WO2008034929A1 - Method for culturing and maintaining mammal multipotential trunk cells and progenitor cells in a non-differentiated state - Google Patents

Abstract

The practice of maintaining multipotential and progenitor cells in a non-differentiated state during culture poses various problems depending on the type, origin and future use of the cells. It is currently possible to culture mammal multipotential cells in systems which maintain their non-differentiated state; however, the spontaneous differentiation which is inherent in culturing is an advanced situation which gives rise to difficulties in the methods for obtaining cells. The method of the present invention allows mammal multipotential and progenitor cells to be maintained and cultured in a non-differentiated state using nitric oxide.

Description

 TITLE

Method for the cultivation and maintenance of pluripotential stem cells and mammalian progenitor cells in an undifferentiated state

OBJECT AND TECHNICAL FIELD OF THE INVENTION.

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.

DESCRIPTION OF THE STATE OF THE TECHNIQUE

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

Numerous studies have shown the importance of the activation of signaling pathways in maintaining the undifferentiated state of the stem cells. In the mouse, the presence of the leukemia inhibitory factor in the culture medium activates the JAK / STAT3 pathway which finally has as target the activation of c-myc [Cartwright, PC; McLean C. et al. "LIF / STAT3 controls EN cell self-renewal and pluripotency by a Myc-dependent mechanism"; Development 132 (5): 885-96 (2005)]. The leukemia inhibiting factor also activates the PI3K and protein pathways of the c-Src family; In these cells the role of these pathways in the regulation of non-differentiation and proliferation have not been fully described. One of the most frequent problems of individualizing signaling pathways in embryonic stem cells is that they present spontaneous differentiation resulting in the generation of heterogeneous populations with different degrees of differentiation, making studies related to non-differentiation due to the diversity of pathways difficult signaling that may be activated. Likewise, the clinical need to produce large amounts of embryonic stem cell biomass in bioreactor systems generates a strong pressure towards differentiation on condition that the differentiation protocols start from a mixture of heterogeneous populations with different degrees of differentiation. Therefore, it is necessary to have cultivation methods in which conditions are controlled. 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)]. 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 In addition, 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.

The culture of human embryonic pluripotential cells on an extracellular matrix obtained from mouse sarcomas rich in extracellular matrix (commercially called MATRIGEL) with conditioned medium in the fibroblast monolayers [Xu C; Inokuma M.S. et al. "Feeder-free growth of undifferentiated human embryonic stem cells." Nat Biotechnol 19 (10): 971-4 (2001)] and in unconditioned medium supplemented with high concentrations of the Basic Fibroblast Growth Factor (bFGF) [(Xu C; Rosler E. et al. "Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. "Stem Cells 23 (3): 315-23 (2005)]. Human embryonic stem cell cultures on extracellular matrices of animal origin such as MATRIGEL continue to present the problem of contamination animal and additionally present the problem of supply of conditioned medium.The use of unconditional medium with high concentrations of bFGF presents two basic problems, the first related to the high cost of production of bFGF, making economically unfeasible the production of large amounts of cells and the second problem related to the expression of the receptors of the fibroblast growth factors, since not all the s pluripotential cells express it.

Other problems with the use of MATRIGEL are that it 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)]. The presence of 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 culture of human embryonic stem cells on matrices using a chemically defined medium has been described [Yao S .; Chen S. et al .; "Long-term self-renewal and directed differentiation of human embryonic stem cells ¡n chemically defined conditions." Proc Nati Acad Sci (JSA 103 (18): 6907-12 (2006).) The use of chemically defined media has been proposed as a solution to the use of components of the animal-derived environment; there is still insufficient evidence of the performance of These systems, because the supplements traditionally used have been used in differentiation protocols towards neurons (B27-GIBCO from Invitrogen) It is desirable to have a method for culturing mammalian embryonic pluripotential stem cells that allows reduced differentiation in the absence of fibroblast monolayers and without the use of conditioned media, this can ensure that said cells are not subject to contamination by xenogenic products, can be maintained in optimal growth conditions and reduce the costs of scaling when producing large cell masses.

EXPLANATION OF THE INVENTION

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.

