WO1994009118A1 - Chondrocytes humains immortalises - Google Patents

Chondrocytes humains immortalises Download PDF

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WO1994009118A1
WO1994009118A1 PCT/US1993/009718 US9309718W WO9409118A1 WO 1994009118 A1 WO1994009118 A1 WO 1994009118A1 US 9309718 W US9309718 W US 9309718W WO 9409118 A1 WO9409118 A1 WO 9409118A1
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cells
chondrocytes
collagen
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human
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Mary Goldring
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The General Hospital Corporation
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655Chondrocytes; Cartilage
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00Genetically modified cells
    • C12N2510/04Immortalised cells

Definitions

  • IMMORTALIZED HUMAN CHONDROCYTES This invention relates to the field of chondrocytes. Background of the Invention
  • the chondrocyte is a terminally differentiated cell that functions to maintain the cartilage specific matrix phenotype under normal conditions of low turnover.
  • the chondrocyte is derived from the chondroblast, a mesenchymal derivative.
  • cartilage tissue is composed only of chondrocytes and does not contain nerves, blood vessels or lymphatics.
  • the cartilage tissue is capable of both a positional and interstitial growth which occurs by cell division of the mature chondrocyte and the secretion of an extracellular matrix.
  • the major constituents of the extracellular matrix of mature hyaline cartilage found in diarthroidal joints are type II (with some types IX and XI) collagen and the cartilage-specific, large aggregating proteoglycan.
  • chondrocyte function is altered and there are changes in the composition of the extracellular cartilage matrix (Krane et al., 1986, In: Articular Cartilage Biochemistry. Kuettner et al., eds., Raven Press, New York, p413; Dodge and Poole, 1989, Journal Clinical Invest. .83.:647; Hamerman, 1989, New England Journal of Medicine 320: 1322; Harris, 1990, N. Engl. J. M ⁇ d. 322:1277) .
  • changes in the production and temporal release of cytokines result in altered chondrocyte function.
  • Mature differentiated chondrocytes can be defined by numerous cellular characteristics: the production of predominately type II collagen, the secretion of a specific profile of proteoglycans, the production of link protein and specific cellular responses to the cytokines interleukin-1 (IL-1) and interferon- ⁇ (IFN- ⁇ ) (Goldring and Goldring, 1990, Clinical Orthopedics and Related Research) .
  • IL-1 interleukin-1
  • IFN- ⁇ interferon- ⁇
  • the invention provides an immortalized human chondrocyte cell line having a homogeneous polygonal morphology which, in addition, is further characterized in that it expresses type II collagen, suppresses the expression of type II collagen in response to IL-1 and IFN- ⁇ , expresses collagenase in response to IL-1, and expresses the proteoglycans expressed by human primary chondrocytes, but does not express the proteoglycan Versican not normally found in cartilage.
  • the invention provides an immortalized chondrocyte cell line in insulin-containing serum substitute characterized in that it has the same gene expression profile characterized above and corresponding to that of mature primary chondrocytes in culture.
  • the invention further includes any immortalized cell line with the aforementioned characteristics derived from human primary chondrocytes trahsfected with origin defective SV40.
  • the aforementioned immortalized human chondrocyte cell lines are made by a procedure hich involves isolating human chondrocytes, preferably juvenile primary chondrocytes, and growing them in culture, transfecting the culture with origin-defective SV40 or another immortalizing nucleotide seguence, subculturing the transfected cells, preferably for a period of greater than six weeks, and selecting a cell line that displays a cobblestone morphology and is further characterized in that it a) expresses type II collagen, b) produces less of type II collagen in response to IL-1 and IFN- ⁇ , c) expresses collagenase in response to IL-1, d) secretes a proteoglycan profile corresponding to that of mature human chondrocytes.
  • the cell line may be maintained in medium with insulin-containing serum, for example, as Nutridoma-SP to obtain enhanced expression of primary chondrocyte phenotypes.
  • insulin-containing serum for example, as Nutridoma-SP to obtain enhanced expression of primary chondrocyte phenotypes.
  • the phenotypic selection for immortalized chondrocytes is done following a drug resistance selection which eliminates those cells which do not bear the transfected nucleic acids.
  • the invention also provides a method of treating a degenerative cartilage disease by transfecting cells obtained from a cell line described above with a gene encoding a therapeutic protein, and administering the transfected cells to the patient.
  • This method may be used to treat the degenerative cartilage disease osteoarthritis.
  • the transfection method may also be used to incorporate a gene encoding a cytokine, preferably TGF- ⁇ and/or IGF-I to treat diseases of the cartilage, for example diseases of cartilage degradation.
  • Transfection may also be used as stated above to incorporate a gene encoding a cytokine inhibitor such as IL-1, a similarly acting receptor antagonist, or an IL-1 mutant.
  • the same method of transfection may also be used to transfect genes encoding proteases or protease inhibitors, or extracellular matrix components.
  • Immortalized chondrocytes containing a gene encoding a therapeutic protein may be administered to the patient by localized injection.
  • the invention also provides a method by which the immortalized chondrocytes described above may be used as a source for harvesting antivasicular compounds by a method involving culturing the immortalized chondrocyte cells and purifying from these cells a compound which prevents vascularization.
  • the invention further provides a method of screening for compounds useful in the therapy of cartilaginous diseases. This method involves applying potential therapeutic compounds to the immortalized chondrocyte cells and then monitoring the cells for alterations in gene regulation which ameliorate the pathological condition.
  • Fig. IA is a photograph of the T/C-28a2 cells in culture and Fig. IB a photograph of the C20/A4 cell line in culture.
  • Fig. 2 depicts the expression of type II collagen RNA in the immortalized chondrocyte cell line C20/A-4.
  • Fig. 3A and Fig. 3B depict the expression of type II collagen mRNA in the immortalized chondrocyte cell line T/C-28a2 in a variety of media.
