WO2014122370A1 - Method for purifying cells - Google Patents

Abstract

The present invention concerns a method for isolating, purifying and/or enriching cells, comprising a step of bringing a cell population previously isolated from an individual into contact with at least one marker sensitive to mitochondrial membrane potential and at least one marker for quantifying the number of mitochondria.

Description

 PROCESS FOR PURIFYING CELLS

 The present invention relates to a method of purifying cells.

Although regeneration of nerve tissue exists in mammals within the central nervous system, this self repair capacity remains very low. This reflects the inability of neural stem cells and neural progenitor cells to proliferate and migrate into damaged regions in sufficient quantities to repair and reconstruct neural circuits. Cell transplantation has emerged in the last 20 years as an interesting approach to treat central or peripheral neurological disorders (US Patent 2009/0214484).

Neural cells are derived from the ectodermal layer that gives the epidermis and nerve tissue. There are different types of neural cells such as neurons (dopaminergic, serotonergic, GABAenergic, ...) or glial cells including astrocytes (Dupin et al., Neural crest progenitors and stem cells: From early development to adulthood. Biology 366: 83-95 (2012)).

Deficits in neural cells are responsible for many pathologies including: acute or chronic degenerative diseases such as Parkinson's disease or Hungtington's disease, multiple sclerosis, strokes, visual problems, attacks of the disease. retina, hearing disorders, spinal cord injury, peripheral nerve damage including neurons and motor neural patches, nerves (specialized in the autonomic system or not), sphincters, involvement of the neurosensory system including the visual system, the system auditory, etc ...

Also, it seems important to be able to provide methods to replace or stop the deficit of neural cells to treat these different diseases.

Several methods have been proposed to remedy this problem.

For example, the application US 2009/0068155 describes a method of administering neural cells in the central nervous system. Cell therapy techniques have also been proposed. But all of these techniques do not achieve a satisfactory result. *

The use of neural stem cells has been shown to be promising in the course of therapy. Potential applications of these cells are indeed important in vitro, particularly in the areas of drug screening, neurotoxicology, electrophysiology, cell therapy or regenerative.

However, such stem cells are present in very small amounts in tissues and organs, and it is difficult to isolate and purify them.

Current methods of isolation rely mainly on the detection of surface markers such as CD15 or CD133 (Obermair et al., A novel classification of quiescent and transit amplifying neural stem cells by surface and metabolic markers Stem Cell Research 5: 131-143 (2010)). However, these methods are made difficult and imperfect because there is no specific antigen of neural cells. Indeed, markers of neural cells are not always constitutively expressed during the cell cycle and are generally expressed by other cell lines and by tumor cells.

 The peculiarity of cardiomyocytes and neural cells is to have a high level of mitochondria, whereas the number of mitochondria is low in totipotent embryonic cells.

 The number of mitochondria increases during differentiation and reaches a particularly high number in cardiomyocytes. During development, mitochondria will migrate from the nuclear juxtaposition to other parts of the cytoplasm with, for example, an association at the level of the contractile apparatus for cardiomyocytes.

The shape of the mitochondria changes with the appearance of fusion / fission phenomena. The amount of DNA copies also increases in the mitochondria and the membrane potential changes. The metabolism of these mitochondria during development is particular because the energy demand in these cells is particularly high, and the mitochondria will go from an energetic metabolism to provide an anaerobic glycolytic to an aerobic metabolism with beta fatty acid oxidation. involving the Krebs cycle and the mitochondrial respiratory chain. These modifications will be accompanied by a modification of the potential of the mitochondrial membrane which increases. This difference in potential is particularly marked in cardiomyocytes where energy demand is high because of their active metabolism and work in anaerobiosis during exercise.

The enzymatic "machinery" that accompanies these modifications will also increase the protection systems of these cells in the event of aggression, in particular against free radical-type mitochondrial metabolites, also called ROS (Reactive Oxygen Species), which induce lesions of the skin. Mitochondrial DNA.

During cell differentiation, and with the variation of the membrane potential, there is an increase in the production of free radicals.

 Also, the use of markers of the physiological state of mitochondria could be a good way to isolate stem cells.

Hattori et al. (Nongenetic method for purifying stem cell-derived cardiomyocytes, Nature Methods, vol7 and 61-66, (2010)) have shown that labeling of cells with a mitochondrial potentiometric marker (TMRM) makes possible the purification and isolation of cardiomyocytes. .

 This study shows that cardiomyocyte precursors have a high TMRM binding rate and represent 98% of the cell fraction. For TMRM to enter the cells, there must be a difference in membrane potential and therefore the cells are alive. Cells with low levels of TMRM labeling are mostly dead cells.

Cells isolated by Hattori et al. cardiomyocytes, but these cells could only be isolated at a late stage of cardiac cell development, ie at 10 days of embryonic development, whereas many markers of cardiac cells (including transcriptional markers) are present as early as 3 ^rd day of development and that the cells already have contractile properties on the ^8th day of development. Although mitochondrial markers appear to be promising for cell isolation, there is still a real need for a method of cell purification and isolation, especially at early stages of embryonic development.

One of the objects of the invention is to provide a method for efficient cell purification.

 Another object of the invention is to provide a method for obtaining and purifying several distinct cell types, the cell types being well differentiated.

The invention relates to a method for isolating, purifying and / or enriching cells, comprising a step of contacting a tissue or cells with at least one membrane-sensitive marker of the mitochondria and at least one minus a marker for quantifying the number of mitochondria and / or, if appropriate, increasing the production of free radicals, said at least one membrane-sensitive marker of the mitochondria and said at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals that can correspond to one and the same marker,

 in such a way that

 if said at least one marker sensitive to the membrane potential of the mitochondria and said at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals are used simultaneously, or if said at least one a marker sensitive to the membrane potential of the mitochondria and said at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals correspond to one and the same marker, said method allows the isolation, the purification and / or enrichment of neural stem cells, and

if said at least one marker sensitive to the membrane potential of the mitochondria and said at least one marker making it possible to quantify the number of mitochondria are used separately or spread over time, said method allows the isolation, purification and / or or enrichment of neural stem cells and / or cardiomyocyte cells. The present invention is based on the surprising finding made by the inventors that the use of mitochondrial markers, making it possible to measure the membrane potential of the mitochondria and at least one other mitochondrial marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals makes it possible to purify, isolate and / or enrich cells, especially stem cells, and in particular stem cells at a very early stage of their differentiation, in particular cardiac cells and neural cells.

This method uses particular properties of the mitochondria of neural cells and / or cardiomyocytes during their development and a conjunction of mitochondrial markers.

 Moreover, unlike cell purification methods based on the selection of membrane differentiation markers, or a selection of cells determined by genetic means, the method according to the invention makes it possible to obtain non-tumor cells, which is to say unprocessed cells that have lost none of the characters that the cells of an organism possess (loss of senescence, loss of contact inhibition ...).

 In the invention, the term "stem cells" includes isolated or non-isolated, multipotent or totipotent cells. It can be preparations or tissues. It may be, for example, developing tissue, embryonic tissue or gestational tissue including appendages (umbilical cord, placenta, amniotic fluid), neonatal differentiated tissues or other such as adult tissue.

 In the invention, the terms "neural stem cells" or "neural cells" may be used for each other. Neural stem cells are multipotent cells that can differentiate into certain types of mature cells, such as neurons, glia or even astrocytes.

In the invention, "isolation, purification and / or enrichment" means that it is possible, by means of the method as described,

or to purify the cells, that is to say to obtain specific cells. For example, the purification of neural cells means that the resulting cells are all neural cells, and that the cell population does not contain, or contains a small portion (less than 1%, preferentially less than 0.1%) of cells other than neural cells.

 or to isolate the cells, that is to say to extract from their natural context a cell population where all the cells is of the same phenotype.

 or to enrich in cells, that is to say from a mixed population of cells, which can be isolated and purified, to carry out a selection of a particular subpopulation of cells, so that after enrichment, this sub-population is the majority.

 The process also makes it possible to combine isolation, purification and enrichment together.

 Also, the invention relates to a method of isolating cells as defined above. The invention further relates to a method of cell purification as defined above.

 The invention also relates to a cell enrichment method as defined above.

 The invention also relates to a method for isolating and purifying cells as defined above.

 The invention also relates to a method for isolating and enriching cells as defined above.

 The invention also relates to a method for purifying and enriching cells as defined above.

 Finally, the invention also relates to a method for isolating and purifying and enriching cells as defined above.

The cells or tissues may be of animal origin or not. It may be living tissue, but also cells obtained from postmortem tissue since it appears that the stem cells survive after the death of the subject for some time. It can also be genetically modified cells, fused cells or iPS (irtduced pluripotent stem cell) cells obtained by the genetic reprogramming of somatic cells. These may be embryonic tissues, animal or human embryonic lineages. < ^■

The tissues particularly rich in neural cells are preferred, for example in the central nervous system, the paraventricular regions and the gyrus hypocampal. It is also possible to use isolated cells from tissues such as the dermis, especially in the scalp.

In a particular aspect of the invention, the cell populations from which it is possible to purify, isolate and / or enrich cells using the method according to the invention do not result from the destruction of human embryo.

The method according to the invention can be used either in vitro to isolate cells in culture, or in vitro as a control of the quality of cells during the screening of cells before implantation in animals, especially in humans.

The process is advantageously carried out in vitro.

There are currently cytometric techniques for sorting cells and meeting clinical requirements, compatible with their use in humans (Babetta et al., Flow cytometry: A multipurpose technology for a wide spectrum of global biosecurity applications, Journal of Laboratory Automation vol 14 (3): 148-156 (2009)).

It is also possible to purify the cells, without resorting to a cell sorter, on the basis of magnetic or electromagnetic properties by using ferromagnetic particles coupled to the markers (Corr et al., Multifunctional Magnetic-Fluorescent Nanocomposites for Biomedical Applications , Nanoscale Research Letters 3: 87-104 (2008), Medintz et al., Potential Clinical Applications of Quantum Dots, Int J Nanomedicine 3 (2): 151-167 (2008)).

The method of the invention purifies, isolates or enriches neural cells, or two separate populations of neural cells and cardiomyocyte cells.

The purified neural cells according to the invention can be used for central or peripheral nerve regenerations. In particular, the neural cells obtained according to the process according to the invention are suitable in the context of the treatment of neurodegenerative diseases, stroke, amblyopia and retinal problems, for example macular degeneration, spinal cord trauma and / or or medullary degeneration, peripheral nerve reconstruction, muscle tissue innervation, motor plates, the "tissue engineering" of innervated tissue as a muscle or cardiac tissue, glands, neuroendocrine tissue (such as pituitary, adrenal gland, Langerhans islets), manufacture of smooth muscle tissue or skeletal or innervated heart, treatment of spastic disorder ...

This approach also allows isolation of living heart cells at an early embryonic stage, ie to the ^6th day of development. On the ^tenth day of development, the isolation of the cells is made easier with only one marking. The method according to the invention makes it possible to isolate cardiomyocytes on the basis of their mitochondrial potential, since the selection is made on a potential related to the mitochondrial mass. The isolated cells are not tumorous either.

The method according to the invention can also be used in vitro in order to test the toxicity or the effectiveness of chemical or medicinal products.

The cells isolated according to the process of the invention may be combined in vitro or in vivo with medical and / or surgical supports or devices. The skilled person is able to choose these supports and devices according to his needs, using his technical knowledge.

In the invention, the term "individual" means any animal, especially mammals, in particular humans.

By "mitochondria membrane potential sensitive marker" is defined in the invention a molecule that can detect active mitochondria, which are able to maintain a potential difference between their internal environment and their external environment.

In the invention, the initial binding of the mitochondria membrane potential sensitive marker is dependent on the mitochondrial membrane potential and therefore requires that the mitochondrion be alive. This marker binds covalently to the mitochondrial matrix. The initial fixation is a function of the initial potential of the mitochondria. Once the marker is fixed, if the potential of the mitochondria changes, the fixation of the marker remains unchanged. Fixation of the marker is proportional to the mass of mitochondria in the cell.

