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WO2008034929A1 - Procédé permettant de cultiver et d'entretenir des cellules souches pluripotentes et des cellules souches d'un mammifère à l'état non différencié - Google Patents

Procédé permettant de cultiver et d'entretenir des cellules souches pluripotentes et des cellules souches d'un mammifère à l'état non différencié Download PDF

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WO2008034929A1
WO2008034929A1 PCT/ES2007/000537 ES2007000537W WO2008034929A1 WO 2008034929 A1 WO2008034929 A1 WO 2008034929A1 ES 2007000537 W ES2007000537 W ES 2007000537W WO 2008034929 A1 WO2008034929 A1 WO 2008034929A1
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nitric oxide
cells
mammalian
maintenance
stem cells
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WO2008034929B1 (fr
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Bernat Soria Escoms
Juan Rigoberto Tejedo Huaman
Francisco Javier Bedoya Bergua
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Universidad Pablo de Olavide
Fundacion Progreso y Salud
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Fundacion Progreso y Salud
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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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/03—Compounds acting on the NO pathway, e.g. nitrososarginine

Definitions

  • the present invention describes a new method for the culture and maintenance of mammalian pluripotential cells using nitric oxide.
  • the invention is limited to the field of therapeutic and biotechnological applications of pluripotential stem cells and progenitor cells.
  • Pluripotential stem cells can be obtained from tissues of adult origin, fetal origin and embryonic origin. In the case of pluripotential cells of embryonic origin, they are obtained from the internal mass of the blastocyst. These cells have the ability to proliferate indefinitely in "in vitro" cultures in an undifferentiated state while maintaining their normal karyotype and ability to differentiate into various types of lineages and tissues, becoming an unlimited source of cells potentially useful in Regenerative Medicine. However, for this they are grown on a layer of fibroblasts, so that the currently available cell lines do not meet the necessary standards for use in the clinic and in other biotechnological applications.
  • Embryonic stem cells are widely used in research and cell therapy programs. Their expansion in crops is a critical point for them to maintain their undifferentiated state and their pluripotential capacity.
  • Mouse embryonic stem cells were initially maintained in an undifferentiated state when cultured on a monolayer of inactivated mouse fibroblasts either by mitomycin or irradiation. It was subsequently described that the culture medium supplemented with serum and cytokines, mainly the leukemia inhibitor factor, allows the growth in an undifferentiated state of these cells when they are grown on gelatin coated plates [Williams, R. L 1 DJ Hilton et al .
  • Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336 (6200): 684-7 (1988)], Currently there are commercial lines that can grow on certain types of high adhesion plates without the need for gelatin. However, in the case of human embryonic cells, the leukemia inhibitor factor is not effective, so there are no effective methods to keep human embryonic cells in an undifferentiated state in cultures that do not involve the use of monolayers. fibroblasts
  • Human embryonic stem cells to be cultured for prolonged times maintaining their undifferentiated state require the use of a support formed by other cell types, for example, a monolayer of mouse embryonic fibroblast cells [Thomson, JA; Itskovitz-Eldor J .; "Embryonic stem cell lines derived from human blastocysts”; Science 282 (5391): 1145-7 (1998)] or of cells from human foreskin (Inzunza, J .; Gertow, K. et al. "Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. "Stem CeIIs 23 (4): 544-9 (2005)].
  • mice embryonic fibroblasts The cultivation of pluripotential cells on mouse embryonic fibroblasts implies contamination with animal material reducing the possibility of application of these cells in cell therapy in humans
  • the monolayers of mouse embryonic fibroblasts and the conditioned medium obtained from them do not provide species-specific growth factors for human embryonic stem cells decreasing the efficiency of their growth.
  • Foreskin fibroblasts of human origin solve the problem of animal contamination, but the logistical problems related to the maintenance of parallel cultures of fibroblasts and pluripotential cells make it difficult to scale for the production of large amounts of cells.
