Am J Physiol Regul Integr Comp Physiol 285: R162–R170, 2003.
First published January 30, 2003; 10.1152/ajpregu.00721.2001.
Normal and glucocorticoid-induced development of the
human small intestinal xenograft
N. Nanda Nanthakumar, Corrie E. Klopcic, Isabel Fernandez, and W. Allan Walker
1
Developmental Gastroenterology Laboratory, Combined Program in Pediatric
Gastroenterology and Nutrition, Massachusetts General Hospital, and Department
of Pediatrics, Harvard Medical School, Charlestown, Massachusetts 02129
Submitted 3 December 2001; accepted in final form 17 January 2003
sucrase; lactase; trehalase; intestinal ontogeny; hormonal
regulation
THE PRIMARY FUNCTION OF small intestine is end-stage
digestion and absorption of nutrients and is facilitated
by the expression of specific hydrolases and transporters on the epithelium (4, 14, 19). Among hydrolases,
disaccharidases are responsible for the terminal digestion of carbohydrates (9, 14, 38). With regard to disaccharidases, sucrase, lactase, and trehalase have
unique substrate specificity and are synthesized from a
single gene into a glycosylated enzyme anchored in
differentiated epithelium (9, 14, 38). Expression of sucrase and lactase is specific to small intestine, whereas
trehalase is also expressed in the proximal tubular
This work was presented, in part, as a poster at the Digestive
Disease Week in San Diego, May 2000.
Address for reprint requests and other correspondence: N. N.
Nanthakumar, Developmental Gastroenterology Laboratory, Massachusetts General Hospital-East, 114 16th St.: Rm. 3650, Charlestown, MA 02129 (E-mail: [email protected]).
R162
epithelium of the kidney (9, 29, 38). Because of tissue
and substrate specificity as well as the ease of biochemical quantitation, these enzymes have been used as
classic markers for the last 30 years (11, 14, 19),
providing an excellent tool for delineating functional
development of the small intestine (12, 14, 17, 25, 29).
Functional development of the mammalian small
intestine is well characterized in rats and mice (9, 11,
12, 29, 38) and is expressed in two phases. The first
phase of functional development coincides with morphological development just before birth, as the epithelium becomes a monolayer with proliferating and differentiating cells compartmentalized in well-defined
crypts and villi, respectively (10, 34, 45). At this time
the proteins responsible for digestion and absorption of
milk, such as lactase, are expressed. The second phase
accomplishes the onset of the adult phenotype of the
intestine during the third postnatal week (4, 9, 14, 18,
31). At this time, a number of proteins responsible for
digestion and absorption of solid food are expressed.
Among them, sucrase and trehalase activities rapidly
raise to mature levels (9, 30, 32, 38).
However, in humans, the first phase of functional
development occurs during the end of the first trimester (11, 17, 19, 21, 22, 45). Between 9 and 12 wk of
gestation, stratified epithelium becomes a monolayer
and crypt villus structures become established. By the
end of the first trimester, tissue-specific markers such
as peptidases, hydrolases, and glucose transporters are
expressed in a differentiated villus epithelium. Among
disaccharidases, sucrase activity is expressed at a level
comparable to the mature intestine, whereas lactase is
barely detectable (4, 14, 18, 19, 21). However, lactase
activity increases just before birth to its highest level
in the newborn infant. It has been debated whether the
rise of lactase activity in the human corresponds to the
induction of a second phase of functional maturation of
the gut and whether the second and third trimesters
correspond to the suckling period of rodents (4, 9, 14,
19). A lack of availability of human tissue has precluded testing of this hypothesis as well as identifying
other markers for the late gestational development of
the fetal human intestine. However, the recent demonThe costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6119/03 $5.00 Copyright © 2003 the American Physiological Society
http://www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
Nanthakumar, N. Nanda, Corrie E. Klopcic, Isabel
Fernandez, and W. Allan Walker. Normal and glucocorticoid-induced development of the human small intestinal
xenograft. Am J Physiol Regul Integr Comp Physiol 285:
R162–R170, 2003. First published January 30, 2003;
10.1152/ajpregu.00721.2001.—The aim of this study was to
determine whether intestinal xenografts could recapitulate
human in utero development by using disaccharidases as
markers. Twenty-week-old fetal intestine was transplanted
into immunocompromised mice and was followed. At 20-wk
of gestation, the fetal human intestine was morphologically
developed with high sucrase and trehalase but had low lactase activities. By 9-wk posttransplantation, jejunal xenografts were morphologically and functionally developed and
were then monitored for ⱕ6 mo. Both sucrase and trehalase
activities remained unchanged, but lactase activity increased
in a manner similar to that described in in utero development. Changes in sucrase and lactase activities were paralleled by protein levels. Cortisone acetate treatment at 20-wk
posttransplantation accelerated the ontogeny of lactase but
did not alter sucrase and trehalase activities. Biopsies from
1- and 2-yr-old infant intestine showed that all activities,
except trehalase in the proximal intestine, corresponded to
the levels found in jejunal xenografts at 24 wk posttransplantation. These studies suggest that 20-wk-old fetal intestine
has the extrauterine developmental potential to follow normal intrauterine ontogeny as a xenograft.
ONTOGENY OF THE HUMAN GUT XENOGRAFT
MATERIALS AND METHODS
Chemicals. Ultra-pure D-glucose, sucrose, trehalose, lactose, and bovine serum albumin were obtained from Sigma
(St. Louis, MO). Protein concentrations were measured by
using a BCL kit (Pierce, Rockford, IL) against BSA standards
in a calorimetric assay according to the manufacturer’s protocol. Cell culture media [(DMEM) and Connaught Medical
Research Laboratories (CMRL) media], nonessential amino
acids, methionine, vitamin A, and penicillin were obtained
from GIBCO-BRL (Rockville, MD). All other chemicals were
either of reagent or molecular grade. Stock solutions of cortisone acetate (Cortone; Merck, Sharp and Dohme, West
Point, PA) were purchased from the hospital pharmacy. Glucose was measured by using the Trinder 100 kit from Sigma
as described previously (29, 30).
