Note: Descriptions are shown in the official language in which they were submitted.
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MEDIUM FOR PREPARING DEDIFFERENTIATED CELLS
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to a medium for preparing
dedifferentiated cells and more particularly to a basal
feeding medium for the development, maintenance and
expansion of a dedifferentiated cell population with at
least bipotentiality, which may be used in an in vitro
method for islet cell expansion.
(b) Description of Prior Art
Diabetes mellitus
Diabetes mellitus has been classified as type
I, or insulin-dependent diabetes mellitus (IDDM) and
type II, or non-insulin-dependent diabetes mellitus
(NIDDM). NIDDM patients have been subdivided further
into (a) nonobese (possibly IDDM in evolution), (b)
obese, and (c) maturity onset (in young patients).
Among the population with diabetes mellitus, about 20%
suffer from IDDM. Diabetes develops either when a
diminished insulin output occurs or when a diminished
sensitivity to insulin cannot be compensated for by an
augmented capacity for insulin secretion. In patients
with IDDM, a decrease in insulin secretion is the
principal factor in the pathogenesis, whereas in
patients with NIDDM, a decrease in insulin sensitivity
is the primary factor. The mainstay of diabetes
treatment, especially for type I disease, has been the
administration of exogenous insulin.
Rationale for more physiologic therapies
Tight glucose control appears to be the key to
the prevention of the secondary complications of
diabetes. The results of the Diabetes Complications
and Control Trial (DCCT), a multicenter randomized
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trial of 1441 patients with insulin dependent diabetes,
indicated that the onset and progression of diabetic
retinopathy, nephropathy, and neuropathy could be
slowed by intensive insulin therapy (The Diabetes
Control and Complication Trial Research Group, N.
Engl. J. Med., 1993; 29:977-986). Strict glucose
control, however, was associated with a three-fold
increase in incidence of severe hypoglycemia, including
episodes of seizure and coma. As well, although
glycosylated hemoglobin levels decreased in the
treatment group, only 5% maintained an average level
below 6.05% despite the enormous amount of effort and
resources allocated to the support of patients on the
intensive regime (The Diabetes Control and Complication
Trial Research Group, N. Engl. J. Med., 1993; 29:977-
986). The results of the DCCT clearly indicated that
intensive control of glucose can significantly reduce
(but not completely protect against) the long-term
microvascular complications of diabetes mellitus.
Other therapeutic options
The delivery of insulin in a physiologic manner
has been an elusive goal since insulin was first
purified by Banting, Best, McLeod and Collip. Even in
a patient with tight glucose control, however,
exogenous insulin has not been able to achieve the
glucose metabolism of an endogenous insulin source that
responds to moment-to-moment changes in glucose
concentration and therefore protects against the
development of microvascular complications over the
long term.
A major goal of diabetes research, therefore,
has been the development of new forms of treatment that
endeavor to reproduce more closely the normal physio-
logic state. One such approach, a closed-loop insulin
pump coupled to a glucose sensor, mimicking (3-cell
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function in which the secretion of insulin is closely
regulated, has not yet been successful. Only total
endocrine replacement therapy in the form of a
transplant has proven effective in the treatment of
diabetes mellitus. Although transplants of insulin-
producing tissue are a logical advance over
subcutaneous insulin injection , it is still far from
clear whether the risks of the intervention and of the
associated long-term immunosuppressive treatment are
lower those in diabetic patients under conventional
treatment.
Despite the early evidence of the potential
benefits of vascularized pancreas transplantation, it
remains a complex surgical intervention, requiring the
long-term administration of chronic immunosuppression
with its attendant side effects. Moreover, almost 50%
of successfully transplanted patients exhibit impaired
tolerance curves (Wright FH et al., Arch. Surg.,
1989;124:796-799; Landgraft R et al., Diabetologia
1991; 34 (suppl 1):561; Morel P et al., Transplantation
1991; 51:990-1000), raising questions about their
protection against the long-term complications of
chronic hyperglycemia.
The major complications of whole pancreas
transplantation, as well as the requirement for long
term immunosuppression, has limited its wider
application and provided impetus for the development of
islet transplantation. Theoretically, the
transplantation of islets alone, while enabling tight
glycemic control, has several potential advantages over
whole pancreas transplantation. These include the
following: (i) minimal surgical morbidity, with the
infusion of islets directly into the liver via the
portal vein; (ii) the possibility of simple re-
transplantation for graft failures; (iii) the exclusion
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of complications associated with the exocrine pancreas;
(iv) the possibility that islets are less immunogenic,
eliminating the need for immunosuppression and enabling
early transplantation into non-uremic diabetics; (v)
the possibility of modifying islets in vitro prior to
transplantation to reduce their immunogenicity; (vi)
the ability to encapsulate islets in artificial
membranes to isolate them from the host immune system;
and (vii) the related possibility of using
xenotransplantation of islets immunoisolated as part of
a biohybrid system. Moreover, they permit the banking
of the endocrine cryopreserved tissue and a careful and
standardized quality control program before the
implantation.
The problem of Islet transplantation
Adequate numbers of isogenetic islets
transplanted into a reliable implantation site can only
reverse the metabolic abnormalities in diabetic
recipients in the short term. In those that were
normoglycemic post-transplant, hyperglycemia recurred
within 3-12 mo. (Orloff M, et al., Transplantation
1988; 45:307). The return of the diabetic state that
occurs with time has been attributed either to the
ectopic location of the islets, to a disruption of the
enteroinsular axis, or to the transplantation of an
inadequate islet cell mass (Bretzel RG, et al. In:
Bretzel RG, (ed) Diabetes mellitus (Berlin: Springer,
1990) p.229) .
Studies of the long term natural history of the
islet transplant, that examine parameters other than
graft function, are few in number. Only one report was
found in which an attempt was specifically made to
study graft morphology (Alejandro R, et al., J Clin
Invest 1986; 78: 1339). In that study, purified islets
were transplanted into the canine liver via the portal
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vein. During prolonged follow-up, delayed failures of
graft function occurred. Unfortunately, the graft was
only examined at the end of the study, and not over
time as function declined. Delayed graft failures have
also been confirmed by other investigators for dogs
(Warnock GL et al., Can. J. Surg., 1988; 31: 421 and
primates; Sutton R, et al., Transplant Proc., 1987; 19:
3525). Most failures are presumed to be the result of
rejection despite appropriate immunosuppression.
Because of these failures, there is currently
much enthusiasm for the immunoisolation of islets,
which could eliminate the need for immunosuppression.
The reasons are compelling. Immunosuppression is
harmful to the recipient, and may impair islet function
and possibly cell survival (Metrakos P, et al., J.
Surg. Res., 1993; 54: 375). Unfortunately, micro-
encapsulated islets injected into the peritoneal cavity
of the dog fail within 6 months (Soon-Shiong P, et al.,
Transplantation 1992; 54: 769), and islets placed into
a vascularized biohybrid pancreas also fail, but at
about one year. In each instance, however, histological
evaluation of the graft has indicated a substantial
loss of islet mass in these devices (Lama RP, et al . ,
Diabetes 1992; 41: 1503). No reasons have been
advanced for these changes. Therefore maintenance of an
effective islet cell mass post-transplantation remains
a significant problem.
In addition to this unresolved issue, is the
ongoing problem of the lack of source tissue for
transplantation. The number of human donors is
insufficient to keep up with the potential number of
recipients. Moreover, given the current state of the
art of islet isolation, the number of islets that can
be isolated from one pancreas is far from the number
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required to effectively reverse hyperglycemia in a
human recipient.
