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Definitions

• the present invention relates to a method of preparing induced pluripotent cells (iPS) with improved efficiency and homogeneity of iPS cells obtained. This improvement is achieved by using netrin-1 or a netrin-1 analogue to prevent or block cell death mediated by DCC (Deleted in Colorectal Carcinoma) or Unc5s (Unc5A, Unc5B, Unc5C and Unc5D) receptors. Netrin-1, in the initial stages of the cellular reprogramming process.
• DCC Deleted in Colorectal Carcinoma
• Unc5s Unc5A, Unc5B, Unc5C and Unc5D
• somatic cells can be de-differentiated into cells called induced pluripotent stem cells or iPS, which have similar characteristics to embryonic stem cells.
• iPS induced pluripotent stem cells
• De-differentiation or cell reprogramming methods generally involve expressing one or more key exogenous factors for reprogramming somatic cells to maintain stem cell pluripotency.
• Cell reprogramming methods of culturing somatic cells on a culture medium suitable for rendering and maintaining them pluripotent by expression of different transcription factors have been widely described in the literature, including expression of the family Oct, Sox, Klf and Myc, and especially the use of a mixture Oct4, Sox2, Klf4 and c-Myc or OSKM cocktail.
• Netrin-1 or Ntn1 is a diffusible secretory protein related to laminin.
• Netrin-1 is the DCC receptor ligand (Deleted in Colorectal Cancer) and UNC5A-D (UNCoordinated A, B, C and D).
• DCC receptor ligand deleted in Colorectal Cancer
• UNC5A-D UNCoordinated A, B, C and D.
• Netrin-1 (Sanskrit, "the one that guides”) was the first axon guidance factor characterized for its role in targeting axons of commissural neurons in the spinal cord (Serafini et al., 1994; al., 2004, Serafini et al., 1996). Its key role in the control of axonal guidance and neuronal migration has subsequently been confirmed in other nervous system territories (Moore et al., 2007).
• the present invention consists of a method for the preparation of induced pluripotent stem cells (iPS) by somatic cell culture subjected to a cell reprogramming method, characterized in that the somatic cells are cultured in the presence of netrin-1 or an analogue at least netrin-1 at the beginning of the cellular reprogramming process.
• iPS induced pluripotent stem cells
• netrin-1 or a netrin-1 analogue to prepare induced pluripotent stem (iPS) cells by somatic cell culture subjected to a cell reprogramming method, particularly to improve reprogramming efficiency.
• the invention does not relate to the identification of new systems for the reprogramming of somatic cells in iPS.
• the invention relates to the inhibition of cell death induced by the reprogramming process by acting on the interaction of somatic cell DCC or Unc5s receptors with netrin-1 or a netrin-1 analogue.
• the invention therefore consists in compensating for this decrease in netrin-1 in the culture medium at the beginning of the reprogramming process, at the moment when its expression decreases.
• the invention thus consists in culturing the somatic cells being reprogrammed in the presence of netrin-1 in the culture medium, or a netrin-1 analogue, in order to prevent or block cell death mediated by the DCC receptors. or Unc5s in somatic cells when reprogramming them.
• the present invention thus relates to a process for the in vitro preparation of induced pluripotent stem cells (iPS) by somatic cell culture subjected to a cell reprogramming process, characterized in that the somatic cells are cultured in the presence of netrin. -1 or at least one netrin-1 analogue at the beginning of the cellular reprogramming process.
• iPS induced pluripotent stem cells
• the present invention covers a process for the in vitro preparation of induced pluripotent stem (iPS) cells which comprises the following steps:
• induced pluripotent stem cells or "iPS” means cells obtained by in vitro cellular reprogramming of somatic cells into which stem cell pluripotency factors have been introduced.
• Cell reprogramming refers to a process of dedifferentiation of somatic cells into pluripotent stem cells.
• the inventors have been able to show on the one hand that the presence of netrin-1 increases the efficiency of the somatic cell reprogramming process in iPS. They also showed that blocking or inhibiting the interaction of netrin-1 with its DCC receptor resulted in a decrease in the efficiency of cellular reprogramming of somatic cells in iPS compared to a control method without the addition of Netrin-1 or Netrin-1 interaction inhibitor / DCC.
• the present invention which seeks to promote the survival of somatic cells during cell reprogramming can be implemented regardless of the reprogramming method employed.
• netrin-1 preferably refers to human netrin-1 and in particular to the polypeptide corresponding to the 604 amino acid sequence below as deposited in the Genbank database under the accession number. NP_004813, November 17, 2006, or its mature form which corresponds to amino acids 25 to 604 of this sequence.