 Methods for measuring mitochondrial membrane potential in vertebrates have been reported using a non-specific fluorescence quencher (US application 2010/0209960). It is also possible to measure the membrane potential by modifying molecules of interest to make them fluorescent, for example fluorescent ((Umezu et al., Biochemical characterization of the novel rice kinesin K23 and its kinetic study using fluorescence resonance energy transfer between an intrinsic tryptophan and ATP fluorescent analogue, J Biochem 149 (5), pages 539-550 (2011), to modify the fluorophores themselves as for example by the modification of YFP (Abraham et al., Bidirectional fluorescence resonance energy transfer (FRET) in mutated and chemically modified yellow fluorescent protein (YFP), Bioconj Chem 22 (2): 227-34 (201 1)). It is also possible to use markers specific for mitochondria of a given species (such as the SYTO 18 marker, specific for yeast mitochondria).

In the present invention, the "mitochondrial membrane potential sensitive marker" may correspond to a marker for measuring the activity of the respiratory chain and / or its level of development, in particular aero / anaerobic. It may be, for example, a marker that binds specifically to transcription factors, thus reflecting the development or activity of the respiratory chain.

 ATP mitochondrial aerobic production is a complex mechanism that involves glycolysis, fatty acid oxidation (or "oxidation"), the Krebs cycle ("citric acid cycle"), and oxidative phosphorylation ( OXPHOS, "oxidative phosphorylation"). This method of producing ATP energy is far superior to that of anaerobic respiration.

 Anaerobic glycolysis takes place in the cytoplasm and gives rise to the production of pyruvate, NADH (nicotinamide adenine dinucleotide) and ATP.

Aerobic respiration takes place in the mitochondria, acetyl-CoA is produced from pyruvate from glycolysis and oxidation of fatty acids. Acetyl-CoA enters the Krebs cycle to produce NADH, FADH ₂ (flavin adenine dinucleotide) and ΓΑΤΡ. NADH and FADH ₂ will be used by the mitochondrial respiratory complex (mtETC, Mitochondrial encoded Electron Transport Chain ") to create an electrical and chemical gradient in the mitochondrial membrane. The high energy electrons derived from NADH and FAD¾ are transferred to complex I and II respectively, then to coenzyme Q which will transfer the electrons to complex III. Cytochrome C is the second electron acceptor, it transmits electrons to complex IV ("cytochrome C oxidase COX"). In the final step, the electrons are accepted by the oxygen via the production of H 0. The complexes I, III and IV use the energy released by the transfer of electrons to activate the proton pumps which will transfer protons in the inter-membrane mitochondrial space and generate a gradient of pH and potential.

 ATP is ultimately produced by the return of the protons to the mitochondrial matrix through the V complex, which then uses the energy generated by the electrochemical gradient to produce ΓΑΤΡ from adenosine diphosphate (ADP) and inorganic phosphate. The mitochondrial membrane potential is therefore closely related to the activity of the respiratory chain and its metabolism, especially aerobic, which increases this potential. This chain is a multiprotein complex (complex I (NADH deshydrognease), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase COX), complex V, and two electron carriers: coenzyme Q and cytochrome C).

 There are markers for these different complexes as well as for possible isotypes during maturation or for the transcription factors encoding these complexes.

 By "mitochondrial membrane potential sensitive marker" is also meant markers specifically recognizing metabolites derived directly or indirectly from the metabolism of the respiratory chain or neural differentiation.

Free radicals (or ROS, Reactive Oxygen Species) are a product derived from the activity of the respiratory chain. Free radicals are toxic to the cell and can in particular cause damage to mitochondrial DNA (mtDNA). When activating aerobic metabolism, there is an increase in antioxidant enzymes such as, for example, Cu / Zn superoxide dismutase (Cu / Zn-SOD), glutathione peroxidase 1 (GPx1) and peroxyredoxin (Prx) 1 and 2. Due to the hypermetabolism of oxidative phosphorylation (OXPHOS), the production of free radicals will increase. The localized increase of free radicals will itself increase by self-amplification loops. The ability of cells to use aerobic energy production, and to adapt to the increase in derived products such as free radicals, could be a key factor in cell differentiation.

 The aerobic activity of the respiratory chain will therefore result in a difference in membrane potential, a pH gradient and also an increase in the production of free radicals.

 Thus, detecting a difference in potential or pH or the production of free radicals under certain conditions may be equivalent in the present invention.

 The "membrane potential sensitive marker" TMRM we have used will increase the production of free radicals after excitation.

 In parallel, another marker can be used to detect the local mitochondrial production of free radicals, such as, for example, dihydrorhodamine 123, or to measure the local mitochondrial pH, such as, for example, SNARF 1 (acetyl methyl ester having a structure derived from TMRM spiro). . In the invention, it is possible to combine at least one membrane-sensitive marker of the mitochondria and at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals, which means that one or two, or three, or more markers sensitive to the membrane potential of the mitochondria may be combined with one or two, or three, or more markers to quantify the number of mitochondria and / or increase the production of radicals free.

In one advantageous embodiment, the invention relates to a method as defined above, in which either said at least one membrane-sensitive marker of the mitochondria or said at least one marker making it possible to quantify the number of mitochondria. and / or increase the production of free radicals, or both, are cationic type markers. Thus, in the invention

 the membrane-sensitive marker of the mitochondria is a cationic marker, the marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals that is not cationic,

 the membrane-sensitive marker of the mitochondria is not a cationic marker, the marker for quantifying the number of mitochondria and / or increasing the production of free radicals is a cationic marker,

 either the membrane potential sensitive marker of the mitochondria and the marker for quantifying the number of mitochondria and / or increasing the production of free radicals are cationic markers.

The cationic labels according to the invention are preferably lipophilic and electrically charged markers, in this case positive charge (see cationic markers). The accumulation of cations in the mitochondria will depend on different parameters, including mitochondrial membrane potential, plasma membrane potential and mitochondrial volume ratio relative to cell volume. The markers will therefore penetrate the high potential mitochondria. The marker will be chosen so that the marker does not bind covalently to the mitochondrial matrix. It is actually free, only retained in the mitochondria because of the depolarization of the membrane. If this polarization decreases, then there is gradual release of the marker outside the mitochondria. If the mitochondrion depolarizes completely, the marker is completely released.

In one mode of the invention, these markers may be fluorescent markers or not. These markers have an absorption spectrum in which the marker will be stimulated with a source of energy such as an electromagnetic wave and an emission spectrum in which the release of energy will be analyzed as fluorescence for example. In another advantageous embodiment, the invention relates to a method as mentioned above, wherein either said at least one membrane-sensitive marker of the mitochondria or said at least one marker making it possible to quantify the number of mitochondria and / or increase the production of free radicals, or both, are fluorescent markers.

The use of fluorescent markers is advantageous because it makes it possible to measure and quantify the marker used.

 The fluorescence may be intrinsic (when the marker as such is fluorescent) or extrinsic (when the marker is coupled to a fluorophore).

 Among the fluorescent mitochondrial markers sensitive to the membrane potential used according to the invention, it is possible to list, without being limiting:

 Rhodamine 123 (RI 23);

- the methyl ester of tetramethylrhodamine (TMRM, C _2S Î ₂₅ C1N ₂ 0 _7, tetramethylrhodamine, methylester (xanthylium, 3,6-bis (dimethylamino) -9- (2-

(methoxycarbonyl) phenyl perchlorate)) such as T668;

- tetramethylrhodamine ethyl ester (TMRE, (C ₂₆ H ₂ 7cl o7, tetramethylrhodamine ethylester, perclorate (xanthylium, 3,6-bis (dimethylamino) -9- [2- (etho2çycarbonyl) phenyl] -, perchlorate)) as by example TMRE Ύ669;

 tetramethylrhodamine-5-maleimide

 5,5 ', 6,6'-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide (JC-

1),

 - the JC-9,

 3,3'-dihexyloxacarbocyanine iodide (DiOC6), and

 2- (4- (dimethylamino) styryl) -N-ethylpyridinium iodide) (DASPEI)

 - the Green FM MTG mitotracker, "Mitotracker dye",

 - Mitotracker red CMXRos,

 - Mitotracker orange CMTMRos,

- Dihydrorhodamine 123 (C ₂₁ H ₁₈ N ₂ O ₃ benzoic acid, 2- (3,6-diamino-9H-xanthene)

9-yl) - methyl ester having the same structure as TMRM but without the methylation of aromatic rings) Among the fluorescent mitochondrial markers allowing the detection of the mitochondrial mass, it is possible to list, without being limiting:

 - Mitotracker Green FM (MTG),

 - Mitotracker Red 580nm MitoTracker® Red FM (MTR),

 - the MitoTracker® Deep Red FM (MTDR),

 or, for example, markers which detect an activity of the El alpha pyruvate dehydrogenase sequence (see cell light mitochondria-GFP C010600 Invitrogen®),

or, for example, markers which in contrast make it possible to evaluate the total volume of the mitochondria of the cell (such as the cytoplasmic marker calcein-AM), fluorescent nonyl acridine orange,

 a marker recognizing cardiolipin, whose level of membrane expression of the mitochondria is proportional to the mitochondrial mass

 a marker which specifically recognizes mitochondrial transcription factors coding for the replication or transcription of mitochondrial DNA, for example and in a non-exclusive manner, transcription factors TFAM, TFB2M, POLG.

Other markers such as markers for analyzing enzymatic biological activities, local pH modification, calcium (for example Fluo-5N AM), markers for measuring various activities of oxidative phosphorylation and the Mitochondrial Krebs (see also rho-2 Koopman WJ, Computer-Assisted Live Cell Analysis, Methods 2008 46 (4): 304-311) (Fluo-5N AM C ₅₀ H ₄ 7F ₂ N ₃ O ₂₅ which in chemical terms is derived from TMRM with side chain modification and substitution of aromatic rings metabolites), free radicals, ΓΑΤΡ "turnover", cytochrome oxidase, superoxide activity of antioxidant proteins (such as MnSOD or CuZnSOD), l lactate dehydrogenase activity or the expression of certain mitochondrial regulatory genes may be used. These markers are interesting because their initial fixation does not depend directly on the potential of the mitochondria.

It is also possible to use expression vectors encoding an Elalpha pyruvate dehydrogenase sequence fused to GFP or RFP. Advantageous markers make it possible to measure the amount of free radicals (ROS). It may be mentioned, without being limiting:

 RedoxSensor Red CC-1,

- the MitoTracker Red CM-H ₂ XRos,

 - Dihydrorhodamine 6G, which is not fluorescent,

- MitoTracker® Orange CM-H ₂ TMRos, and

DCFDA type markers, of which CM-H ₂ DCFDA (5- (and-6) -chloromethyl-2 ', 7'-dichlorodihydrofluorescein diacetate, acetyl ester) or DCF-DA or H ₂ DCFDA,

- Dihydrorhodamine 123.

The many fluorophores capable of being coupled to the markers are well known to those skilled in the art. In a nonlimiting manner, mention may be made, for example, of TMR, fluorescein (FITC), calcein, dichlorofluorescein, cyanines, Alexa molecules, or else fluorescent proteins, in particular the CFP, GFP, YFP, DsRed or Flavine. The use of particular fluorophores generally depends on their absorption and emission spectrum. For example, Dichlorofluorescein (DCF) or Calcein can be conveniently coupled to TMRM.

Among the fluorophores capable of being coupled to the markers, it is also possible to use quantum dots (QDot), that is to say nanocrystals whose fluorescence varies according to their diameter. In a nonlimiting manner, mention may be made, for example, of QDot ZnSe, QDot CdSe or QDot CdTe.

In another advantageous embodiment, the invention also relates to a method as defined above, wherein said at least one membrane-sensitive marker of the mitochondria is selected from tetramethyl rhodamine methyl ester (TMRM) and Tetramethyl rhodamine ethyl ester (TMRE).

 Said at least one membrane-sensitive marker may ideally be selected from the list below:

 tetramethyl rhodamine methyl ester (TMRM)

 - Tetramethyl Rhodamine Test (TMRE)

tetramethylrhodamine-5-maleimide Rhodamine 123 (R123),

 5,5 ', 6,6'-tetracoro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide (JC-1),

- the JC-9,

 3,3'-dihexyloxacarbocyanine iodide (DiOC6), and

 2- (4- (dimethylamino) stylyl) -N-ethylpyridinium iodide) (DASPEI).

 These different markers can be chemically modified, bound or associated with other compounds.

Surprisingly, the inventors have found that neural cells have particular properties during their development.