In response, three competing technologies have
been proposed and are under development. First, islet
cryopreservation and islet banking. The techniques
involved, though, are expensive and cumbersome, and do
not easily lend themselves to widespread adoption. In
addition, islet cell mass is also lost during the
freeze-thaw cycle. Therefore this is a poor long-term
solution to the problem of insufficient islet cell
mass. Second, is the development of islet
xenotransplantation. This idea has been coupled to
islet encapsulation technology to produce a biohybrid
implant that does not, at least in theory, require
immunosuppression. There remain many problems to solve
with this approach, not least of which, is that the
problem of the maintenance of islet cell mass in the
post-transplant still remains. Third, is the resort to
human fetal tissue, which should have a great capacity
to be expanded ex vivo and then transplanted. However,
in addition to the problems of limited tissue
availability, immunogenicity, there are complex ethical
issues surrounding the use of such a tissue source that
will not soon be resolved. However, there is an
alternative that offers similar possibilities for near
unlimited cell mass expansion.
An entirely novel approach, proposed by
Rosenberg in 1995 (Rosenberg L et al., Cell
Transplantation, 1995;4:371-384), was the development
of technology 'to control and modulate islet cell
neogenesis and new islet formation, both in vitro and
in vivo. The concept assumed that (a) the induction of
islet cell differentiation was in fact controllable;
(b) implied the persistence of a stem cell-like cell in
the adult pancreas; and (c) that the signals) that
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would drive the whole process could be identified and
manipulated.
In as series of in vivo studies, Rosenberg and
co-workers established that these concepts were valid
in principle, in the in vivo setting (Rosenberg L et
al., Diabetes, 1988;37:334-341; Rosenberg L et al.,
Diabetologia, 1996;39:256-262), and that diabetes could
be reversed.
The well known teachings of in vi tro islet cell
expansion from a non-fetal tissue source comes from
Peck and co-workers (Corneliu JG et al., Horm. Metab.
Res., 1997;29:271-277), who describe isolation of a
pluripotent stem cell from the adult mouse pancreas
that can be directed toward an insulin-producing cell.
These findings have not been widely accepted. First,
the result has not proven to be reproducible. Second,
the so-called pluripotential cells have never been
adequately characterized with respect to phenotype. And
third, the cells have certainly not been shown to be
pluripotent.
More recently two other competing technologies
have been proposed- the use of engineered pancreatic (3-
cell lines (Efrat S, Advanced Drug Delivery Reviews,
1998;33:45-52), and the use of pluripotent embryonal
stem cells (Shamblott MJ et al., Proc. Natl. Acad. Sci.
USA, 1998;95:13726-13731). The former option, while
attractive, is associated with significant problems.
Not only must the engineered cell be able to produce
insulin, but it must respond in a physiologic manner to
the prevailing level of glucose- and the glucose
sensing mechanism is far from being understood well
enough to engineer it into a cell. Many proposed cell
lines are also transformed lines, and therefore have a
neoplastic potential. With respect to the latter
option, having an embryonal stem cell in hand is
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appealing because of the theoretical possibility of
being able to induce differentiation in any direction,
including toward the pancreatic (3-cell. However, the
signals necessary to achieve this milestone remain
unknown.
It would be highly desirable to be provided
with a platform for the preparation of dedifferentiated
intermediate cells derived from post-natal islets of
Langerhans, their expansion and the guided induction of
islet cell differentiation, leading to insulin-
producing cells that can be used for the treatment of
diabetes mellitus.
SUMMARY OF THE INVENTION
One aim of the invention is to provide a
platform for the preparation of dedifferentiated
intermediate cells derived from post-natal islets of
Langerhans, their expansion and the guided induction of
islet cell differentiation, leading to insulin-
producing cells that can be used for the treatment of
diabetes mellitus.
In accordance with one embodiment of the
present invention there is provided an in vitro method
for islet cell expansion, which comprises the steps of:
a) preparing dedifferentiated cells derived from
post-natal islets of Langerhans cells;
b) expanding the dedifferentiated cells; and
c) inducing islet cell differentiation properties
of the expanded cells of step b) to become
insulin-producing cells.
Preferably, step a) and step b) are
concurrently effected using a solid matrix, basal
feeding medium and appropriate growth factors to permit
the development, maintenance and expansion of a
dedifferentiated cell population with at least
bipotentiality.
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Such a medium for preparing dedifferentiated
cells derived from post-natal islets of Langerhans
comprises in a physiologically acceptable culture
medium an effective amount of a solid matrix
environment for a three-dimensional culture, a matrix
protein, and a first and a second factor for
developing, maintaining and expanding the
dedifferentiated cells.
Preferably the first factor induces a rise in
intracellular cAMP, and the second factor is derived
from acinar cells. The acinar cells must be present in
addition to the other three factors in order for the
change to occur. The first factor may comprise one or
more of cholera toxin (CT), forskolin, high glucose
concentrations, a promoter of cAMP, and EGF.
The culture medium may comprise DMEM/12
supplemented with an effective amount of fetal calf
serum, such as 10%.
The matrix protein comprises one or more of
laminin, collagen type I and MatrigelTM.
Preferably, step c) is effected by removing
cells from the matrix and resuspended in a basal liquid
medium containing soluble matrix proteins and growth
factors.
Preferably, the basal liquid medium is CMRL
1066 supplemented with at least 10% fetal calf serum,
wherein the soluble matrix proteins and growth factors
are selected from the group consisting of fibronectin,
IGF-1, IGF-2, insulin, and NGF. The basal liquid medium
may further comprise glucose concentration of at least
11 mM. The basal liquid medium may further comprise
inhibitors of known intracellular signaling pathways of
apoptosis and/or specific inhibitor of p38.
In accordance with another embodiment of the
present invention there is provided an in vitro method
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for producing cells with at least bipotentiality, which
comprises the steps of:
a) preparing dedifferentiated cells derived from
post-natal islets of Langerhans cells from a
patient; whereby when the dedifferentiated
cells are introduced in situ in the patient,
the cells are expanded and islet cell
differentiation properties are induced to
become in situ insulin-producing cells.
In accordance with another embodiment of the
present invention there is provided an in vitro method
for stem cell expansion, which comprises the steps of:
a) preparing dedifferentiated intermediate cells
derived from stem cells;
b) expanding in vitro the dedifferentiated
intermediate cells; and
c) inducing in vitro stem cell differentiation
properties of the expanded cells of step b) to
become stem cells.
Preferably, the stem cells are selected from
the group consisting of muscle, skin, bone, cartilage,
lung, liver, bone marrow and hematopoietic cells.
In accordance with another embodiment of the
present invention there is provided a method for the
treatment of diabetes mellitus in a patient, which
comprises the steps of
a) preparing dedifferentiated cells derived from
post-natal islets of Langerhans cells of the
patient; and
b) introducing the dedifferentiated cells in situ
in the patient, wherein the cells are expanded
in situ and islet cell differentiation
properties are induced in situ to become
insulin-producing cells.
In accordance with another embodiment of the
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present invention there is provided a method for the
treatment of diabetes mellitus in a patient, which
comprises the steps of
a) preparing dedifferentiated cells derived from
post-natal islets of Langerhans cells of the
patient;
b) expanding in vitro the dedifferentiated cells;
c) inducing in vitro islet cell differentiation
properties of the expanded cells of step b) to
become insulin-producing cells; and
d) introducing the cells of step c) in situ in the
patient, wherein the cells produce insulin in
si to .
For the purpose of the present invention the
following terms are defined below.
