Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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EXTRACEILULAR MATRIX INDUCTION METHOD TO PRODUCE
PANCREATIC ISLET TISSUE
BACKGROUND OF THE INVENTION
Embryonic development of the pancreas
involves an epithelial endoderm budding from the primitive gut tube
into surrounding mesenchyme (splanchnic mesoderm). The process is
described in detail in the publication by R. Pictet and W. Rutter,
HhNDBOOK OF PHYSIOLOGY, Vol. 1, Section 7, Endocrinology:
Endocrine Pancreas, D. Steiner and N. Freinkel, edsO, Amer.
Physiol. Soc. L972, pp. 25-660 The sequential
epithelial/mesenchymal interactions results in the formation of
both exocrine and endocrine pancreas. Later in adult life, it is
believed that very little endocrine neogenesis occurs in the
pancreas. This has led to the conclusion of sorne investigators in
the diabetes field to consider the beta cell (the beta cell is the
source cell for insulin) a "terminal" cell. A terminal cell is a
fully differentiated cell that exhibits very little mitosis in
adult life, and consequently, at birth, an individual has the full
complement of these cells. The terminal nature of the beta cell
has far reaching irnplications in the field of diabetes since be~a
cell destruction or functional impairment results in a devastating
clinical situation without any hope of new, healthy~ beta cell
formation occurring to alleviate the disease.
~ In the past, many investigators have studied the effects of
various conditions on the mitotic activity of pre-existing beta
~ cells in the pancreas. These conditions have included the effect
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of diet, hormones, partial pancreatectomy, diabetogenic chemicals,
genetic manipulation, sulfonylureas, insulin antibody, glucose, and
amno acids. See, for example, J. Logothetopoulos, HANDBOOK OF
PHYSIOLOGY~ Vol. 1~ Section 7, Endocrinology: Endocrine Pancreas,
D. Steiner and N. Freinkel, eds., Amer. Physiol. Soc. 1972, pp.
67-76. The crux of these studies has been to increase the mitotic
activity of pre-existing beta cells. None of these studies has
dealt with the concept of the neoformation or regeneration of islet
beta cells from non-islet entities (islets are groups of beta
cells).
~any investigators have found that in vitro culture of fetal
pancreas exhibits a high rate of beta cell mitosis and also some
evidence of neoformation of beta cells from "budding islets" which
seems to result from true neogenetic process (morphogenesis and
histogenesis); et al., Morphological Study of Cultured Pancreatic
Fetal Islets: Diabetes, Vol. 29, Jan. 1980, pp. 16-21. In
addition, Archer and Jai (U~S. Patent No. 4,439,521; March 27,
1984; ~ethod for Producing Self~Reproducing Mammalian Pancreatic
Islet-like Structures) describe a method for producing pancreatic
islet-llke structures using ln vitro culturing techniques. They
have been able to increase islet tissue using standard tissue
culture technology of pancreatic islets, pancreatic duct pieces,
cell clusters of pancreas, cell tissues obtained as by products of
the culturing method, or previously produced islet-like structures.
The invention of this application relates to a method and
composition for regenerating cells in a post natal subject~ cells
which do not normally regenerate apart from fetal development.
Specifically, islet cells of the pancreas may be regenerated.
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SUMMARY OF THE INVENTION
In accordance with this invention, a method is provided which
comprises implanting into a living subject an effective amount of
fetal mesenchyme cells of a type and age capable of eliciting a
desired biological response from other fetal cells. As the result
of such implantation, the biological response is elicited in the
cells of the living subject. Thus, by this method, cells in the
body of an adult may be stimulated by the implantation of the
proper mesenchyme cells to behave in a manner which is abnormal for
adult cells~ but which is typical behavior for precursor cells to
those adult cells in the fetus. By this technique, desired growths
and transformations of living adult cells can take place in a
manner which is analogous to fetal growth; speciFically, the
process involves reawakening dormant adult "precursor" epithelial
cells by use of temporally spacially limited fetal mesenchyme.
For example~ new pancreatic islet cells which secrete insulin
have been formed in accordance with this invention from ductal
epithelial cells of the pancreas, thus providing hope for a cure of
diabetes in patients by means of this invention. Another hopeful
possibility that may be accomplished by the method of this
invention is the eliciting of nerve cell regrowth to repair nerve
damage in patients by the use of an appropriate selection of fetal
mesenchyme cells. Also, wound healing may be s~imula~ed, with
epidermal regeneration, for example. Small body structures such as
parts of the eye, heart, or inner ear may hopefully be regrown in
post-natal subjects by this invention.