  • MATRIGEL is a product whose exact composition is unknown.
  • the conditioned medium prepared with serum or serum substitutes additionally presents the difficulties of the animal origin of its components and the maintenance of a fibroblast culture system to be conditioned. It has been shown that one of the difficulties in using the unconditional medium is that it has high BMP-related activity, which activates differentiation systems in human embryonic stem cells [(Xu RH; Peck RM et al. "Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. "Nat Methods 2 (3): 185-90 (2005)].
  • BMP protein antagonists such as NOGGIN, in the culture medium decreases the differentiating pressure of the medium. The use of this system has been shown to produce extremely low yield and in prolonged cultures the cells differentiate and lose their pluripotential capacity.
  • the object of the present invention is a method for the culture and maintenance of mammalian pluripotential stem cells in an undifferentiated state.
  • the effect of no Differentiation is achieved by adding nitric oxide to said pluripotential stem cell culture.
  • Mammalian pluripotential stem cells can be derived from either adult tissues, fetal tissues, or they can be embryonic stem cells, including human embryonic stem cells.
  • nitric oxide can be carried out by exogenous donors, preferably diethylenetriamine adduct with nitric oxide (DETA / NO), S-nitroso-N-acetyl-D, L-penicillamine (SNAP), SIN-I and others, adding the donor of nitric oxide directly to the culture medium at concentrations between 500 nM and 10,000 nM and optionally mixed with other compounds, preferably with extracellular matrix component proteins as support for cell growth.
  • DETA / NO diethylenetriamine adduct with nitric oxide
  • SNAP L-penicillamine
  • nitric oxide is carried out by endogenous production of nitric oxide, preferably through the overexpression of enzymes involved in the synthesis of nitric oxide, more preferably of nitric oxide endothelial synthase (eNOS).
  • eNOS nitric oxide endothelial synthase
  • a way of performing said endogenous nitric oxide production would be by transfection of pluripotent stem cells with a plasmid mammalian Ia inducing over-expression of eNOS Ia.
  • nitric oxide can be carried out by exogenous donors, preferably diethylenetriamine adduct with nitric oxide (DETA / NO), S-nitro-N-acetyl-D, L-penicillamine (SNAP), SIN-I and others, with the donor being added of nitric oxide directly to the culture medium at concentrations between 500 nM and 10,000 nM and optionally mixed with other compounds, preferably with extracellular matrix component proteins as support for cell growth.
  • DETA / NO diethylenetriamine adduct with nitric oxide
  • SNAP L-penicillamine
  • nitric oxide can be carried out by endogenous production of nitric oxide, preferably through the overexpression of enzymes involved in the synthesis of nitric oxide, more preferably of nitric oxide synthase endothelial (eNOS).
  • eNOS nitric oxide synthase endothelial
  • a way of carrying out said endogenous production of nitric oxide would be by transfection of the mammalian pluripotential stem cells with a plasmid inducing the e-expression of the eNOS.
  • FIGURES Figure 1 Scheme of the Oligo hp ⁇ ct4 eGFP poly A pGK hygro plasmid
  • Figure 2 Expression of non-differentiation markers in Line D3-0ct4
  • Figure 3 Telomerase activity of lines D3-pOCT4 and D3-pOCT4-eNOS.
  • Figure 4 Alkaline phosphatase activity in lines D3-pOct4 and D3-pOct4-eNOS.
  • A. Line D3-pOct4 P17 grown in the presence of LIF for 7 days.
  • B. Line D3-pOct4 P18 grown in the presence of LIF for 9 days.
