatr Surg, 1998. 33(2): p. 243-54.

26. Matthews, J.A., et al., VEGF optimizes the formation of tissue-engineered small intestine. Regen Med, 2011. 6(5): p. 559-67.

27. Misiakos, E.P., et al., Short bowel syndrome: current medical and surgical trends. J Clin Gastroenterol, 2007. 41(1): p. 5-18.

28. Bragg, L.E. and J.S. Thompson, Serosal patching impairs intestinal adaptation following enterectomy. J Surg Res, 1992. 52(2): p. 118-22.

29. Thompson, J.S., et al., Morphologic and nutritional responses to intestinal patching following intestinal resection. Surgery, 1988. 103(1): p. 79-86.

30. Nguyen, B.L. and J.S. Thompson, Growing small-intestinal neomucosa in serosa-lined pouches. Curr Surg, 1990. 47(4): p. 269-71.

31. Salerno-Goncalves, R., A. Fasano, and M.B. Sztein, Engineering of a multicellular organotypic model of the human intestinal mucosa. Gastroenterology, 2011. 141(2): p. e18-20.

32. Pusch, J., et al., The physiological performance of a three-dimensional model that mimics the microenvironment of the small intestine. Biomaterials, 2011. 32(30): p. 7469-78.

33. Spence, J.R., et al., Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature, 2011. 470(7332): p. 105-9.

34. Vacanti, J.P., et al., Selective cell transplantation using bioabsorbable artificial polymers as matrices. J Pediatr Surg, 1988. 23(1 Pt 2): p. 3-9.

35. Evans, G.S., et al., The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J Cell Sci, 1992. 101 ( Pt 1): p. 219-31.

36. Choi, R.S., et al., Studies of brush border enzymes, basement membrane components, and electrophysiology of tissue-engineered neointestine. J Pediatr Surg, 1998. 33(7): p. 991-6; discussion 996-7.

37. Grikscheit, T.C. and J.P. Vacanti, The history and current status of tissue engineering: The future of pediatric surgery. J Pediatr Surg, 2002. 37(3): p. 277-88.

38. Kaihara, S., et al., Long-term follow-up of tissue-engineered intestine after anastomosis to native small bowel. Transplantation, 2000. 69(9): p. 1927-32.

39. Grikscheit, T.C., et al., Tissue-engineered small intestine improves recovery after massive small bowel resection. Ann Surg, 2004. 240(5): p. 748-54.

40. Perez, A., et al., Tissue-engineered small intestine: ontogeny of the immune system. Transplantation, 2002. 74(5): p. 619-23.

41. Tavakkolizadeh, A., et al., Tissue-engineered neomucosa: morphology, enterocyte dynamics, and SGLT1 expression topography. Transplantation, 2003. 75(2): p. 181-5.

42. Speer, A.L., et al., Murine tissue-engineered stomach demonstrates epithelial differentiation. J Surg Res, 2011. 171(1): p. 6-14.

43. Grikscheit, T.C., et al., Tissue-engineered large intestine resembles native colon with appropriate in vitro physiology and architecture. Ann Surg, 2003. 238(1): p. 35-41.

44. Grikscheit, T., et al., Tissue-engineered esophagus: experimental substitution by onlay patch or interposition. J Thorac Cardiovasc Surg, 2003. 126(2): p. 537-44.

45. Winter, H.S., et al., Human Intestine Matures as Nude-Mouse Xenograft. Gastroenterology, 1991. 100(1): p. 89-98.

46. Leapman, S.B., et al., Transplantation of fetal intestine: survival and function in a subcutaneous location in adult animals. Ann Surg, 1974. 179(1): p. 109-14.

47. Zhang, Z., et al., Shigella infection in a SCID mouse-human intestinal xenograft model: role for neutrophils in containing bacterial dissemination in human intestine. Infect Immun, 2001. 69(5): p. 3240-7.

48. Seydel, K.B., et al., Human intestinal epithelial cells produce proinflammatory cytokines in response to infection in a SCID mouse-human intestinal xenograft model of amebiasis. Infect Immun, 1997. 65(5): p. 1631-9.

49. Savidge, T.C., D.C. Lowe, and W.A. Walker, Developmental regulation of intestinal epithelial hydrolase activity in human fetal jejunal xenografts maintained in severe-combined immunodeficient mice. Pediatr Res, 2001. 50(2): p. 196-202.

50. Sattar, A., et al., Expression of growth regulatory genes in a SCID mouse-human model of intestinal epithelial regeneration. J Pathol, 1999. 187(2): p. 229-36.