The expression "post-natal islets of
Langerhans" is intended to mean islet cells of any
origin, such as human, porcine and canine, among
others.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates cell-type conversion from
islet to duct-like structure (human tissues), (a)
Islet in the pancreas, (b) Islet following isolation
and purification, (c) islet in solid matrix beginning
to undergo cystic change, (d-f) progressive formation
of cystic structure with complete loss of islet
morphology.
Fig. 2 illustrates same progression of changes
as in Fig. 1. Cells are stained by immunocytochemistry
for insulin. (a) Islet in pancreas. (b) Islet after
isolation and purification. (c-e) Progressive loss of
islet phenotype. (f) High power view of cyst wall
composed duct-like epithelial cells. One cell still
contains insulin (arrow).
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Fig. 3 illustrates same progression of changes
as in Fig. 1. Cells stained by immunocytochemistry for
glucagon. (a) Islet in pancreas. (b) Islet after
isolation and purification. (c-e) Progressive loss of
islet phenotype. (f) High power view of cyst wall
composed duct-like epithelial cells. One cell still
contains glucagon (arrow).
Fig. 4 A-C illustrate demonstration of cell
phenotype by CK-19 immunocytochemistry. Upper left
panel- cystic structure in solid matrix. All cells
stain for CK-19, a marker expressed in ductal
epithelial cells in the pancreas. Lower panel-
following removal from the solid matrix, and return to
suspension culture. A structure exhibiting both
epithelial-like and solid components. Upper right
panel- only the epithelial-like component retains CK-19
immunoreactivity. The solid component has lost its CK-
19 expression, and appears islet-like.
Fig. 5 A-B illustrate upper panel
Ultrastructural appearance of cells composing the
cystic structures in solid matrix. Note the microvilli
and loss of endosecretory granules. The cells have the
appearance of primitive duct-like cells. Lower panel
ultrastructural appearance of cystic structures removed
from the solid matrix and placed in suspension culture.
Note the decrease in microvilli and the reappearance of
endosecretory granules.
Fig. 6 A-B illustrate in situ hybridization for
pro-insulin mRNA. Upper panel-cystic structures with
virtually no cells containing the message. Lower panel
cystic structures have been removed from the matrix and
placed in suspension culture. Note the appearance now,
of both solid and cystic structures. The solid
structures have an abundant expression of pro-insulin
mRNA.
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Fig. 7 illustrates insulin release into the
culture medium by the structures seen in the lower
panel of Fig. 6. Note that there is no demonstrable
insulin secreted from the tissue when in the cystic
state (far left column). FN-fibronectin; IGF-1-insulin-
like growth factor-1; Gluc-glucose.
Fig. 8 illustrates Islets embedded in collagen
matrix and maintained in DMEM/F12-CT. Photos from
under the inverted microscope (A, C, E) and
corresponding histological sections stained for
pancytokeratin AE1/AE3 by immunocytochemistry (B, D,
F). (A, C, E, x100; B, D, F, x200)
Fig. 9 illustrates Islets at an intermediate
stage of cystic transformation still contain cells that
(A) express the pro-insulin mRNA and that (B)
synthesize and store insulin protein. (x400)
Fig. 10 A illustrates Intracellular level of
cAMP during the time course of islet-cystic
transformation. Note the relatively constant level of
intracelluar CAMP in islets maintained in CMRL 1066
alone.
Fig. 10 B illustrates the integrated amount of
cAMP (area under the curve in A) measured at 120 hours.
There were no differences observed between islets
cultured in DMEM/F12-CT, CMRL-CT and CMRL-forskolin.
Note, however, that islets maintained in CMRL alone had
significantly less intracellular CAMP.
Fig. 10 C illustrates the percentage of islets
undergoing cystic transformation increased over the
time course of the culture period in the DMEM/F12-CT,
CMRL-CT and CMRL-forskolin groups. Islets maintained in
CMRL 1066 had a very low level of cystic transformation
that remained constant. * p<0.05, ** p<0.01, ***
p<0.001
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Fig. 11 illustrates the progressive loss of
tissue insulin content during the time course of cystic
transformation. Note the steep decline in islets
maintained in DMEM/F12-CT, CMRL-CT and CMRL-forskolin,
which corresponds to the early onset of apoptosis by 16
hours. * p<0.03
Fig. 12 illustrates Apoptotic activity (A) and
BrdU labeling index (B) of islets cultured in DMEM/F12-
CT and CMRL 1066 over the time course of cystic
transformation. Note the shift to the left in the onset
of apoptosis in islets in DMEM/F12-CT. *p<0.02;
**p<0.01; ***p<0.001.
Fig. 13 illustrates the effect of integrin-binding
peptides GRGDSP and GRGESP (A), extracellular matrix
proteins laminin and fibronectin (B) and a combination
of GRGDSP or GRGESP and laminin (C) on islet-cystic
transformation. *p<0.05, **p<0.01. ***p<0.001.
Fig. 14 illustrates the effect of extracellular
matrix on islet-cystic transformation in isolated
canine islets.
DETAILED DESCRIPTION OF THE INVENTION
In vivo cell transformation leading to (3-cell
neogenesis and new islet formation can be understood in
the context of established concepts of developmental
biology.
Transdifferentiation is a change from one
differentiated phenotype to another, involving
morphological and functional phenotypic markers (Okada
TS., Develop. Growth and Differ. 1986;28:213-321). The
best-studied example of this process is the change of
amphibian iridial pigment cells to lens fibers, which
proceeds through a sequence of cellular
dedifferentiation, proliferation and finally
redifferentiation (Okada TS, Cell Diff. 1983;13:177-
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183; Okada TS, Kondoh H, Curr. Top Dev. Biol.,
1986;20:1-433; Yamada T, Monogr. Dev. Biol., 1977;13:1-
124). Direct transdifferentiation without cell division
has also been reported, although it is much less common
(Beresford WA, Cell Differ. Dev., 1990;29:81-93). While
transdifferentiation has been thought to be essentially
irreversible, i.e. the transdifferentiated cell does
not revert back into the cell type from which it arose,
this has recently been reported not to be the case
(Danto SI et al., Am. J. Respir. Cell Mol. Biol.,
1995;12:497-502). Nonetheless, demonstration of
transdifferentiation depends on defining in detail the
phenotype of the original cells, and on proving that
the new cell type is in fact descended from cells that
were defined (Okada TS, Develop. Growth and Differ.
1986;28:213-321).
In many instances, transdifferentiation
involves a sequence of steps. Early in the process,
intermediate cells appear that express neither the
phenotype of the original nor the subsequent
differentiated cell types, and therefore they have
been termed dedifferentiated. The whole process is
accompanied by DNA replication and cell proliferation.
Dedifferentiated cells are assumed a priori to be
capable of forming either the original or a new cell
type, and thus are multipotential (Itoh Y, Eguchi G,
Cell Differ., 1986;18:173-182; Itoh Y, Eguchi G,
Develop. Biology, 1986;115:353-362; Okada TS, Develop.
Growth and Differ, 1986;28:213-321).
Stability of the cellular phenotype in adult
organisms is probably related to the extracellular
milieu, as well as cytoplasmic and nuclear components
that interact to control gene expression. The
conversion of cell phenotype is likely to be
accomplished by selective enhancement of gene
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expression, which controls the terminal developmental
commitment of cells.
The pancreas is composed of several types of
endocrine and exocrine cells, each responding to a
variety of trophic influences. The ability of these
cells to undergo a change in phenotype has been
extensively investigated because of the implications
for the understanding of pancreatic diseases such as
cancer and diabetes mellitus. Transdifferentiation of
pancreatic cells was first noted nearly a decade ago.
