WO2009004664A2 - Lignée cellulaire immortalisée de cellules souches mésenchymateuses murines, son procédé de préparation et ses utilisations - Google Patents

Lignée cellulaire immortalisée de cellules souches mésenchymateuses murines, son procédé de préparation et ses utilisations Download PDF

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WO2009004664A2
WO2009004664A2 PCT/IT2008/000439 IT2008000439W WO2009004664A2 WO 2009004664 A2 WO2009004664 A2 WO 2009004664A2 IT 2008000439 W IT2008000439 W IT 2008000439W WO 2009004664 A2 WO2009004664 A2 WO 2009004664A2
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cells
mtert
cell line
msc
cell
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WO2009004664A3 (fr
WO2009004664A8 (fr
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Paolo Di Nardo
Giancarlo Forte
Ornella Franzese
Enzo Bonmassar
Maria Prat
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Universita' Degli Studi Di Roma 'tor Vergata'
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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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • the present invention concerns a new immortalized cell line of mesenchymal murine stem cells, method for preparation and uses thereof, particularly as an experimental model.
  • Telomeres are specialized structures, localized at the ends of the eukaryotic chromosomes, consisting of nucleotide sequence repeat units (TTAGGG) n and various proteins (Moyzis et al., 1988; Smogorzewska et al., 2004), which are necessary to maintain the chromosome integrity and prevent chromosome fusion events. During each cell division, the incomplete replication of the chromosome ends results in the loss of 50-200 bases of telomere repeats (Hathcock et al., 2005).
  • TTAGGG nucleotide sequence repeat units
  • Telomeres can be consider mytotic counters suitable to control the number of cell divisions, they induce the cell cycle stop and senescence when their length cannot longer assure the chromosome integrity (Valenzuela and Effros, 2000). Therefore, primary cultures of human cells display a limited duration, before a definitive growth stop, phenomenon known as replicative senescence.
  • the telomerase complex [RNA template sequence and catalytic subunit of telomerase reverse transcriptase (TERT)] adds repeats of TTAGGG sequence to 3' end of chromosomes, thus compensating the progressive telomere loss occurring during every cell division.
  • Telomerase is constitutively active in cells of stem cell line and in most tumour cells (Kim et al., 1994), while it is not expressed in many human somatic differentiated cells (Urquidi et al., 2000). Moreover, the telomerase activity is shown to be high in some populations of somatic cells, like lymphocytes and haemopoietic stem cells (Bodnar et al., 1996; Morrison et al., 1996; Chiu et al., 1996; Weng et al., 1998). While the RNA template sequence (TR) is present in many different cell types, including those telomerase negative, TERT generally is expressed in cells displaying telomerase activity.
  • TR RNA template sequence
  • telomere enzymatic complex This activity can be restored by means of ectopic expression of TERT gene, which encodes for the catalytic subunit of reverse transcriptase associated to telomerase enzymatic complex.
  • Hyper-expression of human telomerase (hTERT) in various cell lines resulted in a dramatic increase of viability of such cells (Hooijberg et al., 2000; Simonsen et al., 2002; Yang et al., 1999) without increase of the incidence of tumour transformation events (Jiang et al., 1999).
  • hTERT constitutive expression has been associated to the onset of resistance to many forms of apoptosis (Akiyama et al., 2002; Yamada et al., 2002; Nakajima et al., 2003), including those resulting from oxidative stress and those induced by the activation of p53 pathway (Luiten et al., 2003; Rahman et al., 2004), as well to the capacity in preventing some aspects of senescence related cellular dysfunctions, like the functionality loss of cytotoxic T lymphocytes (Dagarag et al., 2003; Dagarag et al., 2004).
  • telomerase in addition to the maintenance of telomere integrity, can play additional roles for the viability and cellular functionality (Cao et al., 2002; Chung et al., 2005). This role has been confirmed by various studies indicating that the inhibition of the telomerase activity by antisense oligonucleotides or hTERT negative dominant variants result in the growth inhibition and increase of the cell death sensitivity (Kushner et al., 2000; Misawa et al., 2002; Liu et al., 2004).
