Method of neuronal cell differentiation from embryo stem (es)

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, particularly to a method of induced differentiation of embryo stem (ES) cells in neuronal precursor cells. The presented method involves ES cell culture by sowing the ES cells of density approximately 0.5 × 105 - 2 × 105 cells in cm2 and dissociating the ES cells 2 days after sowing. Then, cell aggregates (CA) are formed that involves sampling the cells of high proliferative activity of doubling time within 0 to 24 hours and sowing these cells of density approximately 0.5 × 105 - 5 × 105 cells in ml to form the CAs. Further, the cell aggregates are processed with retinoic acid (RA). Then, the CAs are dissociated to prepare a neuronal precursor cell culture.

EFFECT: presented invention allows preparing substantially homogeneous neuron population wherein practically all neurons belong to the same certain neuronal cell differentiation line, to the same phenotype, cell type and to the same differentiation stage.

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The technical field to which the invention relates.

The invention relates to the generation of in vitro neuronal precursor cells, or progenitor cells, or neurons from poly potent cells, especially embryonic stem (ES) cells.

Background of invention

Long established that poly potent cells, for example, embryonal carcinoma cells (CECS) and embryonic stem (ES) cells can be differentiated into neurons in vitro. In principle, work with ES cells allows identification of cells at certain stages of differentiation and describe neuronal precursor cells. ES cells allow us to study the molecular and genetic path of development in vitro, and, in addition, are a powerful source of cells for transplantation into the brain to treat neurological diseases.

However, implementation of these and other applications is difficult because of the heterogeneity of the disorder and often non-repeatable neuronal development in cell culture. Cellular heterogeneity is a serious problem of using ES cells for producing nerve cells (see references 3 and 4 in the list of references). Usually the culture of neuronal cells generated from ES cells contain many different neuronal subtypes, as well as cells of other origin, including glial cells. Neh is Atka sufficiently large number of cells with defined and identical phenotypes is a serious problem in neurobiology. Previously there was no way of receiving neurons from ES cells, which would be formed a stable number of neurons or specific populations of neurons, so it was impossible to obtain a homogeneous population of cells in sufficient quantities to describe the neurons of the brain, using biochemical approaches. In addition, the relationship lines differentiation of neurons with their immediate predecessors remained unclear.

For using neurons derived from ES cells for transplantation, it is desirable to obtain certain precursor cell that gives a certain generation of cells and not a mixture of cells containing those cells that can continue to divide and to form tumors (REFs 3, 4). Heterogeneous cells can also intervene in the trophic and/or directing signals from the host tissue, which stimulate the integration of the implanted tissue in the brain. Functionally important type of implanted cells, for example, dopaminergic neurons may be particularly be useful for the treatment of such diseases as Parkinson's disease, therefore, for such medical purposes desired more control over cells-precursors and subtypes of neuronal cells. The decrease of cell heterogeneity is necessary to reduce unwanted side effects, the risk of the development of the Oia tumors, and to improve therapeutic effects by increasing the content proportion of the cells, neuronal differentiation which goes in the desired direction.

Recently received an important achievement, which consists in the use of inductive signals and transcription factors for significant enrichment of subtypes of neurons, including dopaminergic neurons and motor neurons. Thus, transcription factors, for example, Nurr1 (reference 5), or co-culturing the stem cells with cells of other types (reference 6) significantly increase the generation of dopaminergic neurons, while the addition of extraneous factors, including the processing of sound, enhances the generation of motor neurons, which was also found to integrate into host tissue after transplantation (reference 7). Despite these achievements, there is still very little is known about the generation in vitro of specific precursors of nerve cells, which may increase the specificity of neuronal phenotypes.

There are a number of protocols describing the generation of neuronal and glial cells (REFs 14, 15, 25-31). Earlier cell line embryonic carcinoma P19 was established that the processing clusters pluripotent cells retinoic acid induces neuronal differentiation (reference 32). Subsequently, bloustein, that treatment of cells embryonic bodies (ST), which is derived ES cells, RK also induces the expression of neural genes and repressing the expression of mesoderm genes (reference 33). ST represent three-dimensional clusters of cells arising from the aggregation and proliferation of ES cells. ST can be obtained by culturing ES cells on the basis of which they can't grow, usually in the cups for culturing bacteria (see, for example, reference 41).