51. Nanthakumar, N.N., et al., Normal and glucocorticoid-induced development of the human small intestinal xenograft. Am J Physiol Regul Integr Comp Physiol, 2003. 285(1): p. R162-70.

52. Angioi, K., et al., Xenografted human whole embryonic and fetal entoblastic organs develop and become functional adult-like micro-organs. J Surg Res, 2002. 102(2): p. 85-94.

53. Levin, D.E., et al., Human tissue-engineered small intestine forms from postnatal progenitor cells. J Pediatr Surg, 2013. 48(1): p. 129-37.

54. Barthel, E.R., et al., Human tissue-engineered colon forms from postnatal progenitor cells: an in vivo murine model. p. 807-18.

55. Kedinger, M., et al., Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells. Dev Biol, 1986. 113(2): p. 474-83.

56. Sato, T., et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 2009. 459(7244): p. 262-5.

57. Yen, T.H. and N.A. Wright, The gastrointestinal tract stem cell niche. Stem Cell Rev, 2006. 2(3): p. 203-12.

58. Haegebarth, A. and H. Clevers, Wnt signaling, lgr5, and stem cells in the intestine and skin. Am J Pathol, 2009. 174(3): p. 715-21.

59. Cheng, H. and C.P. Leblond, Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types. Am J Anat, 1974. 141(4): p. 537-61.

60. Zhu, L., et al., Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature, 2009. 457(7229): p. 603-7.

61. Sangiorgi, E. and M.R. Capecchi, Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet, 2008. 40(7): p. 915-20.

62. May, R., et al., Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice. Stem Cells, 2008. 26(3): p. 630-7.

63. Barker, N., et al., Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature, 2007. 449(7165): p. 1003-7.

64. Asai, R., H. Okano, and S. Yasugi, Correlation between Musashi-1 and c-hairy-1 expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouse. Dev Growth Differ, 2005. 47(8): p. 501-10.

65. Gerbe, F., et al., Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium. Journal of Cell Biology, 2011. 192(5): p. 767-780.

66. Dekaney, C.M., et al., Isolation and characterization of a putative intestinal stem cell fraction from mouse jejunum. Gastroenterology, 2005. 129(5): p. 1567-80.

67. Bjerknes, M. and H. Cheng, Clonal analysis of mouse intestinal epithelial progenitors. Gastroenterology, 1999. 116(1): p. 7-14.

68. Crosnier, C., D. Stamataki, and J. Lewis, Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet, 2006. 7(5): p. 349-59.

69. Marshman, E., C. Booth, and C.S. Potten, The intestinal epithelial stem cell. Bioessays, 2002. 24(1): p. 91-8.

70. Fellous, T.G., et al., A methodological approach to tracing cell lineage in human epithelial tissues. Stem Cells, 2009. 27(6): p. 1410-20.

71. Moore, K.A. and I.R. Lemischka, Stem cells and their niches. Science, 2006. 311(5769): p. 1880-1885.

72. Pinto, D., et al., Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev, 2003. 17(14): p. 1709-13.

73. Kuhnert, F., et al., Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc Natl Acad Sci U S A, 2004. 101(1): p. 266-71.

74. Cordero, J.B. and O.J. Sansom, Wnt signalling and its role in stem cell-driven intestinal regeneration and hyperplasia. Acta Physiol (Oxf), 2012. 204(1): p. 137-43.

75. Aberle, H., et al., beta-catenin is a target for the ubiquitin-proteasome pathway. Embo Journal, 1997. 16(13): p. 3797-3804.

76. Orford, K., et al., Serine phosphorylation-regulated ubiquitination and degradation of beta-catenin. J Biol Chem, 1997. 272(40): p. 24735-8.

77. Batlle, E., et al., Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell, 2002. 111(2): p. 251-63.

78. van de Wetering, M., et al., The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell, 2002. 111(2): p. 241-50.

79. Madison, B.B., et al., Epithelial hedgehog signals pattern the intestinal crypt-villus axis. Development, 2005. 132(2): p. 279-89.

80. Karlsson, L., et al., Abnormal gastrointestinal development in PDGF-A and PDGFR-(alpha) deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis. Development, 2000. 127(16): p. 3457-66.

81. Haramis, A.P., et al., De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science, 2004. 303(5664): p. 1684-6.

82. He, X.C., et al., BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nat Genet, 2004. 36(10): p. 1117-21.

83. Gardner-Thorpe, J., et al., Angiogenesis in tissue-engineered small intestine. Tissue Eng, 2003. 9(6): p. 1255-61.

84. Walsh, G., Biopharmaceuticals and biotechnology medicines: an issue of nomenclature. Eur J Pharm Sci, 2002. 15(2): p. 135-8.