  • Figure 8 Effect of exogenously supplied nitric oxide on the expression of non-differentiation marker proteins measured by flow cytometry, fluorescence microscopy and immunofluorescence
  • Figure 9 Effect of nitric oxide supplied exogenously on the expression of non-differentiation marker proteins measured by immunodetection and by RT-PCR
  • Figure 10 Effect of the inhibition of nitric oxide production on the expression of non-differentiation marker proteins measured by flow cytometry, fluorescence and immunofluorescence microscopy
  • Figure 11 Effect of the inhibition of nitric oxide production on the expression of non-differential marker proteins by immunodetection and by RT-PCR
  • Figure 12 Activation of signaling pathways by nitric oxide in lines D3-0ct4 and D3- Oct ⁇ eNOS of mouse embryonic stem cells
  • Figure 13 Effect of the exogenous addition of nitric oxide on the expression of non-differentiation marker proteins by immunodetection and by RT-PCR in human embryonic stem cells HS-181.
  • Nitric oxide can be supplied from exogenous sources (nitric oxide donors such as DETA- NO) or inducing its endogenous production by means of the overexpression of endothelial nitric oxide synthase (eNOS).
  • eNOS endothelial nitric oxide synthase
  • the addition of nitric oxide to the cultures of human pluripotential cells on matrices such as "Matrigel", with medium without conditioning and without supplementing with bFGF allows to keep the cultures in an undifferentiated state, during prolonged cultures.
  • This method has advantages with respect to the culture with conditioned medium since it solves the problem of supply of conditioned medium and with respect to the use of bFGF it reduces the costs in the cultures of cells that express the FGF receptor and allows maintenance in those that do not express.
  • nitric oxide synthase endothelial in pluripotential stem cells solves the problem of nitric oxide supply and eliminates the possibility that the donor ligand may have unpredictable side effects.
  • Example 1 Maintenance of mammalian pluripotential cells in a state not differentiated by nitric oxide; Obtaining and characterizing the D3-p0ct4 and D3-pOct4-eNOS cell lines of mouse embryonic stem cells.
  • This example describes the obtaining and characterization of the D3-pOct4 and D3-pOct ⁇ -eNOS lines of embryonic stem cells, which are part of the tools used for the realization of the present invention.
  • the commercial mouse embryonic stem cell line D3 was obtained from the ATCC (ATCC number: CRL1934). Cells were cultured in undifferentiated state in Dulbecco 's Modified Eagle Medium (DMEM) [Gibco 32430-100], supplemented with 15% fetal bovine serum: FBS (ESC Tested) [Hyclone SH30070-03], 100 U / mL Penicillin, 100 ⁇ g / mL streptomycin [Gibcol5140-122], 2mM L-Glutamine [Gibco25030-032], 1% non-essential amino acids [Gibcol 1140-035], 100 ⁇ M ⁇ -mercaptoethanol (50 mM) [ Gibcol31350-010], 0.1 U / ⁇ L of Leukemia Inhibitory Factor (LIF) 10 6 U / ml (Chemicon ESG-1106).
  • DMEM Dulbecco 's Modified Eagle Medium
  • FBS fetal bo
  • Subculture was performed by separating the cells with a solution of 0.05% trypsin + 0.02% EDTA-4Na of (Gibco, Invitrogen, England) at 37 0 C to 37 ° C for 5 minutes. Trypsin was neutralized with medium and the cell mass was recovered by centrifugation at 1000 rpm for 5 minutes. The cell precipitate was resuspended in culture medium with LIF at the desired concentration, then counting viable cells and then sowing at the desired cell density.
  • Transfection of Jas D3 cells was performed by electroporation. 800 ⁇ L of the suspension of D3 cells in serum-free medium contained in an electroporation cuvette was added 4 ⁇ g of the Oligo hp ⁇ ct4 eGFP poly A pGK hygro plasmid cut with the enzyme Ssp I and purified. Then it was mixed gently and left on ice for 5 minutes. Electroporation was performed at a voltage of 800 V, infinite amperage, 50 ⁇ faraday and 30 milliseconds of time. Before sowing in the culture plate the upper layer is removed and the rest is sown in a culture bottle with complete medium.