  • telomerase in murine stem cells has been evaluated in some studies, which pointed out the presence of high activity in embryonic murine stem cells (mES) and immortalized cell lines, while it diminishes quickly during the terminal differentiation (Sharma et al., 1995). Similar results have been obtained by Lee and co-workers, which were able to demonstrate that the forced hyper-expression of mTERT does not affect the in vitro ES proliferation or differentiation ability, but prevents from cellular death during the differentiation (Lee et al., 2005). mTERT ectopic expression also has shown the ability to confer resistance to oxidative stress induced apoptosis and other genotoxic insults (Lu et al., 2001).
  • telomere activity has been recently studied for haemopoietic and mesenchymal stem cells (HSC and MSC, respectively).
  • HSCs haemopoietic and mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • telomerase activity decrease has been correlated to the loss of proliferative activity and plasticity typical for hMSC senescence (Stenderup et al., 2003).
  • telomeres When compared to ES cells, the greater part of adult stem cells show limited ability to maintain an unaltered length of telomeres and detected telomerase activity does not seem sufficient to prevent the loss of telomeres, suggesting that they do not have indefinite proliferative ability (Allsopp and Weissman, 2002; Ramirez et al., 1997; Yui et al., 1998). Therefore the expression of the telomerase in adult stem cells could play an important role in conferring an advantage for the replication activity.
  • MSC Mesenchymal stem cells
  • MSCs are multipotent precursors suitable to generate in vitro and in vivo the greater part of organism cells.
  • MSCs ability to generate adipocytes, chondrocytes, osteoblasts (Bianco et al., 2001), hepatocytes (Schwartz et al., 2002), smooth muscle, skeletal (Herzog et al., 2003) and cardiac (Forte et al., 2006) cells in vitro has been demonstrated and the possibility that these cells can be used for in vivo transplants appears to be realizable (Ferrari et al., 1998; Duan et al., 2003).
  • an immortalized MSC murine cell line from highly purified murine MSC single cells negative for the expression of differentiated cells markers by means of transfection with mTERT.
  • mTERT-MSC continue to display typical fibroblastoid morphology and high proliferative capacity, preserving characteristic phenotype (Lin-/c- kit+/Sca1+/CD105+/lslet1+/Nucleostemin+/Nestin+/Nanog+) at least until 60st culture passage, contrarily to MSC ex vivo expanded cells which go into senescence after 6-7 passages.
  • mTERT-MSCs maintain in culture the ability to differentiate into adipocytic, osteoblastic, chondrocitic and cardiac phenotypes, when cultured in differentiating media (see materials and methods).
  • This approach represents the first attempt to generate a monoclonal line of adult highly purified stem cells, to be used as a research tool for biology of stem cells (study of differentiation and sprouting in vitro and in vivo processes) and perpetual source of undifferentiated stem cells suitable to result subsequently in appropriate stimuli for organism all cell types.
  • an immortalized cell line of murine mesenchymal stem cells having the following phenotype Lin-/c- kit+/Sca1 +/CD105+/lslet1 +/Nucleostemin+/Nestin+/Nanog+ and characterised in that it expresses mTERT constitutively.
  • the immortalized cell line according to the invention can be further transformed using GFP and/or RFP.
  • a cell line further transformed with marker proteins like GFP and RFP is used, it is possible to provide an experimental model in order to detect the cell differentiation through the staining modification thereof (from green to red).
  • the present invention further refers to the use of the cell line as above defined as an experimental model, particularly for the study of differentiation and sprouting in vitro and in vivo processes.
  • the invention further refers to the use of the cell line as above defined, as a perpetual source of stem cells suitable to transformation as a result of differentiation appropriate stimuli into organism all cell types.
  • MSCs mesenchymal murine stem cells
  • Lin- cell selection b) transfection with mTERT of MSC cells obtained in a) step; c) clone selection and purification by means of limit dilution in order to obtain a Lin-/c-kit+/Sca1+/CD105+/lslet1+/Nucleo stemin+/Nestin+/Nanog+. monoclonal population.
  • mTERT transfected cells have been cultured individually and selected based on the expression of characteristic phenotype of stem cells.
  • said antibodies from a) step are a cocktail of biotin- conjugate monoclonal antibodies against antigens of anti-Ly-6G (Gr-1), 7- 4, TeM 19, CD5, CD45R (B220), CD11 b differentiated cells.