  • the first 5 lanes of Fig. 3A represent type II mRNA expression in primary chondrocytes.
  • Lanes 6-24 of Fig. 3A represent the type II mRNA levels in T/C-28a2 in a variety of media.
  • the autoradiograph shown in Fig. 3A is of a brief exposure of the Northern blot, and Fig. 3B is a longer exposure of the same blot depicted in Fig. 3A.
  • Fig. 4 is a graph showing the detection of expression of the proteoglycans aggrecan, PGI, PGII and versican in addition to collagen IX in diverse media for both normal and immortalized T/C-28a2 chondrocytes.
  • Fig. 5 depicts the expression of collagenase and early gene mRNAs in response to IL-1 in human costal chondrocytes at 3 hours, 6 hours and 24 hours.
  • Fig. 6 depicts the level of c-Jun mRNA expression at 3 and 5 hours after the addition of IL-l ⁇ in C20-A4 cells.
  • Fig. 7 depicts primary costal chondrocytes (day 11 of primary culture) which were preincubated for 24 h alone (control) or with hIL-13 (5 pM) without or with indomethacin (l ⁇ M) .
  • Fig. 8A and Fig. 8B depict costal chondrocytes (see Fig. 7) which were preincubated for 24 h alone (control) or with IFN- ⁇ (10, 100, and 500 units/ml) .
  • Fig. 9 represents the dot hybridization analysis of procollagen mRNA levels in costal chondrocytes treated with IFN- ⁇ and IL-10.
  • Fig. 10 depicts the kinetics of ⁇ l(II) and ⁇ l(I) procollagen mRNA expression in costal chondrocytes treated with IL-13 and indomethacin in the absence and presence of cycloheximide.
  • Fig. 11 is a set of graphs which represent the analysis of the stability of ⁇ l(II) and ⁇ l(I) procollagen mRNAs treated with L-l ⁇ in the absence and presence of cycloheximide (CHX) .
  • Figs. 12A and 12B show the expression of 5'- flanking regulatory sequences of COL2A1 by human costal chondrocytes in different culture conditions.
  • Fig. 13 depicts the dose-dependent inhibition of expression of pCAT-B/4.0 by IL-10 and IL-l ⁇ .
  • Fig. 14 depicts the dose-dependent suppression of pCAT-B/4.0 (PCAT4.0) expression and potentiation of this suppression by indomethacin in human costal chondrocytes.
  • Fig. 15 shows the expression of pCAT-B/4.0 in rat chondroblasts and inhibition by human IL-lj3 and urine IFN- ⁇ .
  • Fig. 16 is a graph demonstrating the expression of pCAT Control, but not pCAT-B/4.0, by human dermal fibroblasts.
  • Fig. 17A is a table representing the CAT activity of plasmids pCAT4.0, pCAT2.9 and pCAT2.0 in both_control and hIL-1/9 serum conditions.
  • Fig. 17B depicts the effect of recombinant IL-1/9 on expression of COL2A1 regulatory sequences in immortalized human chondrocytes.
  • 17(C) depicts the effect of recombinant IFN- ⁇ on expression of CAL2A1 regulatory sequences in immortalized human chondrocytes.
  • A. Isolation of Primary Chondrocytes Human costal cartilage was obtained from ribs removed during pectus excavatum repair of a 5-year-old juvenile male and a 15-year-old female. Chondrocytes were isolated by dispersion with proteases and cultured as described below. Cartilage slices were incubated at 37°C with hyaluronidase (lmg/ml in PBS; bovine testicular from Sigma) for 10 min, followed by 0.25% trypsin (GIBCO/BRL) for 45 min.
  • hyaluronidase lmg/ml in PBS; bovine testicular from Sigma
  • the cartilage was then chopped in small fragments and incubated at 37°C with collagenase (3 mg/ml in serum-free culture medium; clostridial peptidase from Worthington) for 48 h.
  • the dispersed cells were then washed with Ca ++ - and Mg ++ -free PBS and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) with medium changes every 3-4 days, as described previously (Goldring and Krane, 1987, J. Biol. Chem. 262:16724: Goldring et al., 1988, J. Clin. Invest. 82.:2026) .
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • C-20/A4 An immortalized human chondrocyte cell line was generated using pMK/SV40 ori " plasmid transfection and is termed C-20/A4.
  • C-20/A4 was derived from juvenile costal chondrocytes isolated from a specimen of rib cartilage from a 5-year-old male obtained as discarded material after surgery as described above.
  • origin-defective SV40 (20 ⁇ g/flask of pMK/SV40 ori " plasmid DNA) (Small et al., 1982, Nature 298:671) using the polybrene/DMSO method (Chaney et al., 1986, Somatic Cell Molec. Genet.
  • the immortalized cells were obtained by the selection of foci 4-6 weeks after transfection and continuous subculture to produce senescence of the parental cells. Of the 140 foci (10-20 per flask) selected originally, only 20 were subcultured. After 20-40 passages, the C-20/A4 line was selected for further characterization, since these cells continued to grow well and maintain a homogenous polygonal morphology characteristic of chondrocytes rather than fibroblasts. Growth in a insulin-containing media such as Nutridoma-SP (Boehringer Mannheim) was found to be required for the expression of the full range of primary chondrocyte phenotypes by the immortalized chondrocytes in culture.
  • Figure IB depicts the C-20/A4 cells in culture.
  • the C-20/A4 cells have been maintained in culture for over three years, during which time they have continued to grow well and maintain a homogenous polygonal morphology. Additional immortalized cell lines with characteristics similar to those of C-20/A4 may be obtained from the 120 immortalized clones or by replicating the immortalization procedure.