• the present invention also covers the use of amino acid sequences of Ntn1, precursor of 604 amino acids (aa) or mature form (aa25-aa604)) with minor modifications, such as conservative substitutions, as long as those do not significantly affect the activity netrin-1 to prevent or block cell death induced by its receptors, and in particular by DCC.
• Netrin-1 which is present in the culture medium at least at the beginning of the cell reprogramming process may be natural or recombinant, preferably recombinant.
• the culture medium contains the recombinant netrin-1 commercially available from Adipogen (ref: AG-40B-0040-0000).
• Netrin-1 can also be expressed by the somatic cells themselves, in particular by transfection.
• netrin-1 analogue may be present in the culture medium, directly or produced by cells.
• netrin-1 analogue is meant any substance capable of mimicking the biological activity of netrin-1, that is to say any substance capable of exerting the biological activity of netrin-1, and preferably any substance capable of preventing or blocking cell death induced by DCC or Unc5s receptors, particularly cell death induced by the DCC receptor.
• These analogs are therefore netrin-1 agonists, which can, for example, bind to the DCC or Unc5s receptors or else induce the oligomerization of these receptors, and in particular of DCC, and prevent the induction of cell death mediated by these receptors.
• analogs may be selected by any method known to a person skilled in the art for determining the ability of a particular substance to bind to the DCC or Unc5s receptors, such a method may include an ELISA immunoenzymatic test.
• analogs can also be selected by any known method for detecting the ability of a substance to inhibit the attachment of netrin-1 to the DCC receptor or Unc5s receptors, preferably competitively or non-competitively inhibit binding. of netrin-1 to these receptors, more preferably their ability to competitively inhibit the binding of netrin-1 to these receptors.
• analogs may be further selected by any method for determining the ability of a substance to prevent or block cell death induced by the DCC receptor or the Unc5s receptors, examples of which methods are described in this patent application.
• Examples of netrin-1 analogs that may be mentioned include DCC or Unc5s receptor agonist antibodies, netrin-1 polypeptide fragments capable of preventing or blocking DCC or Unc5s-mediated cell death.
• the somatic cell culture medium subjected to cell reprogramming may therefore contain one or more fragments of netrin-1, and in particular fragments of the amino acid sequence deposited in the Genbank database under accession number NP_004813, November 17, 2006, since it (they) retain (s) the property of inhibition or blocking of cell death mediated by its receptors, and in particular by DCC.
• netrin-1 or the netrin analogue are particularly advantageously, netrin-1 or the netrin analogue
• netrin-1 intake on the efficiency of the process depends on the dose that is brought, the higher the dose, the better the result. In the tests carried out by the inventors, the best result was obtained with a netrin-1 concentration of 900 ng / ml.
• concentration of netrin-1 or of the netrin-1 analogue best suited to the process that he will carry out, taking into account both the improvement in the efficiency, but also the the price of returning the process when the amount of netrin-1 or netrin-1 analogue is added.
• the concentration of netrin-1 present in the culture medium is at least 100 ng / ml, preferably greater than or equal to 150 ng / ml.
• netrin-1 analogs those skilled in the art will be able to choose the appropriate amount of this analogue so as to obtain the same efficiency as for netrin-1.
• Netrin-1 or the netrin-1 analogue is advantageously present in the somatic cell culture medium being reprogrammed for at least the first 7 days after initiation of the reprogramming process (J0).
• J0 reprogramming process
• Netrin-1 when added directly to the culture medium, it is added daily, and in particular to a concentration of at least 100 ng / ml, in particular during the changes of medium required during the process. reprogramming.
• the present invention can be implemented regardless of the cell reprogramming method employed.
• the reprogramming method used may include expression in somatic cells of one or more stem cell pluripotency factors selected from the group consisting of Oct, Sox, Klf, and Myc.
• the invention is implemented in a reprogramming process comprising, or even consisting of, the expression of Oct4, Sox2, Klf4 and c-Myc transcription factors, also called OSKM cocktail, which is currently the the most common method employed by those skilled in the art.
• stem cell pluripotency factors can be expressed in somatic cells using different techniques, such as viral infection, transfection using liposomes, electroporation, membrane protein permeability, and the like.
• these stem cell pluripotency factors are introduced by a stage of viral infection of the somatic cells, with viruses allowing the expression of nucleic acid sequences encoding these factors, and especially chosen from lentiviruses, retroviruses, adenovirus, Sendai virus.
• viruses allowing the expression of nucleic acid sequences encoding these factors, and especially chosen from lentiviruses, retroviruses, adenovirus, Sendai virus.
• the method of the invention may further comprise a step of introducing a reporter system into somatic cells to indicate the efficiency of generation and production of iPS, in particular selected from such as GFP, colorimetric systems, antibiotic resistance systems, etc.
• the present invention is implemented by means of a cell reprogramming method which is carried out without genomic integration and / or without expression of oncogenes.