  • the b) step transfection of above mentioned method is carried out employing pCINeo- mTERT expression vector.
  • the transfection is carried out by means of calcium phosphate method although any of transfection methods known to those skilled in the art could be used.
  • step c) for cell clone selection is carried out using G418.
  • mTERT transfected cells selected with G418 antibiotic have been serially diluted to 1 cell/well in a 96 well plate. Then cells have been selected based on the expression of a characteristic phenotype of stem cells by RT-PCR as reported in the example.
  • the method can further comprise a transfecton step of the cell line obtained from step c) using viral vectors encoding for GFP and/or RFP.
  • a transfecton step of the cell line obtained from step c) using viral vectors encoding for GFP and/or RFP can be carried out separately, sequentially or concurrently.
  • mTERT-MSCs will be transformed with a Lentiviral (LV) vector encoding for GFP (fluorescent green protein) under the control of an ubiquitous promoter (preferably PGK or CMV) and then with a LV encoding for RFP (fluorescent red protein) under the control of a specific promoter, active only in a particular cell line (for example, ⁇ -MHX or MLC-2v in the case of cardiomyocytes,).
  • LV Lentiviral
  • GFP fluorescent green protein
  • RFP fluorescent red protein
  • the present invention further refers to an immortalized cell line of murine mesenchymal stem cells obtainable using the method as above defined.
  • Figure 1 shows the insertion of telomerase encoding portion in the MSC cell DNA by means of PCR carried out using genomic DNA extracted from p1-, p7-, and mTERT-MSC mesenchymal stem cells;
  • FIG. 2 shows the mTERT effect on MSC telomerase activity: MSC: telomerase activity (TRAP test) of mTERT transfected Lin-MSCs compared to controls, estimated at different culture times; telomerase activity has been assayed for extracts corresponding to 5 x 10 3 viable cells;
  • MSC telomerase activity (TRAP test) of mTERT transfected Lin-MSCs compared to controls, estimated at different culture times; telomerase activity has been assayed for extracts corresponding to 5 x 10 3 viable cells;
  • FIG. 3 shows that the mTERT transfection is associated to an increase of the TERT protein expression in MSCs; Western Blot analysis of mTERT expressed protein with assay on whole protein extracts of Lin- MSCs transfected with pCINeo-mTERT and relevant controls at different culture times (p1 , p7);
  • Figure 4 shows the effect of the mTERT transfection on the proliferative ability of Lin-MSC cells; the number of cells has been determined every 3 days; the cellular duplications have been quantified as described in Materials and Methods section;
  • Figure 5 shows that the mTERT ectopic expression restores
  • MSC proliferative properties and fibroblastoid morphology upper panel: Iin-p8 MSCs go into senescence and stop to grow as a monolayer, due to the loss of the proliferative ability; mTERT hyper-expression restores the
  • MSCs proliferative ability lower panel: MSC Lin-MSC (p1) cells show fusiform morphology, while p8 MSC assume flat morphology, evidencing the senescence phenomenon; the mTERT expression reverts the senescent phenotype and restores fibroblastoid morphology;
  • FIG. 6 shows that mTERT contrasts the increase of ⁇ -GAL activity in MSCs; lin-MSCs show an increase of ⁇ -galactosidase activity with culture progress, while mTERT expression restores ⁇ -GAL MSC typical activity; Figure 7 shows that mTERT does not affect MSC undifferentiated phenotype; MSC cells display an antigenic stem cell characteristic profile, independently from the culture passage; mTERT transfected cells maintain undifferentiated phenotype;
  • Figure 8 shows that mTERT restores the MSC proliferative activity; p1 MSC cells display high expression of ki67 (20%), that is reduced due to senescence onset (10%); mTERT hyper-expression results in the restoration of ki67 expression (18%).
  • Figure 9 shows the analysis by semi-quantitative RT-PCR of the expression of genes correlated to MSC-mTERT cellular cycle, compared to control cells: mTERT modifies the expression profile of the genes correlated to MSC cellular cycle;
  • FIG. 10 shows FACS (a) and western blot (b) analyses of p16 INK4a expression in MSC p1-, p7- and MSC-mTERT p30: mTERT inhibits the accumulation of p16 cellular cycle inhibitor in senescent MSCs.