 Neural cells indeed have a low and stable mitochondrial membrane potential associated with a high number of mitochondria, as well as a protection system allowing them to resist the toxicity of free radicals.

 Neural cells are therefore able to withstand toxic agents such as free radicals, especially those induced during TMRM stimulation.

 The cardiomyocytes have a high membrane potential, less stable, for a lower number of mitochondria, these cells are therefore less protected from free radicals.

 The totipotent cells, as well as the other cell types, have less mitochondria and are, for their part, much less protected against free radicals.

 Thus, labeling a cell population with a mitochondrial membrane potential sensitive marker, such as TMRM, allows selection of neural stem cell and cardiomyocyte populations. Which populations can then be distinguished from each other by labeling with a marker to quantify the number of mitochondria.

The inventors have also surprisingly found that simultaneous labeling of TMRM with a second mitochondrial marker, such as MTG, results in depolarization and death of cells that are not neural stem cells. Stimulation of TMRM via the second marker could lead to a greater release of free radicals, toxic to other cell types. In this way, simultaneous labeling of a cell population makes it possible to select the neural stem cells that are the most resistant. For this reason, cell populations can also be distinguished by using a marker to increase the production of free radicals.

Thus, in the invention the second marker can be both a marker for measuring the number of mitochondria and a marker which, in the manner used in the invention, will increase the production of free radicals.

For example, activation of MTG results in FRET energy transfer to TMRM, the over-activation of which then leads to an increased production of free radicals. The isolation of neural cells is then facilitated by the property of these cells to be better protected against free radicals compared to other cell types, including cardiomyocytes.

In yet another advantageous embodiment, the invention relates to a method mentioned above, wherein said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals is selected from mitochondrial protein markers. or markers of mitochondrial DNA.

In the present invention two or more mitochondrial markers may be sufficient to isolate the cells of interest, especially if these markers are linked to other non-specific mitochondrial markers. It may also be possible to use different markers of mitochondrial development to isolate neural cells. These markers are markers related to the mass and number of mitochondria, markers for detecting structural proteins, markers for detecting specific enzymatic activities, markers for measuring different copies of mitochondrial DNA, markers metabolites or markers of replication, transcription, packing or repair of mitochondrial DNA (such as TFAM, TFB2M, POLG) ...

In yet another advantageous embodiment, the invention relates to an aforementioned method, wherein the marker for quantifying the number of mitochondria and / or increasing the production of free radicals is green mitochondrial tracer (MitoTracker Green; C34H28Cl5N3O (Benzoxazolium, 2- [3- [5,6-dichloro-1,3- bis [[4- (chloromethyl) phenyl] -1,3-dihydro-2H-benzimidazol-2-ylidene] -1-propenyl] -3-methyl-, chloride)).

In yet another advantageous embodiment, the invention relates to an aforementioned method, wherein the second marker is green mitochondrial tracer (MitoTracker Green, MTG) and the first marker is TMRM or TMRE.

The labels used according to the invention can be chemically modified and can even be combined with one another. They can be connected by a spacer ("branched" or not) that can be used to transfer energy.

In one embodiment, the invention relates to a method as defined above wherein said at least one membrane-sensitive marker of the mitochondria and said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals allows a transfer of energy from one to the other of the markers that can lead to the production of free radicals.

In another advantageous embodiment, said at least one membrane-sensitive marker of the mitochondria and said at least one marker making it possible to quantify the number of mitochondria and / or increase the production of free radicals are fluorescent, and allow a transfer of energy or FRET.

The energy exchange between fluorescent markers is called "FRET"("Fluorescence Resonance Energy Transfer") (Journal: Selvin PR, Nature Structural Biology, vol.7: 9 (2000)). The donor fluorochrome and the energy recipient are considered. This phenomenon generally requires energy compatibility between the molecules. This means that the donor's emission spectrum must at least partially cover the absorption spectrum of the acceptor or be very close. There are possibilities to modify the different markers and to allow this interaction. The FRET mechanism applies by extension to an energy exchange between molecules that are not necessarily fluorescent and the exchange of energy does not necessarily take place in the visible wave domain. There are many acceptors for energy donors. For example, terbium cryptate and europium crypate can be used with Cy5 fluorophores, but they also have the advantage of being able to be associated with fluorescein fluorophores or fluorescent proteins (GFP).

For the MTG, the absorption wavelength is 490 nm and the emission wavelength is 516 nM. For TMRM, the absorption wavelength is 549 nm and the emission wavelength is 574 nM.

Thus, there may be energy transfer between the two markers, or FRET. When there is a FRET mechanism between the two markers, the fluorescence emitted by MTG at 516 nM decreases and there is an increase in fluorescence at 549 nM in the TMRM spectrum. Also, the light emitted by the MTG is at a wavelength corresponding to the excitation wavelength of the TMRM.

 In the present invention, the inventors have shown that FRET between MTG and TMRM makes it possible to identify neural stem cells and to eliminate other cell quotas. For neural stem cells, the cells will therefore remain positive for the first marker because their membrane potential remains stable. In the presence of the second marker, the energy transfer takes place due to the compatibility of the energy transfer. The fluorescence of the second marker will decrease, but the fluorescence of the first marker will be greatly increased: there is FRET phenomenon. With this method, the neural cells will have a very positive labeling for the first marker, which is paradoxically increased because of FRET. The cells will thus be easy to identify by analyzing the fluorescences for the two markers (first marker "high" and "intermediate" for the second marker).

The FRET phenomenon thus allows the positive selection of the cells of interest whose fluorescence is increased, but also allows the decrease of the fluorescence of the other cells, which are not cells of interest, by depolarization and / or cell death. This selection method results in an enrichment in the preparation of the cells of interest that survive more easily and a decrease in other cells more sensitive to toxicity. The first TMRM labeling can be done on isolated cells clustered or not, on an embryo or on tissues for the same duration. In other cases, it is possible to dissociate the cells in an enzyme solution (including for example collagenase) or not. Methods for preparing neural cells from tissue are well known to those skilled in the art.

It is possible to rinse the cells twice in PBS without calcium and magnesium and then in a trypsin EDTA solution which chelates calcium and polarizes the cells. After dissociation, the cells are taken up in the culture medium or in a buffer and then the labeling is carried out for ½ h. at 37 ° C. Two washes cells are then made with the TMRM buffer (116 nM NaCl, 20 mM Hepes, 12.5 mM NaH2P04, 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgS0 ₄ Ph 7.35). The fluorescence is then read directly by flow cytometry.

In addition to the advantageous TMRM MTG combination, other pairs of markers can be used in the invention.

 Without limitation, the following couples may be mentioned:

 - TMRE / MTG,

 JC-1 / Mitotracker red 580 nm,

 Rhodamine 123 / MTG,

 TMRM / BODIPY 11 (in this case the advantageous use is carried out with 1.0 μΜ of BODIPY 11 for 30 min at ambient temperature, and for 30 min with TMRM at 0.10 μΜ),

 - Cy3 / Cy5,

 - GFP / YFP,

 - GFP / DsRed,

 - Alexa 488 / Alexa 568,

 - Alexa 546 / Alexa 594,

 - Alexa 594 / Alexa 647,

 - Mitotracker Orange (CMTMRos) / MTG,

 - TMRM / Quantum Dot having an absorption at less than 500 nm or more than 600 nm and emission in the acceptor spectrum here TMRM (500-600 nm), for example Quantum Dot CdSE / ZnS,

- TMRM / Quantum Dot CdSE / ZnS coupled to naoparticles (y-Fe ₂ 0 ₃ ), TMRM coupled directly to a power source or energy transmission chain,

 TMRM / TMRM modified to have an emission peak in the TMRM absorption peak and serve as an energy donor. (It is possible to modify the aromatic cycles to change the fluorescence for example, by methylation of these cycles or interposition of a carbon chain on the side chain.

Of course, this list is not limiting, and the skilled person is able to adapt the combinations of his choice according to the desired cell types.

According to an advantageous embodiment, one of the markers may be non-fluorescent, but capable of delivering energy to the fluorescent marker and cause the increase in the biological activity of the first marker, resulting for example in the increase of radicals. free toxic. Such an approach facilitates the purification of more resistant cells.

In a preferred embodiment, the invention relates to a method of isolating neural cells, wherein the membrane potential sensitive marker and the marker for quantifying the number of mitochondria and / or increasing the production of free radicals. correspond to a single marker.

Indeed, it is possible to chemically couple a membrane-sensitive marker with:

 a marker making it possible to quantify the number of mitochondria or

 a marker for increasing the production of free radicals, or

 a marker making it possible to quantify the number of mitochondria and to increase the production of free radicals.

For example, it is possible to design a single label composed of a first membrane potential sensitive marker and a second marker that absorbs energy in a given absorption spectrum, and retransmits it into a spectrum of emission compatible with the absorption spectrum of the first marker sensitive to mitochondrial membrane potential. In a nonlimiting manner, such a single marker may be, for example, TMRM chemically coupled to a quantum dot.

 Dot quantum absorbs energy and then emits energy into the absorption spectrum of TMRM by FRET. This energy delivery will be accompanied by a "superactivation" of the TMRM with a very increased production of free radicals compared to the production obtained in the absence of the energy sensor.

The invention also relates to a method for isolating neural and / or cardiomyocyte stem cells, as defined above, said method comprising:

 at. a step of incubation of a cell population with

 i. at least one marker sensitive to the membrane potential of the mitochondria,

 ii. either at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals, b. a step of incubating the labeled cell population in step a), with

 i. at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, if at least one membrane-sensitive marker of the mitochondria has been used in step a),

 ii. or at least one membrane-sensitive marker of the mitochondria, if at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals has been used in step a), and

 vs. a step of isolating the neural and / or cardiomyocyte stem cells.

The invention also relates to a method for isolating neural and / or cardiomyocyte stem cells, as defined above, said method comprising:

at. a step of incubating a cell population with at least one membrane-sensitive marker of the mitochondria, in particular TMRM or TMRE, b. a step of incubating the cell population labeled in step a), with at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, in particular MTG, and

 vs. a step of isolating the neural and / or cardiomyocyte stem cells.

The invention also relates to a method for isolating neural and / or cardiomyocyte stem cells, as defined above, said method comprising:

 at. a step of incubating a cell population, with at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, in particular MTG, and b. a step of incubating a cell population previously removed with at least one marker sensitive to the membrane potential of the mitochondria, in particular TMRM or TMRE,

 vs. a step of isolating the neural and / or cardiomyocyte stem cells.

In yet another advantageous embodiment, the invention relates to a method of isolating neural stem cells mentioned above, said method comprising:

 at. a step of incubating a cell population with:

 i. simultaneously at least one membrane-sensitive marker of the mitochondria, and

at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals,

 or

 ii. if said at least one membrane-sensitive marker of the mitochondria and said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals correspond to one and the same marker, with this alone and same marker,

 and

b. a step of isolating the neural stem cells. It is also possible in the invention to carry out a preliminary TMRM labeling step before the aforementioned step a). This step makes it possible to isolate cells labeled with TMRM. The cells are then cultured and / or washed in an appropriate buffer for a period of about 1 hour to about 48 hours, preferably from about 1 hour to about 24 hours.

The cells are then incubated according to step a) mentioned above.

Advantageously, the invention relates to a method for isolating neural stem cells as defined above, said method comprising:

 at. a first step of incubating a cell population with at least one membrane-sensitive marker of the mitochondria, in particular TMRM or TMRE, and

 b. a second step of screening the cell population with positive labeling for said mitochondria membrane potential sensitive marker, preferably at least about 100-fold higher than the labeling level of the cells considered unmarked by said marker, and

 vs. a third incubation step of the cell population selected in step b) with at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, in particular MTG,

said third step making it possible to obtain a population marked by both said membrane-sensitive marker of the mitochondria and by said marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, and . a step of isolating the neural stem cells having a positive labeling for said mitochondria membrane potential sensitive marker, preferably at least about 100 times the labeling level of the cells considered unmarked by said marker, and a labeling positive for said marker for quantifying the number of mitochondria and / or increasing the production of free radicals, preferably at least about 100 times greater than the level of labeling of the cells considered to be unmarked by said marker.

In one advantageous embodiment, the invention relates to a method for isolating neural stem cells as defined above, in which the cell population selected in step b) is incubated with a marker sensitive to the mitochondrial membrane potential, simultaneously or separately from step c).