• somatic cells can be reprogrammed into iPS in the context of the method of the present invention, and in particular fibroblasts, keratinocytes, T cells, hepatocytes, umbilical cord cells, fat cells, intestinal cells and cells. blood cells, and advantageously fibroblasts.
• the present invention finds a very particular application for preparing mammalian iPS.
• the somatic cells employed in the context of the method of the invention may be chosen from the group consisting of rats, mice, rabbits, pigs, sheep, goats, cows, monkeys and humans.
• the somatic cells used are human somatic cells, and in particular human fibroblasts.
• the somatic cells used are human intestinal epithelium.
• the present invention also relates to the use of netrin-1 or a netrin-1 analogue to prepare induced pluripotent stem (iPS) cells by somatic cell culture subjected to a cell reprogramming method. , in particular to improve the efficiency of cellular reprogramming of somatic cells in iPS.
• iPS induced pluripotent stem
• FIG. 1 represents the expression level of netrin-1 and its DCC receiver (FIG. 1A), and Unc5B and Unc5C receptors (FIG. 1B) during the MEF (Mouse Embyronic Fibroblasts) reprogramming process, at days 0, 2, 4 and 6 after infection with retroviral particles encoding OSKM.
• Figure 2 shows the effect of inactivation of netrin-1 expression on the reprogramming of MEFs infected with OSKM lentiviral particles ( Figure 2A) and on the reprogramming of intestinal epithelium induced to reprogram by treatment with doxycycline ( Figure 2B).
• Figure 3 shows the effect of increasing doses of Ntn1 on the efficiency of cell reprogramming ( Figure 3A) and reprogramming of intestinal epithelium induced to reprogram by doxycycline treatment ( Figure 3B). .
• Figure 4 shows the effect of sequential Ntn1 delivery on cell reprogramming efficiency (dO-7: first 7 days, d7-14: day 7 to day 14 and dO-14: first 14 days) .
• Figure 5 shows the effect of Ntn1 blocking antibody on cell reprogramming efficiency (dO-7: first 7 days, d7-14: day 7 to day 14 and dO-14: first 14 days).
• Figure 6 shows the effect of inactivating DCC (A) receptor expression and Ntn1 (B) expression on the efficiency of cell reprogramming.
• FIG. 7 represents the alkaline phosphatase activity (A) and the DESeq (B) hierarchical clustering analysis of iPS cell clones derived under "control" conditions or in the presence of recombinant Nétrin-1.
• Figure 8 shows the analysis of major chromosomal alterations between control (white histogram) and derivative iPS lines in the presence of Ntn1 (black histogram).
• FIG. 9 represents the histological analysis of the teratomas induced by the in vivo injection of iPS clones derived in the presence of Ntn1 (Scale of size: 100 ⁇ ).
• FIG. 10 shows the effect of recombinant Netrin-1 (black histogram) on the emergence of SSEA4 positive colonies from human foreskin fibroblasts cultured on mitomycin C-treated fibroblasts 7 days after infection (bar scale corresponds to 200 ⁇ ) relative to the control (white histogram).
• Mouse embryonic fibroblasts are derived from E13.5 embryos of different strains.
• the pre-iPS, iPS and ES cells are cultured on irradiated MEFs or gelatin as previously described.
• 293T and flat-E cells are cultured in DMEM supplemented with 10% FCS and penicillin / streptomicin.
• the iPS cells are cultured in KSR + LIF or KSR 2i + LIF medium.
• the shRNA experiments are performed with pLKO.1 vectors and Sigma siRNAs (SHCLNG-NM-008744 for Ntn1, SHCLNG-NM-007831 for Dec and EMU022741 for Mbd3).
• the antibodies used in this study for immunofluorescence and FACS are: anti-Oct4 (Santa Cruz, C10), anti-Netrin-1 (R & D Systems, mab1109), anti-thyl (Ebiosciences, 53-2.1) , anti-SSEA1 (Stem cell technologies, 60060PE) and NL493 conjugated anti-SSEA4 antibody (R and D Systems SC023).
• the recombinant netrin-1 used is commercially available from Adipogen (ref: AG-40B-0040-0000).
• E-flat cells were used to produce retroviral particles from pMX-s vectors encoding the cDNAs of Oct4, Sox2, Klf4 and c-Myc, as previously described by the inventors.
• Virus particles encoding mcherry were used to monitor the infection efficiency of MEFs.
• 293T cells were used to produce the lentiviral particles.
• MEF cells were passed into 6-well dishes. Twelve hours later, these cells were infected with equivalent amounts of each retroviral particle in the presence of polybrene at a concentration of 8 ⁇ g / ml polybrene.