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Mesoderm (source of this mesenchyme) is that tissue which
derives i~s origin from the middle layer of the early embryo. It
has been previously observed that~ during the development of the
embryo, mesenchyme at various times exerts an influence on other
cells to cause the other cells to multiply and/or transform into
new tissues in a manner which appears to be induced by the
mesenchyme.
In accordance with this invention, the broad concept is to
select and implant mesenchyme from a fetus of the exact type and
the exact age at which such cells are exerting their in~luence on
nearby fetal epithelial cells to exhibit a normal biological
response as another step in the development of the fetus. For
example, the formation of islet cells out of pancreatic ductal
epithelium, or the growth of nerves, are two possiblities for
biological responses that appear to be governed by certain fetal
mesenchyme tissues at certain times in the normal development of
the fetus. By this invention, the very fetal mesenchyme which
elicits such a response from other tissues is thus transplanted
into a post-fetal living subject, with the fetal mesenchyme being
transplanted into a site in the patient where they can survive for
a long time enough to perform their influence on tissues of the
living subject, which tissues are preferably adjacent the
transplantation site.
Alternatively, equivalent to mammalian fetal mesenchyme
include tissue from lower animal forms on the phylogenetic scale
that have known regenerative capability, such as salamander,
li~ard9 or crayfish, for example for limb regeneration.
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As stated above, it has been found that new pancreatic islet
cells can be formed from ductal epithelium in adult living subjects
(specifically rats) and that such islet cells can function normally
to suppress symptoms of diabetes in such subjects.
Specifically, the fetal mesenchyme used herein may comprise
pancreatic bud tissue from a fetus, (composed of fetal mesenchyme
plus fetal epithelium) or, alternatively, fetal mesenchyme cells
from an 11 to 14 day old ~etus, i.e.S those particular cells which
are believed by embryologists to have a role in the formation of
~0 the pancreatic islet cells in the rat fetus.
Additionally, fetal mesenchyme may be implanted while in
recombinant, a relationship of fetal mesenchyme with adult
epithelial cells of the living subject which are capable of
exhibiting the biological effect as elicited by the fetal
mesenchyme. The fetal mesenchyme is isolated at such time in
development when the inductive activity of the mesenchyme is
available. Specifically, 11 to 14 day old rat fetus mesenchyme may
be formed into recombinants with pancreatic adult ductal epithelial
tissue. After the recombinant tissue body has formed, they may be
transplanted subcutaneously into a nude mouse, creating a living
cell body in the mouse that forms insulin-containing beta cells.
The location of the implantation is preferably adjacent to
the cells of the living subject in which the biological response is
to be elicited. Accordingly, where nerve regrowth is desired, the
implantation shoud take place at the injury site, from which scar
tissue is preferably cleared away to provide an open path for the
nerve regrowth and rejoining. In the instan~e where it is desired
to form new islet tissue in the pancreas, the fetal mesenchyme may
be implanted in the omentum of the living
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subject. The importance of where fetal mesenchyme is implanted
raises the distinct possiblity of an inducting~ trophic, and/or
mitogenic factor being secreted from the mesenchymal tissue. This
factor could be isolated and injected into the individual directly
without the need to implant the cellular mesenchymal component.
Consequently, this invention additionally directs itself towards
regenerating pancreatic islets by injecting an isolated mesenchymal
factor.
The present invention is also directed toward a method to
cause the neo-formation or regeneration of pancreatic islet tissue
or other tissues using the induction capacity of an extracellular
matrix upon a ductal epithelium. The extracellular matrix is
isolated at such time in development when the inductive activity of
the mesenchyme is available. One purpose of the method is to
provide a treatment for diabetes by replacing beta cells that have
either been destroyed or are functionally impaired. The method is
unique in that all diabetics have ductal epithelium in their
pancreas that is capable of being induced by an extracellular
matrix to produce islet tissue.
A further purpose of the invention is to cause neoformation
or regeneration of pancreatic islet tissue that is histocompatible
with the diabetic patient so that no immune response or rejection
occurs when the regenerated islet tissue is subsequently
transplanted back into the diabetic patient. Present
transplantation procedures for the treatment of diabetes have the
rejection of the transplanted islet tissue as a major obstacle to
overcome. The described method minimizes the issue of immune
rejection since the regenerated islet tissue comes from the
diabetic patient's own epithelium and consequently should have the
same genotype and/or histocompatible antigens, thus alleviating the
immune response.