In yet another advantageous embodiment, the invention relates to a method for isolating neural stem cells as defined above, said method comprising:

 at. a first step of incubating a cell population with at least one membrane-sensitive marker of the mitochondria, in particular TMRM or TMRE, and

 b. a second step of screening the cell population with positive labeling for said mitochondria membrane potential sensitive marker, preferably at least about 100-fold higher than the labeling level of the cells considered unmarked by said marker, and

 vs. a third incubation step of the cell population selected in step b) with at least one marker making it possible to quantify the number of mitochondria and / or increase the production of free radicals, in particular MTG,

said third step being delayed by at least several hours, preferably about 24 hours, in order to attenuate the labeling of the cell population by said mitochondria membrane potential sensitive marker used in step a), preferably by about 65%, and d. an isolation step of the neural stem cells having a positive labeling for said marker making it possible to quantify the number of mitochondria and / or to increase the free radical production by at least 10 000 times greater than the labeling level of the cells considered as unmarked by said marker. Advantageously, the invention relates to a method for isolating cardiomyocyte cells as defined above, said method comprising:

 at. a first step of incubating a cell population previously removed with at least one marker sensitive to the membrane potential of the mitochondria, in particular TMRM or TMRE, and

 b. a second step of screening the cell population with positive labeling for said mitochondria membrane potential sensitive marker, preferably at least about 100-fold higher than the labeling level of the cells considered unmarked by said marker, and

 vs. a third incubation step of the cell population selected in step b) with at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, in particular MTG,

 said third step being delayed by at least several hours, preferably about 24 hours, in order to attenuate the labeling of the cell population by said mitochondria membrane potential sensitive marker used in step a), preferably by about 65%, and d. a step of isolating the cardiomyocyte cells having a positive labeling for said marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals by approximately 100 to 1000 times greater than the labeling level of the cells considered as unmarked by said marker.

Advantageously, the invention relates to an isolation method as defined above, in which either the step of selecting a cell population, or the step of isolating the cells of interest, or the two steps, are performed. using a flow cytometry apparatus, preferably a cell sorter.

Another aspect of the invention relates to a method as defined above wherein said at least one mitochondria membrane potential sensitive marker, or said at least one marker for quantifying the number of mitochondria. and / or increase the production of free radicals, or both, are coupled to at least one magnetic particle.

If the first and / or second marker is coupled to a magnetic particle, it follows that positive or double positive cells exhibit greater magnetism. These can be selected from other cell populations using a magnetic field, for example via a magnet affinity column. The dead cells, meanwhile, will release the markers and their magnetism will decrease, they will not be selected.

In the case where TMRM and MTG are coupled to magnetic particles, the neural stem cells, which are labeled with TMRM and MTG, have the greatest magnetism and will be selected preferentially.

Fixation by the markers being reversible over a few days, the particles are released and the magnetism of the cells will decrease thereafter.

In this form the invention is particularly suitable for a clinical application because cell sorting devices, using magnetic columns or magnetic fields, prevent contact between the cells and the device.

It is possible to sort the labeled cells with a magnetic marker using the method described below.

 1) The cell population is incubated with the magnetic marker.

 2) The incubated cell population is placed in a magnetic or electromagnetic field of low or high intensity.

 3) The cells marked with the magnetic marker are charged and will thus be fixed on the walls of the support.

 4) The sample is washed, and uncharged cells, which are not attached to the walls, are removed.

 5) Medium is added to the sample;

6) The magnetic field is stopped and the marked cells are resuspended in the medium. In a flow system where the cells are in motion, the cells labeled with a magnetic marker can be deflected according to their charge, and therefore according to their marking, in order to be isolated. Without limitation, the heart (core) of the magnetic particles usually consists of magnetite Fe ₃ 0 ₄ such as cobalt ferrite, magnetite or _an F 0 ₃ as the manganese ferrite.

 In an advantageous embodiment, the invention relates to a method as defined above wherein said minus one magnetic particle is a nanoparticle having superparamagnetic properties.

In the form of nanoparticles, ferromagnetic metals can become superparamagnetic. The nanoparticles are no longer permanently magnetic, but become so only in the presence of a magnetic field.

The use of these superparamagnetic nanoparticles therefore limits the appearance of aggregates of particles and cells, and allows a more uniform marking.

The coupling of superparamagnetic nanoparticles to fluorescent markers is known to those skilled in the art. For example, superparamagnetic iron oxide nanoparticles can be coupled to TMRM (Maurizi et al., Langmuir, 25 (16), 8857-8859 (2009)). Magnetic nanoparticles can also be coupled to quantum dots, for example. A ferromagnetic nanoparticle that can be coupled to a mitochondrial marker can be for example SPIO (SuperparaMagnetic Iron Oxide). Advantageously, the invention relates to an isolation method as defined above, in which either the step of selecting a cell population, or the step of isolating the cells of interest, or the two steps, are performed. using a magnetic sorting apparatus, preferably a magnetized column. Another aspect of the invention relates to a kit comprising at least one marker sensitive to the mitochondrial membrane potential and at least one marker for quantifying the number of mitochondria and / or, where appropriate, increasing the production of free radicals, said at least one marker sensitive to the potential of membrane of the mitochondria and said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals that can correspond to a single marker,

 in such a way that

 if said at least one marker sensitive to the membrane potential of the mitochondria and said at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals are used simultaneously, or if said at least one a marker sensitive to the membrane potential of the mitochondria and said at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals correspond to one and the same marker, said method allows the isolation, the purification and / or enrichment of neural stem cells, and

 if said at least one marker sensitive to the membrane potential of the mitochondria and said at least one marker making it possible to quantify the number of mitochondria are used separately or spread over time, said kit allows isolation, purification and / or or enrichment of neural stem cells and / or cardiomyocyte cells.

Advantageously, the invention relates to a kit as defined above in which either said at least one membrane-sensitive marker of the mitochondria, or said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals, or both, are fluorescent markers. The invention will be better illustrated by the following Examples and Figures. BRIEF DESCRIPTION OF THE FIGURES

1A-1D represent the stamping TMRM ES-D3 embryonic embryonic development in the 10 ^th day.

Figure 1A shows flow cytometric analysis of ES-D3 cells. The Y axis represents cell granulosity (SSC) and the X axis represents cell size. The PI window represents the selected cells.

Figure 1B shows the TMRM labeling intensity of cells from the PI window, after 30 min labeling, at 37 ° C with 50 nM TMRM. The X axis represents the TMRM fluorescence, in arbitrary logarithmic units, and the Y axis represents the number of cells.

Figure 1C shows the intrinsic green fluorescence of cells not labeled with TMRM. The X axis represents the TMRM fluorescence, in arbitrary logarithmic units, and the Y axis represents the number of cells.

The frame indicates the intensity of auto fluorescence.

Figure 1D shows TMRM labeling as a function of green self-fluorescence. Only cells with low green fluorescence (FITC) are selected. The X axis represents intrinsic green fluorescence, in arbitrary logarithmic units, and the Y axis represents TMRM fluorescence in arbitrary logarithmic units.

Frames represent cells marked strongly with TMRM (high), medium (mid) and low (low).

Figures 2A-2C show the characterization of strongly or moderately labeled cells with TMRM.

2A shows the variation of expression of MHC (cardiac marker) cells labeled strongly TMRM (second column) or medium conspicuousness ^(3rd column). The first column represents the total marking in cells ^10th day of differentiation.

Figure 2B depicts strongly or moderately labeled TMRM1 cells which have been cultured. The clusters correspond to the re-aggregation of the cells that adhered to the culture plate. FIG. 2C represents the result of the re-culturing of cells weakly labeled with TMRM. It can be seen that no cell is able to proliferate. The cells are dead. Figures 3A-3C show the characterization of neural stem cells, strongly labeled with TMRM, after double labeling TMRM and MTG, sequential or concomitant, from ES-D3 cells after 10 days of development.

Figure 3A shows flow cytometric analysis of ES-D3 cells after TMRM labeling. The X axis represents the intrinsic green fluorescence, corresponding to P autofluorescence, the Y axis represents the TMRM marking. The frame selects the strongly labeled TMRM cells.

Figure 3B shows the cells from the frame mentioned in Figure 3A, and labeled 1 hour later with MTG. The X axis represents the MTG mark, the Y axis represents the TMRM mark. The cells surrounded by the ellipse in the frame indicated "2x pos" are neural cells and represent 1, 2% of the population (zone Q2). The remaining cells are essentially dead cardiomyocyte cells representing 80% of the cell population (zone Q3), or depolarized or dying cells which represent 18.4% of the cell population (zone Q4).

Figure 3C shows cells from the frame shown in Figure 3A, and labeled 1 hour later with MTG and TMRM. The X axis represents the MTG mark, the Y axis represents the TMRM mark. The cells surrounded by the ellipse continues in the frame indicated "2x pos" are neural cells (zone Q2). Their TMRM labeling is increased by FRET phenomenon (increase in red fluorescence and decrease in green fluorescence). The fluorescence level without FRET is indicated by the discontinuous ellipse.

The majority of the cells are now in the Q3 zone corresponding to the dead cells, the new labeling with TMRM / MTG did not affect the size of the neural population (zone Q2). The predominantly cardiomyocytic population has practically disappeared (<1%) and is found in dead cells (98%) (zone Q3). The fluorescence level without FRET is indicated by the discontinuous ellipse. Figures 4A-4C show the characterization of neural stem cells, moderately labeled with TMRM, after double labeling TMRM and MTG, sequential or concomitant, from ES-D3 cells after 10 days of development.

Figure 4A shows flow cytometric analysis of ES-D3 cells after TMRM labeling. The X axis represents the intrinsic green fluorescence, corresponding to Γ autofluorescence, the Y axis represents the TMRM marking. The frame selects the moderately marked cells at the TMRM.

Figure 4B shows the cells from the frame mentioned in Figure 3A, and labeled 1 hour later with MTG. The X axis represents the MTG mark, the Y axis represents the TMRM mark. The cells surrounded by the ellipse in the frame indicated "2x pos" are neural cells and represent 0.8% of the cell population (zone Q2). The remaining cells are dead cells representing 96.6% of the cell population (zone Q3) or dying (2.9%) (zone Q4). Figure 4C shows the cells from the frame shown in Figure 3A, and labeled 1 hour later with MTG and TMRM. The X axis represents the MTG mark, the Y tax represents the TMRM mark. The cells surrounded by the ellipse continues in the frame indicated "2x pos" are neural cells (zone Q2). Their TMRM marking is increased by FRET phenomenon. The fluorescence level without FRET is indicated by the discontinuous ellipse.

The other cells are mainly dead cells representing 97.9% of the cell population (Q3 zone). It is found that the TMRM is also particularly toxic vis-à-vis non-cardiomyocyte cells of the preparation. Figures 5A-5F show the evolution of TMRM marking during development. FIGS. 5A, C and E respectively represent the TMRM fluorescence intensity of ES-D3 cells after 0, 5 or 10 days of development, said cells not being labeled with TMRM. The X axis represents the TMRM fluorescence, in arbitrary logarithmic units, and the Y axis represents the number of cells.

FIGS. 5B, D and F respectively represent the TMRM fluorescence intensity of ES-D3 cells after 0, 5 or 10 days of development, said cells being labeled with 50 nM TMRM, 30 min at 37 ° C. The X axis represents the TMRM fluorescence, in arbitrary logarithmic units, and the Y axis represents the number of cells.

Figures 6A-6F ^'show the evolution of MTG marking during development.

FIGS. 6A, C and E respectively represent the MTG fluorescence intensity of ES-D3 cells after 0, 5 or 10 days of development, said cells not being labeled with MTG. The X axis represents the MTG fluorescence, in arbitrary logarithmic unit and the Y axis represents the number of cells.

FIGS. 6B, D and F respectively represent the MTG fluorescence intensity of ES-D3 cells after 0, 5 or 10 days of development, said cells being labeled with 50 nM MTG, 30 min at 37 ° C. The X axis represents the MTG fluorescence, in arbitrary logarithmic unit and the Y axis represents the number of cells.

FIG. 7 represents the double TMRM / MTG labeling of the ES-D3 cells at 0 days of development. The X axis represents the MTG mark, the Y axis represents the TMRM mark.