Animals. Four-week-old homozygous scid and nu/nu mice
were housed in a specific pathogen-free facility and maintained on rodent laboratory chow 5001 (Ralston Purina, St.
Louis, MO) and water ad libitum. The animals were raised in
air-conditioned quarters at 21 ⫾ 1°C on a 12:12-h light/dark
cycle with lights on at 0600. Sterilized food (rodent laboratory chow 5001) and deionized water were provided ad libitum from the day of arrival until the completion of the
experiments. To avoid circadian influences, all animals were
killed between 1100 and 1300. Surgery and postsurgical care
were carried out according to the approved animal protocol of
the research animal care committee of the Massachusetts
General Hospital (MGH) as well as the guidelines published
by American Physiological Society (1).
Human intestinal tissue. Twenty-week-old human small
intestines were obtained from prostaglandin/saline-induced
aborted fetuses with informed consent according to the regulations of the Committee for the Protection of Human Subjects from Research Risks at the Brigham and Women’s
Hospital and the Human Investigation Committee at MGH.
To maintain sterility, tissues were collected only from fetuses
in which the abdomen had not been previously opened. These
tissues were then transported to the laboratory in ice-cold
fresh CMRL 1066 media containing glutamate, nonessential
amino acids, penicillin and gentamicin as described before
(28). Tissues were processed as described below. Fresh samples of intestine were frozen and assayed for disaccharidase
activities along with xenograft tissue.
Small intestinal (duodenal) mucosal biopsies from infants
and older children were obtained with informed consent by
endoscopy in the Pediatric Endoscopy Suite at MGH when
these children were endoscoped for diagnostic purposes. Only
biopsies from patients without any histological abnormalities
or pathological diagnosis were used in these studies.
Fetal small intestinal xenograft model. Sterile fetal small
intestine in 2-cm segments stripped of its mesentery was
implanted subcutaneously into homozygous nude or scid
mice as described previously (36, 37, 48). Clear and meconium-free jejunal tissues were used for xenotransplantation,
because tissue could be easily obtained with minimal handling before surgery. On the day of xenotransplantation mice
were anesthetized by avertin at a dose of 200 mg/kg body wt.
Avertin consists of 1 g of 3-bromoethanol and 0.62 ml of
2-methyl-2-butanol dissolved in 79 ml of deionized-distilled
sterile water. Under aseptic conditions, a small incision was
made in the proximal and distal end of the dorsal surface of
each recipient mouse and an air sac was formed. Then, in
each air sac, 2-cm segments of fetal intestine were inserted
along the midline and sutured in place at the cranial and
caudal ends. The skin was closed with clips, and the animals
were returned to their cages. Subsequently, mice were evaluated for the next 3 days and once weekly, thereafter. Earlier
experiments were carried out by using nude mice as the host.
However, we were able to improve the success rate of xenotransplantation from 45 to 75% when we used scid mice
without any difference in the development of the xenografts.
Only successful grafts were used for analysis. Animals were
killed subsequently and human xenografts were collected
and frozen for subsequent biochemical analysis and a section
of tissue was processed for histochemistry. Treatment with
glucocorticoids was carried out by a single subcutaneous
injection of cortisone acetate (CA) suspension at a dose of 50
mg/100 g body wt.
Disaccharidase activities. The region of the small intestine
from the stomach to the ligament of treitz was defined as the
duodenum, the proximal half of the reminder of the gut was
defined as jejunum, and the distal half as ileum for subsequent analyses. The lumen of each section was rinsed with
sterile DMEM before freezing for subsequent analysis. Various regions of both human gut and xenograft were used to
determine the level of sucrase, lactase and trehalase as
described previously (29, 30). Tissues were excised and
flushed with ice-cold saline and homogenized in 9 volumes of
0.15 M KCl by using a Potter-Elvehjem Teflon-glass homogenizer. The tissue homogenate was then assayed in duplicate
by using ultrapure sucrose, lactose, and trehalose as substrates. The amount of glucose liberated by these disaccharidases was quantitated by using a glucose-oxidase method
(Trinder 100 kit from Sigma) as described previously (29, 30).
Protein concentrations were measured by using a BCL kit
(Pierce) against BSA standards according to the manufac-
AJP-Regul Integr Comp Physiol • VOL
285 • JULY 2003 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
stration of disaccharidase expression in the xenograft
model (36) has provided an opportunity to test this
hypothesis.
In rodents, both endogenous and exogenous glucocorticoids are capable of precociously inducing the second
phase of functional development during the first two
postnatal weeks (9, 11, 12, 14, 15, 29). If the second and
third trimesters of the fetal human intestine correspond to the suckling period of the rodent gut, then the
human gut that corresponds to this period should undergo precocious maturation after exposure to exogenous steroids. With the use of lactase as a marker,
sensitivity to glucocorticoids had been demonstrated in
short-term organ cultures of 12- to 14-wk-old human
fetal small intestine (2, 40). However, the absence of a
suitable in vivo model system for a dynamic study of
the developing human intestine has precluded an understanding of normal development as well as the role
of glucocorticoids in functional development.
With the establishment of a xenograft model, fetal
human gut subcutaneously transplanted into immunocompromised mice has the capability of regenerating
and surviving in these hosts for extended periods of
time (36, 37, 48). However, there has been a lack of
systematic evaluation of xenografts as a function of
time posttransplant to determine whether this model
could be used to study the ontogeny of the human
intestine in the later stages of gestation. In this report,
we have begun to evaluate the potential role of xenografts in recapitulating in utero development and the
glucocorticoid effect in modulating the ontogeny of the
human intestine by using disaccharidases as developmental markers.