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The method described herein dif~ers from previous
attempts to increase islet beta cell numbers by (a)
employing the use of fetal mesenchyme acting upon an
epithelium in order to redirect the differentiation
process in a manner that results in the neoformation of
i~let tissue and (b) using a non-islet entity ductal
epithelium from which to rsgenerate the islet tissue.
Cther aspects of this invention are as follows:
A method for producing functional pancreatic islet
tissue comprising interacting fetal mesenchyme with a
substantially pure ductal epithelium under conditions
necessary for growth or maintenance of mammalian cells,
said conditions necessary for growth or maintenance of
mammalian cells comprising an ln vitro culture system.
A method of obtaining substantially pure pancreatic
epithelium which comprises digesting a selected
pancreas, separating the duct from the pancreas,
digesting the duct, and separating the epithelium from
the æurrounding connective tissue to remove any adherent
adult stroma and thereby obtain a substantially pure
epithelium.
A composition which generates pancreatic islet
cells comprising ~etal mesenchyme and substantially pure
adult ductal epithelium.
A composition which generates pancreatic islet
cells in an adult living subject comprising fetal
mesenchyme o~ a type and age to alicit a biological
response in other fetal cells, for implanting into said
living subject to elicit the same biological response in
said adult living subject.
BRIEF D~SCRIPTION O~ T~E DRAWINGS
The invention will be better understood by
reference to the followi~g detailed description when
considered in connection with the accompanying drawings
wherein:
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7a
Figure 1 is a schematic flow diagram of a method of
producing regeneration of pancreatic islet tissue.
Figure 2 is a schematic flow diagram of a method of
producing regeneration of pancreatic islet tissue in situ.
DETAIL~D D~SCRIPTION OF T~ INVENTI~N
A specific embodiment of the present invention
involves the interaction of an extracellular matrix,
such as ~etal mesenchyme with a pancreatic ductal
epithelium to produce pancreatic islet tissue. It is
applicable, however, to any tis~ue having a ductal
element and ~ontemplates using a patient's own tissue as
a source of transplantable differentiated tissua. The
extracellular matrix or mesenchymal can be obtained from
a fetal source, a lower phylogenetic source, or from
chemically defined material, isolated from biological
sources, chemically synthesized or genetically
engineered.
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More specifically, in a method of producing pancreatic islet
tissue, the method of the invention involves the recombination of
substantially pure adult ductal epithelium with fetal duodenal
mesenchyme as the extracellular matrix. Mesenchyme is an embryonic
tissue consisting of mesenchymal cells and an extracellular matrix
which is composed of fibers, proteins, proteoglycans and
glycosaminoglycans. The mesenchymal cells are supported in the
extracellular matrix of the mesenchyme. An extracellular matrix
may consist of any or all of the following: a protein lattice
network; differentiation factors secreted by cells or tissues
involved in directing such differentiation, or cells or tissues
which, by virtue of cell to cell contact, direct differentiation.
Although it is hypothesized that a pool of islet precursor
cells exists throughout life, it is believed that the islet
cytodifferentiation from pancreatic ductal epithelium by means of
the described method is a consequence of a true reawakening of
fetal differentiation. It has been determined that the presence of
a substantially pure pancreatic ductal epithelium is essential for
islet cytodifferentiation to occur. Any residual adult stroma
adhering to the epithelium was found to prevent
cytodifferentiation. It is believed the adherent adult stroma
acted as a barrier and inhibited interaction between fetal
mesenchyme and adult epithelium, thus preventing
cytodifferentiation. Even if an islet progenitor cell population
is present in adult ductal epithelium, islet renewal does not
significantly occur in the adult. Electron dense cells~ considered
to be endocrine progenitors, occur in ducts of developing pancreas,
but no such cells have been observed in adult pancreatic ductal
epithelium, Since only epithelium stripped cleanly from adult
stroma ("substantially pure epithelium") was
recombined with mesenchyme, the chance of pre-existing islets
attached to the periphery of the duct contaminating the epithelium
is minimal.
The invention further provides a method of preparing
substantially pure epithelium, factilitating the removal of adult
stroma which could block tissue differentiation as well as a method
of identifying and preparing adult ductal epithelium suitable for
use in tissue recombination.