  • the SV-40 large T-antigen encoding DNA was inserted in the pZip Neo SV(X) vector (Psi-2 U19-5 plasmid) (Williams et al., Mol. Cell Biol. 8.:3864) and freshly isolated cultures of juvenile human costal chondrocytes from a specimen of rib cartilage from a 15- year-old female obtained as discarded material after surgery (pectus excavatum repair) at day 5 of primary culture were used for immortalization. Cells were cultured for more than 8 weeks before the initial G418 drug resistance selection. After selection of transfected cells in G418, the uncloned cell populations (designated T/C-28) were established through several subcultures following G418 selection.
  • T/C-28 chondrocyte lines were accomplished most readily after further selection of neomycin-resistant celis by agarose suspension culture for 2 to 4 weeks, as described for chick embryo chondrocytes by Castagnola et al. (J. Cell Biol. 102:2310-2317, 1986) .
  • the non-cloned T/C-28 chondrocytes at passage 20 were incubated with trypsin-EDTA and the resulting cell suspension was washed and transferred to plates that had been coated with 1% agarose.
  • chondrocytes aggregated and began within 24 h to form clumps that increased in size and number through the first week, as we have observed in agarose suspension cultures of normal human chondrocytes. After 10 to 14 days of agarose culture, the clumps began to disaggregate, become smaller and form a single cell suspension. When this suspension was replated in tissue culture dishes, a more homogeneous monolayer of polygonal cells was generated that proliferated readily in culture medium containing 10% FCS and reached confluence within one week. Confluent cultures formed nodules reminiscent of the original transformed foci.
  • agarose-selected cells passaged weekly at subconfluence, have maintained stable morphology and proliferative capacity throughout a further 40 passages and have been used in the studies described below. Further cloning by limited dilution of the agarose-selected cells was easily accomplished directly after replating the cells on tissue culture plastic. Characteristics of both the uncloned population selected on agarose (T/C-28a2) and several of the cloned lines (e.g., 1-2 and D-8 clones) are described below.
  • 10% FCS suppresses the capacity of the normal human chondrocytes to express regulatory sequences of the type II collagen gene in transient transfection experiments and removal of serum and addition of an insulin-containing serum substitute is permissive for expression of either endogenous type II collagen or the transfected type II collagen gene regulatory sequences even in subcultured chondrocytes.
  • the immortalized chondrocytes display these features as well; the C-20/A4 and T/C-28 chondrocyte lines can be grown and subcultured continuously in culture medium containing 10% FCS, and they exhibit high expression of type II collagen mRNA after one to several days of culture in serum-free defined medium containing 1% Nutridoma-SP (Boehringer- Mannheim) .
  • cartilage-specific collagen mRNAs including those for type IX and type XI can also be observed under these conditions and addition of IGF-I and/or TGF-3 may permit optimal expression of type II collagen mRNA.
  • These immortalized cells also express cartilage-specific aggrecan mRNA, as well as the small proteoglycan biglycan and decorin mRNAs appropriately. Expression of mRNA encoding versican, a small proteoglycan not found in cartilage, has not been observed under any condition of culture.
  • T/C- 28 chondrocytes synthesize and deposit in the extracellular matrix proteins immunoreactive with anti- proteoglycan (chondroitin-4-sulfate and large proteoglycan, from Seikagaku, Tokyo, Japan) and type II collagen (from Chemicon, Temecula, CA) monoclonal antibodies.
  • anti- proteoglycan chondroitin-4-sulfate and large proteoglycan, from Seikagaku, Tokyo, Japan
  • type II collagen from Chemicon, Temecula, CA
  • Type II collagen mRNA may be detected using the hgColII probe, which is a 3.8 kb genomic fragment of the human ⁇ l(II) procollagen gene encoding amino acids 892 to the end of the C-propeptide (-1150 nucleotides of exon material) (Sandell, L.J. J. Cell Biol. 99:405, 1984; Goldring et al., J. Biol. Che . 261: 9049-9055, 1986; Goldring et al., J. Clin. Invest. 82: 2026-2037, 1988; Sandell et al., Conn.
  • pkTh!330 probe which is a -600-bp fragment encoding a helical portion of the type II collagen chain near the C-propeptide and was cloned using a human costal chondrocyte library available from Dr. T. Kimura, Osaka, Japan and Dr. M. Goldring, Boston, MA) ; also useful is pCAT-B/4.0. which includes the 5'-flanking regulatory sequences (-577 to +3428) of the human type II collagen gene including the promoter and an enhancer in the first intron (Ryan et al., Genomics 8: 41-48, 1990; Goldring et al., Arthritis Rheum. 34: S106, 1991; and Sandell et al., Orthopaed. Res. Trans. 17: 1, 1992b.
  • Type IX collagen mRNA may be detected using the pkTh!23 probe which includes 600 bp cDNA encoding a portion of the human ⁇ l(IX) procollagen subunit, prepared using a cDNA library prepared from a human costal chondrocyte mRNA (Goldring et al., J. Clin. Invest. 82: 2026-2037, 1988; Kimura et al., Eur. J. Biochem. 179: 71-78, 1989).
  • Type XI collagen transcription may be detected using the pkThl ⁇ l probe which is a 1265 bp cDNA encoding a helical portion of the human 2(XI) procollagen subunit (Kimura et al., J.
  • Collagens which are not present in articular cartilage but associated with chondrogenesis and/or growth plates include Type X and Type IIA collagens.
  • Type X collagen probes are used to assess chondrogenic potential of cloned lines (Sandell et al., Orthopaed. Res. Trans. 17: 1992)) and Type IIA probes which reveal alternatively spliced type II collagen mRNA may also be used (Sandell et al., J. Cell Biol 114: 1307-1319, 1991; Sandell et al., Articular Cartilage and Osteoarthritis. 1992) .
  • Type I collagen and Type III collagen are detected only in dedifferentiated chondrocytes. Transcription from the Type I collagen gene may be detected using the Hf677 probe which is a 1500 bp cDNA encoding part of the 0:1(1) procollagen subunit (Chu et al., Nucleic Acids Res. 10: 5925-5934, 1982; Goldring et al. , J. Biol. Che .