The addition of 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.

Alternatively, the addition of 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). ^{'Specifically,} 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.

It is also an object of the present invention a method for the cultivation and maintenance of mammalian progenitor cells in an undifferentiated state, in which the effect of non-differentiation is achieved by adding nitric oxide to said progenitor cell culture. The addition of 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.

Alternatively, the addition of 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). Specifically, 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.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Scheme of the Oligo hpθct4 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.

C. Line D3-p0ct4 P18 cultured in the presence of LIF and 2 μM DETA / NO for 9 days.

D. Line D3-pOct4-eNOS P21 grown in the presence of LIF for 9 days.

E. Line D3-pOct4-eNOS P21 cultured in the presence of LIF and L-NMiVlA 400 μM for 9 days. Figure 5: Typical karyotype of D3-pOct4 P 18 and D3-pOct4-eNOS P21 cells Figure 6: Expression of non-differentiation markers in line D3-0ct4-eN0S Figure 7: Expression of the eNOS protein

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- OctΦeNOS 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.

DETAILED DESCRIPTION AND MODE OF EMBODIMENT OF THE INVENTION

The present invention describes the use of nitric oxide in the cultivation and maintenance of the undifferentiated state of mammalian pluripotential cells and progenitures. 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). 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.

The addition of exogenous nitric oxide donors to the culture medium under conditions that promote the differentiation of mammalian pluripotential and progenitor cells slows down the differentiation process. The endogenous generation of nitric oxide through the overexpression of 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-pOctΦ-eNOS lines of embryonic stem cells, which are part of the tools used for the realization of the present invention. Growing conditions

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 μg / mL streptomycin [Gibcol5140-122], 2mM L-Glutamine [Gibco25030-032], 1% non-essential amino acids [Gibcol 1140-035], 100 μM β-mercaptoethanol (50 mM) [ Gibcol31350-010], 0.1 U / μL of Leukemia Inhibitory Factor (LIF) 10 ⁶ U / ml (Chemicon ESG-1106). 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. Transfection was performed by electroporation. 800 μL of the suspension of D3 cells in serum-free medium contained in an electroporation cuvette was added 4 μg of the Oligo hpθct4 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 μfaraday 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. The next day, the medium is changed by adding the hygromycin at a concentration of 400 μg / ml. D3-pOct4-eNOS cells were obtained by transfection of D3-pθct4 cells with plasmid pCDNA3.1-eNOS (Department of Medicine of the University of Toronto, Canada). Transfected cells were selected with medium containing 400 μg / ml of hygromycin and 500 μg / ml of neomycin.

The 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 ™ 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

One of the fundamental tests that show the culture in the undifferentiated state is the analysis of the expression of pluripotential markers such as Oct3 / 4, Nanog, Sox2, FoxD3. Together, these transcriptional factors activate the expression of undifferentiation genes and repress the expression of early markers of cell differentiation to maintain the capacity of self-renewal and the pluripotential state of the cells when they are cultured under conditions of undifferentiation. To evaluate the expression of said pluripotential markers in the transfected lines, they were cultured for 7 days in the presence of LIF and subsequent analysis of the expression by RT-PCR. The sequence of the primers used is indicated in Table 1. Table 1. Sequence of the primers used for the RT-PCR

Determination of Telomerase Activity

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

Karyotype analysis.

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 μg / 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. Excessive digestion in trypsin causes an effect of "chromosome fraying" or "feathery chromosomes" due to excessive digestion of the proteins that pack the DNA fibers in the chromosomes. Karyotypes of at least 20 cells were analyzed. All plaques are counted at least five times until a reliable estimate of the total number of chromosomes is established. Results

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. The results show that 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.

Karyotype The karyotype of the cells of the D3-0ct4 line is shown in Figure 5, panel A and of the D3-0ct4-eN0S in Figure 5, panel B. The results show that they have a stable karyotype XX.

Determination of eNOS by immunodetection. The expression of the eNOS protein, by immunodetection, by the cell lines is shown in Figure 7. It is shown that both line D3 and D3-Oct4 express low levels of eNOS and that line D3-

0ct4-eN0S over-expresses the eNOS protein.