R163
R164
ONTOGENY OF THE HUMAN GUT XENOGRAFT
RESULTS
Sucrase, lactase, and trehalase activities in the 20wk-old human fetal gut. Various regions of the gut
were separated into duodenum, proximal and distal
jejunum, and proximal and distal ileum, flushed with
ice cold saline and stored at ⫺20°C until assayed. The
level of sucrase, lactase, and trehalase activity was
determined as described above (Fig. 1). Sucrase activity showed a gradient of expression with the lowest
levels in the duodenum and distal ileum and the highest levels of activity in the distal jejunum, whereas
trehalase activity showed a decreasing gradient along
the length of the small intestine. The distribution of
Fig. 1. The gradient of sucrase (A), trehalase (B), and lactase (C)
activities along the length of prexenotransplanted 20-wk-old fetal
small intestine. Tissue was divided into duodenum (Du), proximal
jejunum (PJ), distal jejunum (DJ), proximal ileum (PI), and distal
ileum (DI) before being assayed. Enzyme activity was expressed as
micromoles per hour per milligram protein. Results are given as the
means ⫾ SE. Six fetal tissues were used to obtain each data point. An
absence of error bars indicates that the SE is smaller than the
symbol.
both sucrase and trehalase activities along the anteriorposterior axis of the small intestine was similar to that
described for rats and mice (9, 11, 19, 21). The distribution of trehalase in humans has not previously been
described but appears to be similar to that of the
rodent (14, 30). Overall lactase activity was very low
throughout the small intestine, barely being detectable
in the duodenum and uniformly expressed at the lower
levels in the remaining regions of small intestine. As
AJP-Regul Integr Comp Physiol • VOL
285 • JULY 2003 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
turer’s protocol. Enzyme activity was expressed as micromoles of substrate hydrolyzed per hour per milligram of total
protein.
Processing of tissue for immunoprecipitation. The xenograft and biopsy samples were biosynthetically labeled as
previously described (27). Tissues were labeled continuously
with 100 ␮Ci of [35S]methionine EXPRE35S 35S Protein labeling mix (⬎1,000 Ci/mmol; New England Nuclear Life
Sciences, Boston, MA) for 4 h, after incubating in methionine-free media for 2 h in organ tissue culture dishes (Becton Dickinson, Lincoln Park, NJ) and in a tissue culture
incubator as described previously (28). After the labeling
period, specimens were washed three times in media with the
labeling mix before being homogenized at 4°C in 250 ␮l of
extraction buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1%
NP-40 with protease inhibitor cocktail [Sigma, St. Louis,
MO]). The tissue homogenate was then cleared of debris by
centrifugation at 1,000 g for 30 min at 4°C and the supernatant was processed for immunoprecipitation as described
previously (27). Briefly, 100 ␮g of protein were precleared
twice with 25 ␮l of protein G-Sepharose beads before being
incubated with antibody and then protein G-Sepharose complex for 1 h, respectively, at 4°C with shaking. The beads
were then spun down in the cold room and washed three
times at 4°C before being denatured in a SDS-sample buffer
and fractionated in SDS-PAGE gels. The gels were then
incubated in Autofluor (National Diagnostics, Atlanta GA)
for 45 min at room temperature before being dried and
exposed to Kodak X-Omat films. The specific antibodies for
lactase (HBB 4/14), sucrase (HIS 4/34/4/1), CDX-1 (CPSP)
and CDX-2 (CNL) were generous gifts of Drs. Buford Nichols
(Houston, TX) and Debra Silberg (Philadelphia, PA) (35, 39).
Antibodies for GAPDH were obtained from RDI Research
Diagnosis (Flanders, NJ) and all other reagents, including
secondary antibodies were obtained either from Santa Cruz
Biotech (Santa Cruz, CA) or Jackson ImmunoResearch (West
Grove, PA).
Histology. Specimens were fixed in 4% paraformaldehyde
for 4 h at 4°C and then overnight in PBS containing 40%
sucrose at 4°C before being embedded either in paraffin or in
Tissue Tek optimum cutting temperature (O.C.T.) compound
for frozen sections. The paraffin and frozen sections were cut
at 5- and 7-␮m increments, respectively, for staining as
described previously (28).
Statistics. Results are presented as means ⫾ SE. Effects of
age and treatment on enzyme activities were analyzed by a
two-way analysis of variance. After the overall significance
was confirmed, post hoc tests for individual variables were
performed by a two-tailed unpaired t-test. Differences with a
P value ⬍0.05 were considered significant.
ONTOGENY OF THE HUMAN GUT XENOGRAFT
within paneth cells can be clearly seen, demonstrating
the differentiation of proliferative cells migrating toward the crypt base.
To determine the functional development of these
tissues, after taking organ culture samples for metabolic labeling, we homogenized and assayed the remaining tissues for sucrase, lactase, and trehalase
activities. These data from jejunal xenografts are
shown in Fig. 3. Nine weeks posttransplantation, sucrase, trehalase, and lactase activities in intestinal
xenografts were not significantly different from that of
the 20-wk-old (Fig. 1) fetus before xenotransplantation
(P ⬎ 0.3). In addition, the level of sucrase activity was
not significantly different between 9 and 24 wk (P ⬎
0.45), reflecting the normal, predicted ontogeny of this
enzyme in the jejunum. Trehalase activity in xenograft
tissues reflects a developmental profile similar to sucrase. Between 9 and 24 wk, trehalase activity in the
jejunal xenograft was not significantly different from
the 20-wk-old (Fig. 3) prexenograft fetal jejunum (P ⬎
0.3). In contrast, lactase activity was expressed at very
low levels by 9 wk and was comparable to the prexenograft fetal jejunum. However, lactase activity
gradually increased between weeks 9 and 24 posttransplantation (P ⬍ 0.03) reaching the level of the newborn
and thus appears to recapitulate the predicted ontogeny for the human small intestine in utero.
