Stimulated Endocrine Cell Proliferation and Differentiation in Transplanted Human Pancreatic Islets
Effects of the ob Gene and Compensatory Growth of the Implantation Organ
Björn Tyrberg,
Jarkko Ustinov,
Timo Otonkoski, and
Arne Andersson
From the Department of Medical Cell Biology (B.T., A.A.), Uppsala
University, Uppsala, Sweden; the Transplantation Laboratory (J.U., T.O.),
Haartman Institute; and the Hospital for Children and Adolescents (T.O.),
University of Helsinki, Helsinki, Finland.
Address correspondence and reprint requests to
Björn Tyrberg, UCSD Cancer Center, Burnham
Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. E-mail:
btyrberg{at}ucsd.edu
.
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ABSTRACT
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Neogenesis is crucial for the maintenance of ß-cell mass in the human
pancreas and possibly for the outcome of clinical islet transplantation. To
date, no studies have reported a stimulation of human ß-cell neogenesis
in vivo. Therefore, we investigated whether human
-, ß-, and duct
cell growth can be stimulated when human islets are xenotransplanted to obese
hyperglycemic-hyperinsulinemic ob/ob mice immuno-suppressed with
anti-lymphocyte serum. Moreover, we wanted to study whether ß-cell growth
and duct-to-ß-cell differentiation were induced in the hepatocyte growth
factor (HGF)-dependent compensatory kidney growth model. For that purpose, we
evaluated human islets grafted to nude (nu/nu) mice before
uninephrectomy of the contralateral kidney for DNA-synthesis and duct cell
expression of the ß-cell-specific transcription factor Nkx 6.1 as an
estimate of differentiation. Human islet grafts were well preserved after 2
weeks when transplanted to ob/ob mice during anti-lymphocyte
immunosuppression. Both human ß-cells (P < 0.01) and duct
cells (P < 0.001) were growth stimulated when islets were
transplanted to ob/ob mice. We also observed a correlation between
increased duct cell proliferation and increased organ donor age (P =
0.02). Moreover, duct (P < 0.05) and ß-cell (P <
0.05) proliferation, as well as duct cell Nkx 6.1 expression (P <
0.05), were enhanced by the compensatory kidney growth after uninephrectomy.
We conclude that it is possible to stimulate human ß-cell neogenesis in
vivo, provided that the recipient carries certain growth-stimulatory traits.
Furthermore, it seems that duct cell proliferation increases with increasing
organ donor age. Altogether, these data and previous results from our
laboratory suggest that human ß-cell neogenesis becomes more dependent on
differentiation and less dependent on proliferation with increasing age.
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INTRODUCTION
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Human ß-cell neogenesis (i.e., differentiation from precursor cells
and proliferation from pre-existing ß-cells) is crucial for the
maintenance of ß-cell mass in the native human pancreas and possibly for
the outcome of clinical islet transplantation. Due to the limited supply of
human islets for transplantation, any possibility to stimulate the growth
and/or the differentiation of ß-cells would be of interest. We and others
have previously reported that adult human ß-cells have a limited capacity
to proliferate both in vitro and in vivo
(1,2,3).
Because glucose was found to have a stimulatory effect on ß-cell
proliferation
(3,4),
investigators have extensively searched for other possible ß-cell
growth-stimulating agents
(5,6,7).
Also, there are a few animal models with extensive islet cell growth, and one
of these is the obese hyperglycemic-hyperinsulinemic ob/ob mouse,
which has an abundance of enlarged native islets
(8). The growth factors
involved in this model have not been characterized in detail, although it has
been suggested that glucose, insulin, glucagon-like peptide 1 (GLP-1), and
C-peptide play a role
(4,9,10).
We have previously observed that this islet enlargement is also induced when
islets isolated from lean mice have been transplanted to obese recipients
(11,12).
In the present study, we investigated whether human
-, ß-, and
duct cell growth could be further stimulated in vivo, when human islets were
transplanted to immunosuppressed obese hyperglycemic-hyperinsulinemic
ob/ob mice and followed for 2 weeks before killing and evaluating
DNA-synthesis.