Example 2: Maintenance of mammalian pluripotential cells in a state not differentiated by nitric oxide: Nitric oxide decreases the pressure towards differentiation in conditions of withdrawal of LIF in D3-p0ct4 cells.

Using the D3-0ct4 line, experiments were carried out to observe the effect of the addition of nitric oxide produced from exogenous donors on the capacity of non-differentiation in conditions of LIF withdrawal from the culture medium that favors the differentiation of embryonic stem cells mouse

Experimental conditions

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 μg / 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 µM of DETA-NO (Sigma) added. For flow cytometry experiments the cells after the indicated treatments were individualized with trypsin, washed twice with PBS, filtered on a 70 μM diameter filter and resuspended at concentration of 10 cells / mL in PBS-EDTA, GFP positive cells were detected in BD-FACSAria ™ cytometer.

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

In Figure 8, 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 μM 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. Figure 8, panel B shows the photographs of representative colonies under the conditions described above taken with fluorescence microscopy. In Figure 8, 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 expression of the Oct4, Sox 2 and NANOG proteins was also analyzed by immudetection and by RT-PCR (Figure 9, panels A, B). Panel A shows the expression of the proteins. The results show that the expression of Oct4, Sox 2 and Nanog does not disappear when the cells are cultured in the absence of LIF plus nitric oxide. Likewise, the mRNA of these proteins is maintained under these conditions (Figure 9, panel B). Example 3: Maintenance of mammalian pluripσtential cells in a state not differentiated by nitric oxide: The inhibition of nitric oxide production increases the pressure towards differentiation in conditions of LIF withdrawal in D3-p0ct4-eN0S cells.

Using the D3-0ct4-eN0S line, experiments were carried out to measure the role of endogenously produced nitric oxide on the non-differentiation capacity of mouse embryonic stem cells. 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).

Experimental conditions

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 µM NMMA (Sigma) added. Results

In Figure 10, 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 µM 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). The results show that the expression of Oct4 and SSEAl is completely abolished when NMMA is added to the medium in the absence of UF. The expression of the 0ct4, Sox 2 and NANOG proteins was also analyzed by immunodetection and by RT-PCR (Figure 11, panels A, B). Panel A shows the expression of proteins. The results show that the expression of 0ct4, Sox 2 and Nanog disappears when the cells are cultured in the absence of LIF plus NMMA. Likewise, the mRNA of these proteins maintains the same behavior in these conditions (Figure 11, panel B).

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.

In this example it is shown that nitric oxide promotes the maintenance of the undifferentiated state of mouse embryonic stem cells through the activation of signaling pathways.

Experimental conditions.

In addition to the methods described above in Examples 1, 2 and 3, the following methods were used: Protein Immunoprecipitation in Sepharose-Protein G: Immunoprecipitates were used to detect with specific antibodies that recognize phosphorylated forms of proteins by "western blot ".

Results

In Figure 12, 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.

Experimental conditions. Embryonic stem cell culture of human origin.

Human embryonic stem cells were cultured on foreskin fibroblasts inactivated with mitomycin. For this, 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-β-Mercaptoethanol (Invitrogen 31350-010) , 8 ng / mL of b-FGF (R&D Systems 234-FSE-025).

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 μg / 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. Additionally, 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. In the present example the commercial matrix "Matrigel" (BD Matrigel ™ 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 μM 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.

Human embryonic stem cells grown on Matrigel in conditioned medium supplemented with bFGF, maintain their non-differentiation characteristics for long periods of culture (20 passes). When grown in unconditioned medium in the presence of 8 ng / mL of bFGF they maintain their non-differentiation characteristics for three passes; in longer periods of cultivation they differ. For this reason the bFGF withdrawal experiments were performed between passes 1 and 3 on Matrigel in unconditional medium. In Figure 13, 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 μM of DETA-NO prevents the appearance of these areas; In addition, 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 μM 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. In Figure 13, panel B shows the immunodetection of the expression of the transcription factors markers of non-differentiation, Oct4, Sox 2 and Nanog. The expression of these non-differentiation factors decreases markedly when cells are cultured in the absence of bFGF. The addition of DETA-NO under conditions of absence of bFGF prevents the disappearance of the transcription factors. It is important to note that in the presence of nitric oxide the expression of 0ct4, Sox 2 and Nanog is greater than that found for the condition of medium supplemented with bFGF. In Figure 13, panel C, the presence of messenger RNA of the transcription factors detected by RT-PCR is shown; The results show that the levels of 0ct4 messenger RNA remain constant, however, the Sox 2 and Nanog transcripts decrease in the bFGF withdrawal condition and that the addition of nitric oxide prevents their disappearance.