The interaction of extracellular matrix and ductal epithelium
can occur ln vitro or in vivo under conditions necessary for growth
and/or maintenance of the mammalian cells. An in vitro culturing
system would consist of a medium, serum, serum substitute and/or
other ingredients to promote the growth and maintenance of
mammalian cells. The culturing system may consist of a vessel
treated with a variety of substances that will create a lattice or
environment on which the interaction and/or regeneration of islet
tissue may occur. In vivo, a transplantation site into a human or
animal would serve to supply the nutrients, gases, blood, nerve
supply, and any other ingredients necessary for the growth or
maintenance of mammalian cells. The human or animal may be a
nondiabetic or diabetic host.
The human or animal donating the epithelium to be induced by
the extracellular matrix to produce pancreatic islet tissue may
then be transplanted back with the regenerated islets, and the
islets will be functional and substantially ~ree from immune
rejection.
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EXAMPLE 1
The method for the regeneration of pancreatic islet tissue
using an extracellular matrix and epithelium can be described as
follows and with reference to the flow diagram of Figure 1. A pure
adult rat ductal epithelium was isolated by digesting the chopped
adult rat pancreas with collagenase (3 mg/pancreas~ in Hank's
buffered saline salt solution, pH 7.4, containing 0.02% fat-free
BSA, 0.01% soybean trypsin inhibitor, and 0.5% EDTA. The pancreas
was shaken for 15 minutes at 37 degrees C. or incubated without
shaking for one hour at 4 degrees C. Subsequently9 both large and
small ducts were picked from the chopped digested tissue and
incubated in 1% Diffco trypsin in Hank's buffered salt solution (no
additions) for one hour at 4 degrees C. The duct was cut open and
the epithelium carefully stripped away from the surrounding
connective tissue thereby obtaining a "pure" epithelium.
The extracellular matrix used in this method was derived from
fetal mesenchyme. The fetal mesenchyme was isolated by dissecting
the 14-day rat fetus in 20% fetal bovine serum (FBS) and incubating
the stomach-duodenal portion in l% Diffco trypsin for 3 hours at 4
degrees C. After incubation, the trypsin solution was removed and
50% FBS was added. After combining the fetal mesenchyme and
epithelium together (hereafter termed recombinant)g the recombinant
was placed in a 95~ air/5~ C02 humidified atmosphere at 37
degrees C. for 48 hours on a 1% bactoagar gel culture containing
RPM1 1640 culture medium. The recombinant was marked with bone
charcoal to aid in recovery and transplanted subcutaneously into a
nude mouse for a six-week period. Mesenchyme alone, duc~al
epithelium alone, intact fetal pancreatic bud, and
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isolated adult pancreatic islets were also transplanted into other
nude mice as controls, After six weeks, transp1anted tissue was
harvested and (a) fixed in Bouin's for light microscopy and
immunocytochemistry, (b) fixed in 2% paraformaldehyde-2.5%
glutaraldehyde in 75 mM cacodylate buffer for one hour on ice
followed by 1% osmium tetroxide in 100 mM cacodylate buffer for
electron microscopy, or (c) sonicated and extracted in acid ethanol
for 24 hours at 4 degrees C for radioimmunoassay of insulin. No
tissue other than the transplanted tissue was analy7ed.
A total of 57 recombinants were processed. Twelve of 23
(52%) contained immunoassayable insulin; 10 out of 17 (59%) showed
by light microscopy histological evidence of extracellular
matrix/epithelium activation with development of islets; and 3 of
the above 10 (30%) were confirmed to be islets as indicated by the
presence of insulin-containing beta cells. The remaining 17
recombinants were processed for electron microscopy and they
indicated intimate association of the extracellular matrix with the
epithelium, thus producing pancreatic islet tissue.
The range oF weights and insulin content of the harvested
tissue are recorded in Table 1. The weight and insulin content of
the transplanted tissue were highly variable and probably reflects
the amount of tissue that was originally isolated and
transplanted. Extracts of 12 recombinants were assayed at various
dilutions and the insulin content was found to be parrallel with
the standard curve for rat insulin.
Light microscopic evaluation of the recombinants showed
clusters of cells closely resembling islet tissue and containing
insulin immunoreactivity as indicated by immunocytochemistry.
Electron microscopic analysis of the r~combinants demonstrated an
interaction between the extracellular matrix and the epithelium
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which seems to be a hallmark for islet tissue regeneration in this
method and cells containing secretory granules typical of islet
beta cells.
TABLE 1
Transplanted ~et ~eight (mg) Insulin (ng/mg of
Tissue N Range Tissue) Range
Recombinants 12 1.1 - 15.4 0.11 - 20.4
Islets 7 4.8 - 20.0 0.01 - 2.67
Pancreatic bud 3 0.8 -- 17.1 0.05 - 11.9
Mesenchyme 7 0.~ - 13.7 0*
Duct 6 0* 0**
* No insulin detectable by RIA
** No identifiable transplanted tissue could be recovered for
weight and extraction after 6 weeks.