Fig. 2. Morphological development of human fetal jejunum xenografted subcutaneously into a nude (nu/nu)
mouse after a 6-wk and 6-mo period. A: early development of the xenograft in which undifferentiated cells
are proliferating and covering the lumen. The tissue
lacks a complex villus and crypt structure. Cells also
lack brush-border and goblet cells indicating an undifferentiated state. B, C, and D: spatial compartmentalization of proliferative crypts (C) and differentiated
villus (V) epithelium with goblet cells (*) in both compartments separating the lumen (L) from the mucosa,
the appearance of brush-border (arrow) and paneth
cells (P) containing multiple granules. (A–D: ⫻63; toluidine blue).
AJP-Regul Integr Comp Physiol • VOL
285 • JULY 2003 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
reported previously (11, 21), a gradient of lactase activity had not been established by 20 wk of gestation.
Development of the fetal jejunum as a xenograft.
Twenty-week-old fetal jejunal sections were xenotransplanted into immunocompromised mice and followed
for 24 wk. Successful grafts revascularized and developed into small intestine. A minimum of six xenografts
was harvested at 9, 19, 22, and 24 wk after transplantation. Only those tissues that showed an intact gut
morphology were used for further biochemical analysis.
A representative morphological section of xenograft
is shown in Fig. 2. The jejunal xenograft after 6 (Fig.
2A) and 24 wk (Fig. 2, B–D) was fixed, sectioned, and
stained with toluidine blue. Figure 2A shows a developing xenograft 6 wk after implantation. The lumen is
covered with an undifferentiated epithelial monolayer,
without crypt-villus orientation. Figure 2, B and C
clearly depicts the xenograft 6 mo after transplantation with a well developed crypt-villus architecture. A
clearly differentiated epithelium with enterocytes, endocrine, and goblet cells can be seen in the villus. A
well-differentiated glycocalyx can also be seen on the
apical surface of enterocytes. The frequency of the
goblet cells is much higher than in normal tissue and
as expected, they can be seen in both the villus and
crypt compartments. Figure 2D is a representative
section through the base of a crypt, where granules
R165
R166
ONTOGENY OF THE HUMAN GUT XENOGRAFT
tase expression increases in 14- to 22-wk-old xenografts, but the levels of sucrase and CDX-1 and -2
remained unchanged. The level of sucrase and lactase
protein follows the profile of enzyme activity and suggests that the development of the jejunal xenograft
follows a predicted ontogeny even in the absence of
luminal stimuli. Tissue-specific transcription factors,
CDX-1 and -2, appear unlikely to play a role in these
developmental changes in human intestinal xenografts.
The effects of cortisone acetate on jejunal xenografts.
At 20 wk posttransplantation, half of the animals with
jejunal xenografts were either injected with a CA suspension or a saline vehicle. Seven days after treatment,
xenografts were harvested and processed for histology
and biochemical analysis, as described previously. The
data for sucrase, trehalase, and lactase activities from
jejunal xenografts are shown in Fig. 5. In a manner
similar to that of 20-wk-old fetal intestine (Fig. 3),
saline-treated tissue expressed high sucrase and trehalase activities but low lactase activity. Treatment
with CA had no significant effect on sucrase (P ⬎ 0.47)
Fig. 3. The development of sucrase (A), trehalase (B), and lactase (C)
activities in jejunal xenografts. Jejunum was assayed before xenotransplantation (Age: 0) and then subsequently harvested at 9, 19,
and 24 wk posttransplantation. Enzyme activities are expressed as
micromoles per hour per milligram protein. Results are reported as
means ⫾ SE. The number of xenografts for each data point are from
6 specimens from 5 different experiments.
These enzyme activities for sucrase and lactase were
confirmed by immunoprecipitation after the tissue was
labeled with [35S]methionine (Fig. 4). The lactase protein was absent in 12-wk-old xenograft, but present at
low levels in 20 wk and was comparable to the enzyme
levels found in 24-wk-old xenografts. However, the
level of sucrase protein was unchanged at all stages
(Fig. 4A). Because CDX-1 and -2 are two unique transcription factors expressed only in the gut and have
been implicated in the expression of these disaccharidases, the level of these proteins along with lactase and
sucrase was determined (Fig. 4A). Again, the low lac-
Fig. 5. Effect of cortisone acetate (CA) on the developing intestinal
xenograft. Twenty-week posttransplanted jejunal xenografts were
injected either with a single injection of CA at 50 mg/100 g body wt
or a identical volume of saline vehicle (VEH). Seven days after
treatment xenografts were harvested and assayed for sucrase, trehalase, and lactase activities. Enzyme activities were expressed as
micromoles per hour per milligram protein. Results are reported as
means ⫾ SE. Six xenografts were used to obtain each data point.
AJP-Regul Integr Comp Physiol • VOL
285 • JULY 2003 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
Fig. 4. Developmental expression of sucrase (Suc), lactase (Lac), and
GAPDH protein in human xenografts by immunoprecipitation. A:
ontogeny of lactase and sucrase in 12-, 20-, and 24-wk-old human
xenografts was compared with an infant biopsy (Bp) after metabolic
labeling studies. B: expression of CDX-1 and CDX-2 was determined
along with lactase and sucrase in 14- and 22-wk-old human xenografts. Unlike lactase, the levels of sucrase, CDX-1, and CDX-2
protein were unchanged. Results of preliminary experiment with
reproducible results are shown.
R167
ONTOGENY OF THE HUMAN GUT XENOGRAFT
Table 1. Specific activity of disaccharidases from
biopsies and mature xenografts of proximal
small intestine
Sucrase
Lactase
Trehalase
Biopsy
Xenograft
4.82 ⫾ 0.56
2.21 ⫾ 0.48
4.62 ⫾ 0.39
5.32 ⫾ 0.35
2.41 ⫾ 0.23
2.6 ⫾ 0.19
Values are means ⫾ SE of ␮mol 䡠 h⫺1 䡠 mg protein⫺1.
