U.S. patent application number 10/386759 was filed with the patent office on 2003-09-18 for platform for the differentiation of cells.
This patent application is currently assigned to McGill University. Invention is credited to Rosenberg, Lawrence.
Application Number | 20030175963 10/386759 |
Document ID | / |
Family ID | 22380754 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175963 |
Kind Code |
A1 |
Rosenberg, Lawrence |
September 18, 2003 |
Platform for the differentiation of cells
Abstract
The present invention relates to an in vitro method for islet
cell expansion, which comprises the steps of: a) preparing
dedifferentiated cells derived from cells in or associated with
post-natal islets of Langerhans; b) expanding the dedifferentiated
cells; and c) inducing islet cell differentiation the expanded
cells of step b) to become insulin-producing cells.
Inventors: |
Rosenberg, Lawrence; (Cote
St.-Luc, CA) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
McGill University
Montreal
CA
|
Family ID: |
22380754 |
Appl. No.: |
10/386759 |
Filed: |
March 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10386759 |
Mar 11, 2003 |
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09890717 |
Aug 3, 2001 |
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09890717 |
Aug 3, 2001 |
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PCT/CA00/00105 |
Feb 2, 2000 |
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60118790 |
Feb 4, 1999 |
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Current U.S.
Class: |
435/375 ;
435/455 |
Current CPC
Class: |
C12N 2506/22 20130101;
C12N 2501/33 20130101; A61P 5/48 20180101; A61P 3/10 20180101; A61K
35/12 20130101; C12N 5/0676 20130101; C12N 2501/13 20130101; C12N
2501/105 20130101; C12N 2501/58 20130101 |
Class at
Publication: |
435/375 ;
435/455 |
International
Class: |
C12N 005/02; C12N
015/85 |
Claims
What is claimed is:
1. An in vitro method for producing cells with at least
bipotentiality, which comprises the step of: a) inducing cystic
formation in cells in or associated with post-natal islets of
Langerhans obtained from a patient to obtain a duct-like
structure.
2. A method for the treatment of diabetes mellitus in a patient,
which comprises the steps of: a) inducing cystic formation in
post-natal islets of Langerhans cells obtained from said patient to
obtain a duct-like structure; and b) introducing said duct-like
structure cells in situ in said patient, wherein said cells are
expanded in situ to become insulin-producing cells.
3. A method for the treatment of diabetes mellitus in a patient,
which comprises the steps of: a) inducing in vitro cystic formation
in post-natal islets of Langerhans cells obtained from said patient
to become insulin-producing cells; and b) introducing said cells of
step a) in situ in saidpatient, wherein said cells produce insulin
in situ.
Description
BACKGROUND OF THE INVENTION
[0001] (a) Field of the Invention
[0002] The invention relates to an in vitro method for islet cell
expansion; an in vitro method for producing multi bipolar cells; an
in vitro method for stem cell expansion; and a method for the
treatment of diabetes mellitus in a patient.
[0003] (b) Description of Prior Art
[0004] Diabetes Mellitus
[0005] 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.
[0006] Rationale for More Physiologic Therapies
[0007] 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 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.
[0008] Other Therapeutic Options
[0009] 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.
[0010] A major goal of diabetes research, therefore, has been the
development of new forms of treatment that endeavor to reproduce
more closely the normal physiologic state. One such approach, a
closed-loop insulin pump coupled to a glucose sensor, mimicking
.beta.-cell 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 injections, 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.
[0011] 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 F H et al., Arch. Surg.,
1989;124:796-799; Landgraft R et al., Diabetologia 1991; 34 (suppl
1):S61; Morel P et al., Transplantation 1991; 51:990-1000), raising
questions about their protection against the long-term
complications of chronic hyperglycemia.
[0012] 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 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.
[0013] The Problem of Islet Transplantation
[0014] 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 R G, et al. In: Bretzel R G,
(ed) Diabetes mellitus. (Berlin: Springer, 1990) p.229).
[0015] 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 (Warnock G L
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.
[0016] 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 (Lanza R P, 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.
[0017] 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 required to effectively reverse
hyperglycemia in a human recipient.
[0018] 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.