  • D3-pOct4-eNOS cells were obtained by transfection of D3-p ⁇ ct4 cells with plasmid pCDNA3.1-eNOS (Department of Medicine of the University of Toronto, Canada). Transfected cells were selected with medium containing 400 ⁇ g / ml of hygromycin and 500 ⁇ g / ml of neomycin.
  • transfected colonies were selected by the plaque depletion method and finally from a stable colony the positive GFP cells were selected by "cell sorter" on a BD-FACSAria TM cytometer. Characterization of transfected cells.
  • the characterization was carried out using standard methods for the production of non-differentiation markers.
  • Protein immunodetection was performed by "western blot" from the total homogenates obtained from cultured cells under the conditions described by breaking with the RIPA buffer supplemented with phosphatase inhibitors and protease inhibitors (Sigma); The protein separation was performed by electrophoresis in SDS-PAGE and subsequently transferred by wet transfer to PVDF membranes; for immunodetection, the membranes were successively incubated in a blocking solution for 2 hours, primary antibodies diluted in TBST at concentrations and times previously standardized for each of them, washed 3 times with TBST for 10 minutes, secondary antibodies conjugated with peroxidase diluted in TBS at a concentration of 1: 20,000 for 30 minutes, washed 3 times with TBS for 10 minutes. Finally, the peroxidase activity was detected by the chemiluminescence reaction using standard methods. Expression of non-differentiation markers by RT-PCR
  • Telomerase activity was evaluated using the Telo TAGGG Telomerase PCR Elisa PLUS commercial kit (Roche 12 013 789 001), following the manufacturer's instructions. Determination of the alkaline phosphatase activity.
  • Alkaline phosphatase activity was measured following the instructions of the SIGMA 86C kit
  • the cells were induced to form metaphases by incubating them with a solution of colchemide (SIGMA C-9754) at the final concentration of 0.02 ⁇ g / mL for 3 hours.
  • the Nuclear cell membranes were broken with a hypotonic solution following the standards (International System for Human Cytogenetic Nomenclature, ISCN).
  • G - banding of the chromosomes were performed by treating the slides with trypsin for 8 seconds at 37 0 C. Trypsin was prepared in PBS 0.05% and previously tempered at 37 0 C for at least 30 minutes.
  • Figure 2 shows that line D3-0ct4 expresses the indifferentiation markers Oct4, Sox 2, Nanog, and Fox D3, indicating that it is an undifferentiated population.
  • the expression of indifferentiation markers of the D3-Oct4-eNOS cell line is shown in Figure 6. It is observed that this line expresses 0ct4, Nanog, Sox 2 and Fox D3.
  • Telomerase activity One of the fundamental characteristics that define embryonic stem cells is their high capacity for self-renewal. This capacity is supported by high telomerase activity. Telomerase activity decreases during the differentiation process in mouse embryonic stem cells.
  • the telomerase activity of lines D3, D3-0ct4, D3-0ct4-eN0S and their respective embryonic bodies are shown in Figure 3.
  • line D3 has a teiomerase activity less than line D3-pOct4, indicating that the population of the transfectant line is homogeneous.
  • the telomerase activity of D3-0ct4-eN0S is greater than that of lines D3 and D3-p0ct4, which indicates that the continuous production of nitric oxide due to overexpression of the eNOS, increases the telomerase activity and improves the characteristics of non-differentiation of these cells.
  • Alkaline phosphatase activity One of the tests commonly used to show the cell culture in its undifferentiated state is the presence of alkaline phosphatase activity. Alkaline phosphatase is active when cells remain undifferentiated and this activity is lost as the culture ages and differentiated cells appear.
  • Figure 4 shows the alkaline phosphatase activity of lines D3- 0ct4 and D3-Oct4-eNOS. It is observed that both lines have a robust alkaline phosphatase activity.
  • Nitric oxide decreases the pressure towards differentiation in conditions of withdrawal of LIF in D3-p0ct4 cells.
