Note: Descriptions are shown in the official language in which they were submitted.
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A MEDIUM TO PROMOTE ISLET CBLL ~UKVlVAL
RA~K~ROUND OF THE INVBNTION
(a) Field of the Invention
The invention relates to a culture medium which
promote islet cell survival, which may be transplanted
to reverse hyperglycemia.
(b) DescriPtion of Prior Art
Adequate numbers of isogeneic 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 months. 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.
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 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 and primates. 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,
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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. Unfortunately, micro-
encapsulated islets injected into the peritoneal cavityof 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 (Lanza RP et al., Diabetes, 1992,
41:1503). Histological evaluation indicates a
substantial loss of islet mass in these devices. No
reasons have been advanced for these changes.
Only whole pancreas transplantation is capable
of permanently reversing the diabetic state and
preventing the secondary complications of diabetes.
This suggests that there is a fundamental biological
difference between grafts of whole pancreas and those
of purified islets.
It is perhaps significant that the major
emphasis in islet transplantation has been the
enhancement of the purity of the islet preparation to
promote engraftment and reduce immunogenicity. After
more than 20 years of concerted research and more than
200 attempts at human islet transplantation, the
achievement of insulin independence remains elusive.
Contamination of the islet preparation by non-endocrine
cells does not impair function or engraftment, nor
enhance immunogenicity. In a canine model, pancreatic
tissue fragments autotransplanted into the spleen
appeared to survive better than islets refluxed into
the liver (Kretemom NM and Warnock GL, Transplantation,
1990, 49, 679). The survival advantage was attributed
to the specific site, but pancreatic fragments may
actually have an inherent survival advantage compared
to purified islets. In people, insulin-independence
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has been obtained after intraportal injection of
unpurified autologous islets. Fewer impure islets were
more successful than many more purified ones. A
similar experience was repeated with allotransplants,
with unpurified islets from a single human pancreas
successfully reversing hyperglycemia (Gores PF et al.,
Lancet, 1993, 341:19). From these and other reports,
it is apparent that unpurified islets from one pancreas
may survive as well as pure islets prepared from
multiple donors.
To date, the only known roles of apoptosis, or
programmed cell death, in the regulation of islet cell
mass are in the involution of ~ cell mass after
pregnancy and perhaps in remodeling during development
as recently suggested by Finegood (Finegood OT et al.,
Diabetes, 1995, 44:249). These situations, however,
are physiologic in nature and it remains to be
determined under what unusual circumstances islets may
also be induced to undergo apoptotic cell death. The
recent report of a selective decrease in ~ cell mass
following transplantation of human islets into diabetic
nude mice is therefore of considerable interest. The
induction of apoptosis in relation to islet isolation
and transplantation has not been reported previously.
We have original observations on human islets after
isolation. Light microscopic examination of these
islets just prior to culture demonstrates that at least
15% of the cells have morphological evidence of
apoptosis (pyknotic nuclei). Tissue transglutaminase
(TG) expression is a fundamental event in the induction
of apoptosis. TG is a calcium-dependent enzyme whose
activity is well established in many mammalian tissues,
including pancreas. It is involved in the cross-
linking of intracellular proteins that precedes the
irreversible ultrastructural changes characterizing
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cells undergoing apoptosis. The appearance of
apoptotic bodies parallels an increased expression of
TG. Our pilot studies have shown TG activity to be
elevated immediately following islet isolation, with
continued increase up to at least 1 week. These
findings correlated with data from a cell death ELISA
that detects histone-associated DNA fragments, another
characteristic of the apoptotic process (Paraskevas S.
et al., Transplant Proc., 1997, 29:750). These
fragments result from the activity of an endonuclease
that cleaves DNA at internucleosomal sites. These data
form the basis for investigating the role of apoptosis
in islet survival after isolation.
It would be highly desirable to be provided
with a means to prevent apoptosis of islet cells,
thereby allowing transplantation with the successful
reversal of hyperglycemia.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide
a culture medium which promote islet cell survival,
which may be transplanted to reverse hyperglycemia.
In accordance with the present invention there
is provided a culture medium for promoting the survival
of mammalian islet cells, which comprises at least an
effective amount of one or more growth factor having
anti-apoptosis effect of islet cells in a
physiologically acceptable culture medium.
The preferred growth factors include, without
limitation, NGF, IGF-I and insulin. Other growth
factors include TGF~, IGF-II and HGF.
The preferred NGF concentration is between
about 10 to about 100ng/ml of medium.
The preferred IGF-I concentration is between
about 10 to about 100ng/ml of medium.
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Preferably, the culture medium of the present
invention includes a immunosuppressant.
The preferred immunosuppressants are selected
from the group consisting of FK506 and a peptide having
NGF potentiating effect, and therefore possibly a
trophic effect on islet cells.
The preferred immunosuppressant concentration
is about 1 micromolar.
More preferably, the culture medium of the
present invention further include insulin. The
preferred insulin concentration is from about 10 to
about 100ng/ml of medium.
The term "physiologically acceptable culture
medium" is intended to mean any commercially available
culture medium including, without limitation, CMRL
1066, RAM 1640 and DMEM/F12.
The medium of the present invention may also be
used to isolate islet cells or to irrigate the site of
transplant to promote in si tu islet cell survival.