  • Hfll31 which is a 1500 bp cDNA probe encoding part of the ⁇ 2(I) procollagen subunit (Bernard et al., Biochemistry 22: 1139-1145, 1983; Goldring et al., J. Biol. Chem. 262: 16724-16729, 1987; Goldring et al., J. Clin. Invest. 82: 2026-2037, 1988) .
  • Cartilage- specific proteoglycan synthesis may be detected using either nucleic acid probes or specific immunoreactive antibodies to Aggrecan (CSPG, large aggregating proteoglycan) , Link protein, Biglycan (PG-I, also called PG-40) , and Decorin (PG-II) .
  • CSPG Aggrecan
  • PG-I Biglycan
  • PG-II Decorin
  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe may be used to monitor the general status of transcription of genes required for cellular maintenance functions (MacNaul et al., J. Biol. Chem.. 265: 17238- 17245, 1990).
  • Proteinase and proteinase inhibitor probes to detect chondrocyte cell line responses to cytokines Collagenase gene expression is monitored using XHF-L which is a cDNA probe for human procollagenase which is a 1.5 kb Xbal cDNA fragment (Stephenson et al., Biochem. Biophvs. Res. Com un. 144: 583-590, 1987; MacNaul et al., J. Biol. Chem.. 265: 17238-17245, 1990). Stro elysin gene expression is monitored using a 1.8 kb PstI cDNA fragment (MacNaul et al., J. Biol. Chem..).
  • Tissue inhibitor of metalloproteinases is monitored using 0.9 kb Hindi cDNA fragment (MacNaul et al., J. Biol. Chem.. 265: 17238-17245, 1990).
  • the above three cDNA probes are provided by Dr. Nancy I. Hutchinson, Merck, Sharp & Dohme Research Laboratories, Rahway, NJ. Plasminogen activator and PA inhi itor probes, are also generally available.
  • c-jun transcription was monitored using a cDNA (pH540) encoding human c-Jun (Nakabeppu et al., Cell 55: 907-915, 1988); Jun B transcription was monitored using a 1800 bp cDNA (p465.20) encoding mouse Jun B (Ryder et al., Proc. Natl. Acad. Sci. USA 85: 1487-1491, 1988); E ⁇ r-1 transcription was monitored using a 3.1 kb EcoRl cDNA fragment (OC3.1) (Sukhatme et al., Cell 53: 37-43, 1988); and c-fos was monitored using a cDNA provided by Dr. R. Tijian, Berkeley, CA (Rauscher et al., Science, 240: 1011-1016, 1988) .
  • the following probes are available for the characterization of chondrocytes.
  • hon-164 human
  • p!236 human laminin Bl
  • p!237 human laminin B2
  • p!239 cDNA encoding ⁇ 2(IV) collagen chain of human type IV collagen
  • cytokine mRNAs For the assessment of cytokine mRNAs the following probes may be used: p3ACSFRI (human M-CSF) ; probes for human GMCSF; probes for human G-CSF; pXM209 (human IL-6) ; and pXMt2 (human IL-7) . These probes are available from Dr. S. Clark, Genetics Institute, Andover, MA and Dr. Steven Goldring, Massachusetts General Hospital, Boston, MA) . In addition, human IL-l ⁇ and IL-1 / 3 probes available from Dr. P. Auron may be used for the identification of IL-1 gene expression.
  • Immortalized human chondrocytes express type II, type IX and type XI collagen RNA.
  • the C-20/A4 line expressed chondrocyte-specific type II collagen mRNA when the cells were cultured in the presence of Nutridoma ( Figure 2) .
  • the probe used to detect the presence of type II collagen RNA was the ⁇ l(II) procollagen cDNA probe (pkThl330) (Kimura et al., 1989, Eur. J. Biochem. 179:71) .
  • the cell line T/C-28a2 also expresses type II collagen mRNA (Fig. 3A and 3B) .
  • RNA for dot blots was prepared by the method of White and Bancroft (1982, J. Biol. Chem 257:8569) and as described previously (Goldring and Krane, 1987, supra; Goldring et al., 1988, supra; and Goldring et al., 1986, J. Biol. Chem. 261:9049).
  • Total RNA for Northern blots was extracted according to a method adapted from Cho czynski and Sacchi (Anal Biochem, 1987, 162:156) .
  • Chondrocytes >1 X 10 6 ) from 6- or 10-cm plates were lyzed directly in 500 ⁇ l (final volume) of Solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1M 2-mercaptoethanol) and sheared with a 27-gauge needle either before or after freezing at -80°C.
  • the RNA was then extracted at 4°C by sequential addition of 2 M sodium acetate (50 ⁇ l) , pH 4.0, water saturated phenol (500 ⁇ l) , and chloroform/isoamyl alcohol (49:1; 100 ⁇ l) and precipitation of the aqueous phase with an equal volume of isopropanol.
  • RNA pellets were dissolved in 50 ⁇ l of DEP-treated 0.01 M HEPES. Alternatively, total RNA from >10 X 10 6 cells was extracted as described by Sandell and Daniel (supra) . The final preparations gave yields of approximately 10 ⁇ g of RNA per 1 X 10 6 cells with the appropriate A 260 :A 280 ratio of approximately 2.0.
  • Northern blots were prepared on either nitrocellulase or BAS-85 membranes (Schleicher & Schuell) as described previously (Goldring and Krane, 1987, supra; Goldring et al., 1988, supra; and Goldring et al., 1986, supra).
  • Type II collagen is a hallmark of mature differentiated chondrocytes.
  • the pKThl23 and pKThl ⁇ l probes were used to monitor the expression of type IX and type XI collagen gene expression, respectively, in immortalized human chondrocytes.
  • Interleukin-1 a product predominantly of monocytes, increases the synthesis and release of procollagenase and pro ⁇ taglandin E 2 by mesenchymal target cells such as synovial fibroblasts and articular chondrocytes, an effect mimicked by some phorbol esters, i) Primary chondrocyte responses.
