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WO1997010348A1 - Procedes d'induction de types cellulaires selectionnes - Google Patents

Procedes d'induction de types cellulaires selectionnes Download PDF

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Publication number
WO1997010348A1
WO1997010348A1 PCT/US1996/014781 US9614781W WO9710348A1 WO 1997010348 A1 WO1997010348 A1 WO 1997010348A1 US 9614781 W US9614781 W US 9614781W WO 9710348 A1 WO9710348 A1 WO 9710348A1
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WIPO (PCT)
Prior art keywords
cell
tissue
cells
epithelial
mesenchymal
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PCT/US1996/014781
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English (en)
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Joshua H. Lipschutz
Peter F. Young
Gerald R. Cunha
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The Regents Of The University Of California
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Priority to AU72387/96A priority Critical patent/AU7238796A/en
Publication of WO1997010348A1 publication Critical patent/WO1997010348A1/fr

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    • 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
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous 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
    • C12N2502/24—Genital tract cells, non-germinal cells from gonads
    • C12N2502/246—Cells of the male genital tract, non-germinal testis 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
    • C12N2502/25—Urinary tract cells, renal cells

Definitions

  • the present invention relates to the regeneration of tissue.
  • it relates to the use of mesenchymal cells to induce differentiation in terminally differentiated adult tissues, thereby producing selected cells or tissue.
  • Mesenchyme is embryonic connective tissue involved in the development of the integumental, gastrointestinal, skeletal, and urogenital systems. Mesenchyme induces a broad spectrum of epithelial forms, including simple epithelial ducts and tubules; branched ductal networks; planar epithelial surfaces and unique epithelial patterns such as those found in teeth, feathers and sense organs (Wessells, N.K. Tissue interactions and development (1977) 276; Sawyer, R.H. In Epithelia l-Mesenchymal Interactions in Development (1983) 1 15-146; Cunha, G.R. et al. J. Andrology (1992) 13:465-475).
  • kidney forms as a result of interactions between the mesenchymal metanephric mesoderm and the epithelial ureteric bud (which also forms the ureter) (Saxen, L. Organogenesis ofthe kidney (1987) Cambridge University Press, New York).
  • Mesenchymal-epithelial tissue recombinants can be made in which the mesenchyme induces the epithelium in a permissive or instructive manner.
  • Tissue interactions characterized as permissive inductions are those in which mesenchyme permits the epithelium to express its normal developmental fate.
  • Instructive inductions are those in which mesenchyme induces an epithelium to express an enurely new developmental fate specified by the mesenchyme.
  • the epithelium is not irreversibly committed and, therefore, retains the ability to respond to a heterologous mesenchymal inductor.
  • urogenital sinus mesenchyme can elicit prostatic development in epithelia of the female urogenital sinus, postnatal vagina, and fetal or postnatal bladder (Cunha, G.R. et al. J. Exp. Zool. (1978) 205: 181-194;
  • the epithelial ducts induced by the urogenital sinus mesenchyme express histological, ultrastructural, and functional features indicative of prostate: Androgen receptors, prostate- specific antigens; androgen dependency for DNA synthesis; and prostatic secretory proteins (Cunha, G.R. et al. J. Cell Biol. (1983) 96: 1662-1670; Cunha, G.R. et al. Biol. Reprod. (1980) 22: 19-43; Cunha, G.R. et al. Endocrinology (1980) 107: 1767-1770; Neubauer, B.L. et al. J. Cell Biol. (1983) 96: 1671-1676).
  • stroma constitutes the non-epithelial component of an organ.
  • the predominant cells of the stroma are fibroblasts and smooth muscle.
  • the role of stromal-epithelial interactions has received considerably less attention than that of mesenchymal-epithelial interactions, probably due to the facUhat adult epithelial cells were believed to be irreversibly determined and terminally differentiated (Ursprung, H. The Stability of the Differentiated State (1968) Springer- Verlag, NY; Slack, J.M.W. From Egg to Embryo: Determinative Events in
  • mesenchymal or stromal cells have elicited changes in the morphology, differentiation, or growth of adult epithelial cells (Cunha, G.R. et al. J. Cell Biol. (1983) 96: 1662-1670; Dudek, R.W. et al. Diabetes (1988) 37:891-900; Sakakura, T. et al. Develop. Biol. (1979) 72:201-210.)
  • the changes elicited by urogenital sinus mesenchyme in the epithelium of the adult urinary bladder provides one example of a mesenchyme-induced alteration in adult epithelial differentiation.
  • embryonic urogenital sinus mesenchyme induced the epithelial cells of the adult urinary bladder to undergo prostatic differentiation. This resulted in formation of a simple columnar secretory epithelium, morphogenesis of a branched ductal network, marked stimulation in epithelial proliferation, and expression of several prostate-specific markers (Donjacour,
  • This invention demonstrates that an instructive induction can be achieved in situ, e.g. , mby direct implantation of mesenchyme into suitably prepared sites of adult animals.
  • This invention demonstrates for the first time that new functional organs can be created in situ by mesenchymal induction of adult epithelial cells.
  • the methods of the invention can be used in place of organ transplants in patients with chronic or irreversible organ failure.
  • This invention also demonstrates that differentiation induced by mesenchymal cells can reduce the tumorigenicity of tumor cells.
  • a mesenchymal cell is contacted and incubated with a second cell (typically an epithelial cell).
  • the second cell then produces differentiated cells corresponding to the tissues from which the mesenchymal cells were isolated.
  • the cells differentiate to form tissues or organs.
  • the mesenchymal cells are selected from cells which direct the production of differentiated cells in the embryo which correspond to the differentiated cells to be produced in the adult (i.e. , cells of the selected phenotype).
  • this process is performed in vivo, producing differentiated tissues or organs in situ at the site of mesenchymal cell introduction.
  • differentiated cells are removed from an adult animal and cultured in vitro in the presence of isolated mesenchymal cells.
  • the resulting differentiated cells are optionally reintroduced into the adult animal where they provide a therapeutic benefit.
  • the differentiated cells are pancreatic, the cells are optionally reintroduced into the adult animal at a site which is in contact with the blood stream, so that the cells can produce and deliver insulin to the adult animal.
  • the mesenchymal cell and the second cell are preferably from organisms of the same species, but optionally from organisms of different species.