Presently, the hepatocyte growth factor (HGF) is the most interesting
factor in the context of human ß-cell growth. In vitro, HGF has been
shown to effectively stimulate human fetal ß-cell proliferation
(13). It has also been
reported to stimulate the proliferation of adult human ß-cells
(2) or the islet-associated
ductal cells, which may represent a population of endocrine precursor cells
(13). Beattie et al.
(14) showed that HGF
stimulates human ß-cell proliferation, but the cells de-differentiate to
pancreatic duodenal homeobox gene 1 (Pdx-1)-positive cells not expressing
insulin. Such a de-differentiation of ß-cells has also been observed
during stimulation with other growth factors
(5,6)
and is, therefore, important to overcome. HGF also plays an important role in
organ regeneration after injury
(15,16).
Previously, we have shown that such compensatory growth processes during organ
regeneration stimulate pancreatic islet cell growth. Thus, the growth of mouse
islets transplanted to liver or kidney before partial hepatectomy or
uninephrectomy is considerably stimulated by the organ resection
(17). Because HGF seems to
primarily have de-differentiating effects on human ß-cells in vitro
(14), in contrast to actually
increasing the ß-cell number in HGF overexpressing mice
(18), we also studied whether
human ß-cell de-differentiation is overcome in vivo, in the HGF-dependent
(16) compensatory kidney
growth model. Therefore, we transplanted human islets to nude (nu/nu)
mice and performed uninephrectomy 2 weeks after transplantation. These mice
were killed 1-5 days thereafter, and the islet grafts were evaluated for DNA
synthesis and the presence of ß-cell precursors, i.e., cells positive for
both the ductal cell marker cytokeratin 19 and the ß-cell-specific
transcription factor Nkx 6.1
(19).
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RESEARCH DESIGN AND METHODS
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Islet isolation and culture. Human islets were isolated from 29
heart-beating organ donors (21 for the ob/ob study and 8 for the
nephrectomy study) at the Central Unit of the ß-cell Transplant,
Brussels, Belgium, and were transported by air to Uppsala, Sweden. The age of
the organ donors (mean ± SE) was 39 ± 3 years (range 8-59).
Islet isolation and characterization by light and electron microscopy
regarding cell viability (95 ± 0.6%) and cellular composition (52
± 2.2% insulin positive cells, 14 ± 1.7% glucagon positive
cells, 29 ± 2.6% nongranulated cells, i.e., predominantly duct cells,
and 1.1 ± 0.8% exocrine cells) were performed in Brussels as previously
described (20). On arrival in
Uppsala, islets were kept in culture in RPMI-1640 medium supplemented with 5.6
mmol/l glucose, 10% fetal calf serum, 0.17 mmol/l benzylpenicillin, and 0.17
mmol/l streptomycin (a favorable culture condition for human islets)
(20,21)
for 4-5 days before further experiments were performed.
Porcine islet-like cell clusters (ICC) were prepared by collagenase
digestion from pancreas anlage of 68- to 74-day pig fetuses (full term
115 days) as described in detail elsewhere
(22). After repeated washings,
the digest was resuspended and cultured in RPMI-1640 medium (11 mmol/l
glucose) supplemented with 10% heat-inactivated pooled human serum (The Blood
Center, Huddinge Hospital, Huddinge, Sweden), 10 mmol/l nicotinamide, 0.17
mmol/l benzylpenicillin, and 0.17 mmol/l streptomycin for 4-5 days before
transplantation.
Animals. Male lean (+/?) or obese ob/ob littermates of both
sexes (C57Bl/6 local colony; Biomedicum, Uppsala, Sweden) or male nude
nu/nu mice (C57Bl/6J; Bomholtgaard, Ry, Denmark) 3-6 months of age
were used as islet graft recipients. In the ob/ob mice and their
littermates, body weight, blood glucose, and serum insulin concentrations were
measured the day before transplantation and on the day of killing. Glucose
concentrations were analyzed by means of a glucometer (ExacTech; MediSense
Sverige, Gothenburg, Sweden) and insulin concentrations by means of a
radioimmunoassay (Pharmacia, Uppsala, Sweden).