  • Primary cultures of human articular chondrocytes were preincubated with recombinant human interleukin I ⁇ or the phorbol ester, phorbol 12-myristate 13-acetate, in the presence or absence of the cyclooxygenase inhibitor, indomethacin.
  • Interleukin I ⁇ or phorbol ester increased the levels of procollagenase (assayed after trypsin activation) and the labeling of several medium proteins by cells incubated with [ 35 S]methionine, independent of prostaglandin synthesis. The labeling of 55 kD protein immunocomplexed with antibodies to procollagenase was also increased. The increased synthesis of procollagenase was paralleled by increased cellular levels of procollagenase mRNA, determined with a cDNA probe coding for human procollagenase. Thus the increased synthesis of procollagenase in response to the inflammatory mediator, interleukin 1, is controlled at a pretranslational level, possibly at the level of transcription.
  • the time courses of expression of the immediate early genes eg/r-1, c-jun and Jun B, the collagen genes ⁇ l(II) and ⁇ l(I) procollagens, and the metalloproteinase genes collagenase and stromelysin were monitored in the same experiments using primary cultures of human chondrocytes, and the kinetics of their induction or suppression by IL-l were compared.
  • the C-20/A4 or T/C- 28a2 condrocytes were plated and grown for three days in culture medium containing 10% FCS. The cells were then made quiescent by serum starvation for 16-24 h in culture medium containing 1% Nutridoma.
  • IL-l was then added in the absence or presence of the protein synthesis inhibitor cycloheximide (CHX; 10 ⁇ g/ml) , the incubation continued for 24 hours and the cells harvested at various time points for extraction of total RNA and Northern blot analysis.
  • CHX protein synthesis inhibitor
  • Type II collagen mRNA was detected in both immortalized chondrocyte cultures and increased with time throughout the 24 h of incubation in 1% Nutridoma. At 1 h, IL-l had no effect on type II collagen mRNA levels in the C-20/A4 chondrocytes, but a slight suppression by IL- 1 was evident in the T/C-28a2 chondrocytes at both 1 and 6 h. A >60% suppression was achieved by 24 h. CHX could be observed to suppress type II collagen mRNA levels, particularly at 24 h, but it did not reverse or enhance the effect of IL-l.
  • egr-l mRNA was expressed in a somewhat reciprocal manner compared with type II collagen mRNA. This was particularly apparent in the T/C-28a2 cells that demonstrated low constitutive expression of egr-1.
  • the C-20/A4 cells consistently expressed relatively high constitutive levels of egr-1 mRNA at 1 h and lower levels of type II collagen mRNA compared with the T/C-28a2 cells.
  • the immortalized chondrocytes were incubated in the presence of FCS, however, the expression of type I collagen mRNA with little type II collagen mRNA was favored.
  • c-jun mRNA was detected and upregulated by IL-l, while egr-l and jun B were barely detectable and only slightly upregulated in the presence of IL-l.
  • Constitutive levels of stromelysin mRNA were observed in both the C-20/A4 and T/C-28a2 chondrocytes.
  • Nuclear run-on transcription of the type II collagen gene is inhibited by IL-l and IFN- ⁇ in primary and immortalized human chondrocytes.
  • the nuclear run-off transcription assays were done as follows: Human immortalized costal chondrocytes were incubated for 42 h in the absence o presence of IL-ljB or IFN- ⁇ in medium containing 10% FCS.
  • the cells were washed in cold hypotonic buffer (20mM HEPES, pH 7.5, 5mM KCl, 0.5 mM MgCl 2 , 0.5 mM DTT) containing 0.2M sucrose, resuspended in hypotonic buffer containing no sucrose, placed on ice for 10 min and lysed with 5-8 strokes in a Dounce homogenizer.
  • cold hypotonic buffer (20mM HEPES, pH 7.5, 5mM KCl, 0.5 mM MgCl 2 , 0.5 mM DTT) containing 0.2M sucrose, resuspended in hypotonic buffer containing no sucrose, placed on ice for 10 min and lysed with 5-8 strokes in a Dounce homogenizer.
  • the resulting nuclear pellet after centrifugation at 500 X g for 5 min was resuspended in storage buffer containing 40% glycerol, 50mM Tris-HCl, pH 8.0, 5 mM Mg acetate and O.lmM EDTA and stored in 100 ⁇ l aliquots containing 1 to 5 X 10 7 nuclei at -70°C.
  • the nuclei were then labeled once with ⁇ - 32/ P-U T P for 30 min at 30°C.
  • the labeled RNA was extracted once with water- saturated phenol-chloroform (2:1) and unincorporated nucleotides were removed from the RNA by 2 sequential precipitations with 70% ethanol.
  • Hybridizations were preformed at 42°C for 48 h using 1 X 10 6 cpm of labeled RNA and 2 ⁇ g of each respective linearized plasmid DNA immobilized on one strip of nitrocellulose per condition. It has been reported previously that human recombinant preparations of either IL-13 or IFN- ⁇ could suppress the synthesis of type II collagen in cultured human adult articular or juvenile costal chondrocytes (Goldring, M.B., et al., 1988, J. Clin. Invest... 82:2026- 2037, Goldring, M.B., et al., 1986, J. Biol. Chem.. 261:9049-9055).
  • IFN- ⁇ also decreased transcription of the type II collagen gene ( Figure 8A and 8B) . This effect was somewhat dose-dependent with maximal (>95%) suppression at a relatively high concentration of 500 units per ml of IFN-V8.7 ( Figure 8A) . Transcription of the ⁇ 2(I) and ⁇ 2(III) procollagen genes was also inhibited by IFN- ⁇ . Suppression by IFN- ⁇ of transcription of the ⁇ l(I) and ⁇ l(II) procollagen genes was observed when the labeled transcripts were hybridized to the respective DNAs which has been run on agarose gels prior to blotting on nitrocellulose ( Figure 8B) .