• the culture medium is replaced 24 hours after infection and 48 hours after infection, the MEF are collected and cultured on fibroblasts irradiated in culture medium for iPS cells. The culture medium was then replaced daily with fresh medium and emerging colonies were counted or collected 12-14 days post-infection.
• the intestinal epithelium is dissociated and the epithelium fragments cultured on a carpet of irradiated feeder cells.
• the pluripotent reprogramming process is induced here by treating the cells at doxycycline (g / ml, for 14 days) since the mice used have an expression cassette of reprogramming factors Oct4, Sox2, Klf4 and inducible c-Myc. Generation of human iPS cells.
• the experiments were carried out with 5 ⁇ 10 5 human foreskin cells (HFF, Millipore) cultured in a well of a 6-well plate and in the presence of medium FibroGRO TM -L medium (Millipore) until delivery. in culture on fibroblasts.
• the cells were infected overnight with 4 Sendai viruses encoding Oct4, Sox2, Klf4 and c-Myc (Cytotune TM, Life Technologies) respectively at an MOI of 3.
• the culture medium is changed the next day and replaced with medium. containing or not recombinant netrin-1 at 150 or 750 ng / mL.
• the medium is then changed daily.
• the cells are dissociated with express tryple (Life Technologies), counted and replated on Mitomycin C-treated MEFs (Sigma Aldrich) at a cell density of 5x10, 1x105 or 2x10 5 HFF per plate.
• the medium is replaced with DMEM / F12 (Life Technologies) supplemented with 20% PluriQ TM Serum Replacement (GlobalStem), 0.1 mmol / L non-essential amino acids, 1 mmol / L L-glutamine, 0.1 mmol / 2-mercaptoethanol, penicillin / streptomycin (Life Technologies), 12.5 ng / ml human basic fibroblast growth factor (Milytenyi Biotec) in the presence or absence of recombinant Nétrin-1.
• the medium is then changed daily.
• reprogrammed colonies were manually stitched and transferred to new MEFs treated with mitomycin C for amplification.
• mice 5x10 6 iPS cells were injected under the 7 week immunodeficient mouse kidney (SCID) capsules (CB17 / SCID, Charles River). After 3 weeks, the mice were euthanized and the tumors surgically removed and fixed in 4% formalin or in PFA for cryosections. The same procedure was used with injection of 5x10 5 iPS cells into the testes.
• SCID 7 week immunodeficient mouse kidney
• Murine embryonic fibroblasts were infected with retroviral particles encoding OSKM for 6 days.
• the expression of netrin-1, DCC, Unc5B and Unc5C receivers were measured over time (OJ, J2, J4, and J6 for Netrin-1 and DCC, and OJ, J3 and J6 for Unc5B and Unc5c).
• Q-RTPCRs display the expression profiles of Ntn1, Dec, Unc5B and Unc5C during the first 6 days of cell reprogramming to the pluripotent state and in established iPS cells.
• the data are normalized to the RS17 and L19 household genes, represented relatively to the level of expression in MEFs and correspond to the mean ⁇ SEM of 3 independent experiments.
• the effect of inactivation of Ntn1 expression on cell reprogramming was analyzed by quantitation of positive alkaline phosphatase or nanog positive colonies.
• the AP positive cells are characterized by a test demonstrating the enzymatic activity of the cells, the nanog positive cells are characterized by immunofluorescence using nanog specific antibodies.
• MEFs were infected with lentiviral particles encoding three different shRNAs that target Ntn1 two days prior to OSKM transduction. The number of colonies produced in the control condition of MEFs infected with shscrambled particles comprising a control shRNA that does not target netrin-1 is increased to 100% for each experiment. Three different shRNAs were used (Ntn1 sh # 1, Ntn1 sh # 2 and Ntn1 sh # 3). The data presented corresponds to the mean ⁇ SEM of three independent experiments carried out with batches of MEF and different viral particles.
• Example 3 Effect of the compensation of the Ntn1 deficiency on cellular reprogramming
• Example 5 Use of Ntn1 as a Soluble Factor for Improving Reprogramming of Murine and Human Somatic Cells in iPS
• Oct4-GFP murine iPS clones derived under standard conditions (control) or in the presence of 600 ng / ml Ntn1 for the first 7 days of cell reprogramming were individually subcultured and cultured.
• the differentiation potential of murine iPS derived in the presence of Ntn1 was studied in vivo by histological analysis of teratomas obtained from the injection of iPS cells derived in the presence of recombinant Netrin-1 (Fig. 9) and in vitro. by the formation of embyooid bodies (EB), demonstrating the emergence of derivatives of the three types of embryonic leaves (endoderm, mesoderm and ectoderm) (results not annexed). It has also been confirmed that murine iPS cells derived in the presence of Ntn1 integrate appropriately into host blastocysts (non-appended results).

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