85. Walsh, G., Biopharmaceutical benchmarks 2010. Nat Biotechnol, 2010. 28(9): p. 917-24.

86. Pereira, P.M. and J.E. Bines, New growth factor therapies aimed at improving intestinal adaptation in short bowel syndrome. J Gastroenterol Hepatol, 2006. 21(6): p. 932-40.

87. Booth, C., G.S. Evans, and C.S. Potten, Growth factor regulation of proliferation in primary cultures of small intestinal epithelium. In Vitro Cell Dev Biol Anim, 1995. 31(3): p. 234-43.

88. de Lau, W., et al., Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature, 2011. 476(7360): p. 293-7.

89. Drucker, D.J., L. DeForest, and P.L. Brubaker, Intestinal response to growth factors administered alone or in combination with human [Gly2]glucagon-like peptide 2. Am J Physiol, 1997. 273(6 Pt 1): p. G1252-62.

90. Duncan, M.D., L.Y. Korman, and B.L. Bass, Epidermal growth factor primes intestinal epithelial cells for proliferative effect of insulin-like growth factor I. Dig Dis Sci, 1994. 39(10): p. 2197-201.

91. Fukamachi, H., Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture. J Cell Sci, 1992. 103 ( Pt 2): p. 511-9.

92. Jones, J.I. and D.R. Clemmons, Insulin-Like Growth-Factors and Their Binding-Proteins - Biological Actions. Endocrine Reviews, 1995. 16(1): p. 3-34.

93. Kim, K.A., et al., Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science, 2005. 309(5738): p. 1256-9.

94. Marchbank, T., et al., Luminal Epidermal Growth-Factor Is Trophic to the Small-Intestine of Parenterally Fed Rats. Clinical Science, 1995. 89(2): p. 117-120.

95. Ohneda, K., et al., Enhanced growth of small bowel in transgenic mice expressing human insulin-like growth factor I. Gastroenterology, 1997. 112(2): p. 444-54.

96. Potten, C.S., et al., Stimulation and Inhibition of Proliferation in the Small-Intestinal Crypts of the Mouse after in-Vivo Administration of Growth-Factors. Gut, 1995. 36(6): p. 864-873.

97. Read, L.C., et al., Insulin-like growth factor-I and its N-terminal modified analogues induce marked gut growth in dexamethasone-treated rats. J Endocrinol, 1992. 133(3): p. 421-31.

98. Steeb, C.B., et al., Systemic infusion of IGF-I or LR(3)IGF-I stimulates visceral organ growth and proliferation of gut tissues in suckling rats. American Journal of Physiology-Gastrointestinal and Liver Physiology, 1997. 272(3): p. G522-G533.

99. Steeb, C.B., et al., Prolonged Administration of Igf Peptides Enhances Growth of Gastrointestinal Tissues in Normal Rats. American Journal of Physiology, 1994. 266(6): p. G1090-G1098.

100. Swaniker, F., et al., Delayed effects of epidermal growth factor after extensive small bowel resection. Journal of Pediatric Surgery, 1996. 31(1): p. 56-60.

101. Ulshen, M.H. and R.H. Raasch, Luminal epidermal growth factor preserves mucosal mass of small bowel in fasting rats. Clinical Science, 1996. 90(5): p. 427-431.

102. Cheeseman, C.I., Upregulation of SGLT-1 transport activity in rat jejunum induced by GLP-2 infusion in vivo. Am J Physiol, 1997. 273(6 Pt 2): p. R1965-71.

103. Ramsanahie, A., et al., Effect of GLP-2 on mucosal morphology and SGLT1 expression in tissue-engineered neointestine. Am J Physiol Gastrointest Liver Physiol, 2003. 285(6): p. G1345-52.

104. Jeppesen, P.B., et al., Glucagon-like peptide 2 improves nutrient absorption and nutritional status in short-bowel patients with no colon. Gastroenterology, 2001. 120(4): p. 806-15.

105. Haderslev, K.V., et al., Short-term administration of glucagon-like peptide-2. Effects on bone mineral density and markers of bone turnover in short-bowel patients with no colon. Scand J Gastroenterol, 2002. 37(4): p. 392-8.

106. Mikos, A.G., et al., Prevascularization of porous biodegradable polymers. Biotechnol Bioeng, 1993. 42(6): p. 716-23.

107. Uyama, S., et al., Delivery of whole liver-equivalent hepatocyte mass using polymer devices and hepatotrophic stimulation. Transplantation, 1993. 55(4): p. 932-5.