  • Indomethacin was added in these experiments because we showed previously that increased concentrations of PGE 2 prevent the stimulatory effect of IL-l on type I collagen gene expression.
  • incubation of chondrocytes for 24 h with IL-l increased the levels of type I collagen mRNA in either the absence or presence of indomethacin.
  • the level of type I collagen mRNA was decreased after incubation for 24 h with IL-l alone, but coincubation with indomethacin reversed this suppression and unmasked the stimulatory effect of IL-l. The apparent discrepancy between the two experiments may be accounted for by the different concentrations of serum used.
  • the stability of type I and type II collagen mRNA's following IL-l administration may be determined for immortalized chondrocytes. This determination may be made using the techniques described in Section (E) , above, on candidate cell lines.
  • the complete DNA sequence of the 5' portion of the human type II procollagen gene (COL2A1) has been published (Ryan, M.C., et al., 1990, Genomics, 8:41-48).
  • Availability of the COL2A1 promoter/first intron construct (pCAT-B/4.0) has allowed examination of its expression for. the first time in human chondrocytes.
  • This construct contains potential regulatory sequences that are homologous to silencer sequences responsive to fibroblast-specific nuclear factors in the upstream promoter region and a tissue-specific enhancer element in the first intron (Ryan, M.C., et al., 1990, Genomics, 8.:41-48).
  • We used the pCAT-B/4.0 construct to determine the conditions required for its expression in human chondrocytes and to determine whether its expression could be inhibited by IL-l and IFN- ⁇ .
  • the pCAT-B/4.0 plasmid construct was prepared in the following manner: A plasmid containing 4.0 kb of DNA sequences of C0L2A1, including -577 bp of upstream promotor sequence and 3428 bp downstream of the mRNA start site (242 bp of exon 1 and approximately 2/3 of the first intron) , was constructed using the pCATTM-Basic plasmid (Promega) . This 4.0 kb Pst I fragment was cloned into the PstI site located immediately upstream of the CAT gene and the plasmid was transfected into the JM109 bacterial strain and grown in the presence of ampicillin. The transient expression assay was performed using immortalized chondrocytes from the C-20/A4 line.
  • Immortalized chondrocy.es were plated at 0.5 X 10 6 cells per 100-mm dish in Ham's F12/DMEM (1:1) containing 10% FCS and allowed to settle down for 24 hr. The medium was then changed and the cells transfected with 10 ⁇ g of plasmid DNA by the calcium-phosphate method (4h) followed by glycerol shock (2 min) . After washing, the cells were allowed to recover in culture medium containing Ham's F12/DMEM/10% FCS. The medium was then changed to Ham's F12/DMEM containing 1% Nutridoma-SP (Boehringer- Mannheim) .
  • IL-l or IFN- ⁇ were added after 16 hours of incubation in Ham's F12/DNEM/1% Nutridoma (Recovery experiments) or IL-ljB or IFN- ⁇ were added immediately following glycerol shock, and washing, in the concentrations shown in Ham's F12/DMEM/1% Nutridoma (No Recovery Experiments) .
  • the cultures were incubated for 24-48 h and harvested for CAT assay. Cell extracts were assayed for CAT activity by the fluor diffusion method (Neumann et al., Biotechniques (1987) .5:444 and Eastmann, Biotechniques (1987) .5:730) . Data points for graphs were selected from a time point on the linear part of the curve.
  • Monolayer cultures of chondrocytes isolated from two separate costal cartilage specimens were plated either on agarose, as described by Castagnola et al. (Castagnola, P., et al., 1986, J. Cell Biol.. 102:2310-2317), or tissue culture plastic in Ham's F12/DMEM containing 10% FCS and cultured for 2 weeks with weekly medium changes.
  • the cells on agarose were then transferred to tissue culture plastic and those on plastic were passaged to clean dishes at l ⁇ 6 cells per 100-mm dish and allowed to settle down for 24h.
  • the medium was then changed and the cells transfected with 10 ⁇ g of plasmid DNA by the calcium-phosphate method (4h) followed by glycerol shock (2 min) . After washing, the cells were allowed to recover overnight in 10% FCS. Separate cultures were then incubated in culture medium containing 10% FCS, 1% Nutridoma-SP or 1% ITS+ without or with hIL-0 (100 pg/ml) for 24 h and harvested for CAT assay. The pCAT-B/4.0 plasmid did not express in chondrocytes incubated in the presence of 10% FCS, whether or not they had been passaged over agarose.
  • the pCAT-B/4.0 plasmid did express in chondrocytes after either agarose or monolayer culture in the presence of an insulin-containing serum substitute, particularly in 1% Nutridoma.
  • an insulin-containing serum substitute particularly in 1% Nutridoma.
  • Another insulin-containing serum substitute, ITS+ was also permissive for expression of pCAT-B/4.0 in chondrocytes but was more effective in cultures that had been redifferentiated on agarose than in those that had been subcultured only on tissue culture plastic. All further transient expression assays were performed, therefore, in culture medium containing 1% Nutridoma.
  • pCAT-B/4.0 The expression of pCAT-B/4.0 was tissue-specific but not species-specific, since it expressed in rat chondroblasts but not in human dermal fibroblasts. Moderate expression of pCAT-B/4.0 could be observed in the conditions, although expression was lower than in human chondrocytes; this expression was inhibited by human IL-13 and by human IFN- ⁇ ( Figure 15) . Human IL-l ⁇ also was an effective inhibitor of pCAT-B/4.0 expression in the rat chondroblasts (data not shown). However, murine IFN- ⁇ was used n the rat cultures, since IFN- ⁇ is relatively species specific. No expression of pCAT-B/4.0 could be observed in human dermal fibroblasts ( Figure 16) .
  • FIG. 17B shows the expression from the pCAT-B/4.0 vector in response to the addition of either IL-10 or IFN- ⁇ .
