EP0273039A1 - Procede utilisant la capacite d'induction d'une matrice extracellulaire pour produire des tissus d'ilots pancreatiques - Google Patents

Procede utilisant la capacite d'induction d'une matrice extracellulaire pour produire des tissus d'ilots pancreatiques

Info

Publication number
EP0273039A1
EP0273039A1 EP87902297A EP87902297A EP0273039A1 EP 0273039 A1 EP0273039 A1 EP 0273039A1 EP 87902297 A EP87902297 A EP 87902297A EP 87902297 A EP87902297 A EP 87902297A EP 0273039 A1 EP0273039 A1 EP 0273039A1
Authority
EP
European Patent Office
Prior art keywords
mesenchyme
fetal
cells
tissue
epithelium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP87902297A
Other languages
German (de)
English (en)
Other versions
EP0273039A4 (fr
Inventor
Ronald W. Dudek
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of North Carolina at Chapel Hill
East Carolina University
Original Assignee
University of North Carolina at Chapel Hill
East Carolina University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of North Carolina at Chapel Hill, East Carolina University filed Critical University of North Carolina at Chapel Hill
Publication of EP0273039A1 publication Critical patent/EP0273039A1/fr
Publication of EP0273039A4 publication Critical patent/EP0273039A4/fr
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/22—Coculture with; Conditioned medium produced by pancreatic cells

Definitions

  • beta cell the beta cell is the source cell for insulin
  • 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 implications in the field of diabetes since beta 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.
  • 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.
  • a method 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.
  • the biological response is elicited in the cells of the living subject.
  • 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.
  • 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.
  • wound healing may be stimulated, 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.
  • Mesoderm source of this mesenchyme
  • Mesoderm source of this mesenchyme is that tissue which derives its 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 me
  • 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 influence on nearby fetal epithelial cells to exhibit a normal biological response as another step in the development of the fetus.
  • mesenchyme from a fetus of the exact type and the exact age at which such cells are exerting their influence on nearby fetal epithelial cells to exhibit a normal biological response as another step in the development of the fetus.
  • 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.
  • the wery 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.
  • fetal mesenchyme equivalent to mammalian fetal mesenchyme include tissue from lower animal forms on the phylogenetic scale that have known regenerative capability, such as salamander, lizard, or crayfish, for example for limb re ⁇ eneration.
  • tissue from lower animal forms on the phylogenetic scale that have known regenerative capability, such as salamander, lizard, or crayfish, for example for limb re ⁇ eneration.
  • 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.
  • 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 fetus, i.e., those particular cells which are believed by embryo!ogists to have a role in the formation of the pancreatic islet cells in the rat fetus.
  • 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.
  • 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.
  • the fetal mesenchyme may be implanted in the omentum of the living 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.
  • the method described herein differs 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 islet tissue and (b) using a non-islet entity ductal epithelium from which to regenerate the islet tissue.
  • 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.
  • a specific embodiment of the present invention involves the interaction of an extracellular matrix, such as fetal mesenchyme with a pancreatic ductal epithelium to produce pancreatic islet tissue. It is applicable, however, to any tissue having a ductal element and contemplates using a patient's own tissue as a source of transplantable differentiated tissue.
  • 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.
  • 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.
  • 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.
  • the invention further provides a method of preparing substantially pure epithelium, facilitating 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 in 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.
  • 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 free from immune rejection.
  • 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.
  • 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 1% 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), the recombinant was placed in a 95% air/5% C0 2 humidified atmosphere at 37 degrees C. for 48 hours on a 1% bactoagar gel culture containing RPM1 1640 culture medium.
  • FBS fetal bovine serum
  • 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, ductal epithelium alone, intact fetal pancreatic bud, and isolated adult pancreatic islets were also transplanted into other nude mice as controls.
  • transplanted 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 M cacodylate buffer for electron microscopy, or (c) sonicated and extracted in acid ethanol for 24 hours at 4 degrees C for radioim unoassay of insulin. No tissue other than the transplanted tissue was analyzed. A total of 57 recombinants were processed. Twelve of 23
  • 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.
  • 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 regeneration 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 glucose 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.
  • 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 normalization of blood glucose levels within seven days of the transplantation.
  • both serum insulin and in situ pancreas insulin levels were elevated in comparison to a control diabetic rat.
  • This example is directed toward a method of in 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.
  • PB pancreatic buds
  • M mesenchyme
  • 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 95% air/5% carbon dioxide humidified atmosphere at 37 degrees C. for approximately 48 hours on a 1% bactoagar gel culture containing RPMI 1640 culture medium. - 16
  • 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 levels.
  • One added rat received a transplant of duct epithelium from a post-fetal rat, and showed no signs of normalization of diabetic symptoms.

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  • Health & Medical Sciences (AREA)
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  • Chemical & Material Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Physiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nutrition Science (AREA)
  • Public Health (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Le procédé et la composition ci-décrits servent à régénérer des tissus en particulier des tissus d'îlots pancréatiques présentant les propriétés histologiques et l'aptitude à contenir de l'insuline propres aux îlots qu'on trouve chez des animaux nouveaux-nés ou adultes. Les tissus d'îlots régénérés sont produits par un mésenchyme foetal, qui agit sur un épithélium, de telle sorte qu'un message inducteur permette à l'épithélium de se différencier en tissus d'îlots.
EP19870902297 1986-04-03 1987-03-27 Procede utilisant la capacite d'induction d'une matrice extracellulaire pour produire des tissus d'ilots pancreatiques. Ceased EP0273039A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US84741386A 1986-04-03 1986-04-03
US847413 1986-04-03

Publications (2)

Publication Number Publication Date
EP0273039A1 true EP0273039A1 (fr) 1988-07-06
EP0273039A4 EP0273039A4 (fr) 1990-01-08

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP19870902297 Ceased EP0273039A4 (fr) 1986-04-03 1987-03-27 Procede utilisant la capacite d'induction d'une matrice extracellulaire pour produire des tissus d'ilots pancreatiques.

Country Status (4)

Country Link
EP (1) EP0273039A4 (fr)
JP (1) JPH01500957A (fr)
CA (1) CA1298551C (fr)
WO (1) WO1987005931A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4544299A (en) * 1998-05-29 1999-12-13 Cythera, Inc. Promotion of cell differentiation by initially passaged cells

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4642292A (en) * 1979-10-29 1987-02-10 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Method for isolation of connective tissue biomatrix
IL68218A (en) * 1983-03-23 1985-12-31 Univ Ramot Compositions for cartilage repair comprising embryonal chondrocytes
US4609551A (en) * 1984-03-20 1986-09-02 Arnold Caplan Process of and material for stimulating growth of cartilage and bony tissue at anatomical sites

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8705931A1 *

Also Published As

Publication number Publication date
JPH01500957A (ja) 1989-04-06
CA1298551C (fr) 1992-04-07
EP0273039A4 (fr) 1990-01-08
WO1987005931A1 (fr) 1987-10-08

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