108. Giacca, M. and S. Zacchigna, VEGF gene therapy: therapeutic angiogenesis in the clinic and beyond. Gene Ther, 2012. 19(6): p. 622-9.

109. Nusse, R. and H. Varmus, Three decades of Wnts: a personal perspective on how a scientific field developed. EMBO J, 2012. 31(12): p. 2670-84.

110. Ching, W. and R. Nusse, A dedicated Wnt secretion factor. Cell, 2006. 125(3): p. 432-3.

111. Zimmerman, Z.F., R.T. Moon, and A.J. Chien, Targeting Wnt pathways in disease. Cold Spring Harb Perspect Biol, 2012. 4(11).

112. Carmon, K.S., et al., R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc Natl Acad Sci U S A, 2011. 108(28): p. 11452-7.

113. Glinka, A., et al., LGR4 and LGR5 are R-spondin receptors mediating Wnt/beta-catenin and Wnt/PCP signalling. EMBO Rep, 2011. 12(10): p. 1055-61.

114. Fairbanks, T.J., et al., Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice. 2004. p. 360-5; discussion 360-5.

115. Kanard, R.C., et al., Fibroblast growth factor-10 serves a regulatory role in duodenal development. 2005. p. 313-6.

116. Ohuchi, H., et al., FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. 2000. p. 643-9.

117. Veltmaat, J.M., et al., Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes. 2006. p. 2325-35.

118. Tai, C.C., et al., Induction of fibroblast growth factor 10 (FGF10) in the ileal crypt epithelium after massive small bowel resection suggests a role for FGF10 in gut adaptation. 2009. p. 294-301.

119. Liang, R., et al., Hedgehog signaling displays a biphasic expression pattern during intestinal injury and repair. p. 2251-63.

120. Barrett, T.A., Developmental biology. Intestinal wound healing requires a Wnt balancing act. p. 51-2.

121. Konsavage, W.M., G. Jin, and G.S. Yochum, The Myc 3' Wnt-responsive element regulates homeostasis and regeneration in the mouse intestinal tract. p. 3891-902.

122. Liu, L., et al., Activation of Wnt3a signaling stimulates intestinal epithelial repair by promoting c-Myc-regulated gene expression. p. C277-85.

123. Zhang, L., A.D. Lander, and Q. Nie, A reaction-diffusion mechanism influences cell lineage progression as a basis for formation, regeneration, and stability of intestinal crypts. p. 93.

124. Torashima, Y., et al., Fgf10 overexpression enhances the formation of tissue-engineered small intestine. J Tissue Eng Regen Med. 2013. Epub ahead of print

Tables and Figures

Figure 1. Human TESI made from post-natal intestinal tissue is capable

of forming a fully differentiated mucosa resembling that of the native

small intestine. Staining: Villin: enteroendocrine cells; Chr A

(Chromogranin A): enteroendocrine cells; Mucin-2: goblet cells;

Lysozyme: Paneth cells. Arrows: insets with high power detail.

Figure 2. Main players acting in the small intestine epithelial-mesenchymal crosstalk. TCF:

transcription factor; Shh: sonic hedgehog; Ihh: indian hedgehog; BMP: bone morphogenetic protein;

BMPR1A: bone morphogenetic protein 1A; PDGFA: platelet-derived growth factor A; PDGFR:

platelet derived growth factor receptor.

Table 1. Candidate peptide growth factors for intestinal epithelium

Effector molecule Effect Context References EGF Epithelial cell proliferation,

increased villus height, increased intestinal maltase activity

In vitro and in vitro [86, 90, 91, 94, 96, 100, 101]

IGF-I Epithelial cell proliferation In vitro and in vitro [87, 89, 90, 92, 95, 96]

LR3IGF-I* Increased crypt depth and villus height, increased length and weight of small bowel, thickening of muscularis externa

In vivo [97-99]

IGF-II Epithelial cell proliferation, increased villus height

In vivo [96]

TGF-α Epithelial cell proliferation In vitro and in vitro [87, 96] PDGF Epithelial cell proliferation In vitro [87] TNF-α Epithelial cell proliferation In vitro [87] h[Gly2]GLP-2* Epithelial cell proliferation In vivo [89] R-Spondin Epithelial cell proliferation

(including Lgr5+stem cells) In vitro and in vivo [88, 93]

SHH Epithelial cell proliferation, repair of intestinal epithelium

In vivo [79, 119]

Wnts Epithelial cell proliferation, wound healing, crypt stability, intestinal regeneration

In vivo, in silico model [74, 120-123]

*more potent IGF-I analog

**degradation-resistant GLP-2 analog