DISCUSSION
In utero development, especially during the second
and third trimester of gestation in the human small
intestine is largely unknown because of the lack of
available tissue and the lack of an appropriate model
system that can recapitulate the ontogeny. In rats and
mice, the in utero development lasts only 21 days and
thus the pups are born just after the gut has undergone
morphological development. This stage in rodents cor-
Fig. 6. The biphasic model for the functional development of the human small
intestine in utero (A) compared with the
actual development in the xenograft
model (B). Arrows indicate the expected
time at birth. Ontogeny of sucrase and
lactase activity was recapitulated by the
xenograft according to the theoretical
model.
AJP-Regul Integr Comp Physiol • VOL
285 • JULY 2003 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
and trehalase (P ⬎ 0.7) activities in jejunal xenografts.
However, CA treatment significantly increased lactase
activity (P ⬍ 0.05).
Disaccharidases levels in intestinal biopsies from infants. To evaluate the level of sucrase, lactase, and
trehalase activities from the infant small intestine,
duodenal biopsies were assayed. As diagnostic biopsies
were obtained, additional tissue were collected from
the small intestine with informed consent from parents
according to the recommended procedure of the Human
Studies Committee at the Massachusetts General Hospital. Table 1 shows the enzyme activity in 2- to 6-moold proximal intestine from infants and the level of
activity from 24-wk posttransplanted jejunal xenografts. Except for trehalase activity, all other enzyme
activities were comparable to xenografts at 24 wk posttransplantation. The overall results again suggest that
by 24 wk posttransplantation, xenograft tissue had
attained developmental levels that correspond to those
of young infants. The discrepancy in trehalase activity
could be due to the location of biopsy tissue. The proximal biopsy tissues were obtained before the ligament
of treitz (duodenum), whereas tissues from jejunal
xenografts were used as 20-wk-old fetal intestinal
tissue.
responds to the 12th wk of human development. For
the first time, in this report, we provide preliminary
evidence that human xenografts may be used as a
simulated in vivo model for the developing human
intestine. Using immunocompromised mice as the host
for xenotransplantation of 20-wk-old fetal human gut,
we have begun to systematically characterize the maturation of jejunal xenografts by using disaccharidases,
a classic developmental marker. In this study, development of the xenograft has been shown to recapitulate
the predicted in utero development of the human small
intestine (9, 11, 14, 21, 22) (Fig. 6). Unlike the shortterm organ culture model (2, 23, 24, 40), xenografts
provide an attractive long-term approach to delineating complex physiological and genetic signals that may
regulate the development of the human intestine. Furthermore, this technique may also provide an appropriate model to study the effect that luminal components, such as amniotic fluid, nutrients, and microbes,
may have on the developing human gut.
In these studies, we have used 20-wk-old fetal tissues for xenotransplantation, whereas other studies
(36, 37, 48) have used 10- to 14-wk-old fetal tissue.
Thus it was not clear whether 20-wk-old tissues were
capable of regenerating as a xenograft. The amount of
tissue and availability is the primary reason for choosing 20-wk-old fetal gut. Moreover, previous studies
used fragmented intestine in which the specific region
of the transplanted gut could not be identified, whereas
by using 20-wk-old tissue, we could transplant sections
of duodenum, jejunum, and ileum separately. Because
sterile fetal gut from the intact fetus at 20 wk could be
obtained, there is much less chance of infecting the
vulnerable scid mouse host. However, in both cases the
transplanted tissue undergoes initial degeneration followed by angiogenesis and reepithelialization of the
luminal surface of the xenograft. When the xenograft
reepithelializes, it does so naturally as pseudostratified epithelium resembling the gut before morphological development (that occurs before 10 wk) of fetal life
(data not shown). By 6 wk postxenotransplantation,
the epithelium of the xenograft becomes a monolayer
without any sign of cytodifferentiation (Fig. 2A). After
these changes, crypts and villi are formed with proliferative and differentiated cells compartmentalized at
these sites, respectively. Thus regardless of the age of
R168
ONTOGENY OF THE HUMAN GUT XENOGRAFT
this approach Fig. 2, B–D, shows fully developed jejunal xenografts 6 mo posttransplantation with a differentiated epithelium displaying all four major cellular
lineages including the presence of enterocytes, goblet
cells, and endocrine cells in the villi and paneth cells in
the crypt base suggesting the establishment of bidirectional epithelial differentiation. This report is consistent with previous studies (36, 37, 48) despite using
older tissue for xenotransplantation suggesting that
20-wk-old human fetal gut possesses the same regenerative capacity as the younger tissues. These data on
the ontogeny of sucrase and lactase activities in the
xenograft suggest that the fetal human intestine reestablished its ontogenic identity after xenotransplantation and then proceeded to mature according to its
predisposed genetic program. Because intact gut of the
20-wk-old fetus can be obtained, the xenograft could
potentially provide a model to study the development
as well as the pathophysiology of disease in the preterm infant gut.
Both endogenous and exogenous glucocorticoids are
capable of accelerating the second stage of functional
development in the rodent small intestine (4, 5, 7). Due
to the lack of a model system, the long-term effect of
glucocorticoids in human gut development is not
known. However, by using 12- to 14-wk-old fetal tissue
for 5 days in organ culture, it has been shown that
glucocorticoid is capable of stimulating an increase in
lactase activity but not sucrase or trehalase activities
(2, 40). A similar effect with EGF was observed for
lactase in this same organ culture system (22, 23). The
mean level of serum cortisol decreases from 8.4 to 4
ng/ml from the 16th to the 20th week of gestation, but
gradually increases ⱕ20 ng/ml by 35–36 wk and then
rapidly reaches 45 ng/ml during the last 4 wk before
birth (26). This surge in circulating cortisol in the
human fetus precedes a rapid increase of lactase activity in the developing fetal small intestine (11, 22).