Immunosuppression. To develop a suitable immunosuppressive regimen
for ob/ob mice, as well as their littermates, lean C57Bl/6J mice were
transplanted with porcine ICC or human islets as detailed below. In the
initial studies, porcine ICCs were used because of the limited supply of human
islets. Mouse anti-lymphocyte serum (MALS) (Accurate Chemical and Scientific,
Westbury, NY), 0.2 ml per animal, titrated on the basis of animal mortality,
was injected intraperitoneally the day before transplantation (day -1) and
then at different time points during the observation period
(Table 1). The grafts were
removed and processed for histology, and rejection was evaluated with a
semiquantitative ranking after hematoxylin-eosin staining, where 0 represents
total rejection with only connective tissue and mononuclear cells present, 1
represents fulminant rejection with massive mononuclear cell infiltration and
with very few endocrine or epithelial cells left, 2 represents some endocrine
or epithelial cells left with a clear mononuclear cell infiltration, 3
represents mainly an intact graft, but with some mononuclear cells present,
and 4 represents no sign of rejection.
Islet transplantation. A graft consisting of
0.6 µl human
islets or 1 µl porcine ICC was transplanted to the left renal subcapsular
space of lean or ob/ob mice as previously described
(23). Either 2 or 4 weeks
after islet transplantation, the recipients were injected intraperitoneally
with 1 µCi/g body wt (200 µl) 3H-thymidine (Amersham
Pharmacia Biotech, Uppsala, Sweden) and killed 2 h later by cervical
dislocation. The graft-bearing kidneys were dissected, fixed in formalin,
embedded in paraffin, and prepared for histology as previously described
(3). The nu/nu mice
received a human islet graft of 0.3-0.6 µl under the renal capsule of the
left kidney. Then, 2 weeks posttransplantation, these mice were anesthetized
again, and the right kidney was extirpated as described elsewhere
(17). Sham-operated controls
were anesthetized, opened, and the kidney was handled without being removed.
Between 1 and 5 days after the nephrectomy, the animals were injected with
3H-thymidine, killed 2 h later, and the remaining kidney was
prepared for histological evaluation as previously described. The
sham-operated controls were killed on either the same day after surgery as the
nephrectomized animals or on day 5.
Immunohistochemistry and autoradiography. Sections of grafts were
stained for insulin (antibovine insulin; ICN, Irvine, CA), glucagon
(antiporcine glucagon; NOVO, Copenhagen) or the human pancreatic duct cell
marker cytokeratin 19 (24)
(Dako, Glostrup, Denmark), the latter after pretreatment with 0.2% trypsin
(Sigma, St. Louis, MO) in 0.2% CaCl2 for 10 min. Antibody binding
was detected with the LSAB system (Dako) giving a red color precipitate at the
antigenic site. Wet slides were then dipped in 50% film-emulsion
(autoradiography emulsion; Kodak, New York) in 0.75 mol/l ammonium acetate and
stored in a light proof chamber to dry overnight. The films were then exposed
for 3 weeks at 4°C before being developed and fixed and counter-stained
with Mayer's hematoxylin. Sections were also stained for cytokeratin 19 in
combination with Nkx 6.1 (anti-mouse gst-Nkx 6.1; obtained from Dr. O.D.
Madsen, Gentofte, Denmark) after pretreatment with 0.1% pepsin (Sigma) in 0.1
mol/l HCl for 5 min and microwaving (500 W) for 5 min in 10 mmol/l citric acid
(pH 6.0). Cytokeratin 19 antibody binding was detected with a biotinylated
donkey anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA) and the
ABC method (Vectastain ABC-AP; Vector Laboratories, Burlingame, CA), which
gave a blue color precipitate (Blue Alkaline Phosphatase Substrate Kit III;
Vector) at the antigenic sites; Nkx 6.1 antibody binding was detected with a
biotinylated donkey anti-rabbit antibody (Jackson ImmunoResearch); the ABC
method (Vectastain ABC-HRP) and amino-ethylcarbazole (Sigma), which gave a
brownish-red color precipitate.