One of the ways the generation of neuronal cells from ES cells presented in the work of Bain and others (reference 14) and Li and others (reference 10), includes the following steps:

culturing ES cells

the formation of the ST,

processing ST retinoic acid (RK),

dissociation ST and

seeding and culturing cells dissociated ST.

Usually the initial culture of ES cells receive on a supporting layer of feeder cells (inactivated fibroblasts) to maintain the ES cells in the form colonies pluripotent undifferentiated ES cells. Suggest that fibroblasts maintain ES cells in an undifferentiated stage. The suppression factor of leukemia (FPL) can be introduced into the culture medium to inhibit differentiation. However, it was found that even in the presence of FPL some ES cells tend to differentiate, and that when f is frmirovanii ST may contain cells of different lines of differentiation (references 3, 34).

In the methods proposed by Bain and others, and Li and others (references 10, 14, 15), cultured ES cells were treated with trypsin and/or pulverized to obtain small clusters of cells and then were inoculated on the non-adhesive cell culture for the formation of the Mat. Cells were cultured for 4 days without RK, then for 4 days in medium with RK, then ST dissociatively and were sown in cups coated with laminin. Inoculated cells were cultured in media containing serum.

Bain and others have reported that using the described method was given culture, consisting of populations of flat cells firmly attached to coated with laminin substrate, and populations of neuron-like cells, mainly located over the flat cells. It is noted that approximately 38% of the cells after two days of cultivation had neuronal morphology. These cell culture was mixed composition of neurons of different types, mainly GABAergic neurons.

Some methods are based on the use of selective markers of neuronal precursor cells, helps eliminate thus, during the differentiation process of cells, non-neuronal precursor cells. Precursor cells nerve cells derived from ES cells, primarily identified by expression of markers of the tick filaments, for example, nestin (reference 9), or transcription factors, for example, sox genes (reference 10). Li et al. applied the selection generation differentiating cells for enrichment heterogeneous population of cells expressing Sox2, due to the elimination Sox2-negative cells (reference 10). Although selective methods have shown their effectiveness in enriching a population of cells of neuronal cells predecessors, they are ineffective in cases where the selected precursor cells you want to apply for the generation of specific neuronal phenotypes. From the results of Li and others, it follows that Sox-positive cells can give rise to cells of most types found in the Central nervous system (CNS), and not the offspring of a particular subpopulation. Thus, the selection of a Sox-positive cells may increase the proportion of neuronal precursor cells in a population of cells derived from ES cells, it seems unlikely that such selection can be used to improve the content in the cell population specifically one subtype of neuronal precursors or neurons.

In other known methods of RK does not apply. For example, in the methods used Okabe and others (REFs 27, 43), RK is not used, but included an intermediate stage of cultivation formed Mat on edges the main substrate in a special environment with subsequent dissociation. Abe and others (reference 30) also used an intermediate stage, after which grown ST are transferred to the substrate to which they can cling. Then ST is cultivated in an adhesive condition, and then subjected to dissociation by trypsin.

In some ways, for example, Abe and others (reference 30) and Okabe and others (reference 27)included the application of basic fibroblast growth factor. Abe, etc. then apply mitotic inhibitors, which cause the death of neurons and cells that is not related to neuronal (reference 30).

Description of the invention

The present invention describes the means by which the differentiation of ES cells into neural cells can be optimized to obtain amazing benefits, expressed in the generation of generations of certain nerve cells and in the homogeneity of the populations of nerve cells. Thus, the present invention describes improved methods of induced and/or activated development and/or differentiation of ES cells into neurons or neuronal precursor cells, or progenitor cells to generate neural cells from ES cells in vitro.

In preferred embodiments of implementing the present invention, the methods of the present invention allow to obtain a substantially homogeneous population of neural cells, in which nerve cells do the CE belong to one specific line of differentiation of neuronal cells, one phenotype, cell types and/or stages of differentiation.