  • the bar graph at the left indicates the level of CAT in response to increasing concentrations of IL-10.
  • the bar graph on the right indicates the level of CAT expression in response to IFN- ⁇ .
  • TL-l ⁇ was obtained as described above.
  • Recombinant preparations of human IFN- ⁇ (2 X 10 6 or 2.65 X 10 7 units/mg) or mouse IFN- ⁇ (17.4 X 10 6 or 5.2 X 10 7 units/mg) were obtained from Genentech, Inc. or they were purchased from AMGEN, Thousand Oaks, CA (hlFN- ⁇ , > 1 X 10 7 units/mg) or from Genzyme, Boston, MA (mIFN- ⁇ , 9 X 10 6 units/mg) .
  • Type II collagen is a cartilage specific gene and the suppression of type II collagen gene expression which is observed in chondrocytes in response to both TL-l ⁇ and IFN- ⁇ is a phenotype of fully differentiated chondrocytes.
  • the finding provides a further demonstration of the fully differentiated phenotype of the C-20/A4 cell line. Similar results are obtained with the T/C-28a2 cell line.
  • IL-l suppresses transcription of the type II collagen gene (COL2A1) in human chondrocytes.
  • a plasmid containing various portions of the promoter, exon 1 and intron 1 of COL2A1 fused the chloramphenicol acetyl transferase (CAT) reporter gene were transfected in the immortalized chondrocyte lines C- 20/A4 and T/C-28a2.
  • High levels of expression of pCAT- B/4.0 with C0L2A1 sequences spanning -577 to +3428 were observed in both lines in the presence of 1% Nutridoma- SP. This expression was inhibited by IL-l by 50-80%.
  • the pCAT-P/0.5 construct (-577/-132 in the pCAT-Promoter vector) and a pCAT-B/0.7+1.1 construct (-577/+63 ⁇ ->+2369/+3426) containing the putative C0L2A1 enhancer also expressed at levels of 85% and 25%, respectively, of pCAT-B/4.0 expression and responded to IL-l.
  • Embodiments I Use of immortalized chondrocytes as a source of transplantation tissue and/or as drug delivery vectors.
  • the immortalized chondrocytes of the invention may be used as a source of transplantation tissue to replace, restore, or reconstruct cartilage tissues in a patient.
  • these cells may be transplanted for use as site-specific delivery vectors for polypeptides of therapeutic use, particularly for the delivery of those polypeptides useful in the treatment of cartilaginous diseases.
  • Allogenic transplantation techniques may be performed using the methods and artificial matrices disclosed in Vacanti et al., United States Patent, SN 5,041,138, hereby incorporated by reference. Using these techniques allogenic cells may be transfected with one or more desirable nucleic acid sequences, and seeded and cultured into a shaped matrix, and implanted into the patient.
  • the immortalized chondrocytes of the invention may be used for the isolation of inhibitors of vascularization. Such factors have pharmacological value as suppressors of tumor growth.
  • Immortalized chondrocytes which will endure and divide for large periods of time in culture offer advantages over the standard methods of isolating such compounds from harvested primary chondrocytes.
  • the method provided by Kuettner et al. in United States Patent SN 4,356,261 is an example of a technique which can be used to isolate such substances from the chondrocytes of the current invention.
  • One skilled in the art will appreciate that other techniques are available and may be used for this purpose.
  • III Construction of a cell line with the phenotype of diseased cartilaginous tissues.
  • Model cell lines for the study of arthritis and other diseases of the cartilage may be constructed by the introduction of vectors which overexpress a preferred protein or by the engineering of null mutations in genes where decreased expression is considered a possible cause of the pathological phenotype. These cell lines may be constructed using one of the immortalized cell lines in combination with known vectors and cloned genes. Such cell lines may be used to elucidate which of those factors altered in various cartilaginous diseases are actually causative in nature. Goldring (1992, In: Cartilage Degradation: Basic Research and Clinical Implications. Eds: F. Woessiner and D. Howell, Marcel Dekker, Inc.), Heinegard and Oldberg (1989, FASEB
  • Hardingham et al. (1991, British Journal of Rheumatology 30(SUPP1 1) : 32-37), Eyre et al. (1991, British Journal of Rheumatology 30(suppl 1) : 10-15) , and Murphy et al. (1991, British Journal of Rheumatology 30(suppl 1) : 25- 31) describe the known gene regulation and phenotypic phenomenon displayed in tissues affected by a variety of cartilaginous diseases.
  • the cells of the invention may be used to screen for synthetic compounds or naturally occurring peptides which mitigate these pathological events.
  • Such compounds may be screened for and detected by applying the compounds of interest to immortalized chondrocytes in culture and monitoring the effects such compounds on gene regulation and protein synthesis using the probes described above in Section Ila of the Preferred Embodiments.
  • the useful pharmaceutical compound is one which alters the gene expression or protein production in a manner which mitigates one or more of the pathological phenotypes of a cartilaginous disease.

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Abstract

Dans les domaines concernant la culture de chondrocytes et l'utilisation, en biologie, de chondrocytes pour dépister et traiter des maladies du cartilage, s'est toujours posé le problème d'obtenir des chondrocytes immortalisés qui gardent leurs charactéristiques de différenciation. L'invention concerne un procédé permettant d'immortaliser des chondrocytes de sorte qu'ils conservent leurs charactéristiques de différenciation, et les cellules ainsi immortalisées. L'invention concerne en outre des procédés d'utilisation de ces cellules dans la thérapie génique de maladie du cartilage, ainsi que des procédés d'utilisation de ces cellules pour dépister des agents affectant la fonction des chondrocytes. L'utilisation des chondrocytes pour produire des agents inhibant la vascularisation est également décrite.