 The cells in the frames Q1 + Q '1, Q2 + Q'2, Q3 and Q4 represent respectively 0.7%, 1.1%, 43.1% and 45.2%.

The frames Q1 and Q2 represent the high markings in the TMRM, the frames Q '1 and Q'2 represent the intermediate TMRM markings. Figure 8 shows the double TMRM / MTG labeling of ES-D3 cells at 5 days of development. The X axis represents the MTG mark, the Y axis represents the TMRM mark.

 The cells in the frames Q1 + Q '1, Q2 + Q'2, Q3 and Q4 represent respectively 1, 7%, 19.1%, 67% and 12.2%.

 The frames Q1 and Q2 represent the high markings in the TMRM, the frames Q 'I and Q'2 represent the intermediate TMRM markings. The cells indicated by an ellipse are living neural cells. FIG. 9 represents the double TMRM / MTG labeling of ES-D3 cells at 8 days of development. The X axis represents the MTG mark, the Y axis represents the TMRM mark.

 The cells in the frames Q1, Q2, Q3 and Q4 represent respectively 1.7%, 18.2%, 78% and 2.1%.

Frames Q1 and Q2 represent intermediate TMRM markings. The cells indicated by an ellipse are living neural cells.

 Q1 + Q3 + Q4 cells are enriched with markers MHC and GATA4, cardiac markers. Only Q1 cells are culturable.

 Q2 cells are living neural cells and strongly express the MASH1 marker.

Figures 10A-10F show cells from fractions Q1-Q4 described in Figure 9. Figure 10A shows cells from fractions Q1, Q3 and Q4 after one day of culture. The cell membrane is altered and the cells are not adherent, they are dead cells.

 Figure 10B shows cells from fractions Q1, Q3 and Q4 after two days of culture. The cell membrane is altered and the cells do not proliferate.

Figure 10C shows cells from fraction Q2, after a day of culture. Round refractive cells are characteristic of embryonic cells. Refractive elongated cells are dividing cells. 0018

35

 Figure 10D shows cells from fraction Q2, after two days of culture.

 FIG. 10E represents cells derived from fraction Q2, after one day of culture in two dimensions. The arrow indicates a dendrite type extension.

Figure 10F shows cells from fraction Q2, after two days of culture in two dimensions. The arrow indicates an axon type extension.

Figures 11A-11D show the method of selection of cardiomyocyte and neural cells.

Figure 11A shows the selection of cells based on the TMRM marker (see Figure 1D). The X axis represents intrinsic green fluorescence, in arbitrary logarithmic units, and the Y axis represents TMRM fluorescence in arbitrary logarithmic units.

Figure 11B shows the number of cells from the population with high TMRM labeling and still showing high labeling after culture for 24 hours without TMRM. They represent 34.7% of the population. The X axis represents the TMRM marking in arbitrary logarithmic unit and Y tax represents the number of cells.

Figure 11C shows the MTG labeling of the TMRM positive cells of Figure 11B. The cells indicated by ellipse 1 are dead, those indicated by ellipse 2 are cardiac cells, and those indicated by ellipse 3 are neural cells. The X axis represents the MTG marking in arbitrary logarithmic unit and the Y tax represents the number of cells.

Figure 11D shows the MTG staining of cells from cells with intermediate TMRM tagging and still showing after culture labeling for 24 hours without TMRM. The cells indicated by ellipse 1 are dead, by ellipse 2 are a mixed population of cardiac cells and other cell types, and by ellipse 3 are neural cells. The X axis represents the MTG marking in arbitrary logarithmic unit and the Y axis represents the number of cells.

EXAMPLES

Example 1: Use of intra-mitochondrial labeling related to the global membrane potential of mitochondria for the isolation of cardiomyocytes from mouse embryonic cells.

This protocol can be transposed to the isolation of cardiomyocytes of other origins, including the isolation of human cardiomyocytes from human embryonic cells (ES cells) or from differentiated cells reprogrammed at a totipotency stage (iPS). This protocol can also be applied to tissues or cells isolated from tissue. In the following experiments the concentrations of TMRM used were 50 nM but this is only indicative. Lower concentrations of 10 nM or even 0.1 nM can be used and some authors report the use of concentrations of TMRM and MTG up to 500 nM or even 1000 nM. Incubation times are also indicative and depend on the temperature, the type of cell, the buffer used, the will or not to saturate or not the fixing of the marker to have a more homogeneous distribution or not. When the marker concentrations increase, the toxicity increases, the specificity decreases especially for their relationship or not with the cell membrane potential. There is also cumulative toxicity for these markers.

As previously reported (see Hattori et al., Nature Methods 2010 vol7 and 61-69), the inventors labeled ES-D3 mouse embryonic cells with a methylester tetramethylrhodamine cationic fluorescent marker (TMRM), for example T668 Invitrogen ™ at 10 days of embryonic development.

ES-D3 cells (ATCC Biological Resource Center (Manassas, Virginia) were cultured and amplified and maintained undifferentiated on culture plates.

In the presence of complete culture medium that associates DMEM medium (high glucose Dulbecco's modified eagle media, Gibco) with 0.1 mM non-essential amino acids (Gibco), 0.1 niM betaJVtercaptoethanol (Sigma), 100 microg / ml penicillin and streptomycin (Invitrogen) and a Leukemia Inhibiting Factor (LIF) (1000 IU / ml) and low concentration of fetal calf serum (5%).

 Differentiation is initiated by forming embryonic bodies that are "clusters" of cells.

 The LIF is then removed and the concentration of fetal calf serum is increased to 20%. The beginning of the induction is considered as day 0 (EBS day 0);

 The cells in the preparation are undifferentiated. These are totipotent cells that express markers of stem cells but not markers of differentiated cells (Puceat M., Protocols for cardiac differentiation of embryonic stem cells, Methods 2008, Leschik J., Cardiac commitment of primate embryonic stem cells, Nature Protocol , 2008).

The initial formation of this 3D structure as the embryonic bodies is a classic technique to obtain an induction of the 3 layers of embryonic development. However, there are other alternatives for differentiating cells without forming embryonic bodies by directly differentiating cells in 2D culture (Puceat M., Protocols for cardiac differentiation of embryonic stem cell, Methods 2008). The undifferentiated cells in culture are suspended by treating them for 5 minutes with trypsin EDTA before being taken up in the culture medium. The embryonic bodies are obtained by making drops of cells and medium (see 800 cells for drops of 20 microliters) which are placed on an inverted lid culture box for 48 hours. Embryonic bodies are transferred to non-adherent 2D plates on the second day of embryonic development (EBS-D2). On ^Day 5, the embryonic body will be transferred on very adherent plates due to the presence of gelatin. The cells will then be cultured until ^Day 10 or more.

Using this technique, the first cardiac transcription markers appear on the ^3rd day with differentiated cells. These cells exhibit contractile properties at EBS-day7-8 due to the expression of proteins of the contractile apparatus of cardiomyocytes.

As proposed by Hattori, the inventors labeled the cells at EBS day 10 with TMPvM. The ^10th day is already at a late stage of cell differentiation cardiac since many heart cells are already beating. The TMRM labeling can be done on the embryonic body, the whole embryo or after dissociation of the embryo into cells in suspension using enzyme solutions or not.

 In this case, the cells were rinsed with PB S buffer without calcium, without magnesium at pH 7.4, the cells were then dissociated for 15 min at 37 ° C with trypsin EDTA.

The cells are taken up in the culture medium for at least half an hour to allow them time to repair the damage caused by the trypsin treatment. The time of dissociation will depend on the state of aggregation of the cells. The viability of the cells measured by trypan blue labeling is more than 80%. The cells will then be incubated with the TMRM marker (50 nM) at 37 ° C for 30 min in the incubator. This concentration is the one previously used by Hattori et al. This is a non-toxic concentration for the cells. The labeling can be done in small tubes with 500 microliters of medium per 1 million cells. At the end of the incubation, the cells are washed twice with TMRM buffer (1 16 nM NaCl, 20 mM Hepes, 12.5 mM NaH ₂ PO _4, 5.6 mM glucose, 5.4 mM KCl 0.8 mM MgSO ₄ , pH 7.35) before being analyzed and optionally sorted by flow cytometry.

 Irradiation of the cells at the TMRM absorption peak at 549 nM and analysis at its emission peak at 574 nM. The incubations can be carried out in culture medium or in buffers approximating in ionic composition and elements of the culture media such as, for example, the TMRM buffer.

 Some cells have autofluorescence in FITC green. In the Hattori study, cardiomyocytes were therefore TMRM cells with a high fluorescence level and FITC negative cells. By performing the TMRM ESD-3 markings at EBS day 10, the inventors obtained TMRM-labeled cells (TMRM positive) and unmarked cells (TMRM-negative). Among the labeled cells, some have a very high level of labeling, but no clear limit separates the strong markings from the intermediate markings (see Figure 1).

To find out where the limit is, we used concomitant labeling performed on isolated cells from mouse myocardial tissue collagenase digestion. Using mouse adult cardiomyocyte preparations or human cardiomyocyte cell lines, the inventors have defined a limit for differentiating between cells strongly labeled with TMRM and cells with intermediate labeling. The compensations determined can then be used for other experiments.

Using a flow cytometer, the cells were sorted and cultured separately,

 (see Figure 2).

The negative TMRM cells are dead cells.

 Intermediate and high TMRM cells are cells that, in gelatin plate culture, have the ability to self-aggregate, which is a hallmark of embryonic cells.

 The proportions of mRNA for certain cardiac cell markers such as MHC relative to the amount of mRNA evaluated for a nonspecific marker of a given cell type, such as GAPDH, is increased in the high TMRM fraction by compared to the intermediate TMRM fraction more than 5 times.

The high TMRM fraction is therefore enriched in cardiac cells (see Figure 2). According to Hattori, 98% of the cells in this population are cardiomyocytes. Using this technique, Hattori were not able to isolate cardiomycytes at an earlier time with good discrimination, regardless of the species considered.

Tagging using a single membrane-sensitive mitochondrial marker to isolate cardiomyocytes remains difficult labeling and does not readily isolate progenitors from cardiomyocytes since isolation is relatively late.

Example 2: Method of isolating neural stem cells involving energy transfer between the different markers.

In this example, the inventors show how to enrich a population of interest by using an intracellular marker to eliminate, by depolarization and / or cell death, the cells that are not searched for and, on the contrary, select the cells. This example also shows that it is possible to isolate neural stem cells at a stage of embryonic development, where these cells are not predominant. This purification is based on the observation made by the inventors that neural cells have particular properties during embryonic development. As noted above, because of their high number of mitochondria and stable mitochondrial membrane potential, neural cells resist the free radical toxicity induced by TMRM stimulation, while cardiomyocytes are less protected. Totipotent cells are even less protected against free radicals.

This example also illustrates the FRET phenomenon between the MTG and TMRM markers.

The ES-D3 cells at the ^10th day of embryonic development with a marking TMRM "high" were isolated.

This cell fraction is composed mainly of cardiomyocytes.

 The inventors carried out a second labeling on these cells to quantify the mitochondrial mass of these cells, which marking was carried out sequentially over time (see FIG.

 To evaluate the mitochondrial mass, the inventors used a fluorescent mitochondrial probe MTG, which is a cationic probe that diffuses freely across the mitochondrial membrane to bind in the mitochondria depending on the initial potential of the mitochondria. However, unlike TMRM, the MTG probe used once becomes mitochondrially inside the mitochondria so that even if the potential of the mitochondria changes, the labeling will not change.

 The retention of MTG in the mitochondria depends mainly on the initial potential of this mitochondria and the number of mitochondria. The binding of MTG (Mitotracker Green MTG, such as mitotracker Green FM M-7514 Invitrogen ™) is a function of the mitochondrial mass while that of the TMRM is a function of the membrane potential.

For TMRM, the initial diffusion is the same as MTG for membrane potential, but once in the mitochondria, there is no covalent attachment, so that if the potential changes, there is release of the TMRM. The distribution of TMRM and MTG within the mitochondria is identical. This spatial proximity participates in FRET energy exchange between the two markers. The MTG is therefore proportional to the mitochondrial mass while the TMRM is proportional to the membrane potential. If the cell dies, there is loss of membrane potential and destruction of mitochondrial membranes with release of MTG and TMRM. On the other hand, if the cell depolarizes, there is then a decrease in the membrane potential with a loss of the TMRM, but the MTG fixation does not change.