  • EXAMPLE 1 Generation of an immortalized MSC cell line MATERIALS AND METHODS
  • MSC cells have been obtained from femurs of 6 week old C57/BI female mouse, using a modification of Friedenstein protocol (Friedenstein et al., 1976).
  • the medullary channel has been washed with cold PBS and obtained pellet has been re-suspended with 5 ml 5 mM EDTA for 30 sec and then one volume of 1.8 % NaCI has been added thereto in order to block hypotonic lysis.
  • Lin pos fraction has been discarded according to immunomagnetic separation protocol (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
  • the cells from the marrow have been incubated with a cocktail of biotin-conjugated monoclonal antibodies (CD5, CD45R (B220), CD11b, anti-Ly-6G (GM) 1 7-4, and TeM 19) against differentiated cell antigens and then separated using secondary antibodies-conjugated nanometric beads against biotin.
  • An aliquot of each obtained sub-population (named Lin- and Lin+) has been labelled with anti-biotin fluorophore-conjugated secondary antibodies and analysed with FACS.
  • Lin- fraction has been re-suspended in complete IMDM medium (Iscove's Modified Dulbecco medium, Cambrex Bio Science, Verviers, Belgium) supplemented with foetal calf serum 10 % (FCS), 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin. Final cell concentration has been adjusted at 0,5X10 6 /cm 2 . After 4-6 days, plate adhering Lin- MSC cell population appeared. Medium has been replaced in order to eliminate the suspended cells and after 7-12 days, the cells have been used for the experiments.
  • complete IMDM medium Iscove's Modified Dulbecco medium, Cambrex Bio Science, Verviers, Belgium
  • FCS foetal calf serum 10 %
  • telomerase activity has been evaluated using "telomeric repeat amplification protocol" (TRAP).
  • the enzyme activity has been assayed on whole cellular extracts prepared in correspondence to times as indicated in results section.
  • the cells have been washed with PBS and ice lysated using lysis buffer containing 0.5 % 3-[(colamidopropyl)- dimethyl-ammonium]-1-propan sulfonate, 10 mM Tris-HCI (pH 7,5), 1 mM MgCI 2 , 1 mM EGTA, 5 mM ⁇ -mercapto ethanol, 0,1 mM [4 (2-aminoethyl)- benzene sulfonyl fluoride] hydrochloride, and 10 % glycerol.
  • the extracts obtained from 5 X 10 3 cells have been used for TRAP assay in 50 ⁇ l reaction buffer [20 mM Tris-HCI (pH 8,3), 68 mM KCI, 1 ,5 mM MgCI 2 , 1 mM EGTA, 0.05 Tween 20, 0.1 ⁇ g TS (AATCCGTCGAGCAGAGTT) primer, 0,5 mM T4 gene protein 32, 10 mM deoxynucleotide triphosfate, Taq polymerase 2 units (Promega, Madison, Wl, USA), and ( ⁇ - 32 P) dCTP 2 ⁇ Ci (3000 Cl/mmol; DuPont NEN Research Products, Boston, BUT)].
  • reaction buffer 20 mM Tris-HCI (pH 8,3), 68 mM KCI, 1 ,5 mM MgCI 2 , 1 mM EGTA, 0.05 Tween 20, 0.1 ⁇ g TS (AATCCGTCGAGCAGAGTT) primer
  • CCTTTA CX oligonucleotide
  • 3CCCTAA Biogen, Rome, Italy
  • IC internal control
  • the cells have been seeded at a concentration of 2 X 10 4 /cm 2 in every passage.
  • the cell viability has been quantified using Trypan blue exclusion assay every two days.
  • the proliferative ability has been calculated as number of "population doublings", using the formula logi 0 (total/initial number)/log 2 .
  • mTERT expression vector For the construction of mTERT expression vector, pcINeo plasmid (Promega, Madison, Wl) has been digested with EcoRI enzyme, and the sequence encoding for mTERT, obtained from pGRN190 plasmid
  • Resulting vector is named pCINeo-mTERT.
  • the cells have been seeded at 50% confluence on the first day
  • Vector transfected cells have been cultured over a long time in the presence of G418, taking advantage of the presence in the vector of the resistance gene to this antibiotic: only the cells wherein the vector had been effectively inserted were viable. These cells have been further purified by means of limit dilution in the order to obtain a monoclonal population.