Microscopical evaluation. Insulin-, glucagon-, and cytokeratin
19-positive cells were counted in a light-microscope (400x) and cells
with
10 black-silver grains over the nuclei were considered to be in the
S-phase of the cell cycle
(25). There was a clear
difference between background thymidine incorporation (generally <3
grains/nucleus) and labeled cells (
10 grains/nucleus). During the
examinations, the observer was unaware of the sample identity. The fraction of
labeled cells at a certain time point was determined and expressed as a
labeling index (LI) (number of labeled cells x 100/total number of
cells). Because the S-phase of rat ß-cells is 6.4 h
(26), we believe that the 2 h
labeling is short enough to show only the fraction of cells that are entering
or have entered the S-phase at that moment. Nkx 6.1 and cytokeratin 19
double-positive cells were also counted in a light microscope, and state of
differentiation was expressed as percent of Nkx 6.1-positive duct cells of all
duct cells. To obtain accurate measurements of LI and state of
differentiation, 4,757 ± 326 ß-cells (n = 42), 2,788
± 375
-cells (n = 10), and 1,101 ± 147 duct
cells (n = 30) in each graft were counted for the LI measurements,
and 415 ± 59 (n = 22) duct cells were counted for the
measurements of state of differentiation (n reflects both the number
of donors and all of the experimental conditions combined).
Calculations. The cell birth rate (CBR), the production of new cells
per 24 h, was calculated as previously described
(26): CBR = LI x
24/st, where st is the duration of the S-phase, i.e.,
6.4 h. The cell-population doubling time (x), the time it would take
for a cell population to double without any reduction in the cell number by
cell death, was also calculated: P = (1 + CBR)x,
where P is the growth index, i.e., 2
(3).
Ethics. The local animal ethics committee approved all animal
experiments, and the Project Management Group of the ß-cell Transplant
(Brussels), approved the use of human islets for the present study.
Statistical analysis. Data are means ± SE. One human pancreas
donor was regarded as one observation, unless otherwise indicated. The paired
experiments were analyzed using Wilcoxon's signed-rank test. The coefficients
of correlations (R) were obtained using simple linear regression, and
the statistical significances of correlations were evaluated using analysis of
variance (ANOVA). Mouse characteristics were compared using ANOVA followed by
group comparisons using paired Student's t test, and the P
values were correlated for multiple comparisons using the Bonferroni method
(27).
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RESULTS
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Immunosuppression. To develop an immunosuppressive regimen for
successful islet xenotransplantation, we injected MALS intraperitoneally to
lean C57Bl/6 mice grafted with porcine ICCs or human islets. Mice receiving no
immunosuppression completely rejected the grafts
(Table 1). With just one
(Table 1) or a few (data not
shown) intraperitoneal injections of 0.2 ml during the observation period,
there were no signs of tolerance induction. However, a regimen with injections
every day from the day before to the day after transplantation and then every
second day resulted in an extensive graft preservation for 10 days of porcine
ICC grafts and for 14 days of human islet grafts
(Table 1 and
Fig. 1). With this regimen,
human islets were well preserved even up to 4 weeks after transplantation,
whereas the porcine ICC grafts were not so well preserved beyond 10 days. The
total mortality was low (11%) during the first 2 weeks, but increased to 30%
during the following 2 weeks. Based on duration of graft survival in
combination with recipient mortality data, we chose to use observation periods
of 2 weeks for the cell proliferation studies.

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FIG. 1. Micrograph of a discordant xenograft of human islets 14 days
posttransplantation to the renal subcapsular space of a C57Bl/6 mouse treated
with 0.2 ml MALS intraperitoneally on days -1, 0, 1, 3, 5, 7, 9, and 12
(original magnification x200).
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Transplantations to ob/ob mice. The ob/ob mice
were slightly hyperglycemic, severely hyperinsulinemic, and obese throughout
the observation period (Table 2).
During the 2-week observation period, there were no major changes in body
weight or glycemic or insulinemic status due to the MALS regimen or the
transplantation, although minor deviations in blood glucose (lean mice) and
serum insulin (ob/ob mice) were observed
(Table 2).