EXAMPLE 2
This example is directed toward a method of in situ
regeneration of islet tissue by transplanting recombinant tissue
into an appropriate transplantation site. This method for
reyeneration of pancrecitic islet tissue can be described as
follows and with reference to the flow diagram of Figure 2.
A diabetic colony of Sprague-Dawley rats was established by
injecting 50 mg/kg body weight of alloxan intravenously~ The blood
ylucose values were monitored periodically to document the
hyperglycemic condition. All rats had elevated blood glucose
values for a least one week before receiving a transplant.
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Four noninbred alloxan diabetic rats have been transplanted
with recombinant tissue made in the manner of Example 1. The
tissue was implanted into the omentum of each rat~ After such
transplantation, all four of the diabetic rats showed norma1ization
of blood glucose levels within seven days of the transplantation.
In addition, both serum insulin and ~n situ pancreas insulin levels
were elevated in comparison to a control diabetic rat. Thus, it
appears that the above process resulted in the creation of new
islet cells in the rats which resulted in the alleviation of their
diabetes. Furthermore, after 10 days, the transplant was removed
again from the diabetic rats with the results shown with respect to
days 14 and 16.
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The results of this experiment are summarized in Table II
below~
TABLE II
Days after Insulin Levels Found
Transplantation in:
1* 7 ]0** 14 16ng/ml ng/mg ng/ml
In situ Recomb
Rat # Blood Glucose mg/dl Serum Pancreas Tissue
51 386 71 97 86 85 19.6 52.1 2.2
52 369 77 1q5 109 85 7.9 5~.3 17.4
53 370.167 158 89 183 5.6 42.5 ***
54 496 80 76 112 142 4.1 17.3 ***
Control Diabetic Rat:
524 447 420 422 401 3.0 4.2
_
* Transplant
** Removed transplant
*** Taken for morphological analysis
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Additionally, light microscopic evidence was found of the
formation of new islet cells in the in situ pancreas of diabetic
rats that have received a recombinant transplant.
Immunocytochemical staining for insulin demonstrated insulin
positive cells within the duct epithelial lining, as well as small
islets forming in close association with the ducts. This indicates
that the increased insulin production and secretion in the diabetic
rats was due to a regeneration of pancreatic islets from ductular
epithelium.
EXAMPLE 3
This example is directed toward a method of ln situ
regeneration of islet tissues from the host's existing unisolated
pancreatic ductular epithelium by transplanting pancreatic buds or
mesenchyme cells into an appropriate transplantation site.
Noninbred alloxam diabetic rats were transplanted with pancreatic
buds (PB) from a fetal rat, or alternatively mesenchyme (M) from a
fetal rat of 11 1/2 to 13 1/2 days gestation. The site of the
transplant was the omentum of each rat? in the vicinity of the
pancreas.
Fetal pancreatic buds were obtained by dissecting the
stomach-duodenal portion of a 14 day old rat fetus in 20% fetal
bovine serum. The developing pancreatic bud was identified and
removed from the other tissues. The isolated pancreatic bud was
placed in a ~5% air/S% carbon dioxide humidified atmosphere at 37
degrees C. for approximately 48 hours on a 1% bactoagar gel culture
containing RPMI 1640 culture medium.
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Two out of three of the diabetic rats that received a
pancreatic bud transplant demonstrated reduced blood glucose values
and showed elevated in situ pancreas insulin levels. One diabetic
rat received a mesenchyme transplant, and also showed reduced blood
glucose levels and a slight increase of in situ pancreas insulin
levelsO
The data of this experiment is summarized by Table III below:
TABLE III
Days Insulin
1 7 10 14 16 ng/ml ng/mg
In situ
Rat # Blood Glucose mg/dl Serum Pancreas
.
53 (PB) 646 435 408 450 415 2.7 9.3
54 (PB) 492 256 266 162 134 10.4 31.4
58 (PB) 418 74 76 89 78 1.2 32.2
59 (M) 373 152 269 101 91 8.1 16.0
Control
Diabetic 380 ~ 373 323 5~1 12.1
Rat:
: One added rat received a transplant of duct epithelium from apost-fetal rat, and showed no signs of normalization of diabetic
symptoms.
The above has been offered for illustravtive purposes only,
and is not intended to limit the scope of the invention of this
application, which is as defined in the claims below.