Hepatocyte-like cells, which are normally not present
in the pancreas, were observed following the
administration of carcinogen (Rao MS et al., Am. J.
Pathol., 1983;110:89-94; Scarpelli DG, Rao MS, Proc.
Nat. Acad. Sci. USA 1981;78:2577-2581) to hamsters and
the feeding of copper-depleted diets to rats (Rao MS,
et al., Cell Differ., 1986;18:109-117). Recently,
transdifferentiation of isolated acinar cells into
duct-like cells has been observed by several groups
(Arias AE, Bendayan M, Lab Invest., 1993;69:518-530;
Hall PA, Lemoine NR, J. Pathol., 1992;166:97-103; Tsap
MS, Duguid WP, Exp. Cell Res., 1987;168:365-375). In
view of these observations it is probably germane that
during embryonic development, the hepatic and
pancreatic anlagen are derived from a common endodermal
Factors which control the growth and functional
maturation of the human endocrine pancreas during the
fetal and post-natal periods are still poorly
understood, although the presence of specific factors
in the pancreas has been hypothesized (Pictet RL et al.
In: Extracellular Matrix Influences on Gene Expression.
Slavkin HC, Greulich RC (eds). Academic Press, New
York, 1975, pp.l0).
Some information is available on exocrine
growth factors. Mesenchymal Factor (MF), has been
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extracted from particulate fractions of homogenates of
midgestational rat or chick embryos. MF affects cell
development by interacting at the cell surface of
precursor cells (Butter WJ. The development of the
endocrine and exocrine pancreas. In: The Pancreas.
Fitzgerald PJ, Morson AB (eds). Williams and Wilkins,
London, 1980, pp.30) and thereby influences the kind of
cells that appear during pancreatic development
(Githens S. Differentiation and development of the
exocrine pancreas in animals. In: Go VLW, et al. (eds).
The Exocrine Pancreas: Biology, Pathobiology and
Diseases. Raven Press, New York, 1986, pp.21). MF is
comprised of at least 2 fundamental components, a heat
stable component whose action can be duplicated by
cyclic AMP analogs, and another high molecular weight
protein component (Butter WJ, In: The Pancreas.
Fitzgerald PJ, Morson AB (eds). Williams and Wilkins,
London, 1980, pp.30). In the presence of MF, cells
divide actively and differentiate largely into non
endocrine cells.
Other factors have also been implicated in
endocrine maturation. Soluble peptide growth factors
(GF) are one group of trophic substances that regulate
both cell proliferation and differentiation. These
growth factors are multi-functional and may trigger a
broad range of cellular responses (Sporn & Roberts,
Nature, 332:217-19, 1987). Their actions can be
divided into 2 general categories- effects on cell
proliferation, which comprises initiation of cell
growth, cell division and cell differentiation; and
effects on cell function. They differ from the
polypeptide hormones in that they act in an autocrine
and/or paracrine manner (Goustin AS, Leof EB, et al.
Cancer Res., 46:1015-1029, 1986; Underwood LE, et al.,
Clinics in Endocrinol. & Metabol., 15:59-77,1986).
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Specifics of their role in the individual processes
that comprise growth need to be resolved.
One family of growth factors are the
somatomedins. Insulin-like growth factor-I (IGF-I), is
synthesized and released in tissue culture by the (3
cells of fetal and neonatal rat islets (Hill DJ, et
al., Diabetes, 36:465-471, 1987; Rabinovitch A, et al.,
Diabetes, 31:160-164,1982; Romanus JA et al., Diabetes
34:696-792, 1985) . IGF-II has been identified in human
fetal pancreas (Bryson JM et al., J. Endocrinol.,
121:367-373,1989). Both these factors enhance neonatal
(3-cell replication in vitro when added to the culture
medium (Hill DJ, et al., Diabetes, 36:465-471, 1987;
Rabinovitch A, et al., Diabetes, 31:160-164, 1982).
Therefore the IGF's may be important mediators of (3-
cell replication in fetal and neonatal rat islets but
may not do so in post-natal development (Billestrup N,
Martin JM, Endocrinol., 116:1175-81,1985; Rabinovitch
A, et al., Diabetes, 32:307-12, 1983; Swenne I, Hill
DJ, Diabetologia 32:191-197, 1989; Swenne I,
Endocrinology, 122:214-218, 1988; Whittaker PG, et al,
Diabetologia, 18:323-328, 1980). Furthermore,
Platelet-derived growth factor (PDGF) also stimulates
fetal islet cell replication and its effect does not
require increased production of IGF-I (Swenne I,
Endocrinology, 122:214-218, 1988). Moreover, the
effect of growth hormone on the replication of rat
fetal B-cells appears to be largely independent of IGF-
I (Romanus JA et al., Diabetes 34:696-792, 1985; Swenne
I, Hill DJ, Diabetologia 32:191-197, 1989). In the
adult pancreas, IGF-I mRNA is localized to the D-cell.
But IGF-I is also found on cell membranes of ~3- and A-
cells, and in scattered duct cells, but not in acinar
or vascular endothelial cells (Hansson H-A et al., Acta
Physiol. Scand. 132:569-576, 1988; Hansson H-A et al.,
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- 19 -
Cell Tissue Res., 255:467-474, 1989). This is in
contradistinction to one report (Smith F et al,
Diabetes, 39 (suppl 1):66A, 1990), wherein IGF-I
expression was identified in ductular and vascular
endothelial cells, and appeared in regenerating
endocrine cells after partial pancreatectomy. It has
not been shown that IGF's will stimulate growth of
adult duct cells or islets. Nor do the IGF's stimulate
growth of the exocrine pancreas (Mossner J et al., Gut
28:51-55, 1987). It is apparent therefore, that the
role of IGF-I, especially in the adult pancreas, is far
from certain.
Fibroblast growth factor (FGF) has been found
to initiate transdifferentiation of the retinal pigment
epithelium to neural retinal tissues in chick embryo in
vivo and in vitro (Hyuga M et al . , Int. J. Dev. Biol.
1993;37:319-326; Park CM et al., Dev. Biol.
1991;148:322-333; Pittack C et al., Development
1991;113:577-588). Transforming growth factor-beta
(TGF-(3) has been demonstrated to induce
transdifferentiation of mouse mammary epithelial cells
to fibroblast cells [20]. Similarly, epithelial growth
factor (EGF) and cholera toxin were used to enhance
duct epithelial cyst formation from among pancreatic
acinar cell fragments (Yuan S et al., In vitro Cell
Dev. Biol., 1995;31:77-80).
The search for the factors mediating cell
differentiation and survival must include both the cell
and its microenvironment (Bissell MJ et al., J. Theor.
Biol., 1982; 99:31), as a cell's behavior is controlled
by other cells as well as by the extracellular matrix
(ECM) (Stoker AW et al. Curr. Opin. Cell. Biol.,
1990;2:864). ECM is a dynamic complex of molecules
serving as a scaffold for parenchymal and
nonparenchymal cells. Its importance in pancreatic
WO 01/32839 CA 02388208 2002-04-23 PCT/CA00/01284
- 20 -
development is highlighted by the role of fetal
mesenchyme in epithelial cell cytodifferentiation
(Bencosme SA, Am. J. Pathol. 1955; 31: 1149; Gepts W,
de Mey J. Diabetes 1978; 27(suppl. 1): 251; Gepts W,
Lacompte PM. Am. J. Med., 1981; 70: 105; Gepts W.
Diabetes 1965; 14: 619; Githens S. In: Go VLW, et al.