Both ß-cell and duct cell proliferation rates were considerably
increased in human islets transplanted to ob/ob mice when compared
with data from the lean recipients (ß-cell LI [n = 9-10] 0.10
± 0.02 lean and 0.17 ± 0.03 obese; duct cell LI [n = 6]
0.57 ± 0.17 lean and 1.25 ± 0.22 obese). On the other hand,
-cell proliferation was unaffected (
-cell LI [n = 5]
0.17 ± 0.04 lean and 0.16 ± 0.03 obese). When paired comparisons
were made between growth of cells grafted to obese and to lean recipients,
these observations were statistically confirmed
(Fig. 2). Moreover, duct cell
proliferation seemed to correlate to the age of the human islet donors, i.e.,
there was an increased proliferation with increased donor age (P =
0.02), whereas ß-cell proliferation tended to decrease with increased
donor age, but the difference was not statistically significant
(Fig. 3).

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FIG. 2. LI of human ß-, -, and duct cells 14 days after
transplantation to lean or obese ob/ob C57Bl/6 mice. LI expressed as
percent of control (lean mice) and compared with paired Wilcoxon's signed-rank
test.
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FIG. 3. Duct and ß-cell LI correlated to age of the human islet donors 2
weeks after transplantation to C57Bl/6 mice immunosuppressed with MALS
according to the regimen described in Table
1. The statistical significances of correlation were evaluated
with ANOVA ( with continuous line, duct cell LI versus age, R =
0.88, P = 0.02 and x with dashed line, ß-cell LI versus
age, R = 0.54, P = 0.1)
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Influence of nephrectomy on ß-cell neogenesis. In the
nu/nu mice, human ß-cell LI in the subcapsular islet grafts
increased 3 days after contralateral nephrectomy and remained elevated 2 days
later, when compared with values for the sham-operated controls
(Fig. 4). Duct cell LI
increased 1 day after nephrectomy, an increase that was further exaggerated 3
days after nephrectomy (Fig.
4). Differentiation of duct cells to cells expressing a
ß-cellspecific transcription factor was studied with double
immunostaining for the duct cell marker cytokeratin 19 and the ß-cell
transcription factor Nkx 6.1 (Fig.
5). It was found that the frequency of Nkx 6.1-positive duct cells
in the grafts was 2.2 ± 0.36% in sham-operated controls, 4.3 ±
1.2% 1 day after nephrectomy, 6.4 ± 1.4% (P < 0.05 vs.
sham) 3 days after nephrectomy, and 7.0 ± 1.6% (P < 0.05
vs. sham) 5 days after nephrectomy (n = 4-7).

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FIG. 4. LI of human ß-cells (x) and duct cells ( ) transplanted
to the renal subcapsular space of nu/nu mice. Contralateral
nephrectomy (uninephrectomy) was performed 2 weeks after transplantation, and
the mice were killed 1, 3, or 5 days thereafter. LI is expressed as percent of
sham-operated controls and compared with paired Wilcoxon's signed-rank test
(in the controls, ß-cells LI was 0.15 ± 0.03% and duct cell LI was
0.65 ± 0.10%; n = 3-8).
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FIG. 5. Part of a human islet graft 3 days after nephrectomy, with some cells
( ) stained for both cytokeratin 19 (blue) and Nkx 6.1 (red). Original
magnification x200.
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DISCUSSION
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Discordant xenotransplantation of human islets to C57Bl/6 mice requires
constant immunosuppression to achieve longterm survival of the grafted islets.
Because administration of cyclosporine and steroids has been demonstrated to
negatively affect islet cell function and proliferation
(4,28,29),
we wanted to use an immunosuppressive regimen without such side effects.
Previously, anti-lymphocyte serum has been successfully used in allogeneic
transplantation studies obtaining 2-week graft survival after one single
injection (30). In concordant
and discordant xenogeneic islet transplantation, multiple injections have been
used with varying results
(31,32).