BRIEF nRS~RTPTION OF TH_ DRAWINGS
Fig. 1 illustrates an Agarose gel demonstrating
DNA laddering in islets incubated in a control medium
(control lane), compared to islets incubated with
insulin or NGF;
Fig. 2 illustrates TUNEL staining of porcine
islets, (A) islets incubated in the standard medium and
(B) islets incubated with insulin; and
Fig 3 illustrates a graph of the stimulation
of insulin secretion by a glucose challenge in vitro.
D_TATTRn DR-~C~TPTION OF TH_ INV_NTION
Growth factors act in an autocrine and/or
paracrine manner to mediate a broad range of cellular
responses, including ECM formation, cell proliferation
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and differentiation. The role of these factors in
islet cell survival in the pancreas is relatively
unexplored.
Hepatocyte growth factor (HGF) has effects on a
wide variety of epithelial cell types, including
pancreas, where it has been detected in acinar and
islet cells. It has no known role in the adult
pancreas. In comparison, Insulin-like growth factors
(IGFs) effect cell growth and differentiation in both
the fetal and adult pancreas. During induced islet
cell neogenesis in the adult hamster, we have found
that ~ cell differentiation from the ductal epithelium
was associated with a transient increase in IGF-II
expression, recapitulating the fetal environment as
characterized by Hill (Rafacloff R et al., Diabetes,
1993, 42(suppl.1):137A). This suggests a direct role
for IGF-II in the differentiation process in the adult.
Evidence for IGFs as survival factors has also been
reported. Similarly, recent findings support a role
for Nerve growth factor (NGF) in normal development,
morphogenesis and functional maturation of islets.
Greater uncertainty surrounds reg, a gene that is over-
expressed within pancreatic cells after pancreatitis or
resection, and particularly during islet cell
regeneration. We and others have found that the reg
gene is induced in our model of islet neogenesis.
Although it has been hypothesized that reg is an
important paracrine factor in the maintenance and
growth of ~-cells, the true significance of this gene
remains to be determined. Transforming Growth Factor
(TGF)-a is a mitogen expressed in pancreatic duct and
acinar cells, whose role in maintaining cell survival
is undefined. TGF-~ is also a potent regulator of cell
proliferation, but its major activity is to stimulate
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the synthesis and deposition of various ECM proteins
and to increase the expression of integrins. TGF-
~
expression in vivo may be important in the re-
establishment of basement membrane following its loss
during islet isolation, and hence in islet cell
survival.
Growth factors may also be inhibitory in almost
all situations in which apoptosis, or programmed cell
death, is positively stimulated. This suggests that
regulation of growth factor levels is not only
important in the control of cell proliferation but also
in maintaining viability of cells susceptible to
apoptosis. These data suggest, that for the success of
islet transplantation, the activity of survival factors
may be fundamental to the long term maintenance of
graft function.
The preferred Islet Survival Medium of the present
invention
The formulation of the preferred medium of the
present invention takes advantage of (1) newly
recognized effects of known growth factors on
inhibiting the induction of apoptosis in islet cells,
~2) the combination of these factors to achieve a
synergistic beneficial effect on islet cell survival,
and (3) the potentiation of this synergistic effect by
the addition of the drug FK506 (ProGraf~, Fujisawa).
The growth factors that have been combined are
NGF (nerve growth factor), IGF-I (insulin-like growth
factor-l) and insulin. None has been previously known
to play a role in the prevention of apoptosis.
Certainly, the role of NGF on islet tissue has only
recently been recognized with respect to islet cell
differentiation during normal fetal development. Its
known trophic effects on cells have been solely in
relation to neuronal cells. Similarly, IGF-I is active
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in islet cell differentiation during fetal development,
but a role in apoptosis has never been reported.
FK506 is a macrolide immunosuppressive agent
which acts by inhibiting T-cell activation. FK506 is a
ligand for the immunophilin FKBP12. The FK506-FKBP12
complex binds several targets, one of which is the
calcium calmodulin dependent phosphatase, calcineurin.
FK506 has been shown to potentiate the neurotropic
effects of NGF. The exact mechanism by which FK506
facilitates NGF activity is still unclear.
We have demonstrated that a CMRL 1066 medium (a
conventional islet culture medium) supplemented with
the above substances can significantly reduce the
amount of apoptotic cell death sustained by insulin-
producing ~-cells following islet isolation and
purification.
The evidence is based on four assays.
First, Western blots demonstrate that the
balance between activation of stress-activated protein
kinases (SAPK) including JNKl/JNK2 and p38 relative to
erkl/erk2, is altered. The former two pathways are
known to be associated with the induction of the
apoptotic program, while the latter may be protective.
Second, there is a significant reduction on
agarose gels of the DNA laddering that is
characteristic for the DNA fragmentation that is the
outcome of apoptosis (Fig. 1).
Third, TUNEL staining (another technique to
visualize apoptotic events) of actual isolated islets,
demonstrates a significant reduction in the number of
apoptotic cells (Fig. 2).
Finally, in vitro functional studies
demonstrate that islets cultured in the presence of
IGF-I, NGF and insulin have a much improved insulin
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g
secretory response to a glucose challenge (the hallmark
of ~-cell function) (Fig. 3).
While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any varia-
tions, uses, or adaptations of the invention following,
in general, the principles of the invention 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.