(eds) The Exocrine Pancreas: Biology, Pathobiology and
Disease. (New York: Raven Press, 1986) p. 21). ECM is
found in two forms- interstitial matrix and basement
membrane (BM). BM is a macromolecular complex of
different glycoproteins, collagens, and proteoglycans.
In the pancreas, the BM contains laminin, fibronectin,
collagen types IV and V, as well as heparan sulphate
proteoglycans (Ingber D. In: Go VLW, et al (eds) The
Pancreas: Biology, Pathobiology and Disease (New York:
Raven Press, 1993) p. 369) . The specific role of these
molecules in the pancreas has yet to be determined.
ECM has profound effects on differentiation.
Mature epithelia that normally never express
mesenchymal genes, can be induced to do so by
suspension in collagen gels in vitro (Hay ED. Curr.
Opin. in Cell. Biol. 1993; 5:1029). For example,
mammary epithelial cells flatten and lose their
differentiated phenotype when attached to plastic
dishes or adherent collagen gels (Emerman JT, Pitelka
DR. In vitro 1977; 13:316). The same cells round,
polarize, secrete milk proteins, and accumulate a
continuous BM when the gel is allowed to contract
(Emerman JT, Pitelka DR. In vitro, 1977; 13:316). Thus
different degrees of retention or re-formation of BM
are crucial for cell survival and the maintenance of
the normal epithelial phenotype (Hay ED. Curr. Opin. in
Cell. Biol. 1993; 5:1029).
During times of tissue proliferation, and in
the presence of the appropriate growth factors, cells
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are transiently released from ECM-determined survival
constraints. It is now becoming clear that there are
two components of the cellular response to ECM
interactions- one physical, involving shape changes and
cytoskeletal organization; the other biochemical,
involving integrin clustering and increased protein
tyrosine phosphorylation (Ingber DE. Proc. Natl. Acad.
Sci. USA, 1990;87:3579; Roskelley CD et al., Proc.
Natl. Acad. Sci. USA, 1994;91:12378).
In addition to its known regulatory role in
cellular growth and differentiation, ECM has more
recently been recognized as a regulator of cell
survival (Bates RC, Lincz LF, Burns GF, Cancer and
Metastasis Rev., 1995;14:191). Disruption of the cell-
matrix relationship leads to apoptosis (Frisch SM,
Francis H. J. Cell. Biol., 1994;124:619; Schwartz SM,
Bennett MR, Am. J. Path., 1995;147:229), a
morphological series of events (Kerr JFK et al., Br. J.
Cancer, 1972;26:239), indicating a process of active
cellular self destruction.
In accordance with one embodiment of the
present invention, the platform technology is based on
a combination of the foregoing observations,
incorporating in a basal feeding medium the following
components that are necessary and sufficient for the
preparation of dedifferentiated intermediate cells from
adult pancreatic islets of Langerhans:
1. a solid matrix permitting "three dimensional"
culture;
2. the presence of matrix proteins including but not
limited to collagen type I and laminin; and
3. the growth factor EGF and promoters of CAMP,
including but not limited to cholera toxin and
forskolin.
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The preferred feeding medium is DMEM/F12 with
10% fetal calf serum. In addition, the starting tissue
must be freshly isolated and cultured without absolute
purification.
The use of a matrix protein-containing solid
gel is an important part of the culture system, because
extracellular matrix may promote the process of
transdifferentiation. This point is highlighted by
isolated pancreatic acinar cells, which
transdifferentiate to duct-like structures when
entrapped in Matrigel basement membrane (Arias AE,
Bendayan M, Lab Invest., 1993;69:518-530), or by
retinal pigmented epithelial cells, which
transdifferentiate into neurons when plated on laminin-
containing substrates (Reh TA et al., Nature
1987;330:68-71). Most recently, Gittes et al. demon-
strated, using 11-day embryonic mouse pancreas, that
the default path for growth of embryonic pancreatic
epithelium is to form islets (Gittes GK et al.,
Development 1996; 122:439-447). In the presence of
basement membrane constituents, however, the pancreatic
anlage epithelium appears to programmed to form ducts.
This finding again emphasizes the interrelationship
between ducts and islets and highlights the important
role of the extracellular matrix.
This completes stage 1 (the production of
dedifferentiated intermediate cells) of the process.
During the initial 96 h of culture, islets undergo a
cystic transformation associated with (Arias AE,
Bendayan M, Lab. Invest., 1993;69:518-530) a
progressive loss of insulin gene expression, (2) a loss
of immunoreactivity for insulin protein, and (3) the
appearance of CKA 19, a marker for ductal cells. After
transformation is complete, the cells have the
ultrastructural appearance of primitive duct-like
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cells. Cyst enlargement after the initial 96h is
associated, at least in part, with a tremendous in-
crease in cell replication. These findings are
consistent with the transdifferentiation of an islet
cell to a ductal cell (Yuan et al., Differentiation,
1996; 61:67-75, which showed that isolated islets
embedded in a collagen type I gel in the presence of a
defined medium undergo cystic transformation within 96
hours ) .
Stage 2- the generation of functioning (3-cells,
requires a complete change of the culture conditions.
The cells are moved from the digested matrix and
resuspended in a basal liquid medium such as CMRL 1066
supplemented with loo fetal calf serum, with the
addition of soluble matrix proteins and growth factors
that include, but are not limited to, fibronectin (10-
ng/ml), IGF-1 (100 ng/ml), IGF-2 (100 ng), insulin
(10-100 ~,g/ml), NGF (10-100 ng/ml). In addition, the
glucose concentration must be increased to above 11 mM.
20 Additional culture additives may include specific
inhibitors of known intracellular signaling pathways of
apoptosis, including, but not limited to a specific
inhibitor of p38.
Evidence for the return to an islet cell
phenotype includes: (1) the re-appearance of solid
spherical structures; (2) loss of CK-19 expression; (3)
the demonstration of endosecretory granules on electron
microscopy; (4) the re-appearance of pro-insulin mRNA
on in situ hybridization; (5) the return of a basal
release of insulin into the culture medium.
The present invention will be more readily un-
derstood by referring to the following examples which
are given to illustrate the invention rather than to
limit its scope.
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EXAMPLE I
Preparation of a basal feeding medium
The purpose of this study was to elucidate the
mechanisms involved in the process of
transdifferentiation.
Canine islets were isolated using Canine
LiberaseTM and purified on a Euroficoll gradient in a
Cobe 2991 Cell Separator. Freshly isolated islets were
embedded in collagen type I gel for up to 120 hr and
cultured in (i) DMEM/F12 plus cholera toxin (CT); (ii)
CMRL 1066 supplemented with CT; (iii) CMRL 1066
supplemented with forskolin, and (iv) CMRL 1066 alone.
At 16 hr, intracellular levels of cAMP (fmol/103
islets), determined by ELISA, were increased in Groups
(i)-(iii) (642~17, 338~48, 1128~221) compared to Group
iv (106~19, p<0.01). Total intracellular cAMP at 120 hr
(integrated area under the curve) coincided with the
of islets undergoing transdifferentiation (63~2, 48~2,
35~3, 8~1), as determined by routine histology,
immunocytochemistry for cytokeratin AE1/AE3, and by a
loss of pro-insulin gene expression on in situ
hybridization.
To evaluate the role of matrix proteins and the
3-D environment, islets were embedded in collagen type
I, MatrigelTM and agarose gel and cultured in DMEM/F12
plus CT. Islets in collagen type I and MatrigelTM
demonstrated a high rate of cystic transformation
(63~2% and 71~4o respectively), compared to those in
agarose (0~Oo, p<0.001). In addition, islet cell
transdifferentiation was partially blocked by prior
incubation of freshly isolated islets with an RGD
motif-presenting synthetic peptide.