In the present study, we used MALS with low recipient mortality and good human
islet graft preservation for at least 2 weeks. It is unlikely that the MALS
treatment alone affects islet cell proliferation, because the antibodies are
specific for lymphocytes and should not bind to other cell types. However, we
cannot rule out that secondary effects from the immunosuppression might affect
the islets in some way. Indeed, we observed minor changes in recipient blood
glucose and serum insulin concentrations during the 2-week observation period,
but this did not seem to antagonize the stimulation of islet cell
neogenesis.
In the present study, we have demonstrated that stimulation of human
ß-cell neogenesis can be achieved not only in vitro
(2,3,14),
but also in vivo. Thus, both human ß-cells and duct cells were growth
stimulated when islets were transplanted to ob/ob
hyperglycemic-hyperinsulinemic mice during MALS immunosuppression. Moreover,
duct and ß-cell proliferation, as well as duct cell differentiation, was
enhanced by the compensatory kidney growth after uninephrectomy. An increased
proliferation is expected to be accompanied by an increased volume of the cell
population studied, provided that cell death is presumed to be negligible. The
human islet graft volume 2 weeks after transplantation to ob/ob mice
was estimated to be 4% greater than in grafts residing in lean littermates.
Such calculations (and those performed below) were based on LI values from the
present study combined with previously published islet cell cycle data
(26) and formulas
(3,26).
It would not be possible to demonstrate this small increase with an estimated
bias of
10% in the original volume of the grafts at the day of
transplantation. A measurable volume increase might have been induced by 4
weeks. However, it was not possible to carry out such studies because of the
increased recipient mortality and the decreased immunosuppressive efficiency
at that time (previously described). Nephrectomy leads to a pulse of
growth-factor production and release for only a few days
(33). Again, the short
time-span of the growth stimulation probably induces only a minute increase
that is difficult to assess in islet volume. However, if persistent, an LI
increase of 100-500%, as seen in the obese or nephrectomized animals, should
lead to an impressive increase of the ß-cell mass. Thus, assuming
ß-cell growth could be constantly stimulated over time, the ß-cell
population doubling time (see RESEARCH DESIGN AND METHODS) would dramatically
decrease (Fig. 6).

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FIG. 6. Estimated ß-cell population doubling times (see RESEARCH DESIGN AND
METHODS). Average ß-cell LIs were used from human islets transplanted to
lean C57Bl/6 or sham-operated nu/nu mice (human control LI 0.12%),
ob/ob C57Bl/6 mice (human [ob] LI 0.17%), and nu/nu
mice 3 days after nephrectomy (human [nephrectomy {nx}] LI 0.53%). The
estimations were based on the presumption that these LIs were constant over
time.
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Concerning the underlying mechanisms of the presently demonstrated human
ß-cell growth stimulation, one might anticipate that the leptin
deficiency of the ob/ob mice
(34) might have beneficial
effects on islet cell growth. To test this hypothesis, we aimed to transplant
human islets to leptin receptordeficient db/db mice that
overexpress the functional leptin protein
(35,36).
However, the MALS regimen designed for the ob/ob mice was not
applicable in the db/db mice. Thus, the overall survival of the
recipients was markedly decreased, and the graft survival was markedly
attenuated. However, it is likely that the metabolic syndrome in the
ob/ob mice is responsible for the observed growth stimulation of
human ß- and duct cells. Besides the leptin deficiency, the combination
of high serum concentrations of glucose
(4) and insulin
(9), perhaps combined with
overproduction of proposed growth factors such as GLP-1 and C-peptide
(10), are likely to be of
importance. The ob/ob environment might also have affected the
differentiation state of the duct cells. However, due to the lack of
specimens, this was not possible to assess.
Shortly after an organ injury, a regeneratory process is initiated in which
both hyperplasia and hypertrophy of different cell types continue for several
days
(15,37).
Under such circumstances, HGF is of great importance, particularly in kidney
and liver regeneration
(15,16).
Thus, after injury (partial hepatectomy, uninephrectomy, or toxin-induced
injury), HGF is rapidly mobilized from the extracellular matrix of stromal and
epithelial cells
(38,39),
followed by 6-12 h post-nephrectomy upregulation of HGF mRNA transcription and
translation (40). The HGF
receptor (c-Met) is known to be expressed in various cells of epithelial
origin, among other pancreatic islets
(13) and duct cells
(41). The growth stimulatory
effects of HGF on both fetal and adult human islet cells have been scrutinized
in vitro
(2,13,14,42,43).