  • a plurality of differentiated cells are produced in the adult, thereby forming an epithelial tissue, wherein the tissue is selected from the group of tissues consisting of kidney tissue, pancreatic tissue, bladder tissue, lung tissue, intestinal tissue, liver tissue, and reproductive tissue.
  • the invention provides methods of producing a selected cell from a terminally differentiated cell, by incubating the terminally differentiated cell with a mesenchymal cell.
  • the cell is an epithelial cell selected from the group consisting of kidney tissue, pancreatic tissue, bladder tissue, lung tissue, intestinal tissue, liver tissue, and reproductive tissue.
  • the invention provides for the regeneration of a defective kidney in an animal by incubating a terminally differentiated ureter cell with kidney mesenchyme.
  • the ureter cell gives rise to kidney tissue in an anatomically correct arrangement relative to the urinary system.
  • a clonal population of cells is used to give rise to a fully differentiated organ.
  • a primary culture of epithelium is subjected to clonal selection.
  • the clonal cells in culture which may represent epithelial stem cells, are used in conjunction with mesenchyme to grow a corresponding organ in vivo.
  • a single cell (or a population of cells) from a clonal population of cells is grown into a prostate in vivo by placing the cell in close proximity to, or contact with, urogenital sinus mesenchyme in a host animal.
  • the invention also provides kits comprising mesenchymal cells and a container.
  • the kits optionally further comprise terminally differentiated cells, animals, instructional materials and the like.
  • the invention provides cells produced by incubating a terminally differentiated or adult cells with an embryonic mesenchymal cell. These cells are optionally in cell culture or in vivo, and are optionally incorporated into tissues or organs.
  • the invention provides mesenchymal-epithelial tissue recombinants which are models for assessing carcinogenesis and tissue differentiation.
  • the tissue recombinants are a host animal with a mesenchymal cell and an epithelial cell.
  • the host animal, epithelial cell and mesenchymal cell are all from different sources, e.g. , the host animal is optionally a mouse (such as an athymic mouse), the mesenchymal cell is optionally from a rat embryo and the epithelial cell is optionally a human cell.
  • the epithelial cell is a tumor cell.
  • the tissue recombinants can be used to study the effects of test compounds on differentiation or carcinogenesis.
  • the test compound is applied to the tissue recombinant host, typically so that the test compound contacts the interacting epithelial-mesenchymal cell.
  • the effect of the test compound on a measurable marker such as differentiation or cell growth are then assessed.
  • the ability to screen test compounds for their affect on cell growth provides a valuable new model for studying differentiation and tumorigenesis.
  • Figure 1 is a schematic drawing illustrating the placement of neonatal rat seminal vesicle mesenchyme on the adult mouse ureter epithelium.
  • Figure 2 shows an induced mouse seminal vesicle at the severed end of the ureter with the platinum wire visible (small arrow).
  • Phenotype refers to the detectable physical characteristics of a cell or tissue or organ.
  • a "selected" phenotype is a phenotype which shares physical characteristics with a given known cell, tissue or organ.
  • epidermal is used to refer to the cellular layer covering all free surfaces in a mammal, including cutaneous, mucous, and serous layers, as well glands and other structures derived from them.
  • the term "mesenchyme” is used to refer to embryonic connective tissue, usually found between the epithelia and tissue masses of major organs.
  • the term "instructive induction' is used to refer to those processes by which mesenchyme induces an epithelium to express an entirely new developmental fate specified by the mesenchyme. These processes are contrasted with tissue interactions characterized as “permissive inductions" in which mesenchyme permits the epithelium to express its normal developmental fate.
  • mesenchymal-epithelial tissue recombinants can be made in which the mesenchyme induces the epithelium in a permissive or instructive manner.
  • the ureter is derived from the metanephric diverticulum which, along with the derivatives of the metanephric mesoderm, forms the kidney.
  • the left ureters of adult male athymic mouse hosts were severed below the kidney, and mesenchyme from neonatal rat seminal vesicles (SVM) was grafted to the cut end of the ureter, thus bringing adult mouse ureter epithelium (URE) in contact with neonatal rat SVM.
  • SVM neonatal rat seminal vesicles
  • URE adult mouse ureter epithelium
  • the in situ tissue recombinants were harvested, and the epithelial secretory proteins recovered.
  • the induced seminal vesicle epithelium also expressed androgen receptors (AR) which are not seen in urothelial tissue. Staining with Hoechst dye 33258, which can distinguish cells of mouse and rat origin, further demonstrated that the induced SVE was indeed of mouse origin and not a contaminant of the inducing rat SVM.
  • neonatal mouse vaginal mesenchyme was grafted in situ beneath the bladder mucosa of adult male mice, and the host animals were killed after 3 months. The vaginal mesenchyme implanted into the bladders induced prostate-like acini, indicating that the above reprogramming of adult organs in situ is not an isolated occurrence.
  • This invention demonstrates that an instructive induction can be achieved in situ by direct implantation of SVM into suitably prepared sites of adult animals. Since the embryonic Wolffian ducts normally give rise to the epithelia of the epididymis, seminal vesicle, ureter, and ductus deferens, this system provides an excellent model in which the mo ⁇ hological and functional aspects of the reprogramming of adult epithelia can be studied in situ, and the results described in the examples below are generally applicable to other organ systems. This invention demonstrates for the first time that new functional organs can be created in situ by mesenchymal induction of adult epithelial cells. In vitro Uses of The Invention
  • a differentiated cell is a cell with a characteristic mo ⁇ hology or phenotype which expresses a specific set of differentiation markers, e.g. , uroplakin for urothelial cells, or PSA for prostate cells.
  • a specific set of differentiation markers e.g. , uroplakin for urothelial cells, or PSA for prostate cells.
  • Certain cell types e.g. kidney or liver epithelial cells, are difficult to grow in primary culture.
  • production of tissue-specific factors from these cells is facilitated by incubating embryonic mesenchymal cells associated with the corresponding embryonic tissue type in the presence of more easily isolated differentiated cells such as epithelial cells.
  • the secreted products, gene transcripts, and the cells themselves are more readily available for study and commercial application.
  • cell products amenable to this type of production are human serum albumin or clotting factors from liver epithelium, steroids from adrenal cortical epithelium and insulin from pancreatic tissue.
  • this culture system facilitates gene isolation and cloning.