In conclusion, these studies confirm the
potential of freshly isolated islets to undergo
epithelial cell transdifferentiation. Elevated levels
of intracellular cAMP and matrix proteins presented in
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a 3-dimensional construct are necessary for this
transformation to be induced. The precise nature of the
resulting epithelial cells, and the reversibility of
the process remain to be determined.
EXAMPLE II
Factors mediating the transformation of islets of
Langerhans to duct epithelial-like structures
MATERIALS AND METHODS
Islet Isolation and Purification
Pancreata from six mongrel dogs of both sexes
(body weight 25 - 30kg) were resected under general
anesthesia in accordance with Canadian Council for
Animal Care guidelines (Wang RN, Rosenberg L (1999) J
Endocrology 163 181-190). Prior to removal, the
pancreatic ducts were cannulated to permit intraductal
infusion with Liberase CI~ (1.25mg/ml) (Boehringer
Mannheim, Indianapolis, IN, USA) according to
established protocols (Horaguchi A, Merrell RC (1981)
Diabetes 30 455-461; Ricordi C (1992) Pancreatic islet
cell transplantation. pp99-112. Ed Ricordi C. Austin:
R. G. Landes Co.). Purification was achieved by density
gradient separation in a three-step EuroFicoll gradient
using a COBE 2991 Cell Processor (LOBE BCT, Denver,
CO., USA) (London NJM et al. (1992) Pancreatic islet
cell transplantation. pp113-123. Ed Ricordi C. Austin:
R. G. Landes Co.). The final preparation consisted of
95% dithizone-positive structures with diameters
ranging from 50 to 500~,m.
Experimental Design
To evaluate the role of intracellular cAMP,
freshly isolated islets were embedded in type 1
collagen gel (Wang RN, Rosenberg L (1999) J
Endocrology 163 181-190) and cultured in: (i) DMEM/F12
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(GIBCO, Burlington, ON, CANADA) supplemented with 10%
FBS, EGF (100 ng/ml) (Sigma, St. Louis, St. Louis, MO,
USA) and cholera toxin (100ng/ml) (Sigma, St. Louis,
MO, USA); (ii) CMRL1066 (GIBCO) supplemented with
10%FBS and cholera toxin (100ng/ml) and 16.5mM D-
glucose; (iii) CMRL1066 supplemented with 10%FBS and
2~M forskolin (Sigma, St. Louis, MO, USA), and (iv)
CMRL1066 supplemented with 10% FBS. Approximately 3000
islets per group per time point were used. Islets were
cultured in 95% air / 5% COZ at 37°C, and the medium
was changed on alternate days. Representative islets
from each group were examined after isolation (0 hour),
and then on hours of 1, 16, 36, 72 and 120 using the
following investigations.
The following series of experiments were
conducted to evaluate the role of cell-matrix
interactions in the process of cystic transformation.
First, to determine whether the process required a
solid gel environment, islets were cultured in
suspension in DMEM/F12 with 10% FBS plus CT and EGF.
To determine whether a solid gel environment and
extracellular matrix proteins were independent
requirements, islets were embedded in 1.5% agarose gel
and maintained in DMEM/F12 with loo FBS plus CT and
EGF. Alternatively, islets were cultured in suspension
with in DMEM/F12 with 10% FBS plus CT and EGF in the
presence of soluble Laminin (50~g/ml) or Fibronectin
(50~,g/ml) (Peninsula Laboratories). To determine
whether the process was, at least in part, integrin-
mediated, islets were pre-incubated at 37°C for 60 min
either in the presence of the RGD-motif containing
GRGDSP peptide or the control peptide GRGESP (400~.g/ml)
(Peninsula Laboratories). Finally, to determine whether
cystic transformation was dependent on type 1 collagen
alone, islets were also embedded in Matrigel~
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(Peninsula Laboratories, Belmont, CA, USA).
Morphological Analysis
Immunocytochemistry
Tissue was fixed in 4% paraformaldehyde (PFA)
and embedded in 2o agarose following a standard
protocol of dehydration and paraffin embedding Wang RN,
Rosenberg L (1999) J Endocrology 163 181-190). A set of
six serial sections (thickness 4 Vim) was cut from each
paraffin block.
Consecutive sections were processed for routine
histology and immunostained for pancreatic hormones
(insulin, glucagon and somatostatin, Biogenex, San
Ramon, CA., USA) and the pan-cytokeratin cocktail
AE1/AE3 (Dako, Carpinteria, CA., USA), using the AB
complex method (streptavidin-biotin horseradish
peroxidase; Dako), as described previously (Wang RN et
al. (1994) Diabetologia 37 1088-1096). For cytokeratin
AE1/AE3, sections were pretreated with 0.1% trypsin.
The sections were incubated overnight at 4°C with the
appropriate primary antibodies. Negative controls
involved the omission of the primary antibodies.
In situ hybridization
In situ hybridization for human proinsulin mRNA
(Novocastra, Burlington, ON, Canada) was performed on
consecutive sections of freshly isolated islets and
epithelial cystic structures at 120 h. The sections
were hybridized with a fluorescein labelled
oligonucleotide cocktail solution for 2 h at 37°C.
Slides were then incubated with rabbit Fab anti-FITC
conjugated to alkaline phosphatase antibody (diluted
1:200) for 30 min at room temperature. The reaction
product was visualised by an enzyme-catalysed colour
reaction using a nitro blue tetrazolium and 5'-bromo-4-
chloro-3-indolyl-phosphate kit (Wang RN, Rosenberg L
(1999) J Endocrology 163 181-190, Wang RN et al. (1994)
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Diabetologia 37 1088-1096).
Analysis of Intracellular cAMP Level
Cells were harvested from the collagen gel and
washed in 1mM cold PBS. Following addition of 200.1 of
lysis buffer, each sample was sonicated for 30s, then
incubated for 5min at room temperature. 1001 of cell
lysate was transferred to donkey anti-rabbit Ig coated
plate. The intracellular CAMP content of non-acetylated
samples was measured using a commercially available
cAMP enzyme-linked immunoassay kit (assay range 12.5 -
3200fmo1/well, Ameraham, Little Chalfont, U.K.). The
data are expressed as fmol per 103 islets.
Insulin Content Assay
Cellular insulin content was measured using a
solid-phase radioimmunoassay (Immunocorp, Montreal,
Quebec, Canada) (Wang RN, Rosenberg L (1999) J
Endocrology 163 181-190) with a sensitivity of 26.7
pmol/1 (0.15 ng/ml), an inter-assay variability of <5%,
and an accuracy of 100%. The kit uses anti-human
antibodies that cross-react with canine insulin.
Obtained values were corrected for variations in cell
number by measuring DNA content using a fluorometric
DNA assay (Yuan S et al. (1996) Differentiation 61, 67-
75). The data are expressed as ~g per ~g DNA.
Cell Death And Proliferation
Cells cultured in DMEM/F12-CT and CMRL1066 were
harvested from the gel using collagenase XI (0.25
mg/ml) (Sigma, Montreal, Que.) and processed for a
specific programmed cell death ELISA, that detects
histone-associated DNA fragments in the cell cytoplasm-
a hallmark of the apoptotic process (Roche Molecular,
Montreal, Que.) (Paraskevas :S et al. (2000) Ann.
Surgery in press). Cells were incubated in lysis buffer
for 30 min, and the supernatant containing cytoplasmic
oligonucleosomes was measured at an absorbance of
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405nm. Variations in sample size were corrected by
measuring total sample DNA content (Yuan S et al.
(1996) Differentiation 61, 67-75).