[0019] 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 signal(s) that would drive the whole process could be
identified and manipulated.
[0020] In a 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.
[0021] The well known teachings of in vitro islet cell expansion
from a non-fetal tissue source comes from Peck and co-workers
(Corneliu J G 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.
[0022] More recently two other competing technologies have been
proposed the use of engineered pancreatic .beta.-cell lines (Efrat
S, Advanced Drug Delivery Reviews, 1998;33:45-52), and the use of
pluripotent embryonal stem cells (Shamblott M J 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 appealing because of the theoretical possibility of being able
to induce differentiation in any direction, including toward the
pancreatic .beta.-cell. However, the signals necessary to achieve
this milestone remain unknown.
[0023] It would be highly desirable to be provided with a platform
for the preparation of dedifferentiated 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
[0024] One aim of the invention is to provide a platform for the
preparation of dedifferentiated cells derived from cells in or
associated with 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.
[0025] 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:
[0026] a) preparing dedifferentiated cells derived from cells in or
associated with post-natal islets of Langerhans;
[0027] b) expanding the dedifferentiated cells; and
[0028] c) inducing islet cell differentiation of the expanded cells
of step b) to become insulin-producing cells.
[0029] 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 or
being multipotent.
[0030] 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.
[0031] Preferably, the basal liquid medium is CMRL 1066
supplemented with 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.
[0032] In accordance with another embodiment of the present
invention there is provided an in vitro method for producing cells
with at least bipotentiality, which comprises the steps of:
[0033] a) preparing dedifferentiated cells derived from cells in or
associated with post-natal islets of Langerhans from a patient;
whereby when the dedifferentiated cells are introduced in situ in
the patient, the cells are expanded and undergo islet cell
differentiation to become in situ insulin-producing cells.
[0034] 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
[0035] a) preparing dedifferentiated cells derived from cells in or
associated with post-natal islets of Langerhans of the patient;
and
[0036] b) introducing the dedifferentiated cells in situ in the
patient, wherein the cells expand in situ and undergo islet cell
differentiation in situ to become insulin-producing cells.
[0037] 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
[0038] a) preparing dedifferentiated cells derived from cells in or
associated with post-natal islets of Langerhans of the patient;
[0039] b) expanding in vitro the dedifferentiated cells;
[0040] c) inducing in vitro islet cell differentiation of the
expanded cells of step b) to become insulin-producing cells;
and
[0041] d) introducing the cells of step c) in situ in the patient,
wherein the cells produce insulin in situ.
[0042] For the purpose of the present invention the following terms
are defined below.
[0043] The expression "post-natal islets of Langerhans" is intended
to mean islet cells and associated cells, such as duct cells, of
any origin, such as human, porcine and canine, among others.
[0044] The expression "dedifferentiated cells" is intended to mean
cells of any origin which are stem-like cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] 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.
[0046] 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. (d-e)
Progressive loss of islet phenotype. (f) High power view of cyst
wall composed duct-like epithelial cells. One cell still contains
insulin (arrow).
[0047] 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. (d-e)
Progressive loss of islet phenotype. (f) High power view of cyst
wall composed duct-like epithelial cells. One cell still contains
glucagon (arrow).
[0048] FIG. 4 illustrates 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-10
immunoreactivity. The solid component has lost its CK-19
expression, and appears islet-like.
[0049] FIG. 5 illustrates 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.
[0050] FIG. 6 illustrates 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.
[0051] 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.
DETAILED DESCRIPTION OF THE INVENTION
[0052] In vivo cell transformation leading to .beta.-cell
neogenesis and new islet formation can be understood in the context
of established concepts of developmental biology.
[0053] Transdifferentiation is a change from one differentiated
phenotype to another, involving morphological and functional
phenotypic markers (Okada T S., 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 T S, Cell Diff.
1983;13:177-183; Okada T S, 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 W A, 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 S I 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
T S, Develop. Growth and Differ. 1986;28:213-321).
[0054] 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 T S, Develop. Growth and Differ,
1986;28:213-321).
[0055] 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 expression, which
controls the terminal developmental commitment of cells.