  • the present invention provides two general strategies for generating tissues and organs in vivo.
  • the tissues are generated ex vivo and transplanted into the animal.
  • the tissues are generated directly in vivo. Ex vivo Production of Selected Cells and Tissues
  • differentiated tissue from a patient is removed from a relatively accessible site, e.g. urethral, bladder or ureter epithelium, and cultured in vitro with the appropriate embryonic mesenchyme to instructively induce formation of the tissue type desired, e.g. , kidney tubular epithelia, and transplanted back into the donor patient upon redifferentiation in vitro.
  • the source of the embryonic inductive mesenchyme is preferably from the animal into which the resulting tissue is to be introduced, i.e. , human embryonic mesenchyme is typically used to produce a human tissue, but alternate mammalian species are also appropriate donors for embryonic mesenchyme.
  • the cells which are induced by the embryonic mesenchyme are isolated from the animal into which the generated tissue is to be introduced, no graft rejection is observed, providing a distinct advantage over donor transplantation, or transgenic xenotransplantation. Because the cells are induced in vitro, the source of the inducing agent—i.e. , the embryonic mesenchymal cells— is not significant from the standpoint of antigenic rejection, or host versus graft rejection.
  • epithelial tissue from the rectum would be obtained from a patient suffering from ulcerative colitis, and cultured with embryonic mesenchyme from the gut in vitro, to produce intestinal tissues which, upon transplantation back into the patient, replace corresponding diseased tissues which are usually removed surgically.
  • a clonal population of cells is used to give rise to a fully differentiated organ.
  • a primary culture of epithelium is subjected to clonal selection.
  • the clonal cells in culture which may represent stem epithelial cells, are used in conjunction with mesenchyme to grow a corresponding organ in vivo.
  • a single cell (or a population of cells) from a clonal population of cells is grown into a prostate in vivo by placing the cell in close proximity to, or contact with, urogenital sinus mesenchyme in a host animal.
  • other clonal epithelial cells e.g. , kidney
  • mesenchyme is placed into a host at a site where tissue, or a replacement organ is to be generated.
  • tissue or a replacement organ is to be generated.
  • This strategy provides one of skill with the ability to replace defective organs or tissues in situ.
  • the replaced organs because they are ultimately produced by the host, do not encounter the usual problems of tissue rejection associated with standard organ replacement therapy.
  • the present invention provides for a powerful new method of regenerating damaged organs and tissues.
  • animals with new tissue derived from the addition of embryonic mesenchyme to the animal are produced by excising the tissue of interest from embryonic, or very young (typically less than 3 days old, preferably less 10 than 1 day old) neonatal animals and separating the mesenchyme from the tissue by tryptic digestion.
  • tissue of interest typically from embryonic, or very young (typically less than 3 days old, preferably less 10 than 1 day old) neonatal animals and separating the mesenchyme from the tissue by tryptic digestion.
  • seminal vesicles were excised from 0- day-old neonatal rats and vaginas were excised from 0-day -old neonatal mice.
  • Mesenchymes were separated from epithelium following tryptic digestion as described (Cunha, O.R. et al. Human Genetics (1981) 58:68-77).
  • the mesenchyme forms into a cohesive mass of tissue which is transplanted into adult animal hosts at a selected site. It is expected that one of skill is familiar with the appropriate surgical techniques for performing tissue transplantation into the host animal. In brief, the animal is anesthetized, the site into which the mesenchyme is to be placed is isolated and surgically exposed, and the mesenchyme is sutured or stapled in place or simply placed in close proximity to the selected site. The surgical field is then closed with sutures or staples or other appropriate means.
  • the mesenchyme and the surgical site for transplantation depends on the organ or tissue to be produced.
  • the mesenchyme is isolated from an embryonic or neo-natal organ which corresponds to the organ or tissue to be produced, and the surgical site of implantation of the mesenchyme is selected to provide an appropriate anatomical arrangement for the growing organ or tissue.
  • a kidney can be induced in vivo by transplanting human metanephric mesoderm to the animal's ureter. The urinary outflow tract is thus placed in an anatomically correct sequence relative to the developing induced kidney. Competent human metanephric mesoderm is found in embryos around embryonic day 35.
  • the selected mesenchymal cells may be difficult to obtain or culture in vitro.
  • techniques for providing a ready supply of the mesenchymal cells is advantageous.
  • immortalized, temperature sensitive mutants transfected with the gene for the large T antigen of SV40 can be used. These cells are capable of dividing at lower temperature (e.g. , about 33°C) but not at body temperature. Thus, the cells will not proliferate in the body, but can be maintained indefinitely at lower temperatures. This illustrates a difference between tumor cells and immortalized cells.
  • Immortalized cells can often be easily controlled by regulation of the factors which make the cell immortal, unlike tumor cells. Immortalized cells can be constituively or inducibly immortalized.
  • the procedures described herein are generally applicable to all mammals, including rodents such as mice, rats, and guinea pigs, primates such as monkeys, baboons, macaques, chimpanzees, gorillas, and especially humans, dogs, cats, sheep, cattle, goats, horses etc.
  • the isolated mesenchyme be isolated from an embryo of the same species into which the organ or tissue is to be induced in order to reduce complications from tissue rejection.
  • the source of mesenchyme is optionally from an animal species different from the animal into which the mesenchyme is to be placed.
  • rat mesenchyme can be used to induce tissue or organ formation in mice.
  • rat mesenchyme can induce tissue formation with human epithelial cells in mice.
  • mesenchyme is used from allogeneic or xenographic source
  • the animal into which the mesenchyme is to be placed is typically put on a regimen of immunosuppressants to inhibit tissue rejection.
  • methods of preventing access of the host animals immune system can be used to prevent allograft rejection.
  • artificial pancreatic devices containing live islets have been designed to avoid immune rejection, by enclosing islets in a semipermeable pouch or matrix which separates the transplanted islets from immunoreactive cells and molecules (see, e.g. , Newgard, U.S. Patent No. 5,427,940 and Bae, et. al.
  • Such methods can be used for preventing immune response against mesenchymal cells, as well.
  • Certain animals are genetically engineered to be immune suppressed (e.g. , nude mice), or to have immune systems derived from other animal species (e.g. , mice expressing human immune genes).