All these results show that 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).

Claims

1.- Method for the cultivation and maintenance of mammalian pluripotential stem cells in an undifferentiated state, characterized by the addition of nitric oxide to said pluripotential stem cell culture. 2, - Method for the culture and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claim 1, characterized in that the mammalian pluripotential stem cells come from adult tissues. 3, - Method for the culture and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claim I ₁ characterized in that the mammalian pluripotential stem cells come from fetal tissues. 4. Method for the culture and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claim 1, characterized in that the mammalian pluripotential stem cells are embryonic stem cells. 5. Method for the culture and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claim 4, characterized in that the embryonic stem cells are human embryonic stem cells. 6. Method for the cultivation and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claims 1-5, characterized in that the addition of nitric oxide is carried out by exogenous donors, preferably diethylenecentriamine adduct with nitric oxide (DETA / NO ), S-nitroso-N-acetyl-D, L-penicillamine (SNAP), SIN-I and others. 7. Method for the cultivation and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claim 6, characterized in that the oxide donor Nitric is added directly to the culture medium at concentrations between 500 nM and 10,000 nM. 8. Method for the cultivation and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claims 6 and 7, characterized in that nitric oxide is added mixed with other compounds, preferably with extracellular matrix component proteins as support for growth. of the cells 9. Method for the cultivation and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claims 1-5, characterized in that the addition of nitric oxide is carried out by endogenous production of nitric oxide. 10. Method for the culture and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claim 9, characterized in that the endogenous production of nitric oxide is carried out by means of the overexpression of enzymes involved in the synthesis of nitric oxide , preferably of the endothelial nitric oxide synthase (elMOS). 11. Method for the cultivation and maintenance of mammalian pluripotential stem cells in an undifferentiated state according to claim 10, characterized in that the endogenous production of nitric oxide is carried out by transfection of the mammalian pluripotential stem cells with a plasmid inducing The overexpression of elMOS. 12. Method for the culture and maintenance of mammalian progenitor cells in an undifferentiated state, characterized by the addition of nitric oxide to said progenitor cell culture. 13. Method for the culture and maintenance of mammalian progenitor cells in an undifferentiated state according to claim 12, characterized in that the addition of nitric oxide it is carried out by exogenous donors, preferably diethylenetriamine adduct with nitric oxide (DETA / IMO), S-nitroso-N-acety! -D, L-penicillamine (SNAP), SIN-I and others. 14. Method for the culture and maintenance of mammalian progenitor cells in an undifferentiated state according to claim 13, characterized in that the nitric oxide donor is added directly to the culture medium at concentrations between 500 nM and 10,000 nM. 15. Method for the culture and maintenance of mammalian progenitor cells in an undifferentiated state according to claims 13 and 14, characterized in that the nitric oxide is added mixed with other compounds, preferably with extracellular matrix component proteins. 16. Method for the culture and maintenance of mammalian progenitor cells in an undifferentiated state according to claim 12, characterized in that the addition of nitric oxide is carried out by endogenous production of nitric oxide. 17. Method for the culture and maintenance of mammalian progenitor cells in an undifferentiated state according to claim 16, characterized in that the endogenous production of nitric oxide is carried out by means of the over-expression of enzymes involved in the synthesis of nitric oxide, preferably of nitric oxide endothelial synthase (eNOS). 18. Method for the cultivation and maintenance of mammalian progenitor cells in an undifferentiated state according to claim 17, characterized in that the endogenous production of nitric oxide is carried out by transfection of the mammalian progenitor cells with a plasmid inducing the envelope. -expression of the eNOS.