There is a cumulative toxicity of the different markers. Up to 100 nM, MTG uptake is little influenced by membrane potential variations. TMRM can measure the overall mitochondria potential of a cell, but without putting this value in relation to the number of mitochondria, it is impossible to know if it is a large number of mitochondria with a weak membrane potential or a more limited number of mitochondria but with a high potential.

 After sorting using a flow cytometer, cells with high TMRM labeling were taken into culture medium for 1 hour in order to allow the cells time to recover.

 This incubation also allows the TMRM to emerge from depolarizing cells, including cardiac cells, and thus protect a number of cells from the combined toxicity of dual TMRM / MTG labeling. After 1 hour, the cells were labeled with 50 nM MTG added to the culture medium for ½ hour at 37 ° C.

 The cells were washed twice with cells in TMRM buffer and analyzed by flow cytometry.

 Two sources of light energy were used: 490nM and 549nM and the fluorescences were analyzed at 516nM and 574nM (see Figure 3).

 Two cell populations are visible after TMRM and MTG labeling at one hour intervals:

 a majority population of TMRM cells that are negative with MTG fluorescence of the intermediate or low type, and

 a cell group representing only a few percent of the total population which is a group of cells with high TMTM fluorescence and also with high MTG fluorescence.

This last group of cells expresses the markers of the neural cells. Negative TMRM and MTG cells are dead cardiomyocytes.

 The negative TMRM and intermediate MTG cells are possibly depolarized cells that are dying.

 All the cells of the preparation were initially cells with high TMRM labeling and corresponding mainly to cardiomyocytes. Cardiomyocytes are therefore the majority cells of the preparation.

The fact that the cells become negative TMRM means that following MTG labeling, the cardiomycytes have depolarized or died.

 It is possible that cardiomyoctes have depolarized spontaneously. The direct effect of MTG labeling can be demonstrated, because if during secondary labeling a combination of TMRM with MTG is used again instead of MTG alone, it can be seen that the depolarization effect is even more pronounced.

est donc due à la présence de MTG dans la préparation sur des cellules préalablement marquée avec du TMRM. Depolarization and death

is therefore due to the presence of MTG in the preparation on cells previously labeled with TMRM.

For the same value of initial TMRM, it is found that the amount of MTG is greater in neural cells, which means that the membrane potential of their mitochondria is lower than that of cardiomyocytes.

 Neural cells have a membrane potential that remains stable since they maintain their TMRM markings even after MTG labeling.

 At the second marking, if instead of only TMRM marking is carried out a simultaneous MTG and TMRM labeling (50 nM for each marker), we see that there exists:

 a population of MTG negative MTG-negative cells which are predominantly dead cardiomyocytes and

 a population of cells whose MTG fluorescence decreases slightly and whose TMRM fluorescent increases considerably.

This second population corresponds to neural cells that remain polarized and are resistant to the cumulative toxicity of double TMRM / MTG labeling. The decrease of the MTG fluorescence in favor of the TMRM fluorescence is due to a transfer of energy between the donor MTG and the accepting TMRM (FRET). In the presence of TMRM the MTG's own fluorescence decreases and there is energy transmission on the TMRM, very spatially close, and whose absorption wavelength is close to the emission wavelength of the MTG. Example 3: Use of intracellular markers to facilitate the isolation and enrichment of a population of interest.

As before, ES-D3 EBS day 10 cells were labeled with 50 nM TMRM for 1/2 hour at 37 ° C in the incubator. Intermediate TMRM intermediate fluorescence level cells were isolated by selection in flow cytometry with cell sorting (see Figure 4). There are currently cytometric techniques to sort cells and meet clinical requirements compatible with their use in humans, there are also purification techniques from magnetic particles coupled to markers.

 Intermediate cells are living cells that do not contain many cardiomyocytes and are in the intermediate TMRF fluorescence fraction. This fraction contains less than 10% of cardiomyocytes and contains other cell types including neural cells. Intermediate TMRM cells will be cultured in culture medium. If sequential labeling with MTG 50 nM is performed after 1 hour, two populations are again appearing, the first is intermediate TMRM and high MTG (zone Q2) and the second is TMRM low and MTG intermediate or negative (zone Q4 or Q3 area).

The intermediate TMRM population corresponds to neural cells. The negative TMRM population corresponds to a population of depolarized or cell-killing cells because the sequential combination of TMRM and MTG after a short time interval is toxic for the majority of cells in the preparation. If now, during the second labeling, the intermediate TMRM cells are labeled with MTG labeling combined with the TMRM, it is found that the harmful effect on a large part of the cells is further accentuated since practically all the cells become TMRM and MTG negative. On the other hand, there is always the group of cells whose MTG fluorescence decreases a little and whose TMRM fluorescence is increased. These are mostly neural cells confirmed after selective sorting. What is advantageous in this isolation method is that although we start from a population with a TMRM fluorescence intermediate initially, we find that the combination of a MTG marking with a transfer of energy allows to increase the TMRM fluorescence of these cells that become high TMRM as are initially cardiomyocytes. The level of TMRM fluorescence observed does not correspond to a high level of membrane potential of these cells but to a fluorescence artificially increased by a mechanism of energy transfer between the different markers used. This increase in fluorescence facilitates the isolation of the neural cells whereas at the same time the TMRM fluorescence of the non-neural cells is decreased (see FIG. 4).

Example 4: Use of intracellular labeling for the isolation of neural cells during embryonic development. Currently, there is no simple method for isolating neural cells within an embryonic cell population.

Totipotent ES-D3 stem cells prior to differentiation (EBS day 0), at ^day 5 or 8 ^th day of embryonic differentiation have been marked or not with intracellular markers TMRM, MTG, or TMRM and MTG concomitantly.

 In order to obtain cells that could be analyzed by flow cytometry, the cells were detached with a trypsin EDTA solution for 5 min. In the case of EBS-day 0 cells, an incubation of 30 minutes is necessary.

For EBS cells at day 5, where the cell clusters are very compact and therefore more difficult to dissociate, the incubation is 10 min.

After dissociation, the cells are passed through a 70 micron filter which makes it possible to retain the undissociated clusters and does not interfere with the flow cytometry analysis. The labeling is carried out in culture medium for 30 min at 37 ° C. with TMRM and MTG at a concentration of 50 nM respectively, then the cells are washed in 2 × TMRM buffer and analyzed by flow cytometry. Irradiation of cells with the respective wavelengths of MTG and TMRM was performed, and fluorescence analysis in the emission spectrum of MTG and TMRM was performed.

 An analysis window was carried out on the cells of the preparation according to their size and their granulosity. >

 Unmarked cells or labeled only with TMRM and MTG makes it possible to define the positivity for each marker, and to carry out the compensations necessary for a correct analysis. It is found that during embryonic development, following the TMRM or MTG markers, an increasing proportion of cells express the markers. For TMRM, this means that a group of cells with high membrane potential is developing, even though this global potential is to be related to the mitochondrial mass to know the exact mitochondrial potential.

Cells with high TMRM markings represent 10% of the population at day 0, 13% at day 5 and 31% at day 8 (see Figure 5). The increase in this fluorescence may be due to the development of cells with many mitochondria with high membrane potential, a low number of mitochondria but with a high membrane potential or a large number of mitochondria with a low membrane potential.

 As cardiomyocytes develop, their metabolism changes from anaerobiosis to aerobiosis and their membrane potential increases.

 The MTG marker makes it possible to quantify cell mass. Cardiomyocytes and neural cells have a high level of mitochondria.

What is important in these experiments is the kinetics of these different events during cell differentiation. It is also found that during differentiation the number of cells with a large mitochondrial mass increases (see Figure 6).

If TMRM and MTG are co-labeled at day 0, day 5, and day 8, it is very clear that from day 0, there is no real visible population (see Figure 7). On the other hand, at day 5 (see FIG. 8), and at day 8 (see FIG. 9), and therefore very early in the embryonic development, a population presenting a double TMRM and MTG labeling appears (fraction Q2). The combined expression of these two markers separates differentiating cells from more immature totipotent cells.

 It is possible to sort these cells, the latter being viable in more than 80% of cases after sorting.

 For the other TMRM positive MTG negative fractions (Ql fraction), MTG positive MTG negative (Q3 fraction) or negative TMRM negative MTG (Q4 fraction) it is not possible to culture the cells in the cardiomyocyte differentiation culture medium Or other.

 The double positive population (fraction Q2) is a fraction composed of living cells enriched in marker of neural cells (see Figure 9). This is confirmed by measuring by quantitative PCR the expression of the marker MASH-1, the latter being expressed more than 20 times compared to non-neural cells This is a very important enrichment which means that in the Q2 fraction, the majority of the cells are neural cells.

Other markers such as MHC and GATA4, specific for cardiac stem cells, are not overrepresented.

 On the other hand, if the other fractions are considered, the neuronal markers are diminished. In fractions Q1, Q3 and Q4 the cardiac markers are enriched with respect to fraction Q2.

The selection technique is interesting because it allows to select only living cells. Indeed, the fact that the cells have a positive TMRM marking means that they have a mitochondrial membrane potential, therefore a functional mitochondria, which implies that they are alive.

The selection method thus makes it possible to eliminate the undesired cells while allowing positive selection of the cells of interest on the basis of the mitochondrial metabolic activity and morphological parameters, such as the mitochondrial mass. There is therefore no need to associate this technique with markings of cell viability to ensure that the markings observed are specific and do not result from non-specific fixation.

However, this method does not allow the selection of totipotent cells. The neural cells sorted according to the process of the invention adhere to a support, such as a conventional culture dish, and without the need to add to the medium proteins allowing cell adhesion (lamin, fibronectin, collagen ...) (see figure 10). Normally, the differentiation of neural cells is difficult to obtain without resorting to very specific cultivation methods.

The cells can be cultured in media suitable for culturing neural cells or for culturing cardiomyocytes.

 Remarkably, nearly 50% of the isolated cells are dividing cells. Some cells are birefringent, characteristic of poorly differentiated embryonic cells.

 After a few days in culture, cytoplasmic expansions characteristic of neuronal-like cells appear while other cells tend to have glial cell characteristics. Some cells tend to reappregate spontaneously.

 After culturing cells on a glass slide and labeling with "Nestin", the majority of the cells are positive for Nestin, which confirms the phenotype of neural stem cells.

It is possible to associate the cells with three-dimensional supports such as collagen supports, modified or not, with support and / or proteoglycan adhesion molecule molecules which may or may not be cross-linked with non-toxic agents such as, for example, chemical crosslinker such as genipin (as described in WO 2009/007531). The mitochondrial markers employed TMRM and MTG are not species specific. Identical results can be obtained with human cells type "ES" and "iPS". Neural cells can also be identified from a differentiated tissue preparation. Example 5: Method of isolating cells using mitochondrial markers to obtain a cell population with reduced malignancy. Generally, cell sorting methods based on the detection of surface markers make it possible to sort not only normal cells, but also tumor cells.

 Genetic manipulation techniques also expose the cells thus transformed to a risk of increased tumorigenicity.

 The tumor risk is a very important risk with embryonic cells and it is interesting to use methods that make it possible not to select tumor cells.

Tumor cells are often found in totipotent cell populations. We must therefore focus on techniques that do not select these cells. The method according to the invention could also be used to control neural stem cell preparations prior to implantation obtained with the other methods. Doubly positive single cells for TMRM and MTG markers were cultured for one week, then aggregated (1.9 x 10 ⁵ cells) and implanted into collagen matrices in the spinal muscles of NOD-SCID immunosuppressed mice. In none of the grafts were tumors observed even after 2 months after injection. In contrast, 85% of mice grafted with undifferentiated totipotent cells developed tumors.

Example 6: Method of concomitantly isolating cardioinyocytes and neural cells, using sequential labeling. The purpose of this method is to protect cardiomyocytes from the toxicity induced by one of the markers, including the toxicity of TMRM when it is associated with MTG.

To do this, the cells are first labeled with TMRM and then sorted.

TMRM labeling, which only lasts about 24 hours, is then eliminated, and TMRM positive cells are labeled with MTG.

In these experiments, the initial positive TMRM population is very largely enriched in cardiomyocytes (high TMRM) as described in Hattori et al. (see Figure 11). After 24 hours, 34% of the cells are still TMRM positive among high-marked TMRM cells.