  • mTERT transfected cells selected using G418 antibiotic, have been serially diluted to 1 cell/well in a 96 well plate. Then cells have been selected based on the expression of a characteristic phenotype of stem cells by RT-PCR. ( Figure 7).
  • MSC differentiation protocols mTERT-MSC and MSC cells from early and late passages have been seeded in 35 mm plates in IMDM medium supplemented with 10 % FCS. Medium has been replaced and the cells cultured in adipogenic and osteogenic medium (code numbers PT3924, PT3102b Cambrex Bio
  • compositions of used media were as following: chondrogenic medium (DMEM 10 % FCS; 50 ⁇ g/ml ascorbic acid; 0.1 ⁇ M Dexamethasone; 40 ⁇ g/ml L-proline; 100 ⁇ g/ml sodium pyruvate; ITS 1X; 10 ng/ml human recombinant TGF- ⁇ 1), osteogenic medium (DMEM 10% FCS; 50 mg/ml ascorbic acid; 10 mM ⁇ - glycerophosphate; 0.1 ⁇ M Dexamethasone), adipogenic medium (DMEM 10% FCS; 1 ⁇ M Dexamethasone; 0,5 mM 3-isobutyl-1-methylxantine; 1 ⁇ g/ml insulin).
  • chondrogenic medium DMEM 10 % FCS; 50 ⁇ g/ml ascorbic acid; 0.1 ⁇ M Dexamethasone; 40 ⁇ g/ml L-proline; 100 ⁇ g/ml sodium
  • the cells After 10 days in osteogenic medium, the cells have been fixed in 4 % paraformaldehyde for 5 min and washed with distilled water 3 times; then incubated with silver nitrate for 60 minutes and exposed to a 40W lamp (Lennon et al., 2001). After the removal of the silver nitrate solution, the cells have been incubated with 5 % sodium thiosulfate and subsequently again washed with distilled water. AdipoRed staining
  • the adipogenic differentiation has been evaluated using
  • AdipoRedTM Lipid Assay Reagent (Cambrex Bio Science, Verviers,
  • RNA has been extracted using TRIZOL reagent (GIBCO).
  • RNA expression has been carried out using 2 ⁇ g RNA for each sample by means of RT M-MLV (Invitrogen, CA, USA) in the presence of random hexamers.
  • Semiquantitative analysis of the RNA expression has been carried out by RT-PCR comparing control and interest transcripts (GAPDH) in the exponential growth phase.
  • GPDH control and interest transcripts
  • the cells For intracellular proteins labelling, the cells have been fixed and permeabilized using IntraprepTM kit (Beckman Coulter), according to the manufacturer instructions and incubated for 30 min with anti-ki67
  • MSC cells from early (passage 1 , p1), and late (passage 7, p7) passages and mTERT-MSC p20 have been seeded at 2X10 4 cells/cm 2 concentration on immunofluorescence slides (Nalge, Nunc International). The cells have been washed with PBS, 4 % paraformaldehyde fixed (PFA) in PBS containing CaCI 2 for 30 min at 4°C and permeabilized with 0.1 % Triton X-100. Filamentous actin (F-actin) has been labelled with Tetra- rodhamine conjugated Phalloidin in 50 % Methanol for 30 min. Nuclei have been labelled with 4',6'-diamidino-2-phenyl indole (DAPI). ⁇ -Galactosidase specific activity
  • the ⁇ -galactosidase specific activity has been evaluated by means of the ⁇ -GAL assay kit (Invitrogen, CA, USA).
  • the cells have been scratched after 1 minute of incubation in trypsin/EDTA and addition of complete medium in order to inhibit the enzymatic activity.
  • the cells have been centrifuged at 250xg for 5 min, resultant pellet re-suspended in 30 ⁇ l of lysis buffer and lysed using the "freeze-and-thaw" method, according to the manufacturer supplied protocol.
  • the solution has been centrifuged at 13000 x g, at 4°C for 5 min, the supernatant transferred in a new centrifuge tube.
  • the reaction has been stopped by adding 500 ⁇ l of stop buffer and the absorbance value at 420 nm (compared to the "blank” sample containing "only” cleavage buffer ONPG, but not lysate) using 50 Cary UV-visible Bio spectrophotometer (Varian).