[0056] 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 M S et al., Am. J. Pathol., 1983;110:89-94;
Scarpelli D G, Rao M S, Proc. Nat. Acad. Sci. USA
1981;78:2577-2581) to hamsters and the feeding of copper-depleted
diets to rats (Rao M S, 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 A E,
Bendayan M, Lab Invest., 1993;69:518-530; Hall P A, Lemoine N R, J.
Pathol., 1992;166:97-103; Tsao M S, Duguid W P, 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.
[0057] An alternative to transdifferentiation, is the possibility
that new islet cells arise from stem cells that persist
post-natally in adult tissue.
[0058] There are two general categories of stem cells (Young H E et
al. PSEBM 1999; 221:63-71; Young H E et al. Wound Rep Regen 1998;
6:65-75). Progenitors are (a) lineage committed (i.e. they will
form only tissues within their respective committed lineages(s));
(b) they prefer to remain quiescent and therefore need to be
actively stimulated or challenged to do anything; (c) their
life-span is approx. 50-70 cell divisions before programmed cell
death intervenes; (d) they are unresponsive to inductive agents
outside their lineage; and (e) they are responsive to progression
agents (e.g. insulin, IGF-1 or IGF-2) which are needed to promote
phenotypic expression into lineage restricted phenotypes only.
Pluripotents, on the other hand, are lineage uncommitted, derived
from the inner cell mass of the blastocyst.
[0059] Within these two broad categories, there are four types of
cells--(1) the totipotent stem cell; (2) the pluripotent stem cell;
(3) the multipotent stem cell, and (4) the unipotent stem cell.
Multipotent cells (committed to two or more cell lineage, e.g.
chondro-osteogenic, adipo-fibrogenic) and unipotent cells
(committed to a single tissue lineage, e.g. myogenic, adipogenic,
osteogenic), are considered to be progenitor cells. To date,
progenitor cells have been identified from six species thus far,
and also from fetal to geriatric aged individuals. It is quite
possible, therefore, that islet cell differentiation post-natally
may occur as a result of the stimulation of a unipotent or
multipotent progenitor cell as opposed to transdifferentiation.
[0060] One example of such a mechanism can be observed in the
liver. Hepatic oval cells are a small sub-population of cells found
in the liver when hepatocyte proliferation is inhibited and
followed by some type of hepatic injury. They are believed to be
bipotential, able to differentiate into into hepatocytes or bile
duct epithelium. They express the same. markers as hematopoietic
stem cells (HSC), and evidence has been obtained that these cells
can be derived from a bone marrow source (Petersen B E, et al.
Science 1999;284:1168-1170). In this context, it is quite possible
that the hepatocyte-like cells identified in the pancreas, to which
we referred above. (Rao M S et al., Am. J. Pathol., 1983;110:89-94;
Scarpelli D G, Rao M S, Proc. Nat. Acad. Sci. USA
1981;78:2577-2581; Rao M S, et al., Cell Differ., 1986;18:109-117),
may have in fact been derived from the equivalent of oval cells in
the pancreas.
[0061] 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 R L
et al. In: Extracellular Matrix Influences on Gene Expression.
Slavkin H C, Greulich R C (eds). Academic Press, New York, 1975,
pp.10).
[0062] Some information is available on exocrine growth factors.
Mesenchymal Factor (MF), has been 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 (Rutter W J. The development of the endocrine and
exocrine pancreas. In: The Pancreas. Fitzgerald P J, Morson A B
(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 (Rutter W J, In: The Pancreas. Fitzgerald P J, Morson A B
(eds). Williams and Wilkins, London, 1980, pp.30). In the presence
of MF, cells divide actively and differentiate largely into
non-endocrine cells.
[0063] 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 A S, Leof E B, et al. Cancer Res., 46:1015-1029, 1986;
Underwood L E, et al., Clinics in Endocrinol. & Metabol.,
15:59-77,1986). Specifics of their role in the individual processes
that comprise growth need to be resolved.
[0064] One family of growth factors are the somatomedins.