  • Example 2 In addition to the methods used in Example 1, the following experimental processes have been used: for the maintenance of D3-pOct4 cells in an undifferentiated state, 200 ⁇ g / mL of hygromycin is added to the described culture medium.
  • the experimental conditions that we use in this example are: a.-control conditions, the cells are cultured in the medium described in example 1 containing the indicated concentration of LIF. b.- differentiation conditions, the cells were cultured in the medium described in example 1 to which the LIF has been removed. c- experimental conditions for the addition of nitric oxide, I to which LIF has been removed and 2 ⁇ M of DETA-NO (Sigma) added.
  • Immuno-fluorescence colony dyeing was performed according to the following protocol: the cells were cultured in appropriate culture plates (Lab-Tek chamber slide w / cover permanox from NUNC). Once the respective treatments were finished, the cells were washed and fixed with 4% formaldehyde for 15 minutes, then they were permeabilized, blocked and incubated with the specific primary antibodies following standard procedures. The detection of the binding of the primary antibodies to their respective antigens present in the cells was carried out by incubation with secondary antibodies conjugated with FITC, TRITC or Rhodamine Red as appropriate. Image capture was performed on a Leica DM 600 microscope. Results
  • panel A shows results of flow cytometry experiments performed with D3-pOct4 cells. It is observed that at 7 days of culture 69.67% of the cells maintain the expression controlled by the 0ct4 promoter of the green fluorescent protein (eGFP) when they are cultured in the presence of LIF. The removal of LIF from culture medium dramatically decreases the number of eGFP positive cells to 27.61%. The addition of 2 ⁇ M of DETA-NO under conditions of LIF withdrawal stops the differentiating pressure, which is expressed in that 45.37% of the cells are eGFP positive.
  • panel B shows the photographs of representative colonies under the conditions described above taken with fluorescence microscopy.
  • panel C shows the expression of non-differentiation marker proteins detected by immuno-fluorescence with antibodies specific for 0ct4 (green) and SSEAl (red). The results show that the expression of 0ct4 and SSEA1 is restored by the addition of nitric oxide when they are cultured in the absence of LIF.
  • the experimental scheme consists in inhibiting the production of nitric oxide by the eNOS using a nitric oxide synthase inhibitor, NMMA (N-Mono-Methyl-L-Arginine).
  • the experimental conditions used in this example are: a.-control conditions, the cells were cultured in the medium described in example 1 containing the indicated concentration of LIF. b, - experimental conditions of presence of nitric oxide in the absence of LIF, the cells cells were cultured in the medium described in example 1 to which the LIF has been removed. c- differentiation conditions, the cells were cultured in the medium described in example 1 to which the UF has been removed and 400 ⁇ M NMMA (Sigma) added. Results
  • panel A is shown by flow cytometry experiments that after 7 days of culture in high adhesion plates without gelatin, 70.9% of the cells keep the expression controlled by the 0ct4 promoter of the protein fluorescent green (eGFP); when cells expressing eGFP are cultured in the absence of UF it decreases to 51.8% and when they are cultured in the absence of LIF with 400 ⁇ M NMMA, cells expressing eGFP is only 16.4%.
  • Panel B shows photographs of representative colonies under the conditions described above taken with fluorescence microscopy.
  • Panel C shows the expression of non-differentiation marker proteins detected by immunofluorescence with antibodies specific for Oct4 (green) and SSEAl (red).
  • Example 4 Maintenance of mammalian pluripotential cells in a state not differentiated by nitric oxide: Study of the activation of protein components of various signaling pathways by nitric oxide in D3-0ct4 and D3-Oct4-eNOS cells.
  • nitric oxide promotes the maintenance of the undifferentiated state of mouse embryonic stem cells through the activation of signaling pathways.
  • panels A and B show that nitric oxide activates the signaling pathway of PI3K / AW and the c-Src protein, in D3-pOct4 and D3-p0ct4-eN0S cells.