These changes may explain the effectiveness of cortisone in premature infants and suggests an important
protective role for this hormone for the developing gut
(3, 6, 7, 13, 41). The increasing circulating levels of
cortisol during fetal development complements the biphasic model of functional maturation of the small
intestine (Fig. 6) during the third trimester. Only 25 or
50 ng/ml but not 12.5 ng/ml of hydrocortisone is able to
induce lactase activity in the organ culture system by
using 12- to 14-wk-old fetal human intestine (2). These
data are consistent with the levels of circulating cortisol during the late second and early third trimester and
capable of precociously inducing lactase activity only
when the level was elevated beyond 20 ng/ml to between 25 and 50 ng/ml. The studies presented here also
suggest that the xenograft model can be used as an in
vivo model system for the developing human intestine
and exogenous glucocorticoids are capable of accelerating the maturation of the immature gut. The induction
of lactase activity is reflected at the level of its mRNA
in organ culture (46). Our preliminary data from the
intestinal xenograft also suggest that the induction of
lactase activity by glucocorticoids is reflected at the
AJP-Regul Integr Comp Physiol • VOL
285 • JULY 2003 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
the fetal tissue used for xenotransplantation, on revascularization, the epithelium regenerates with an appropriate spatiotemporal organization of crypt-villus
units.
Week 12 of human in utero development marks the
completion of morphological development resulting in
a monolayer of epithelial cells. In addition, proliferative and differentiated cells are compartmentalized
into crypts and villi, respectively. These major developmental events are accompanied by the expression of
digestive enzymes on the brush border of villus enterocytes. This first step in functional development is characterized by the induction of sucrase in humans and
lactase in rodents. The second and final stage of functional maturation is characterized by the induction of
lactase in the human at the end of the third trimester
(Fig. 6) and by sucrase in rodents during the third
postnatal week (9, 14, 29, 38). When tissue was collected from the fetus, the anterior-posterior (cephalocaudal) distribution of disaccharidases was determined. Twenty-week-old duodenal tissue was separated and the remaining jejunum and ileum were
further subdivided into anterior and posterior regions.
Specific disaccharidase activities were then determined. At 20 wk, the level of sucrase in human small
intestine had reached almost the level of the newborn
(Table 1), whereas, lactase, as reported previously (11,
14, 21), remained at a very low level. Figure 1 illustrates the anterior-posterior gradient of sucrase activity and by 20 wk, it has reached the distribution characteristics of the adult (21). Sucrase activity was low at
either end of the small bowel with maximal activity
being detected in the middle region of the small intestine. Lactase activity remained at a basal level
throughout the entire small intestine, as reported previously (11, 21).
Development and distribution of human trehalase
activity in the small intestine heretofore has not been
reported. Recently trehalose has been used as a nontoxic cryoprotectant of enzymes, membranes, vaccines,
animal and plant cells, and organs for surgical transplant (33). It has been predicted that trehalose can also
be used as an ingredient for dried and processed food.
With these recent biotechnological applications for the
use of trehalose (33), it becomes important to determine the ontogeny of trehalase activity. In rodents, the
induction of trehalase parallels sucrase during the
third postnatal week (9, 30, 32). In this study, the
ontogeny of trehalase activity, like that of sucrase, was
accomplished during the first phase of functional maturation of the human gut. The activity was maintained
at that level in the newborn that is sufficient to prevent
diarrhea if overly exposed to trehalose after birth.
These data suggest that trehalose and foods such as
mushrooms containing trehalose can be used as a preservative or as a nutrient in infant food without producing a harmful effect. Only the infant with primary
trehalose intolerance would not be able to handle this
sugar.
Fetal human xenografts also recapitulate the complex heterogeneity of the epithelium. With the use of
ONTOGENY OF THE HUMAN GUT XENOGRAFT
ically adding these trophic factors to a defined media
and infusing it into the lumen of xenografts, the role of
luminal signals in the development of the small intestine can be studied. It is well established that glucocorticoids play a pivotal role in gut development in other
systems (42, 43). Thus the xenograft model could be
useful in determining the role of steroids and other
hormones or trophic factors in the development of the
human small intestine in the prevention of age-related
diseases.
We thank the members of the Developmental Gastroenterology
Laboratory for their input and encouragement, Dr. Hassan Naim for
suggesting the labeling studies, and Patrizia Psendorfer, Ji Chen
and Lei Lu for technical assistance. We also thank Drs. Tim Buie and
Esther Israel for their critical help in acquiring biopsies from infants
and children. Antibodies from Drs. Buford Nichols, Eun Ran Suh,
and Debra Silberg are greatly appreciated.
This work was supported by National Institute of Child Health
and Human Development Grants R37-HD-12437 and RO1-HD31852 and National Institute of Diabetes and Digestive and Kidney
Diseases Grants PO1-DK-33506, P30-DK-043351, and P30-DK40561. Corrie E. Klopcic was partially supported by a summer
student fellowship from the American Gastroenterological Association.
Corrie E. Klopcic is presently a medical student at the University
of Wisconsin at Madison.
REFERENCES
1. American Physiological Society. Guiding principles for research involving animals and human beings. Am J Physiol Regul
Integr Comp Physiol 283: R281–R283, 2002.
2. Arsenault P and Menard D. Influence of hydrocortisone on
human fetal small intestine in organ culture. J Pediatr Gastroenterol Nutr 4: 893–901, 1985.
3. Bauer CR, Morrison JC, Poole WK, Korones SB, Boehm
JJ, Rigatto H, and Zachman RD. A decreased incidence of
necrotizing enterocolitis after prenatal glucocorticoid therapy.
Pediatrics 73: 682–688, 1984.
4. Buddington RK and Diamond JM. Ontogenetic development
of intestinal nutrient transporters. Annu Rev Physiol 51: 601–
19, 1989.
5. Carver JD and Barness LA. Trophic factors for the gastrointestinal tract. Clin Perinatol 23: 265–285, 1996.
6. Crowley P. Corticosteroids after preterm premature rupture of
membranes. Obstet Gynecol Clin North Am 19: 317–326, 1992.