However, the present report is the first to suggest that a process involving
HGF upregulation (16) in vivo
is associated with a stimulation of human islet cell growth and
differentiation.
Nevertheless, it is worth noting that in the compensatory kidney growth
after nephrectomy, HGF is not the only growth factor involved. For instance,
insulin-like growth factors, vascular endothelial growth factors,
platelet-derived growth factors, neural growth factors, fibroblast growth
factors, and transforming growth factor
are also produced and released
locally by different cells in the kidney
(33). All of these may
stimulate ß- or duct cell proliferation and/or differentiation
(5,6,7,13,44).
Although the effects of some of these growth factors have only been studied in
ß-cell lines or fetal/neonatal islets, it is likely that this complex
picture of growth stimulants causes the accelerated adult human ß-cell
neogenesis observed after nephrectomy.
Interestingly, the relation between duct cell proliferation and the age of
the organ donors seems to be the reverse of that of ß-cells. In the
present report, human pancreatic duct cells showed an increased proliferative
capacity when the organ donor age increased, whereas human ß-cells
revealed a decreased capacity to proliferate when the organ donor age
increased. The latter could not be statistically confirmed in this study,
probably due to a small number of observations. However, in a previous study
we showed a clear correlation between decreased human ß-cell growth and
increased donor age (3).
Nevertheless, these observations might suggest that human ß-cell
neogenesis is becoming more dependent on differentiation and less dependent on
proliferation in older ages, as duct cell proliferation seems to parallel the
differentiation of duct cells to ß-cells
(45). The latter is confirmed
in the present study by an increased number of duct cells expressing the
ß-cellspecific transcription factor Nkx 6.1
(19) after nephrectomy. There
is a possibility that the observed increase in Nkx 6.1 staining could instead
reflect a de-differentiation process of the human ß-cells toward duct
cells, as suggested by Yuan et al.
(46). However, we could not
find any cytokeratin 19/Nkx 6.1-double positive cells scattered among the
ß-cells, which most likely would have been the case if the ß-cells
were de-differentiating. Instead, the ductal cells were consistently found in
duct-like clusters.
In summary, we have demonstrated that human pancreatic
-, ß-
and duct cells display signs of active proliferation in vivo when transplanted
to MALS immunosuppressed C57Bl/6 mice or nu/nu mice. Moreover, human
duct and ß-cell proliferation is enhanced after transplantation to
ob/ob mice and when these cells are situated in the remaining kidney
of nu/nu mice undergoing contralateral nephrectomy. Finally, human
duct to ß-cell differentiation is also enhanced in the latter
transplantation model. Although human ß-cell proliferation is quite low
compared with the growth rate of most cell types, the proliferative capacity
observed in this study might have significant effects on the outcome of
clinical islet transplantations, provided that proper methods can be developed
to transfer the beneficial effects of these models to human islet grafts.
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ACKNOWLEDGMENTS
|
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This study was funded by a concerted action in Medical and Health Research
of the European Community (BMH4-CT95-1561), grants from the Swedish Medical
Research Council (12X-109,12X-9237, and JD-12813), grants from the Juvenile
Diabetes Foundation International (JD-12813 to A.A. and 1-1999-694 to T.O.),
the Swedish Diabetes Association, the Novo-Nordisk Insulin Fund, the Family
Ernfors Fund, and Svenska Sällskapet
för Medicinsk Forskning (B.T.).
This study made use of human islets prepared by the Central Unit of the
ß-Cell Transplant.
We are grateful to A. Nordin and E. Törnelius
for excellent technical assistance and A. King for linguistic revision.
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FOOTNOTES
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ANOVA, analysis of variance; CBR, cell birth rate; GLP-1, glucagon-like
peptide 1; HGF, hepatocyte growth factor; ICC, islet-like cell cluster; LI,
labeling index; MALS, mouse anti-lymphocyte serum.
Received for publication April 25, 2000
and accepted in revised form October 19, 2000
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