  • the STAT3 pathway is not activated by nitric oxide.
  • Example 5 Maintenance of mammalian pluripotential cells in a state not differentiated by nitric oxide: Nitric oxide promotes the state of non-differentiation under conditions of bFGF withdrawal in embryonic stem cells of human origin.
  • Human embryonic stem cells were cultured on foreskin fibroblasts inactivated with mitomycin.
  • the cells were cultured in the hESC culture medium consisting of DMEM KO (Invitrogen 10829-018) supplemented with 20% serum substitute (SR) (Invitrogen PL 1082028), 2mM L-Glutamine (Invitrogen 25030-024 ), 1% non-essential amino acids (Invitrogen 11140-035), 50U / mL penicillin, 50mg / mL streptomycin (Invitrogen 15140-122), 0.1 mM 2- ⁇ -Mercaptoethanol (Invitrogen 31350-010) , 8 ng / mL of b-FGF (R&D Systems 234-FSE-025).
  • SR serum substitute
  • SR Invitrogen PL 10820208
  • 2mM L-Glutamine Invitrogen 25030-024
  • 1% non-essential amino acids Invitrogen 11140-0
  • the foreskin fibroblasts were cultured in HEF culture medium consisting of Iscove DMEM (Invitrogen 21980-032) supplemented with 10% SBF (Invitrogen 10106-169), 25 u / mL penicillin, 25 mg / mL streptomycin (Invitrogen 15140-122).
  • the fibroblasts are inactivated with 10 ⁇ g / mL of mitomycin for 3 hours and subsequently washed three times with HEF and hESC without bFGF is added and incubated for at least 12 hours;
  • the conditioned medium is removed and filtered.
  • the conditioned medium before use is supplemented with 8 ng / mL of bFGF.
  • fibroblasts can be maintained in culture for up to 42 passes; under these conditions they provide relatively stable environmental conditions to human embryonic stem cells.
  • Embryonic stem cells of human origin can grow maintaining their undifferentiated state in feeder monolayers free systems on solid surfaces such as extracellular matrices in the presence of conditioned culture medium supplemented with 8ng / mL of bFGF.
  • the commercial matrix "Matrigel” (BD Matrigel TM Basement Membrane Matrix - BD Biociencies Cat. 354223) is used.
  • the experimental conditions employed in this example are: a.-control conditions, the cells were cultured in the unconditional hESC medium described above supplemented with 8ng / ml of bFGF. b.- experimental differentiation conditions, the cells were cultured in the hESC medium without conditioning and without the addition of bFGF. c- conditions of presence of nitric oxide in the absence of bFGF, the cells were cultured in the hESC medium without conditioning, without bFGF and with the addition of 2 ⁇ M of DETA-NO. i. Immunofluorescence detection of non-differentiation markers ii. Immunodetection of non-differentiation markers ⁇ . Study of the PCR expression of non-differentiation markers iv. Study of the activation of protein components of various signaling pathways by nitric oxide Results.
  • panel A shows that in the cells grown in the absence of bFGF, the appearance of differentiated areas in the colonies occurs and that under these conditions the addition of 2 ⁇ M of DETA-NO prevents the appearance of these areas;
  • this panel shows that the expression of the non-differentiation marker transcription factor Oct4 is present in the condition in which bFGF has been added, disappears when the bFGF is removed and is present when 2 ⁇ M of DETA-NO is added; It is important to note that the 0ct4 signal in the presence of DETA-NO is greater than in the presence of bFGF.
  • panel B shows the immunodetection of the expression of the transcription factors markers of non-differentiation, Oct4, Sox 2 and Nanog.
  • nitric oxide promotes the maintenance of the state of non-differentiation in human embryonic stem cells.