7. Crowley P. Antenatal corticosteroid therapy: a meta-analysis of
the randomized trials, 1972 to 1994. Am J Obstet Gynecol 173:
322–335, 1995.
8. Fang R, Santiago NA, Olds LC, and Sibley E. The homeodomain protein Cdx2 regulates lactase gene promoter activity
during enterocyte differentiation. Gastroenterology 118: 115–
127, 2000.
9. Galand G. Brush border membrane sucrase-isomaltase,
maltase-glucoamylase and trehalase in mammals. Comparative
development, effects of glucocorticoids, molecular mechanisms,
and phylogenetic implications. Comp Biochem Physiol B 94:
1–11, 1989.
10. Gordon JI and Hermiston ML. Differentiation and self-renewal in the mouse gastrointestinal epithelium. Curr Opin Cell
Biol 6: 795–803, 1994.
11. Grand RJ, Watkins JB, and Torti FM. Development of the
human gastrointestinal tract. A review. Gastroenterology 70:
790–810, 1976.
12. Haffen K, Kedinger M, and Simon-Assmann P. Mesenchyme-dependent differentiation of epithelial progenitor cells in
the gut. J Pediatr Gastroenterol Nutr 6: 14–23, 1987.
13. Halac E, Halac J, Behue EF, Casanas JM, Indiveri DR, and
Petit JF. Prenatal and postnatal corticosteroid therapy to prevent neonatal necrostizing entercolitis: a controlled trial. J Pediatr 117: 132–138, 1990.
AJP-Regul Integr Comp Physiol • VOL
285 • JULY 2003 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
level of lactase mRNA (Nanthakumar and Walker,
unpublished observation). However, for a comprehensive understanding the functional development of the
fetal human intestine, we must include identification
of other regional and developmental-specific markers
in the xenograft model and compare their expression to
that of the biopsies of the infant intestine. Our preliminary studies fail to support a role of tissue-specific
transcription factors CDX-1 and -2 in these developmental changes although they have been implicated in
the expression of both sucrase and lactase gene expression (8, 20, 22, 44). However, recent studies suggest
that the combination of these factors along with other
transcription factors may be more significant in the
development of sucrase and lactase (8, 20, 44). Those
studies are underway to further validate the suitability
of the xenograft as an appropriate model for the developing human gut.
Another important aspect of the glucocorticoid effect
on the developing small intestine is that the steroid is
effective during a brief period. The rat and mouse
small intestine responds to glucocorticoids from the
time of birth until postnatal day 16 (29). If there is a
period of glucocorticoid sensitivity present in the developing human intestine, then glucocorticoids should
be effective only during that period. To support this, a
meta-analysis of randomized controlled trials of corticosteroid treatment to prevent respiratory distress
syndrome in preterm infants (6, 7) suggests that the
effectiveness of this steroid is lost by 34 wk of gestation, suggesting the existence of a glucocorticoid sensitive period in the developing human and presumably
the human intestine during the third trimester. This is
further suggested in studies using corticosteroids to
prevent necrotizing enterocolitis, an inflammatory condition in preterm infants. In these studies, protection
was considerably greater in mothers given corticosteroids in utero earlier than infants treated postnataly
(3, 5, 7). With the use of lactase as marker, studies are
presently underway to determine whether the intestinal xenograft exhibits a restrictive period of glucocorticoid sensitivity.
With the use of the xenograft model system, it may
be feasible to identify other markers of the gut that
display developmental profiles similar to lactase. Furthermore, it will be important to determine the development of other genes responsible for intestinal functions such as the mucosal immune system. Understanding the ontogeny of the epithelium with regard to
the mucosal immune system will also be important in
delineating the pathophysiology of intestinal infectious
diseases in premature infants, such as necrotizing enterocolitis (3) and toxigernic diarrhea (16). Because
xenografts form a lumen, it could be an appropriate
model to assess the role of luminal signals and/or
factors in its ontogeny. During the second and third
trimester, the luminal surface is bathed in amniotic
fluid that is significantly enriched in various trophic
factors (22, 47). In addition, colostrum and mature
breast milk contain similar trophic factors (5) and can
interact with the premature gut surface. By systemat-
R169
R170
ONTOGENY OF THE HUMAN GUT XENOGRAFT
31. OⴕConnor TP and Diamond J. Ontogeny of intestinal safety
factors: lactase capacities and lactose loads. Am J Physiol Regul
Integr Comp Physiol 276: R753–R765, 1999.
32. Oesterreicher TJ, Nanthakumar NN, Winston JH, and
Henning SJ. Rat trehalase: cDNA cloning and mRNA expression in adult rat tissues and during intestinal ontogeny. Am J
Physiol Regul Integr Comp Physiol 274: R1220–R1227, 1998.
33. Paiva CL and Panek AD. Biotechnological applications of the
disaccharide trehalose. Biotechnol Annu Rev 2: 293–314, 1996.
34. Quaroni A and Isselbacher KJ. Study of intestinal cell differentiation with monoclonal antibodies to intestinal cell surface
components. Dev Biol 111: 267–279, 1985.
35. Quaroni A, Nichols BL, Quaroni K, Hurst L, Herrera M,
Weiser M, and Hamilton SR. Expression and different polarity of aminopeptidase N in normal human colonic mucosa and
colonic tumors. Int J Cancer 51: 404–411, 1992.
36. Savidge TC, Lowe DC, and Walker WA. Developmental regulation of intestinal hydrolase activity in human fetal jejunal
xenografts maintained in severe-combined immunodeficient
mice. Pediatr Res 50: 196–202, 2001.
37. Savidge TC, Morey AI, Ferguson DJP, Fleming KA, Shmakov AN, and Phillips AD. Human intestinal development in a
severe-combined immunodeficient xenograft model. Differentiation 58: 361–371, 1995.