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GB2296017A (en) * 1994-12-13 1996-06-19 Bradley Michael John Stringer Chondrocyte cell-lines
WO1996018728A1 (fr) * 1994-12-13 1996-06-20 Bradley Michael John Stringer Lignees cellulaires de chondrocytes
WO1996034955A1 (fr) * 1995-05-03 1996-11-07 Warner-Lambert Company Procede de traitement de maladies du cartilage par chondrocytes modifies genetiquement
WO1997015655A2 (fr) * 1995-10-20 1997-05-01 Michael Sittinger Nouveaux tissus artificiels, procede de fabrication correspondant et utilisation
EP0828518A1 (fr) * 1995-06-06 1998-03-18 University Of Pittsburgh Of The Commonwealth System Of Higher Education Transfert de genes pour traiter le tissu conjonctif d'un mammifere hote
EP1018987A1 (fr) * 1997-04-04 2000-07-19 Barnes-Jewish Hospital Neocartilage et ses methodes d'utilisation
AU743110B2 (en) * 1994-12-13 2002-01-17 Cellfactors Plc Chondrocyte cell-lines
US6558664B1 (en) 1994-12-13 2003-05-06 Cell Factors Plc Chondrocyte cell-lines
US6569172B2 (en) 1996-08-30 2003-05-27 Verigen Transplantation Service International (Vtsi) Method, instruments, and kit for autologous transplantation
US6592598B2 (en) 1996-08-30 2003-07-15 Verigen Transplantation Service International (Vtsi) Method, instruments, and kit for autologous transplantation
US6713247B1 (en) 1996-09-03 2004-03-30 Signal Pharmaceuticials, Inc. Human CNS cell lines and methods of use therefor
US6866668B2 (en) 1998-08-14 2005-03-15 Verigen Transplantation Service International (“VTSL”) AG Methods, instruments and materials for chondrocyte cell transplantation
RU2683277C1 (ru) * 2018-03-02 2019-03-27 Наталья Константиновна Осина Способ использования хондральных клеток для скринирования веществ, обладающих противовоспалительной активностью

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US6413511B1 (en) 1990-12-20 2002-07-02 University Of Pittsburgh Of The Commonwealth System Of Higher Education Cartilage alterations by administering to joints chondrocytes comprising a heterologous polynucleotide
AU743110B2 (en) * 1994-12-13 2002-01-17 Cellfactors Plc Chondrocyte cell-lines
WO1996018728A1 (fr) * 1994-12-13 1996-06-20 Bradley Michael John Stringer Lignees cellulaires de chondrocytes
US6841151B2 (en) 1994-12-13 2005-01-11 Cellfactors Plc Chondrocyte cell-lines
US6558664B1 (en) 1994-12-13 2003-05-06 Cell Factors Plc Chondrocyte cell-lines
GB2296017A (en) * 1994-12-13 1996-06-19 Bradley Michael John Stringer Chondrocyte cell-lines
EP1142989A3 (fr) * 1994-12-13 2001-11-21 Cellfactors Plc Lignées cellulaires de chondrocytes
WO1996034955A1 (fr) * 1995-05-03 1996-11-07 Warner-Lambert Company Procede de traitement de maladies du cartilage par chondrocytes modifies genetiquement
EP0828518A1 (fr) * 1995-06-06 1998-03-18 University Of Pittsburgh Of The Commonwealth System Of Higher Education Transfert de genes pour traiter le tissu conjonctif d'un mammifere hote
EP1932544A1 (fr) * 1995-06-06 2008-06-18 University Of Pittsburgh Of The Commonwealth System Of Higher Education Transfert de gène pour le traitement de tissus conjonctifs chez les mammifères
WO1997015655A3 (fr) * 1995-10-20 1997-06-12 Michael Sittinger Nouveaux tissus artificiels, procede de fabrication correspondant et utilisation
WO1997015655A2 (fr) * 1995-10-20 1997-05-01 Michael Sittinger Nouveaux tissus artificiels, procede de fabrication correspondant et utilisation
US7048750B2 (en) 1996-08-30 2006-05-23 Verigen Ag Method, instruments, and kits for autologous transplantation
US6569172B2 (en) 1996-08-30 2003-05-27 Verigen Transplantation Service International (Vtsi) Method, instruments, and kit for autologous transplantation
US6592598B2 (en) 1996-08-30 2003-07-15 Verigen Transplantation Service International (Vtsi) Method, instruments, and kit for autologous transplantation
US6592599B2 (en) 1996-08-30 2003-07-15 Verigen Transplantation Service International (Vtsi) Method, instruments, and kit for autologous transplantation
US6599301B2 (en) 1996-08-30 2003-07-29 Verrgen Transplantation Service International (Vtsi) Method, instruments, and kit for autologous transplantation
US6599300B2 (en) 1996-08-30 2003-07-29 Verigen Transplantation Service International (Vtsi) Method, instruments, and kit for autologous transplantation
US7137989B2 (en) 1996-08-30 2006-11-21 Verigen Ag Method, instruments, and kit for autologous transplantation
US6713247B1 (en) 1996-09-03 2004-03-30 Signal Pharmaceuticials, Inc. Human CNS cell lines and methods of use therefor
EP1018987A1 (fr) * 1997-04-04 2000-07-19 Barnes-Jewish Hospital Neocartilage et ses methodes d'utilisation
US7087227B2 (en) 1997-04-04 2006-08-08 Barnes-Jewish Hospital Cartilage composites and methods of use
EP1018987A4 (fr) * 1997-04-04 2004-12-15 Barnes Jewish Hospital Neocartilage et ses methodes d'utilisation
US6645764B1 (en) 1997-04-04 2003-11-11 Barnes-Jewish Hospital Neocartilage and methods of use
US6866668B2 (en) 1998-08-14 2005-03-15 Verigen Transplantation Service International (“VTSL”) AG Methods, instruments and materials for chondrocyte cell transplantation
RU2683277C1 (ru) * 2018-03-02 2019-03-27 Наталья Константиновна Осина Способ использования хондральных клеток для скринирования веществ, обладающих противовоспалительной активностью

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