In the present invention are described in detail methods have been developed for homogeneous neuronal precursor cells, which were identified as radial glial cells. During further cultivation formed from ES cells, radial glial cells gradually differentiate in pyramidal neurons. Precursors and neurons obtained by the methods of the present invention, were largely homogeneous, and the percentage of neuronal cells one line of differentiation was higher compared with the results of applying the methods described in the prior art.

Thus, in preferred embodiments of implementing the present invention, the methods of the present invention allow to obtain a substantially homogeneous population of radial glial cells and pyramidal neurons, which are some of the most important populations of neuronal cells of the cerebral cortex and that it was difficult to get from ES cells by methods known in the prior art.

As obtained according to the present invention, the precursor cells of the nervous cells/progenitor cells have a high level of homogeneity, these cells sentence on the us for further differentiation and/or maturation for the formation of neuronal cells to a specific line of differentiation. Precursors of nerve cells/progenitor cells can be differentiated to obtain pyramidal neurons, as described in the present invention method, or can be changed by internal or external factors to generate populations of other neuronal cells.

The advantage in the number and uniformity of the cells provided by the present invention that distinguishes these cells from those cells that receive ways of neurogenesis and differentiation of nerve cells of the prior art, as well as from a limited number of primary neurons, which can be prepared from the brain of a mouse or rat.

Biochemical studies were previously hampered by the limited number of nerve cells, which can be obtained by known methods of the prior art. The present invention facilitates the study of the biochemical and genetic mechanisms involved in the development of nerve cells, especially in the transformation of the precursors of nerve cells in neuronal cells. ES cells are easily amenable to genetic manipulation, and you can get them in unlimited quantities, and the present invention is ideally suitable way to obtain a large number of neurons in certain lines of differentiation for biochemical studies.

To the ome, because embryonic stem (ES) cells can be easily genetically manipulated, or isolate them from mice carrying significant mutations, the present invention facilitates comparisons of neurons in wild-type and mutant neurons and identification of the mechanisms that cause loss of cells of a particular type in neurodegenerative diseases. Although genetic manipulation of ES cells is not complicated manipulation of primary neurons is extremely difficult, especially sustainable manipulation. Using genetic manipulation of ES cells can be obtained homogeneous modified line in which all daughter cells contain the same mutation, for 1-2 months, and getting a line of mice with a stable mutation can take years. Thus, as described in the present invention methods for producing precursor cells and neuronal cells in vitro from ES cells do not require the acquisition of lines of transgenic mice and, therefore, allow us to study mutant neurons at a level that was not previously available and have not been applied in practice.

The methods of the present invention also provides a system study of cells, such as neurons (for example, the elongation of neurites, neuronal cell death, neurogenesis and synaptogenesis). Such research is needed in this is blasti, but their implementation has been limited, because the neurons it was impossible to obtain in sufficient quantities. The present invention allows to obtain the neurons in large quantities and with greater homogeneity in comparison with the previously known methods, which allows for neuronal research.

Neurons and/or neuronal precursor cells/progenitor cells obtained by the method of the present invention, is also suitable for medical applications, for example, for implantation in the brain for the treatment of neurodegenerative diseases or loss of nerve tissue. Due to the increased homogeneity of the nerve cells of the desired subtype obtained according to the present invention, therapeutic effectiveness of the treatment increases, and the risk of tumor development due to implantation is reduced.

Detailed description of the invention

The present invention relates to methods of producing or generating nerve cells, such as neurons and/or neuronal precursor cells /progenitor cells, activating or inducing differentiation of ES cells into neuronal precursor cells or progenitor cells, and to methods of activation or induction of differentiation or maturation of precursor cells or progenitor cells in near the us.

The present invention relates to an improved method that allows in vitro to induce and/or activate the development and/or differentiation of embryonic stem (ES) cells into neuronal precursor cells, or progenitor cells, or neurons, and/or the receipt or generation of nerve cells, and this method includes:

cultivation of ES cells

the formation of embryonic bodies (ST),

processing ST retinoic acid (RK) and

dissociation ST

in combination with one or more additional properties or stages, described below.

In the methods of the present invention cells dissociated ST are neuronal cells predecessors or neuronal progenitor cells. Thus, dissociation of methadone may establish a culture of neuronal cells predecessors or progenitor cells.