To evaluate cell proliferation, cells cultured
in DMEM/F12-CT and CMRL1066 were pre-incubated with
10~M 5-bromo-2'-deoxyuridine (BrdU, Sigma) for 1h at
37°C. Harvested cells was fixed in 4% PFA as described
above. Immunostaining for BrdU was performed using the
AB complex method. The sections were pretreated with
0.1% trypsin and 2N HC1 denatured DNA. A monoclonal
anti-BrdU antibody was used at 1:500 dilution (Sigma).
To calculate a BrdU labeling index, the number of cells
positive for the BrdU reaction was determined and
expressed as a percentage of the total number of cells
counted. For each experimental group and time point, at
least 500 cells were counted per section.
Statistic Analysis
Data obtained from the six different islet
isolations are expressed as mean ~ SEM. The difference
between groups was evaluated by one-way analysis of
variance.
RESULTS
Morphological Changes
Under the inverted microscope, freshly isolated
islets appeared as solid spheroids. At this time,
cytokeratin-positive cells were not demonstrated
(Figs.8A-B).
For islets embedded in type 1 collagen and
cultured in DMEM/F12 plus CT, CMRL 1066 plus CT or CMRL
1066 plus forskolin, duct epithelial differentiation
was first observed coincident with a loss of cells in
the islet periphery, at approximately 16 hours. At this
time, cells lining the cystic spaces were cytokeratin-
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positive (Figs. 8C-D). Fully developed epithelial
structures were present in culture by 72 hours (Figs.
8E-F). Islets cultured in CMRL 1066 alone maintained a
solid spheroid appearance for the duration of the study
and did not undergo epithelial transformation.
Immunocytochemical staining did not demonstrate co-
localization of cytokeratin and islet cell hormones.
This is in keeping with the observation in the intact
pancreas, that cytokeratin staining was only seen on
duct epithelial cells. Pro-insulin gene expression and
insulin protein were progressively lost during the
period of duct epithelial differentiation (Fig. 9)
Intracellular cAMP
After 1 hour, intracellular levels of cAMP of
islets maintained in DMEM/F12-CT, CMRL1066-CT and
CMRL1066-forskolin were significantly elevated compared
to freshly isolated islets or to islets maintained in
CMRL 1066 alone (Fig. 10A) . In fact the intracellular
level of CAMP of islets cultured in CMRL 1066 alone did
not increase at all during the time course of the
study. The total intracellular cAMP measured over 120
hr (integrated area under the curve) was similar for
islets cultured in DMEM/F12-CT, CMRL 1066-CT and CMRL
1066-forskolin (15~3, 16~2, 17~3 respectively),
although the most sustained elevation of cAMP was in
the DMEM/F12-CT islets, which were exposed to both EGF
and CT. In comparison, islets cultured in CMRL 1066
alone had the lowest level of total intracellular cAMP
(4~1, p<0.001) (Fig. 10B), and this translated into the
lowest level of islet-duct transformation (Fig. 10C).
Intracellular Insulin Content
The cellular content of insulin (Fig. 11) was
highest in freshly isolated islets (11~2~g/~g DNA).
After 16 hours in culture, the insulin content of cells
cultured in DMEM/F12-CT, CMRL1066-CT and CMRL1066-
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forskolin declined dramatically, falling to 7% of the
initial value by 120 hours. Islets cultured in CMRL1066
alone did not undergo epithelial transformation, and
maintained a higher level of intracellular insulin
compared to the other three groups (p<0.03, Fig. 11).
Analysis Of Cell Death And Proliferation
To determine whether cell loss during cystic
transformation was due, at least in part, to programmed
cell death, we used a specific cell death ELISA. At 16
hours, cytoplasmic oligonucleosome enrichment was
significantly higher in islets cultured with DMEM/F12-
CT compared to islets cultured in CMRL1066 alone
(p<0.02, Fig. 12A). After 36 hours, there was no
difference between the groups. Looking at the data as a
whole (Fig. 12A), it appears that a wave of apoptosis
occurred in both groups of islets, but that the time
course of cell death was shifted to the left for islets
undergoing cystic transformation in DMEM/F12-CT.
To assess proliferation, cells were labeled
with BrdU. Following isolation, the BrdU cell labeling
index of islets cultured in DMEM/F12-CT was 0.8%
identical to that of islets cultured in CMRL 1066
alone. After 36 hours, however, a wave of cell
proliferation ensued in the DMEM/F12-CT group, with the
labeling index reaching 18% at 120 hours (Fig. 12B). In
comparison, the labeling index for islets in CMRL 1066
remained essentially unchanged throughout the study
period (p< 0 . O1 ) .
The Role Integrin-ECM Interactions
To determine whether elevation of intracelluar
CAMP was sufficient to induce duct epithelial
differentiation, islets were maintained in suspension
culture in DMEM/F12-CT and not embedded in collagen
gel. Under these conditions, epithelial transformation
did not occur. This suggested that an increase in
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intracellular cAMP was a necessary but not sufficient
requirement for transformation, and that the matrix
must also play an important role in the process.
To determine whether it was the solid gel
environment or the presence of extracellular matrix
proteins alone that was necessary, islets were embedded
in agarose gel, type 1 collagen gel or Matrigel~. Only
islets embedded in the latter two gels underwent cystic
transformation (Table 1). Furthermore, islets
maintained in suspension in DMEM/F12-CT supplemented
with either soluble laminin or fibronectin, failed to
undergo ductal transformation. These experiments
indicated that the process of transformation required
the presence of ECM proteins presented in a solid gel
environment.
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Table 1
The effect of extracellular matrix on islet-cystic
transformation in isolated canine islets
Times Matrigel Collagen Agarose Soluble
I laminin/fibronectina
16h 194.7 141.4 - -
36h 493.7 353.9 - -
72h 603.7 421.6 - -
120h 714.5 632.4 - -
To examine the role of integrin-mediated
signaling in the transformation process in a more
direct manner, islets were pre-incubated with the RGD
motif-containing GRGDSP peptide prior to embedding in
collagen. This reduced cystic transformation to 570 of
the control DMEM/F12-CT group (p<0.001) at 72 hours
(Fig. 14A). The control peptide, GRGESP, had little
influence on the transformation process. Pre-treatment
islets with either soluble fibronectin or laminin prior
to embedding, decreased cystic transformation to 500 of
control (p<0.01) at 72 hours (Fig. 14B). Cystic
transformation was further reduced to 330 of control,
when islets were pre-incubated with both GRGDSP and
laminin (p<0.001, Fig. 14C).
DISCUSSION
Differentiated cells usually maintain their
cellular specificities in the adult, where stability of
cellular phenotype is related to a cell's interaction
with its microenvironment. A perturbation or loss of
stabilizing factors, however, may induce cells to
change their commitment (Okada TS (1986) Develop Growth
Diff 28, 213-221). We have reported previously that
isolated islets of Langerhans embedded in type 1
collagen gel can be induced to undergo
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transdifferentiation to duct-like epithelial structures
(Yuan S et al. (1996) Differentiation 61, 67-75).
Little is currently known regarding the
molecular events involved in transdifferentiation.
Hence, the purpose of the present study was to
characterize the factors involved in this
transformation process in order to better understand
the functional relationships that confer morphogenetic
stability on cells in the isolated islet. Given the
rather poor long-term success rate of cell-based
therapies for diabetes mellitus, in particular islet
transplantation (Rosenberg L.(1998) Int'1 J
Pancreatology 24, 145-168), studies such as those
described here, could provide new insight into the
issues surrounding the problem of graft failure.