  • Western blot
  • the cells have been washed two times with cold PBS, scratched from the plate, incubated in solution containing Trypsin and EDTA and lysated with RIPA buffer (150 mM NaCI, 50 mM Tris-HCI [pH 7,4], 1 % Nonidet P-40, 0.25% sodium deoxycholate, 2 mM orto-vanadate and a cocktail of protease inhibitors (Sigma Aldrich, Concorezzo, Italy).
  • RIPA buffer 150 mM NaCI, 50 mM Tris-HCI [pH 7,4], 1 % Nonidet P-40, 0.25% sodium deoxycholate, 2 mM orto-vanadate and a cocktail of protease inhibitors (Sigma Aldrich, Concorezzo, Italy).
  • the cell lysates have been centrifuged at 13000 rpm at 4°C for 15 min, the protein concentration has been quantified with Bradford method (Amresco, Fl, USA) and the extracts (50 ⁇ g) have been analysed on 12.5 % polyacrylamide gel in the presence of SDS (SDS-PAGE), and transferred on PVDF filters.
  • the filters have been blocked with PBS/7% milk and the analysis of the protein expression has been carried out using following primary antibodies against p42-p44, (Cell Signaling Technology, Beverly, BUT), polyclonal antibodies against TERT (Calbiochem, the JoIIa, Ca), p27 kip1 , p21 waf1 (Lab Vision Corporation, Fremont, CA.
  • MSC lin- cells have been transfected with pCINeo-mTERT expression vector or pCINeo control vector using calcium phosphate method, as described in Materials and Methods section.
  • mTERT hyper- expressing cells have been selected using G418, 300 ⁇ g/ml for 2 weeks.
  • Single cells have been isolated and cultured in the same culture medium used for MSC lin- cells, in order to obtain clones of immortalized stem cells deriving from a single cell.
  • the analysis of pCI-NEO-MTERT vector integration in the cell genome has been carried out by means of PCR, using primers specific for Pci-NEO-MTERT vector sequence. This analysis confirmed the presence of mTERT sequence in the MSC mTERT cell DNA ( Figure 1).
  • the cell morphology appears to be significantly modified.
  • the cells acquire a more flattened aspect than controls and display a progressive reduction of the proliferation capacity, as a result of senescence progression.
  • Forced mTERT expression is suitable to stop the generation of fibroblastoid cells ( Figure 5, lower panel).
  • the ⁇ -GAL activity considered as a senescence marker (Vacanti et al., 2005), has proved to be significantly reduced for mTERT-MSC in comparison to MSC p7, and comparable to p1 cell level ( Figure 6).
  • RT-PCR analysis carried out with sternness typical markers indicated that there are no differences in mRNA levels for p1-, p7- and mTERT-MSC ( Figure 7). These characteristics have been maintained for more than 60 passages in the culture used conditions.
  • mTERT hyper-expression modifies the expression pattern of the genes responsible of the cell cycle regulation.
  • MSC cells ex vivo were characterised by a 24 hour duplication time. Under the used experimental conditions cells proceeded towards the senescence within 6 or 7 passages, as indicated from the appearance of flattened morphology cells, proliferation stop and meaningful increase of the ⁇ -GAL activity. The appearance of these characteristics, considered typical for the senescence, was associated to contemporary increase of p 16 iNK4A eX p resS jon, known as a marker of cell cycle stop. mTERT ectopic expression prevented the induction of the senescent phenotype in MSC p7.

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Abstract

La présente invention concerne une nouvelle lignée cellulaire immortalisée de cellules souches mésenchymateuses murines et un procédé pour la préparation et les utilisations de celle-ci, en particulier en tant que modèle expérimental.
PCT/IT2008/000439 2007-07-03 2008-06-27 Lignée cellulaire immortalisée de cellules souches mésenchymateuses murines, son procédé de préparation et ses utilisations WO2009004664A2 (fr)

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ITRM20070372 ITRM20070372A1 (it) 2007-07-03 2007-07-03 Linea cellulare immortalizzata di cellule staminali mesenchimali murine, metodo per la sua preparazione e relativi usi.
ITRM2007A000372 2007-07-03

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