  • rat mesenchyme was placed into immune compromised mice (e.g. , "athymic" mice) which did not require immuno-suppression.
  • transgenic animals which have the same surface antigens as the host into which the mesenchyme is placed are used as mesenchyme donors to alleviate complications due to tissue rejection.
  • the growth medium for primary cell culture will typically be a buffered salt solution containing amino acids, vitamins and optionally includes other nutrients, or serum, or a serum-substitute.
  • growth media are DMEM and RPMI. Additionally, the medium will contain other supplements to enhance cell growth and prevent the death of the culture.
  • Buffered salt solutions are designed either to equilibrate with atmospheric conditions or to equilibrate with a gas phase containing 5 to 10% carbon dioxide. In the present inventive methods, solutions of the latter type are preferred. These media are based on Earle's salts and are buffered with a bicarbonate/ carbonic acid system which maintains the pH in a CO 2 equilibrated incubator.
  • Well-known example media include Dulbecco's Modified Eagle Medium (DMEM) and RPMI.
  • Media supplements are optionally added to reduce the need for serum supplementation.
  • Such supplements typically contain growth promoting additives such as insulin, transferrin, trace elements (such as manganese, molybdenum, vanadium, nickel, or tin), ascorbic acid, non-essential amino acids, L-glutamine and other growth factors.
  • growth promoting additives such as insulin, transferrin, trace elements (such as manganese, molybdenum, vanadium, nickel, or tin), ascorbic acid, non-essential amino acids, L-glutamine and other growth factors.
  • Other additives to the growth medium include antibiotics and antifungal agents.
  • antibiotics such as penicillin, streptomycin, neomycin and polymyxin are used.
  • Preferred antibiotics include penicillin and streptomycin.
  • Preferred antifungal agents are fungizone and nystatin. Particularly preferred is fungizone.
  • the culture chambers and substrates for primary cells are generally plastic culture dishes or flasks. However, many cells will also grow on glass coverslips placed in plastic dishes and on a variety of membranes and fabrics including collagen membranes, elastic membranes, agar, smooth silicone rubber substrata, polyacrylonitrile fabrics, dacron velour and Parylene-C coated polypropylene microfabric.
  • trypsinization to remove adherent cells from a culture surface is well known to those of skill in the art. Briefly, trypsin or trypsin » EDTA is solubilized in a Ca + + and Mg + + free buffered salt solution (i.e. , HBSS) and the pH is adjusted to 7.4-7.6. Any media or serum is removed from the monolayer by washing with Ca + + and Mg + + free buffered salt solution. The trypsin solution is then added to the vessel containing the monolayer in sufficient quantity to cover the monolayer and the mixture is incubated for about 2 minutes at 37°C. The trypsin solution is removed from the vessel and the monolayer is again incubated until the cells detach from the surface.
  • a Ca + + and Mg + + free buffered salt solution i.e. , HBSS
  • Any media or serum is removed from the monolayer by washing with Ca + + and Mg + + free buffered salt solution.
  • the trypsin solution is
  • serum or medium containing serum is added to the vessel to inhibit further trypsin activity which can damage the cells.
  • These cells can be resuspended by gentle pipetting to break up any clumps, and diluted with media for cell counts and secondary culturing.
  • Cells are optionally frozen to avoid loss by contamination and to provide a constant supply for future use.
  • cultures are dissociated with trypsin to provide cell pellets which are suspended in complete medium containing either glycerol or dimethylsulfoxide as a cryopreservative.
  • complete DMEM containing about 10% dimethylsulfoxide (DMSO) is often used.
  • the cell pellet is suspended in the freezing medium at a concentration of about 1 to 5 x 10 7 cells/mL. Aliquots are placed into vials which are cooled to -20°C for 2 hours and then transferred to a liquid nitrogen-containing storage vessel, or -80°C freezer until further use.
  • Frozen cells are fragile and require gentle handling. Frozen cells should be thawed quickly and are typically plated directly into complete growth media. Cells which are sensitive to the added cryopreservative (e.g. , glycerol or DMSO) should be centrifuged, to remove the medium containing the preservative, and then plated into complete growth medium.
  • cryopreservative e.g. , glycerol or DMSO
  • vials containing frozen cells are defrosted in a 37°C water bath for one minute. The cells are transferred to a sterile centrifuge tube, complete growth medium is added, and the cells are centrifuged. The supernatant is discarded and the pellet is resuspended in complete medium and plated, e.g. , into 75 mm tissue culture flasks to establish a secondary culture. Secondary Cultures
  • Secondary cultures can be obtained from previously frozen cells which had become confluent in the primary tissue culture dishes. Centrifugation of the thawed cells provides a pellet which is resuspended in complete medium, counted and plated in tissue culture flasks. Following incubation as described for the primary culture, the cells are typically washed with calcium and magnesium-free phosphate buffered saline solution and are optionally detached from the dishes mechanically or enzymaticaUy (e.g. , by trypsinization) .
  • tissue or organs are monitored at the structural level by physical examination (i.e. , in conjunction with exploratory surgery), ultrasound examination, or the use of imaging dyes.
  • the tissues or organs are monitored by detecting the presence of antigens on the surface of the tissues or organs which are specific to the cell types which comprise the organs. This may be done histologically or by monitoring the formation of antigens, e.g. , in blood or blood serum.
  • tissue-specific dyes, antibodies, and reagents are known in the art.
  • the level of tissue-specific markers such as antigens, or mRNA, in a cell culture, cell or whole organism is measured by means known in the art.
  • the level of marker is measured in a western blot or other immunoassay such as an ELISA, or by performing quantitative PCR on reverse transcribed mRNA.
  • the level of marker is measured by monitoring the amount of a cell epitope or expressed protein by quantifying binding of the protein to an immunogenic reagent such as an antibody.
  • quantitative PCR the level of an mRNA is measured by monitoring PCR amplification products, and comparing the amount of amplified nucleic acid obtained, as compared to amplification products obtained from amplification performed on a known reference nucleic acid sample.
  • Specific monoclonal and polyclonal antibodies and antisera will usually bind a target marker with a K D of at least about .1 mM, more usually at least about 1 ⁇ M, preferably at least about . 1 ⁇ M or better, and most typically and preferably, .01 ⁇ M or better.
  • polypeptides and their corresponding antibodies will be labeled by joining, either covalently or non covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149; and 4,366,241.