  • the presence of higher levels of non-differentiation marker proteins in the conditions in which nitric oxide is added with respect to the conditions of medium supplemented with bFGF corresponds to the longer maintenance time of the undifferentiated state of the cells by these two factors, nitric oxide being more efficient than bFGF (10 passes (around 80 days) for crops in the presence of nitric oxide and 3 passes for cultures with bFGF (around 24 days).

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Abstract

L'entretien en culture des cellules pluripotentes et souches à l'état non différencié présente divers problèmes inhérents au type, à l'origine et à l'utilisation ultérieure des cellules. De nos jours, il est possible de cultiver des cellules pluripotentes d'un mammifère dans des systèmes qui permettent la conservation de leur état non différencié; toutefois, la différenciation spontanée inhérente à la culture est élevée; cette situation entraîne quelques difficultées dans les processus d'obtention de cellules. Le procédé décrit dans cette invention permet d'entretenir et de cultiver des cellules pluripotentes et souches d'un mammifère à l'état non différencié grâce à l'utilisation d'oxyde nitrique.
PCT/ES2007/000537 2006-09-19 2007-09-18 Procédé permettant de cultiver et d'entretenir des cellules souches pluripotentes et des cellules souches d'un mammifère à l'état non différencié Ceased WO2008034929A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017021535A1 (fr) * 2015-08-05 2017-02-09 Fundación Pública Andaluza Progreso Y Salud Milieu de culture cellulaire amélioré de cellules progénitrices humaines (hpc)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5977181A (en) * 1996-11-13 1999-11-02 Cold Spring Harbor Laboratory Therapeutic uses for nitric oxide inhibitors
WO2005090558A1 (fr) * 2004-03-19 2005-09-29 Geron Corporation Procede destine a preparer des preparations cardiomyocytes haute purete utilisees dans la medecine regenerative

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5977181A (en) * 1996-11-13 1999-11-02 Cold Spring Harbor Laboratory Therapeutic uses for nitric oxide inhibitors
WO2005090558A1 (fr) * 2004-03-19 2005-09-29 Geron Corporation Procede destine a preparer des preparations cardiomyocytes haute purete utilisees dans la medecine regenerative

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BERNABE ET AL.: "Sodium nitroprusside-induced mitochondri the apoptotic events in insulin-secreting RINm5F cells are associated with MAP kinases activation", EXPERIMENTAL CELL RESEARCH, vol. 269, 2001, pages 222 - 229, XP002256953, DOI: doi:10.1006/excr.2001.5315 *
COVACU E TAL.: "Nitric oxide exposure diverts neural stem cell fate from neurogenesis towards astrogliogenesis", STEM CELLS, vol. 24, 2006, pages 2792 - 2800 *
KANNO ET AL.: "Nitrix oxide facilitates cardiomyogenesis in mouse embryonic stem cells", PROC. NATL. ACAD. SCI., vol. 101, 2004, pages 12277 - 12281 *
KRUMENACKER ET AL.: "Differential expression of genes involved in cGM-dependent nitric oxide signaling in murine embryonic stem (ES) cells and ES cell-derived cardiomyocites", NITRIC OXIDE, vol. 14, 2006, pages 1 - 11 *
KRUMENACKER Y.M.: "NO-cGMP signaling in development and stem cells. Minireview", MOLECULAR GENETICS AND STEM CELLS, vol. 87, 2006, pages 311 - 314, XP024947021, DOI: doi:10.1016/j.ymgme.2005.10.009 *
TEJEDO ET AL.: "Nitric oxide triggers the phosphatidynilinositol 3-kinase/Akt survival pathway in insulin-producing RINm5F cells by arousing Src to activate insuline receptor substrate-1", ENDOCRINOLOGY, vol. 145, 2004, pages 2319 - 2327 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017021535A1 (fr) * 2015-08-05 2017-02-09 Fundación Pública Andaluza Progreso Y Salud Milieu de culture cellulaire amélioré de cellules progénitrices humaines (hpc)

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