 After 48h, the percentage of cells labeled with TMR decreases further.

Therefore, it is possible to carry out a second marking with MTG (50 nM).

Interestingly, 92% of cells with high TMRM labeling are MTG positive or even very positive for this labeling, with fluorescence levels above 10 ^J. While MTG labeling is performed 1 hour after TMRM labeling, only 18% of cells with high TMRM labeling are positive for MTG.

 If the TMRM and MTG markings are performed concomitantly, only 9.8% of cells with high TMRM labeling are MTG positive.

On the other hand, in the case of sequential labeling, several MTG fluorescence levels which make it possible to isolate the population of cardiomyocytes.

The fact that the isolated cells bind MTG also means that the cells are alive.

 There are other approaches that can be used to reduce cardiomyocyte toxicity, for example the use of mitochondrial depolarizing blockers, superoxide-lowering agents, and / or peroxidized hydrogen compounds. of antiapoptotic agents.

Furthermore, it is possible to combine the method of the invention with methods for increasing the protection of cardiomyocytes by subjecting the cells, prior to labeling, to stress (hypoxia, hypoglycemia, temperature, etc.) aimed at increasing the antiapoptotic agents.

These methods have the advantage of allowing the isolation of cardiomyocytes by decreasing the concentrations of TMRM and / or MTG. It may also be advantageous to protect the cardiomyocytes for example by using only the wavelength corresponding to the second MTG 490 nM marker and analyzed at 516 nM and not irradiating the cells at the wavelength of the 549 nM TMRM during second cell sorting. Secondary cell sorting is then performed only on MTG fluorescence at 516 nM. The order of the markings can be reversed even if the MTG fluorescence is longer than the TMRM fluorescence. The speed of liberation of the different markers based on cell type and differentiation status could be used to identify different populations of interest for a given stage of development. The different methods can be combined.

Example 7 It is possible to associate the isolation process of the invention with conventional methods for detecting antigenic and / or genetic markers of the cells of interest.

 These combinations (markings according to the invention and other markings) may be simultaneous, separated or spread over time, and this repeatedly one or more times as necessary.

 It is possible, for example, to preselect neural stem cells using a determined differentiation marker, and then to implement the method of the invention in order to eliminate the cells that may be tumor recovered during the first labeling. .

 Thanks to these combinations, it is possible to sort the neural or little tumor stem cells, and to select from these cells one or more types of neural cells of different phenotypes (astrocytes, glial cells or particular neurons). for example).

Example 8 Purification of Neural Cells from Stem Cells Isolated from Tissues

The technique of preparing stem cells from tissue is known to those skilled in the art. Preferably, tissues or organ compartments which have been reported to be rich in cells of interest are selected. However, some areas of the central fabric are not necessarily easy to access. Methods of preparing neural cells from more accessible tissues such as dermal tissue have been described in humans (Ernst et al., Exp Dermatol (6): 549-55 (2010)). It is also possible to isolate these cells from pathological tissues such as scar tissue (Yang et al., Tissue Engineering Part C: Methods 4: 619-629 (2010)). It is also possible to favor tissues in which certain cells derive from neural ridges such as nasal faeces or dental pulp, oral mucosa, hair follicles, blood such as umbilical cord blood. It is still possible to isolate Neural stem cells from adipose tissue (Ahmadi et al., Tissue and Cell, Vol 44, 2: 87-94 (2012)) (Vindigni et al Neurol Res, August 5, 2009).

A piece of neural tissue is obtained by biopsy with a scapel and is digested in a mixed solution of Ham's / DMEM F12 (1: 1) containing papain (2.5 IU / ml), DNasel (250 IU / ml) of dispase II (1 IU / ml) incubated at 37 ° C. The stopping solution is a mixed solution of HAM's / DMEM with 10% fetal bovine serum (FBS). After dissociation, cells and debris are passed through a 70 micron filter. Potential enrichment is possible by producing a gradient of Ficoll or Percoll. The myelin debris on the top is aspirated and 2/3 of the fraction between the red blood cells and the surface is collected. The cells are washed in PBS and the rest of the red blood cells can be removed by incubating the preparation with ammonium chloride at room temperature for 5 min.

 The markings can be carried out at the very beginning of preparation, after isolation of the cells or after culture. For reasons of reproducibility, it is preferable to carry out the labeling on the isolated cells.

 The cells are incubated in the buffer with 50 nM TMRM and 50 nM MTG markers for half an hour at 37 ° C., and the cells are then washed before passage into a flow cytometer. The neural population can be isolated.

The other possibility is to put the cells in culture and proceed to cell sorting later on a preparation enriched in neural cells. The preparation can also be enriched in cells by incubating with the markers and irradiating in the MTG spectrum.

 The cells are then cultured in Ham's / DMEM F12 with glutamine, N2 supplement N2, FGF-2 (20 ng / ml), EGF (20 ng / ml) and heparin (5 μg / ml). ml) on plastic culture plates. To obtain the differentiated cells, the cells are cultured in the differentiation medium (N2 supplemented with 0.5% FBS and forskolin 1 μg) on plates covered with a polyornithine / laminin mixture. Example 9: Obtaining pluripotent cells from somatic cells reprogrammed.

Somatic cells can be reprogrammed using a genetic construct to become pluripotent cells. These pluripotent cells are called iPS. These cells can be autologous. They have the ability to form different cell types derived from neural stem cells. The genetic construct for reprogramming can be removed but these cells retain a persistent tumor risk.

 Methods of isolating neural cells from pluripotent cells have been proposed with surface markings using a CD184 + (CXCR4) / CD271- / CD44- / CD24 + population and culturing these cells in the presence of growth factors such as FGF. , EGF (Yuan et al., PLOS one, Vol.6, Issue 3, el7540 (2011)). However, these techniques do not allow to directly isolate neural cells and requires a subculture that can alter the properties of cells and make them tumorous. In addition, the CD184 marker is not specific for neural cells. According to the invention, the neural cells can be isolated within a population of iPS cells, making it possible to eliminate the tumor cells. Example 10: Coupling of a mitochondrial marker to a fluorescent marker such as a quantum dot and / or a magnetic particle.

The mitochondrial markers used in the invention may be linked to both fluorescent molecules, for example to "quantum dots", and / or to ferromagnetic microparticles, preferably nanoparticles, such as for example SPIO (Superpara Magnetic Iron). oxide).

The TMRM marker can be coupled with a Dot quantum that absorbs energy and then emits energy into the absorption spectrum of TMRM and transmits this energy through FRET. This delivery of energy will be accompanied by a "superactivation" of the TMRM with a very increased production of ROS compared to the production obtained in the absence of the energy sensor.

The techniques for producing SPIOs, for example co-precipitation in the aqueous phase, are well known to those skilled in the art (see Mossart R., IEEE trans Magne, 1981 17, 1247-1248, L. Maurizi, Langmuir 2009, 25 (16), 8857-8859, Wang D., University of New Orleans Thesis 2005: Quantum Dots CDE and Luminescent / Magnetic Particles for Biological Applications). In many chemical interactions, a Fe-OH group present on the ferromagnetic particle will be involved. SPIO particles are neutral under physiological conditions and tend to clump together. To limit this agglutination, various hydrophilic polymers can be attached to the surface of the SPIOs (Makhluf S., Nanotechnol 2006, 2829-2840, Kohler N., J. Am Chem Soc 2004, 126, 7206-7211).

Polyethylene glycol (PEG) is one of the polymers commonly used because of its hydrophobicity and biocompatibility. The covalent attachment of PEG to SPIO stabilizes SPIO for a prolonged period under physiological conditions (Lee H., Chem Soc., 2006 126, 7238-7389). Depending on the number of polymers (PEG) n, it is possible to modify the size of the "spacer" between the first marker and the magnetic particle. There are different types of linear PEGs or not, whose ends are chemically modified to interact with interest groups directly or using a crosslinker. It is easy to modify the PEGs to react with sulfuryl, amine, carboxyl or maleimide groups, for example. It is also possible to bond the PEG to the ferromagnetic particles using one end of the PEG having a particular moiety which will spontaneously bond to the metal particle. It is usually PEG with a Thiol group at the end (PEG-Thiol) or Lipoamide (PEG-lipoamide). The thiol group will bind with a metal-S-R bond directly for the PEG-thiol or "bidental metals-S / S" for the lipoamide component. PEG thiol reacts mainly with gold. The lipoamide component interacts with the majority of metals such as silver, gold and other metals.

The other end of the PEG can be chemically protected by a methyl group. However, it is possible to functionalize this methyl group using for example a long chain of polyester type CT (PEG) n with a periodic exposure of carboxylic groups at the end instead of duradical methyl. These groups will be able to be involved in chemical reactions with amino groups using heterobifunctional crosslinkers or not, such as EDC +/- NHS. PEG can also be produced with a thiol or lipoamide group, at one end, and at the other end, a group for reacting with a sulfuryl, amine, carboxyl or other by direct interaction or using a crosslinker. Crosslinkers are known to those skilled in the art, as are the coupling agents conventionally used.

Quantum dots can be prepared by various techniques well known to those skilled in the art (Wang D., Quantum Quantum Dots and Luminescent / Magnetic Particles for Biological Applications, University of New Orleans, 2005 thesis, Boatman et al. Cumulative Quantum Dot Nonocrystals, J Chem Educ, 82: 1697-1699 (2005)).

Dot quantum can have magnetic properties for example. It is possible to use TMRM coupled to modified Quantum Dot CdSE / ZnS with magnetic molecules such as -Fe ₂ 0 ₃ .

The coupling of ferromagnetic particles to fluorescent markers has already been demonstrated. (Massard R., IEEE Trans Magn., 17: 1247-1248, 1981). The supramagnetic properties are interesting because the markers have a better biodistribution and no tendency to clump together.

To bind a marker to a ferromagnetic particle, there are many ways. The first is to functionalize the particle by introducing different groups, especially carboxylic, thiols, maleimides or others.

It is possible to introduce for example one or more "maleimide" groups on ferromagnetic particles to allow interaction with the molecule of interest. This technique is used to functionalize the ferromagnetic particles (Park J., Nat Mater, 3: 891-895, 2004, Sun S., JAm Chem Soc, 126: 273-279, 2004, Xie J., JAm Chem Soc, 130). : 7542-7543, 2008; Wang D., thesis University of New Orleans, CDSE Quantum Dots and Luminescent / Magnetic Particles for Biological Applications, 2005). Recently, J. Xie has described a technique for coupling nanoparticles of very small size (a few nm) to particles of Fe ₃ O ₄ . The surface of the Fe ₃ O ₄ particles was functionalized using "4-methylatechol (4-MC)" as surfactant. 4-MC bound to Fe ₃ O ₄ then bound a -NH 2 group carried by a molecule of interest into using the Mannich reaction in the presence of formaldehyde (Xie, J., Am. Chem. Soc., 130: 7542-7543, 2008).

Another conventional technique is to use agents that will react with the Fe-OH group to introduce thiol or carboxyl groups which are more reactive. A commonly used chemical component is "DMSA" (2,3-dimercaptosuccinic acid) (see Falconer N., J Coloid Interfac Sci, 194: 427-433, 1997, Roger J, Eur Phys J Apply Phys, 5: 321). -325, 1999; Wang D., thesis University of New Orleans, CDSE Quantum Dots and Luminescent / Magnetic Particles for Biological Applications, 2005).

Coupling TMRM to SPIO

The approach, used here for TMRM, can be transposed to another mitochondrial marker.

TMRM can be modified with a donor energy acceptor and / or ferromagnetic particles. The link chain between the TMRM and the energy sensor can be modified to transmit this energy, for example by electron transfer. TMRM can be easily chemically modified for the introduction of reactive chemical groups by those skilled in the art, for example via -COOCH ₃ groups for TMRM.

It is possible to functionalize the SPIO molecule with different groups to specifically bind the TMRM or the fluorescent marker. The quantum thiolization of Dot CdSE / Zn coupled to ferromagnetic particles has been described by Ph.D. by Wang D. (University of New Orleans thesis, CDSE Quantum Dots and Luminescent / Magnetic Particles for Biological Applications, 2005).