  • a particular protein marker can be quantified by a variety of immunoassay methods.
  • immunoassay methods For a review of immunological and immunoassay procedures in general, see
  • the immunoassays of the present invention can be performed in any of several configurations, e.g. , those reviewed in Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Florida; Tijan (1985) "Practice and Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science
  • Antibodies, polypeptides and nucleic acids are detected and quantified by any of a number of means well known to those of skill in the art. These include analytic biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, immunofluorescent microscopy and the like, and various immunological methods such as fluid or gel precipitin reactions, lmmunodiffusion (single or double), lmmunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
  • the detection of nucleic acids proceeds by well known methods such as Southern analysis, northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography.
  • the present invention provides commercially valuable models for studying carcinogenis and a new form of cancer therapy.
  • Cancer is essentially a form of improper cell differentiation. It is now discovered that tumongemcity of cancer cells can be reduced by causing the cells to undergo differentiation in the presence of normal mesenchyme or stroma from tissue corresponding to a tumor site. This process is referred to as differentiation therapy. Heterospecies tissue recombinants are descnbed which provide a model for assessing tissue development and inhibition of tumors in tissues. For example, a heterospecific prostate model provides for the study of prostate development and tumongenesis. Furthermore, differentiation therapy was shown to inhibit prostate tumongenesis in vivo
  • the developing prostatic epithelium induces the differentiation and mo ⁇ hological patterning of smooth muscle in the UGM.
  • UGM grown by itself formed minute amounts of smooth muscle.
  • prostatic ducts developed which were surrounded by sheathes of actin-positive smooth muscle.
  • the inductive nature of the epithelium was emphasized by the observation that when the epithelium was of rodent origin, the smooth muscle sheathes were thin and thus corresponded to the pattern of rodent prostate.
  • tissue recombinants were prepared with rat UGM plus human prostatic epithelium, the resultant prostatic ductal tissue was surrounded by thick sheathes of smooth muscle, the pattem characteristic of the human prostate.
  • the epithelium induces smooth muscle differentiation from the mesenchyme and also specifies its spatial patteming.
  • prostatic smooth muscle A notable feature of prostatic smooth muscle is its dependence upon androgens for development, growth, and maintenance of differentiation in adulthood. These events are presumably dependent upon expression of AR in prostatic smooth muscle.
  • smooth muscle -actin In fetal prostate, smooth muscle -actin is expressed throughout the UGM, which accounts for the peristaltic contractility of the urogenital sinus in organ culture.
  • smooth muscle differentiation markers appear sequentially, and dense smooth muscle sheathes become organized around the epithelial ducts by 20 days postnatally in the rat. The initial differentiation of prostatic smooth muscle is androgen-dependent both in vitro and in vivo.
  • prostatic smooth muscle in the rat prostate is androgen-dependent. Following castration there is a loss of prostatic smooth muscle as the prostatic smooth muscle bundles disintegrate and disappear with loss of many of the prostatic smooth muscle differentiation markers. Thus, androgen-dependent differentiation of prostatic smooth muscle is dependent upon an interaction with epithelium. Likewise, in adulthood, the maintenance of prostatic smooth muscle is dependent upon androgens and a homeostatic interaction with epithelium.
  • Prostate tissue is thus formed and maintained by the reciprocal interaction of epithelium and mesenchyme during prostatic development, followed thereafter by a reciprocal homeostatic interaction of epithelium and smooth muscle in adulthood.
  • This smooth muscle-epithelial cell interaction is perturbed during prostatic carcinogenesis with adverse sequelae occurring in both epithelium and smooth muscle.
  • the following sequence of events occurs: (1) Under the influence of androgens, urogenital sinus mesenchyme (UGM) induces urogenital sinus epithelium to undergo prostatic ductal mo ⁇ hogenesis and differentiation. (2) As prostatic epithelium differentiates, it in turn signals theJUGM to differentiate into smooth muscle cells that closely surround the epithelial ducts. Differentiation of prostatic smooth muscle occurs after an inductive signal from epithelium and androgens. (3) Once formed, prostatic smooth muscle participates in reciprocal homeostatic interactions.
  • Prostatic smooth muscle under the influence of androgens, signals prostatic epithelium to maintain epithelial differentiation and to repress epithelial proliferation, while prostatic epithelium signals to prostatic smooth muscle to maintain smooth muscle differentiation.
  • homeostasis is maintained through reciprocal interactions between smooth muscle and epithelial cells, with minimal proliferation of either cell type.
  • Prostatic carcinogenesis which is initiated following, e.g. , genetic damage to prostatic epithelium, involves a sequential disruption in these reciprocal homeostatic interactions with ensuing de-differentiation of both the emerging prostatic carcinoma cells and smooth muscle.
  • the epithelium fails to signal appropriately to the smooth muscle, which then begins to de-differentiate.
  • Neonatal Sprague-Dawley rats and adult athymic mice were obtained from Simonson, Gilroy, CA.
  • C57BL/6J mice were obtained from Jackson labs.
  • Neonatal male rats and female mice were used within 24 hours of birth (day 0). All animals received water and laboratory chow ad libitum and were housed under standard laboratory conditions.
  • vesicles were excised from 0-day-old neonatal rats. Vaginas were excised from 0-day-old neonatal mice. Mesenchymes were separated from epithelium following tryptic digestion as described earlier (Cunha, O.R. et al. Human Genetics (1981) 58:68-77). Following overnight culture of the rat SVM on a 1 % agar substrate, the mesenchyme rounded up into a cohesive mass of tissue which was transplanted into adult male athymic mouse hosts anesthetized with Avertin (tertiary amyl alcohol, plus tribromoethanol).
  • Avertin tertiary amyl alcohol, plus tribromoethanol
  • the transplant site for the SVM was the severed proximal end of the ureter of the adult male athymic mouse host.
  • the grafted SVM was fixed in place by a thin platinum wire which acted as a staple, holding the neonatal rat SVM in contact with the adult mouse ureter.
  • the transplant site for the mouse vaginal mesenchyme was the bladder of the adult male C57BL/6J mouse. Bladders were exteriorized through a small, midventral incision in the body wall. The bladder mucosa was exposed by an oblique incision in the muscular wall of the bladder. The vaginal mesenchyme was then inserted into the incision such that the neonatal mouse mesenchyme was in close proximity to the adult mouse bladder epithelium. Recovery and processing of tissue recombinants
  • Hosts were killed 4-12 weeks after grafting.