Insulin-like growth factor-I (IGF-I), is synthesized and released
in tissue culture by the .beta.-cells of fetal and neonatal rat
islets (Hill D J, et al., Diabetes, 36:465-471, 1987; Rabinovitch
A, et al., Diabetes, 31:160-164,1982; Romanus J A et al., Diabetes
34:696-792, 1985). IGF-II has been identified in human fetal
pancreas (Bryson J M et al., J. Endocrinol., 121:367-373,1989).
Both these factors enhance neonatal .beta.-cell replication in
vitro when added to the culture medium (Hill D J, 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
.beta.-cell replication in fetal and neonatal rat islets but may
not do so in post-natal development (Billestrup N, Martin J M,
Endocrinol., 116:1175-81,1985; Rabinovitch A, et al., Diabetes,
32:307-12, 1983; Swenne I, Hill D J, Diabetologia 32:191-197, 1989;
Swenne I, Endocrinology, 122:214-218, 1988; Whittaker P G, 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 J A et al., Diabetes
34:696-792, 1985; Swenne I, Hill D J, 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 .beta.- 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., 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.
[0065] 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 C M et al., Dev.
Biol. 1991;148:322-333; Pittack C et al., Development
1991;113:577-588). Transforming growth factor-beta (TGF-.beta.) 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).
[0066] The search for the factors mediating cell differentiation
and survival must include both the cell and its microenvironment
(Bissell M J 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 A W 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 development is highlighted by the role of
fetal mesenchyme in epithelial cell cytodifferentiation (Bencosme S
A, Am. J. Pathol. 1955; 31: 1149; Gepts W, de Mey J. Diabetes 1978;
27(suppl. 1): 251; Gepts W, Lacompte P M. 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.
[0067] 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 E D.
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
J T, Pitelka D R. 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 J T, Pitelka D R. 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 E D. Curr.
Opin. in Cell. Biol. 1993; 5:1029).
[0068] During times of tissue proliferation, and in the presence of
the appropriate growth factors, cells 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 D E. Proc. Natl. Acad. Sci. USA, 1990;87:3579; Roskelley C
D et al., Proc. Natl. Acad. Sci. USA, 1994;91:12378).
[0069] 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 R C, Lincz L F, Burns G F, Cancer
and Metastasis Rev., 1995;14:19). Disruption of the cell-matrix
relationship leads to apoptosis (Frisch S M, Francis H. J. Cell.
Biol., 1994;124:619; Schwartz S M, Bennett M R, Am. J. Path.,
1995;147:229), a morphological series of events (Kerr J F K et al.,
Br. J. Cancer, 1972;26:239), indicating a process of active
cellular self destruction.
[0070] In accordance with one embodiment of the present invention,
the platform technology is based on a combination of the foregoing
observations, incorporating the following components that are
necessary and sufficient for the preparation of dedifferentiated
intermediate cells from adult pancreatic islets of Langerhans:
[0071] 1. a solid matrix permitting "three dimensional"
culture;
[0072] 2. the presence of matrix proteins including but not limited
to collagen type I and laminin; and
[0073] 3. the growth factor EGF and promoters of cAMP, including
but not limited to cholera toxin and forskolin.
[0074] 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.
[0075] 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 A E, 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 T A et al., Nature 1987;330:68-71). Most recently,
Gittes et al. demonstrated, using 11-day embryonic mouse pancreas,
that the default path for growth of embryonic pancreatic epithelium
is to form islets (Gittes G K 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.
[0076] 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 A E, 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 cells. Cyst enlargement after the initial 96 h is
associated, at least in part, with a tremendous increase 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).
[0077] Stage 2--the generation of functioning .beta.-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 10% fetal calf serum,
with the addition of soluble matrix proteins and growth factors
that include, but are not limited to, fibronectin (10-20 ng/ml),
IGF-1 (100 ng/ml), IGF-2 (100 ng), insulin (10-100 .mu.g/ml), NGF
(10-100 ng/ml). In addition, the glucose concentration must be
increased to above 11 mM. Additional culture additives may include
specific inhibitors of known intracellular signaling pathways of
apoptosis, including, but not limited to a specific inhibitor of
p38.
[0078] 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.
[0079] The present invention will be more readily understood by
referring to the following examples which are given to illustrate
the invention rather than to limit its scope.
[0080] 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 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.
* * * * *