There were two principal findings. First, we
demonstrated that the process of cystic transformation
requires both an elevation of intracellular CAMP and
the presence of ECM proteins presented as a solid
support. Second, we determined that the formation of a
cystic structure from a solid islet sphere is a two-
staged process that involves a wave of apoptosis of
endocrine cells, followed by cell proliferation of the
new duct-like cells.
Signal transduction during transdifferentiation
has only recently become the subject of study,
therefore detailed information is unavailable. It
appears though, that cAMP-mediated information flow
plays an important role (Ghee M, Baker H, et al.
(1998) Mole Brain Res 55, 101-114; Osaka H, Sabban EL
(1997) Mole Brain Res 49, 222-228; Yarwood SJ et al.
(1998) Mole Cell Endocrinol 138, 41-50). In this study
we found that elevation of intracellular cAMP was a
necessary, but not a sufficient condition, for
induction of islet-to-cyst transformation. However, it
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was not simply the peak value of the increase in
intracellular CAMP that was important, rather it was
the duration of the elevation that was associated with
the highest frequency of duct epithelial
transformation. The increase in CAMP levels, like that
produced by medium supplemented with EGF alone or
forskolin alone, produced a less than maximal
transformation response. The longest duration of CAMP
elevation was obtained in medium supplemented with a
combination of EGF and CT. This is in keeping with Yao
et al. (Yao H, Labudda K, Rim C, et al. (1995) J Biol
Chem 270, 20748-20753), who demonstrated the need for
sustained versus transient signaling in cAMP-mediated
EGF-induced differentiation in PC12 cells. This
finding also serves to highlight the similarities
between pancreatic i~-cells and cells of neuronal origin
(Scharfmann R, Czernichow P (1997) Pancreatic growth
and regeneration. Pp170-182. Ed Sarvetnick N. Austin:
Karger Landes). Therefore, as in other systems (Yao H,
Labudda K, Rim C, et al. (1995) J Biol Chem 270, 20748
20753), the cellular responses of islet cells to growth
factor action may be dependent not only on the
activation of growth factor receptors by specific
growth factors, but on synchronous signals that elevate
intracellular signals like cAMP.
An increase in intracellular CAMP is of
interest too, because a rise in cAMP may form part of
the effector system controlling apoptosis in pancreatic
i3-cells (Loweth AC, Williams GT, et al. (1997) FEBS
Lett 400, 285-288). It is therefore noteworthy, that
cell loss due to apoptosis is the first step we
observed in the process of islet-to-cyst
transformation. That apoptosis should occur during
islet transformation in this system is interesting,
because the islets are embedded in a collagen gel, and
WO 01/32839 CA 02388208 2002-04-23 PCT/CA00/01284
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such a matrix has been reported to help promote or
maintain the differentiated state of different types of
cells in culture ( Foster CS et al. (1983) Dev Biol 96,
197-216; Yang J et al. (1982) Cell Biol Int 6, 969-975;
Rubin K et al. (1981) Cell 24, 463-470). On the other
hand, extracellular matrix may also promote the
process of transdifferentiation. This point is
emphasized by isolated pancreatic acinar cells that
transdifferentiate to duct-like structures when
entrapped in Matrigel~ (Arias AE, Bendayan M (1993) Lah
Invest 69, 518-530) , and by retinal pigment epithelial
cells, which transdifferentiate into neurons when
plated onto laminin-containing substrates (Reh TA et
al. (1987). Nature 330, 68-71). Most recently, Gittes
et al. (Gittes GK et al. (1996) Development 122, 439-
447) demonstrated, using 11-day embryonic mouse
pancreas, that the default path for growth of embryonic
pancreatic epithelium is to form islets. In the
presence of basement membrane constituents, however,
the pancreatic anlage epithelium appears to be
programmed to form ducts. This finding again
emphasizes the interrelationship between ducts and
islets and highlights the important role of the
extracellular matrix. Notwithstanding these
observations, the presence of a solid ECM support
appears to be a necessary, although not sufficient
condition, for the transformation of a solid islet to
a cystic epithelial-like structure, the first stage of
which, involves apoptotic cell death.
Conversion of a solid to a hollow structure is
a morphogenetic process observed frequently during
vertebrate embryogenesis (Coucouvanis E, Martin GR
(1995) Cell 83, 279-287). In the early mouse embryo,
this process of cavitation transforms the solid
embryonic ectoderm into a columnar epithelium
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surrounding a cavity. It has been proposed that
cavitation is the result of the interplay of two
signals, one from an outer layer of endoderm cells that
acts over a short distance to create a cavity by
inducing apoptosis of the inner ectodermal cells, and
the other a rescue signal mediated by contact with the
basement membrane that is required for survival of the
columnar cells (Coucouvanis E, Martin GR (1995) Cell
83, 279-287). The combination of these two signals
results in death of inner cells not in contact with the
ECM and survival of a single layer of outer cells in
contact with the basement membrane. A central feature
of this model is the direct initiation of apoptosis by
an external signal that causes cell death. The second
key feature of the model is a signal that appears to be
mediated by attachment to ECM and rescues cells from
cell death. There is after all, ample precedent for
cell dependence on ECM for survival (Meredith JE et al.
(1993) Mol Biol Cell 4, 953-961; Boudreau N, Sympson
CJ, et al. (1995) Science 267, 891-893). In our model
of islet-cystic transformation, the external death
signal is probably provided by those factors that
increase intracellular cAMP. Moreover, the observation
that cell loss during the process of transformation
occurs preferentially in the center of the islet lends
support to the notion that the ECM acts as a rescue
signal for those cells in the periphery. The precise
role of integrins in this process remains to be more
fully delineated. Integrin-ligand binding per se need
not contribute to the survival signal. For example,
integrins can modulate cell responsiveness to growth
factors (Elliot B et al. (1992) J Cell Physiol 152,
292-301).
One area not explored in the present study was
the reversibility of the process of transformation.
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Reversibility of transdifferentiation has been reported
in other cell systems (Erenpreisa J, Roach HI (1996)
Mechanisms of Aging & Develop 287, 165-182).
Transdifferentiation may involve cell proliferation and
the appearance of a multipotential dedifferentiated
intermediate cell (Yuan S et al. (1996) Differentiation
61, 67-75) which can express markers characteristic of
several alternative phenotypes. It is possible that
this is the case in our system (Yuan S et 'al. (1996)
Differentiation 61, 67-75). Thus, it may be possible to
expand a population of multipotential cells and then
induce guided differentiation to a desired cell
phenotype- in this case a mature insulin-producing f3-
cell. The in-vitro system employed in these studies was
unique for two reasons- it did not require fetal
tissue, and the starting tissue, adult islets, was well
def fined .
In summary, this study extends our previous
observation that adult islets of Langerhans can be
transformed into duct epithelial cystic structures by
a two-step process that involves apoptosis followed by
cell differentiation and proliferation. The precise
biochemical mechanism appears to involve, at least in
part, elevation of intracellular CAMP mediated by a
combination of cholera toxin and EGF, and a survival
signal contributed by a solid ECM support. The
differentiation potential of the cells comprising the
new epithelial structure remain to be fully
elucidated.
While the invention has been described in
connection with specific embodiments thereof, it will
be understood that it is capable of further
modifications and this application is intended to cover
any variations, uses, or adaptations of the invention
following, in general, the principles of the invention
WO 01/32839 CA 02388208 2002-04-23 PCT/CA00/01284
- 39 -
and including such departures from the present
disclosure as come within known or customary practice
within the art to which the invention pertains and as
may be applied to the essential features hereinbefore
set forth, and as follows in the scope of the appended
claims.