  • the grafts were isolated by dissection. Secretions were recovered from the lumina of the recombinants, solubilized in
  • Antibodies against mouse SVS proteins and rat SVS protein IV were used for immunocytochemistry.
  • the antibodies against mouse SVS proteins react with mouse but not rat SVS proteins, while antibodies against rat SVS protein IV reacts with rat but not mouse SVS proteins.
  • the immunocytochemical methodology has been described in detail earlier (Higgins, SJ. et al. Development (1989) 106:219-234; Prins, G. et al.
  • Protein samples prepared in PBS-SDS were analyzed by electrophoresis in polyacrylamide (10-20% linear gradient) slab gels using a discontinuous buffer system (Laemmli, U.K. Nature (1970) 227:680-685) with 0. 1 % SDS throughout (Brooks, D.E. et al. J. Reprod. Fertil. (1980) 59:363-375). Proteins (20-50 ⁇ g per lane) resolved by this method (SDS-PAGE) were visualized by staining for 1 hour in 0J % Coomassie blue in acetic acid: methanol :H,0 (1 :3:6 by vol) followed by prolonged destaining in the same solution without the dye. Androgen Receptors
  • the PG21 antibody was used to detect androgen receptors in frozen tissue sections as described previously (Prins, G. et al. Endocrinology (1991) 129:3187-3199). The tissue sections were then stained using the Biotin/Avidin system (Vectastain kit,
  • rat SV both epithelium and mesenchyme
  • SVE which is not shared with URE, is the expression of androgen receptors (AR).
  • AR androgen receptors
  • epithelial nor stromal cells of the ureter exhibit AR.
  • AR are a prominent feature in both the epithelium and stroma of the SV.
  • rat bladder mesenchyme was transplanted to the severed ends of the ureters of adult male athymic mice as described above. In these tissue recombinants, the urothelial phenotype was maintained, and no SVS proteins were detected.
  • vaginal mesenchyme has been shown to be able to induce prostatic development when experimental tissue recombinants are grown in male hosts (Cunha, G.R.
  • AR appear 2 to 3 days postpartum (Cooke, P.S. Endocrinol. Suppl. (1988) 122:92; Shima, H. et al. Endocrinology (1990) 127:3222-3233), whereas SVS protein synthesis begins after day 10 (Fawell, S.E. et al. Mol. Cell. Endocrinol. (1986) 48:39-49).
  • the inductions described in this paper represent the first examples of complete mo ⁇ hological and functional reprogramming in adult epithelial cells by transplantation of an inductive mesenchyme in situ. In essence, an animal was created with three seminal vesicles. This, obviously, has many clinical implications.
  • organs are created utilizing instructive induction, e.g. , to assume organ function following natural organ failure.
  • instructive induction e.g. , to assume organ function following natural organ failure.
  • generation of lung, pancreas, liver, and kidney tissues are now possible in situ.
  • a kidney can be induced in vivo by transplanting human metanephric mesoderm to the patient's own ureter.
  • the urinary outflow tract is thus placed in an anatomically correct sequence relative to the developing kidney.
  • Competent human metanephric mesoderm is found in embryos around embryonic day 35.
  • Example 2 Characterization of a Novel. Heterospecific Model of Prostate Development
  • a novel model of prostate development was developed which utilized tissue recombinants of adult human prostate epithelial cells (HPRE) and rat fetal urogenital sinus mesenchyme (UGM).
  • HPRE human prostate epithelial cells
  • UPM rat fetal urogenital sinus mesenchyme
  • the recombinants are grafted into adult male nude mouse hosts and the identity of the epithelial cells confirmed by species-specific in situ hybridization.
  • the grafts underwent marked stromal and epithelial proliferation and ductal branching mo ⁇ hogenesis in an orderly fashion. Over the first 4 weeks, the epithelial cells reorganized into branching cords, which subsequently canalized to form ducts with a multilayered epithelium which expressed PSA and PAP.
  • HPRE is receptive to an inductive stroma of rodent origin and implies that different species share common mediators of stromal ⁇ epithelial interactions. Furthermore, there was evidence that the HPRE causes an altered stromal mo ⁇ hology in the rat UGM. This indicates the existence of uniquely human epithelial ⁇ stromal factor(s) which may be responsible for the predominance of stroma in the human prostate. This model is useful for the study of the stromal-epithelial interactions involved in human prostate development and BPH. 24
  • Example 2 To examine more closely the interactions occurring between developing human prostate epithelium and inductive mesenchyme, the in vivo heterospecific tissue recombination model of Example 2 was used. This method uses rat urogenital sinus mesenchyme (rUGM) recombined with fresh adult human prostate epithelium (hPrE) in collagen gels grafted beneath the renal capsule of athymic mouse hosts. The resultant hPRE/rUGM tissue recombinants, when grafted into male athymic nude mice or rats, exhibit extensive epithelial growth and ductal branching mo ⁇ hogenesis. The epithelium initially forms solid cords which grow and branch into the su ⁇ ounding mesenchyme.
  • rUGM rat urogenital sinus mesenchyme
  • hPrE fresh adult human prostate epithelium
  • the solid epithelial cords then canalize, and the epithelium differentiates into basal and luminal cell types each expressing their characteristic cytokeratins.
  • the human prostatic secretory proteins, prostatic acid phosphatase and PSA are then expressed in the luminal epithelial cells.
  • the rat UGM differentiates into AR-expressing smooth muscle.
  • the role of epithelium in dictating the pattem of stromal differentiation is underlined by the observation that the smooth muscle differentiates into thick sheets, thus exhibiting a patte normally found in the human prostate as opposed to the thin sheathes characteristic of the rat from which the UGM was derived.
  • tissue recombinants constructed with epithelium and mesenchyme from widely separated species exhibit both instructive and permissive inductions. This indicates that signaling between epithelium and mesenchyme uses the same language in the rodent and human, since a rodent mesenchyme induced prostatic differentiation in a human epithelium.
  • the chimeric rat-human prostate described above lends itself to analysis of gene expression on a species specific basis.
  • the epithelial component of the heterospecific tissue recombinant is of human origin, the mesenchyme is rat, while the mouse host contributes the vasculature.