38. Semenza G. Anchoring and biosynthesis of stalked brush border membrane proteins: glycosydase and peptidase of enterocytes and renal tubuli. Annu Rev Cell Biol 2: 255–313, 1986.
39. Silberg DG, Swain GP, Suh ER, and Traber PG. Cdx1 and
cdx2 expression during intestinal development. Gastroenterology
119: 961–971, 2000.
40. Simon-Assmann P, Lacroix B, Kedinger M, and Haffen K.
Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells. Early Hum Dev 13: 65–74,
1986.
41. Sinclair JC. Meta-analysis of randomized controlled trials of
antenatal corticosteriod for the prevention of respiratory distress
syndrome: discussion. Am J Obstet Gynecol 173: 335–344, 1995.
42. Smith BT and Sabry K. Glucocorticoid-thyroid synergism in
lung maturation: a mechanism involving epithelial-mesenchymal interaction. Proc Natl Acad Sci USA 80: 1951–1954, 1983.
43. Spencer J and MacDonald T. Ontogeny of human mucosal
immunity. In: Ontogeny of the Immune System of the Gut, edited
by MacDonald T. Boca Raton, FL: CRC, 23–41, 1990.
44. Suh E, Chen L, Taylor J, and Traber PG. A homeodomain
protein related to caudal regulates intestine-specific gene transcription. Mol Cell Biol 14: 7340–7351, 1994.
45. Traber PG and Wu GD. Intestinal development and differentiation. In: Gastrointestinal Cancers: Biology, Diagnosis and
therapy, edited by Rustgi AK. Philadelphia, PA: LippincottRaven, 21–43, 1995.
46. Villa M, Menard D, Semenza G, and Mantei N. The expression of lactase enzymatic activity and mRNA in human fetal
jejunum. Effect of organ culture and of treatment of hydrocortisone. FEBS Lett 301: 202–206, 1992.
47. Weaver LT and Walker WA. Epidermal growth factor and the
developing human gut. Gastroenterology 94: 845–847, 1988.
48. Winter HS, Hendren RB, Fox CH, Russell GJ, PerezAtayde A, Bhan AK, and Folkman J. Human intestine matures as nude mouse xenograft. Gastroenterology 100: 89–98,
1992.
AJP-Regul Integr Comp Physiol • VOL
285 • JULY 2003 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
14. Henning SJ. Functional development of the gastrointestinal
tract. In: Physiology of the Gastrointestinal Tract (2nd ed.),
edited by Johnson LR. New York: Raven, 285–300, 1987.
15. Herbst JJ and Koldovsky O. Cell migration and cortisone
induction of sucrase activity in jejunum and ileum. Biochem J
126: 471–476, 1972.
16. Insoft RM, Sanderson IR, and Walker WA. Development of
immune function in the intestine and its role in neonatal diseases. Pediatr Clin North Am 43: 551–571, 1996.
17. Kedinger M, Simon-Assmann P, Lacroix B, Marxer A,
Hauri HP, and Haffen K. Fetal gut mesenchyme induces
differentiation of cultured intestinal endodermal and crypt cells.
Dev Biol 113: 474–483, 1986.
18. Klein RM and McKenzie JC. The role of cell renewal in the
ontogeny of the intestine. I. Regulation of cell proliferation in
adult, fetal, and neonatal intestine. J Pediatr Gastroenterol Nutr
2: 10–43, 1983.
19. Koldovsky O. Hormonal and dietary factors in the development
of digestion and absorption. In: Nutrition and Development,
edited by Winick M. New York: Wiley, 135–200, 1972.
20. Krasinski SD, Van Wering HM, Tannemaat MR, and
Grand RJ. Differential activation of intestinal gene promoters:
functional interactions between GATA-5 and HNF-1 alpha.
Am J Physiol Gastrointest Liver Physiol 281: G69–G84, 2001.
21. Lacroix B, Kedinger M, Simon-Assmann P, and Haffen K.
Early organogenesis of human small intestine: scanning electron
microscopy and brush border enzymology. Gut 25: 925–950,
1984.
22. Menard D. Development of human intestinal and gastric enzymes. Acta Paediatr, Suppl 405: 1–6, 1994.
23. Menard D, Arsenault P, and Pothier P. Biologic effects of
epidermal growth factor in human fetal jejunum. Gastroenterology 94: 656–663, 1988.
24. Menard D, Corriveau LL, and Arsenault P. Differential
effects of epidermal growth factor and hydrocortisone in human
fetal colon. J Pediatr Gastroenterol Nutr 10: 13–20, 1990.
25. Moog F, Denes AE, and Powell PM. Disaccharidases in the
small intestine of the mouse: normal development and influence
of cortisone, actinomycin D and cyclohexamide. Dev Biol 35:
143–159, 1973.
26. Murphy BEP. Human fetal serum cortisol levels related to
gestational age: evidence of midgestational fall and a steep late
gestational rise, independent of sex or mode of delivery. Am J
Obstet Gynecol 144: 276–282, 1982.
27. Naim HY, Sterchi EE, and Lentze MJ. Biosynthesis and
maturation of lactase-phlorizin hydrolase in the human small
intestinal epithelial cells. Biochem J 241: 427–434, 1987.
28. Nanthakumar N, Fusunyan RD, Sanderson IR, Walker
WA. Inflammation in the developing human intestine: a possible
pathophysiologic basis for necrotizing enterocolitis. Proc Natl
Acad Sci USA 97: 6043–6048, 2000.
29. Nanthakumar NN and Henning SJ. Ontogeny of sucraseisomaltase gene expression in rat intestine: responsiveness to
glucocorticoids. Am J Physiol Gastrointest Liver Physiol 264:
G306–G311, 1993.
30. Nanthakumar NN and Henning SJ. Distinguishing normal
and glucocorticoid induced maturation of intestine using bromodeoxyuridine. Am J Physiol Gastrointest Liver Physiol 268:
G139–G145, 1995.