TMRM belongs to the group of thiol-reactive markers (thiol reactive dye) and will therefore spontaneously bind to -SH groups. Thus, if the SPIOs are modified with -SH groups as after functionalization with DMSA, the TMRM will bind to -SH groups. It is possible to modify the TMRM molecules to introduce chemical groups. TMRM molecules have groups - COOCH3 and -COOCH2CH3 respectively that will be able to be modified to introduce for example -NH2 sites. These sites can then link with sites - NH2 or -SH using appropriate crosslinkers. Different types of crosslinker can be used, such as, for example, heterobifunctional derivatives such as SIA, SPDP or NHS-PEG maleimide. Hetero-bifunctional linkers will recognize a -NH2 or -SH motif. Coupling techniques are well known to those skilled in the art. (cfMK Yu, Targeting Strategies for Multifunctional Nanoparticles in Cancer Imaging and Therapy, Theranostics, 2: 1,3-30, 2012). It is also possible to use "Click Chemistry" or "Hybridization" techniques (see MK Yu, Targeting Strategies for Multifunctional Nanoparticles in Cancer Imaging and Therapy, Theranostics, 2: 1, 3-30, 2012).

₍ The link between the TMRM and the SPIO may involve a spacer such as PEG The TMRM can be linked to the SPIO by means of this "spacer" The molecule attached to it can be a quantum Dot with ferromagnetic particle and a sensor / donor of energy on its surface such as the CdSE / ZnS It is possible through the large spacer to maintain the ferromagnetic particles outside the cell while the TMRM will attach itself to the This type of approach is interesting because it will limit the incorporation of foreign material into the cell When the cell depolarizes, the TMRM will be released and the ferromagnetic particle will be released from the cell.

 Alternatively, it is also possible to use metal nanoparticles, covered with graphite, on which it is possible to covalently attach the mitochondrial markers (Turbobeads).

Coupling TMRM to QuantumDot CdSE / ZnS

The surface of Dot quantum dot CdSE / ZnS can be modified with different carboxylic ligands such as, for example, mercaptoacetic acid or mercaptosuccinic acid, which makes them soluble in water and makes them biologically compatible. The quantum dots can then be chemically modified to introduce reactive groups such as thiol groups for example (-SH). So he is it is possible to fix on these thiol groups chemical molecules of interest, for example the TMRM marker.

It is also possible to make this magnetic quantum dot by interacting with these groups with ferromagnetic molecules such as, for example, y-Fe 0 ₃ type molecules. The TMRM can in this case be coupled to the Quantum Dot type CdSE / ZnS. To create these active groups on the particles, the molecules (γ-Fe ₂ O ₃ ) are taken up in DMSA (meso 2,3-dimercaptosuccinic acid) and in an organic / inorganic solvent (chloroform: methanol: water 10/5/1). ) to obtain carboxylic and thiol groups on the surface of the magnetic particles. It is then possible to fix the TMRM to the Quantum Dot by the thiol group, and then to fix the compound obtained with the magnetic particle, via the residual thiol or carboxylic groups.

Isolation of neural cells using TMRM coupled to quantum dot CdSE / ZnS magnetic

The absorption spectrum of quantum dot CdSe is very wide. CdSe quantum dots have significant absorption at less than 600 nm, this absorption gradually decreases from 350 nm to 600, with a slight increase around 550 nm.

The emission spectrum of quantum dot CdSe is much narrower with emission between 500 and 600 nm (University of New Orleans thesis, CDSE Quantum Dots and Luminescent / Magnetic Particles for Biological Applications, 2005).

 For quantum Dot CdSe (ZnS) -thiol, the emission spectrum is between 525nm and 625 nm, slightly lower than that of magnetic CdSe / ZnS nanoparticles.

The absorption spectrum of TMRM is 500-600 nm and its emission spectrum is 550-700 nm. The TMRM is therefore able to absorb the energy emitted by the quantum dot CdSe.

To isolate the neural cells, the cells are first incubated with the unique marker: TMRM coupled to quantum dot CdSE / ZnS magnetic. It is then possible to sort the positively labeled cells via a magnetized column (Pankhurst et al., Applications of Magnetic Nanoparticles in Biomedicine, J Phys D: Appl Phys, 36: R167-R181, 2003, Zborowski et al., Magnetic cell sorting , Mol Mol Methods, 295: 291-300, 2005; Ito et al., Medical application of functionalized magnetic nanoparticles, J Biosci Bioeng, 100 (1): 1-11, 2005; Sofarik et al., Use of magnetic techniques for the isolation of cells, Journal of Chromatography, 722: 33-53, 1999). The TMRM labeled cells are then irradiated in the absorption spectrum of quantum dot and / or TMRM. The irradiation of the quantum dot TMRM results in a more intense stimulation of the TMRM, which leads to an increase in the production of free radicals, inducing among the TMRM positive cells the death of the cardyomyocytes and a preferential survival of the neural cells.

Cellular sorting by magnetism is therefore not essential and the possibility of simple irradiation may be sufficient to enrich the preparation in neural cells because of a preferential survival of these cells with respect to free radicals compared to other cell types.

Claims

claims A method of isolating, purifying and / or enriching neural cells, comprising a step of contacting a cell population, preferably said cell population preferably not resulting from embryo destruction. human, with:  at least one marker sensitive to the membrane potential of the mitochondria and  at least one marker making it possible to quantify the number of mitochondria and / or, where appropriate, to increase the production of free radicals, said at least one marker sensitive to the membrane potential of the mitochondria and said at least one marker making it possible to quantify the number of mitochondria and / or increase the production of free radicals that can be coupled chemically with each other and correspond to one and the same marker,  in such a way that  if said at least one membrane-sensitive marker of the mitochondria and said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals are used simultaneously, separately or spread in the time, or correspond to a single marker, said method allows the isolation, purification and / or enrichment of neural stem cells. 2. The method of claim 1 wherein:  said at least one marker sensitive to the membrane potential of the mitochondria is chosen from: Rhodamine 123 (RI 23), tetramethylrhodamine methylester (TMRM), tetramethylrhodamine ethyl ester (TMRE), tetramethylrhodamine-5-maleimide, 5,5 ', 6,6'-tetrachloro-1,1', 3, 3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), JC-9, 3,3'-dihexyloxacarbocyanine iodide (DiOC6), 2- (4- (dimethylamino) styryl) -N- ethylpyridinium iodide) (DASPEI), the Green FM MTG mitotracker, the Mitotracker red CMXRos, the Orange Mitotracker CMTMRos and the Dihydrorhodamine 123, and  said at least one marker making it possible to quantify the number of mitochondria and / or, where appropriate, to increase the production of free radicals is chosen from: the Green FM mitotracker (MTG), the Mitotracker Red FM (MTR), the mitotracker deep red FM (MTDR) and nonyl fluorescent orange acridine. The method according to claim 1 or 2, wherein either said at least one membrane-sensitive marker of the mitochondria, or said at least one marker for quantifying the number of mitochondria and / or increasing the production of radicals. both are cationic type markers. 4. Method according to one of claims 1 to 3, wherein either said at least one membrane-sensitive marker of the mitochondria, or said at least one marker for quantifying the number of mitochondria and / or increase the Free radical production, or both, are fluorescent markers. The method according to any one of claims 1 to 4, wherein said at least one mitochondria membrane potential sensitive label is selected from tetramethyl rhodamine methyl ester (TMRM) and tetramethyl rhodamine ethyl ester. (TMRE). The method according to any one of claims 1 to 5, wherein said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals is selected from mitochondrial protein markers, and markers of mitochondrial DNA. The method according to any one of claims 1 to 6, wherein said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals is MTG. The method according to any one of claims 1 to 7, wherein said at least one mitochondria membrane potential sensitive marker and said at least one marker for quantifying the number of mitochondria and / or increasing the production. free radicals allow energy transfer from one to the other markers that can lead to free radical production. The method of isolating neural stem cells according to any one of claims 1 to 8, said method comprising:  at. a step of incubation of a cell population with  i. at least one marker sensitive to the membrane potential of the mitochondria,  ii. either at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals, b. a step of incubating the labeled cell population in step a), with  i. at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, if at least one membrane-sensitive marker of the mitochondria has been used in step a),  ii. or at least one membrane-sensitive marker of the mitochondria, if at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals has been used in step a), and  vs. a step of isolating the neural stem cells. The method of isolating neural stem cells according to any one of claims 1 to 8, said method comprising: at. a step of incubating a cell population with: i. simultaneously at least one membrane-sensitive marker of the mitochondria, and  at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals, or  ii. if said at least one membrane-sensitive marker of the mitochondria and said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals correspond to one and the same marker, with this alone and same marker  and  b. a step of isolating the neural stem cells. 11. A method of isolating neural stem cells according to any one of claims 1 to 10, said method comprising:  at. a first step of incubating a cell population with at least one membrane-sensitive marker of the mitochondria, in particular TMRM or TMRE, and  b. a second step of screening the cell population with positive labeling for said mitochondria membrane potential sensitive marker, preferably at least about 100-fold higher than the labeling level of the cells considered unmarked by said marker, and  vs. a third incubation step of the cell population selected in step b) with at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, in particular MTG, said third step making it possible to obtain a population marked by both said membrane-sensitive marker of the mitochondria and by said marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, and . a step of isolating neural stem cells with positive labeling for said membrane potential sensitive label mitochondria, preferably at least about 100-fold higher than the labeling level of the cells considered unmarked by said marker, and positive labeling for said marker for quantifying the number of mitochondria and / or increasing the production of radicals free, preferably at least about 100 times higher than the labeling level of the cells considered unmarked by said marker. The method of claim 11, wherein the cell population selected in step b) is incubated with a mitochondrial membrane potential sensitive marker simultaneously or separately from step c). 13. The method for isolating neural stem cells according to any one of claims 1 to 9, said method comprising:  at. a first step of incubating a cell population with at least one membrane-sensitive marker of the mitochondria, in particular TMRM or TMRE, and  b. a second step of screening the cell population with positive labeling for said mitochondria membrane potential sensitive marker, preferably at least about 100-fold higher than the labeling level of the cells considered unmarked by said marker, and  vs. a third incubation step of the cell population selected in step b) with at least one marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals, in particular MTG,  said third step being delayed by at least several hours in order to attenuate the labeling of the cell population by said mitochondria membrane potential sensitive marker used in step a), and d. an isolation step of the neural stem cells having a positive labeling for said marker making it possible to quantify the number of mitochondria and / or to increase the production of free radicals at least 10,000 times greater than the labeling level of the cells considered unmarked by said marker. 14. An isolation method according to any one of claims 11 to 13, wherein either the step of selecting a cell population, the isolation step of the cells of interest, or the two steps, are performed using a flow cytometry apparatus, preferably a cell sorter. The method according to any one of claims 1 to 14, wherein either said at least one membrane-sensitive marker of the mitochondria, or said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals, or both, are coupled to at least one magnetic particle. 16. An isolation method according to claim 15, wherein either the step of selecting a cell population, the step of isolating the cells of interest, or the two steps, are carried out using an apparatus for magnetic sort, preferably a magnetized column. 17. Kit comprising:  at least one marker sensitive to the membrane potential of the mitochondria and  at least one marker making it possible to quantify the number of mitochondria and / or, where appropriate, to increase the production of free radicals, for its use for the isolation, purification and / or enrichment of neural stem cells. 18. Kit according to claim 17 wherein either said at least one mitochondria membrane potential sensitive marker, or said at least one marker for quantifying the number of mitochondria and / or increasing the production of free radicals, or both are fluorescent markers. Kit according to claim 17 or 18, in which:  said at least one marker sensitive to the membrane potential of the mitochondria is chosen from:  Rhodamine 123 (RI 23), tetramethylrhodamine methylester (TMRM), tetramethylrhodamine ethyl ester (TMRE), tetramethylrhodamine-5-maleimide, 5,5 ', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), JC-9, 3,3'-dihexyloxacarbocyanine iodide (DiOC6), 2- (4- (dimethylamino) styryl) -N-ethylpyridinium iodide) (DASPEI) , Mitotracker Green FM MTG, Mitotracker Red CMXRos, Mitotracker Orange CMTMRos and Dihydrorhodamine 123, and  said at least one marker making it possible to quantify the number of mitochondria and / or, where appropriate, to increase the production of free radicals, is chosen from: Green FM Mitotracker (MTG), Mitotracker Red FM (MTR), Mitotracker deep red FM (MTDR) and nonyl fluorescent orange acridine.