  • Growth factors and their receptors are highly conserved Jjetween species so in most cases techniques such as Western and Northern blotting did not differentiate between proteins and mRNA derived from different species. However, other techniques such as RNase protection assays are able to distinguish messages by species of origin.
  • species specific RT-PCR assays for a number of growth factors and their receptors were developed.
  • RT-PCR primers For most growth factors, which have highly conserved mRNA sequences between species, a single set of RT-PCR primers can amplify a given sequence for multiple species.
  • the amplified fragments of such a reaction were virtually the same size and not distinguishable by gel electrophoresis. However, due to minor variability in their nucleotide sequences, amplified fragments from each species contained unique restriction endonuclease cleavage sites.
  • the amplified fragments were distinguished by restriction digestion of the amplified fragment followed by gel electrophoresis of the products of the digestion.
  • SS-RT-PCR heterospecific tissue recombinants were analysed to determine the species (and thus the tissue type) producing the mRNA under investigation.
  • PCR primers which amplify TGF-/53 cDNA from human, rat or mouse were developed. These primers produced an amplified fragment which contained restriction endonuclease sites which are unique for each species, such that specific digestions can be used to identify the species of origin (and thus tissue layer) of the signal. Detail of the primer sequences, digestion enzymes and product sizes are shown below. In this tissue recombinant, digestion with the enzymes Dde 1 and Msp 1 , which digest human and mouse product, respectively, leads to a small amount of species specific digestion product. The enzyme Bsp 12861 digests the amplified product from all three species; however, it produced a 252bp fragment which is unique to the rat.
  • SS-RT-PCR is used to analyze the expression of growth factors and growth factor receptors in the interacting epithelium and stroma of rat UGM + human prostauc epithelium and stroma of rat UGM + human prostatic epithelium tissue recombinants growing in vivo.
  • carcinoma cells are induced to differentiate with a concomitant reduction on growth rate.
  • Vimentin is initially widely expressed and during development becomes localized to the interducial connective tissue and excluded from the differentiating muscle.
  • the markers of smooth muscle differentiation are expressed sequentially, in a proximal to distal manner along the growing ducts.
  • Example 4 Smooth Muscle Re ulates Prostatic Homeostasis And Malignant Growth While mesenchymal-epithelial interaction is a term used to describe mo ⁇ hogenetic cell-cell interactions during development, the term stromal-epithelial interaction is used for such cell-cell interactions in adulthood.
  • Stroma is a term that denotes the non-epithelial component of an organ. For most intemal organs, the predominant cell types in stroma are fibroblasts and smooth muscle.
  • the principle stromal interaction of adult prostatic epithelium is with the surrounding smooth muscle.
  • smooth muscle cells are organized into thin sleeves of 3 to 4 layers of AR-positive smooth muscle cells interspersed with a few AR-negative fibroblasts.
  • the composition and relative amount of smooth muscle and fibroblasts su ⁇ ounding the epithelial ducts varies proximal-distally from the urethra out to the ductal tips.
  • the stromal sheath is thickest and predominantly of smooth muscle.
  • the stromal sheath is formed of a discontinuous layer of smooth muscle cells with interspersed fibroblasts.
  • the stromal sheath is composed of a thin continuous layer of smooth muscle cells.
  • Prostatic smooth muscle cells are in intimate contact with the basement membranes of epithelial ducts as epithelial and smooth muscle cells are separated by only about 300nm.
  • interaction of adult prostatic epithelium with its immediate stromal environment is generally a smooth muscle-epithelial interaction even though the "acinar capsule" surrounding individual epithelial ducts also contains a small number of fibroblasts.
  • prostatic stroma Since AR-positive smooth muscle constitutes about 22% of the human prostate, a major cell type in cultures of "prostatic stroma” is, in fact, de-differentiated smooth muscle cells. Indeed, prostatic stroma of the guinea pig contains only 5% fibroblasts, with smooth muscle being the major cell type. This is not easily recognized in cell culture, because smooth muscle cells are known to rapidly dedifferentiate into fibroblast-like cells when grown in vitro. Once prostatic smooth muscle differentiates from UGM, the smooth muscle cell becomes an interactive element in the epithelial micro-environment and in homeostatic cell-cell interactions in the adult prostate. In adulthood, normal prostatic epithelial growth and differentiation are regulated by reciprocal smooth muscle-epithelial cell interactions mediated by the local synthesis and action of growth factors and other paracrine signaling molecules.
  • UGM induces prostatic ductal mo ⁇ hogenesis and epithelial differentiation, and developing prostatic epithelium in turn induces smooth muscle differentiation in UGM.
  • prostatic smooth muscle After differentiation of prostatic smooth muscle, reciprocal smooth muscle-epithelial interactions play a homeostatic role in maintaining prostatic structure and function.
  • smooth muscle is proposed to maintain prostatic epithelial structure and function, while prostatic epithelium in turn maintains smooth muscle differentiation.
  • the initiated epithelium begins to diverge from its normal phenotype, and thus signaling from prostatic epithelial cell to smooth muscle becomes aberrant.
  • prostatic smooth muscle cells begin to de-differentiate, resulting in abnormal smooth muscle ⁇ epithelial signaling.
  • Abnormalities in smooth muscle ⁇ epithelial signaling may either actively promote carcinogenesis or permit the progression to anaplasia through the loss or restriction of normal homeostatic/growth inhibitory controls. The result is a cycle of sequential loss of differentiation in both epithelial and smooth muscle compartments, leading to promotion and progression of the tumor, and ultimately culminating in clinical cancer.
  • Prostatic epithelium is an inducer of smooth muscle differentiation, while UGM induces prostatic epithelial differentiation.
  • a prediction of this hypothesis is that normal (but not transformed) prostatic epithelium should be capable of inducing smooth muscle differentiation in UGM. This prediction was verified.
  • Tissue recombinants were prepared with 18 day embryonic rat UGM associated with various normal and neoplastic prostatic epithelia. In all cases, when normal embryonic or adult prostatic epithelia were utilized, the resultant prostatic ductal structures were surrounded by compact sheathes of smooth muscle cells that developed from the UGM. Conversely, when anaplastic prostatic epithelia were used, smooth muscle failed to differentiate in the tissue recombinants, and instead the UGM maintained its mesenchymal nature. The one prostatic carcinoma cell line capable of inducing smooth muscle differentiations was Dunning tumor epithelium, which is a highly differentiated cell. Thus, normal prostatic epithelium can induce smooth muscle differentiation in UGM, while transformed prostatic epithelium
  • prostatic carcinoma cells are incapable of inducing and maintaining smooth muscle differentiation as indicated by the tissue recombinant experiments described above. It is noteworthy that a feature of the Dunning prostatic adenocarcinoma is the absence of smooth muscle in association with the aberrant epithelial ducts. Thus, the process of prostatic carcinogenesis entails aberrations in the interactions of the prostatic epithelium with its smooth muscle micro-environment resulting in reciprocal dedifferentiation of both the emerging carcinoma cells as well as prostatic smooth muscle.
  • stromal changes associated with carcinogenesis may be related to bona fide fibroblasts
  • smooth muscle cells constitute a significant proportion of the fibromuscular stroma of certain organs such as the prostate and thus, stromal changes during carcinogenesis involve smooth muscle cells as well.
  • One of the stromal changes associated with prostatic carcinogenesis is increased agglutinability by concanavalin A of human peritumoral prostatic fibroblasts.
  • prostatic fibroblasts described are in fact de-differentiated smooth muscle cells which in culture resemble fibroblasts.
  • the carcinogenetic process appears to encompass progressive and reciprocal changes in both epithelial and stromal/smooth muscle components.
  • a corollary of cell-cell interactions in the prostate is that for some prostatic adenocarcinomas it is possible to reconstitute a more regular tissue architecture and a slower growth rate through interaction of the carcinoma cell with normal stroma.
  • Stromal-epithelial interactions in the Dunning prostatic adenocarcinoma (DT) are clearly abnormal, as the basement membrane interposed between the neoplastic epithelia ducts and the "stroma" is frequently discontinuous and/or multi-layered.
  • the mesenchyme In grafts of UGM + DT, BUGM + DT, or SVM -I- DT, the mesenchyme induced the undifferentiated DT epithelial cells to differentiate into tall columnar secretory epithelial cells assembled into large cystic ducts. These mesenchyme-induced changes in DT histodifferentiation were associated with changes in neoplastic growth. When SVM-induced differentiated DT cells were tested for their ability to grow as tumors, tumorigenesis was remarkably diminished.

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Abstract

Cette invention porte sur des procédés faisant intervenir une induction directive ou permissive pour induire des types cellulaires sélectionnés chez un animal. Le procédé consiste, généralement, à implanter directement du mésenchyme dans des sites convenablement préparés d'animaux adultes. Il en résulte la création de nouveaux organes fonctionnels in situ par induction mésenchymateuse de cellules épithéliales adultes. Il est, de la sorte, possible d'utiliser les procédés de l'invention pour remplacer des transplantions d'organes chez des patients souffrant d'une défaillance chronique ou irréversible d'un organe. Cette invention, qui porte également sur des modèles destinés à l'étude de cancer ainsi que sur des procédés de réduction de l'action tumorigène, concerne aussi des cellules, des recombinés tissulaires et des matériels.
PCT/US1996/014781 1995-09-14 1996-09-13 Procedes d'induction de types cellulaires selectionnes WO1997010348A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000029002A2 (fr) * 1998-11-13 2000-05-25 Osiris Therapeutics, Inc. Transplantation intra-uterine de cellules embryonnaires mesenchymateuses humaines
WO2002014469A2 (fr) * 2000-08-15 2002-02-21 Geron Corporation Utilisation de cellules souches totipotentes pour reprogrammer des cellules de façon à renforcer l'aptitude à la différentiation
EP1393623A1 (fr) * 2001-05-09 2004-03-03 Taiho Pharmaceutical Co., Ltd. Modele animal de prostatisme interstitiel

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ACTA ANAT., 1995, Vol. 152, CUNHA et al., "Mammary Phenotypic Expression Induced in Epidermal Cells by Embryonic Mammary Mesenchyme", pages 195-204. *
DEVELOPMENT, July 1995, Vol. 121, No. 7, DONJACOUR et al., "Induction of Prostatic Morphology and Secretion in Urothelium by Seminal Vesicle Mesenchyme", pages 2199-2207. *
EPITHELIAL CELL BIOLOGY, 1992, Vol. 1, CUNHA et al., "Developmental Response of Adult Mammary Epithelial Cells to Various Fetal and Neonatal Mesenchymes", pages 105-118. *
EPITHELIAL CELL BIOLOGY, 1993, Vol. 2, HAYASHI et al., "Permissive and Instructive Induction of Adult Rodent Prostatic Epithelium by Heterotypic Urogenital Sinus Mesenchyme", pages 66-78. *
JOURNAL OF ANDROLOGY, November/December 1994, Vol. 15, No. 6, TSUJI et al., "Effect of Mesenchymal Glandular Inductors on the Growth and Cytodifferentiation of Neonatal Mouse Seminal Vesicle Epithelium", pages 565-574. *
KIDNEY INTERNATIONAL, January 1996, Vol. 49, LIPSCHUTZ et al., "Urothelial Transformation into Functional Glandular Tissue In Situ by Instructive Mesenchymal Induction", pages 59-66. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000029002A2 (fr) * 1998-11-13 2000-05-25 Osiris Therapeutics, Inc. Transplantation intra-uterine de cellules embryonnaires mesenchymateuses humaines
WO2000029002A3 (fr) * 1998-11-13 2000-10-05 Osiris Therapeutics Inc Transplantation intra-uterine de cellules embryonnaires mesenchymateuses humaines
WO2002014469A2 (fr) * 2000-08-15 2002-02-21 Geron Corporation Utilisation de cellules souches totipotentes pour reprogrammer des cellules de façon à renforcer l'aptitude à la différentiation
WO2002014469A3 (fr) * 2000-08-15 2002-06-13 Geron Corp Utilisation de cellules souches totipotentes pour reprogrammer des cellules de façon à renforcer l'aptitude à la différentiation
EP1393623A1 (fr) * 2001-05-09 2004-03-03 Taiho Pharmaceutical Co., Ltd. Modele animal de prostatisme interstitiel
EP1393623A4 (fr) * 2001-05-09 2005-12-21 Taiho Pharmaceutical Co Ltd Modele animal de prostatisme interstitiel

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