Optional method also includes seeding in cups cells dissociated ST, resulting in receiving culture surface growth of neuronal cells predecessors or progenitor cells.

The method of the present invention may include culturing neuronal cells predecessors or progenitor cells for receiving neurons. Thus, in the methods of some embodiments of the present invention including the ENES crops in Cup and culturing cells dissociated ST for receiving neurons.

The methods of the present invention optionally include one or more properties/stages, as described below. Any property or stage can be used alone or in combination with any other property or stage, if the context shows otherwise.

Culture of ES cells without using feeder cells

Preferably the present method comprises culturing ES cells in the absence of feeder cells (usually inactivated fibroblasts). The methods can include the initial culturing of ES cells with the supply cells with subsequent cultivation without feeder cells. Power cells can be diluted and removed by repeated passage of ES cells. Preferably, if there was at least one, more preferably, at least two reseeding without feeder cells before the formation of the embryonic phone Thus feeding the cells preferably are absent in the culture of ES cells used to generate ST. "Passage" includes the dissociation of cells and re-seeding of a number of cells. For example, the passage may include disconnection or dissociation of cells from the culture grown in the cups (usually with the use of trypsin), dissociation of aggregates of cells, re-seeding of the number of dissociated ES cells (adherently culture and cultivation of ES cells.

Suitable crops the global cell described in the present invention. The suppression factor of leukemia (FPL) may not necessarily be included in the composition of culture medium for ES cells.

The selection and seeding ES cells to form ST

It was found that the proliferation of ES cells affects their pluripotentiality, and that the density of the cells cultured in this way, affects their ability and tendency to differentiation. It was found that by selecting and seeding proliferating ES cells at the same density as in the control, the greater number of neuronal precursor cells, related to a specific line of differentiation of the cells can be obtained, and cells are less heterogeneous.

The methods of the present invention preferably involve the selection of highly proliferative and/or morphologically homogeneous ES cells for the formation of the Mat. Preferably the methods include seeding measured/assessed/specific/a certain number or density of these ES cells for the formation of the Mat. Preferably the present method involves the selection of measured, estimated, or certain pre-set number of EC cells seeding for receiving methadone treatment.

The present method preferably includes the measurement, evaluation, monitoring or definition:

status proliferation of ES cells (which can be measured or estimated by determining the Oia doubling time of the cells, increasing the number of cells or other reasonable means),

the morphology of ES cells and/or

the number or density of ES cells seeded for the formation of the Mat.

Thus, preferably seeded cells counted defined or specified density. Calculation, valuation or determination of the number of cells can be conducted by any known in the field of methods, for example, by counting cells in a certain specified area using microscopy or by using a conventional cell counters, for example, Casy®1 (firm Schärfe System GmbH). The morphology of the cells studied using microscopy.

Each of these points is discussed below.

Obtaining and selection of cells with high proliferative activity

As cells with high proliferative activity can be applied to cells obtained by a certain method of cultivation described in this invention. It was found that the proliferation of ES cells can vary during the implementation of this method of cultivation of ES cells.

Cultivation of ES cells or their passage preferably result in cells with high proliferative activity. Preferably the passage is repeated approximately every 2 days, and the culture of ES cells preferably includes at least two passage to supply the cells with sleduyschimi, at least two passages without feeder cells. ES cells should be removed from feeder cells when they are at the stage of high proliferative activity, for example, by separation in a Cup with a diameter of 10 cm ES cells with feeder cells and reseeding of ES cells in medium without feeder cells (for example, to take1/4volume of cell suspension and re-plant in a new environment, an amount equal to the original), which should again lead to 60% confluence of the culture of ES cells on the following day. The passage without the use of feeder cells may include about 0.5×105cells/cm2.

Preferably, the culturing of ES cells includes measuring, evaluating or determining the number or density of ES cells seeded to obtain a cell culture.

ES cells with high proliferative activity can be ES cells obtained by cultivation or reseeding ES cells essentially as follows (in norm without feeder cells):

seeding ES cells with a density of approximately 0.3×105up to 4×105cells/cm2for example, approximately 0.5-2×105preferably about 1×105cells/cm2and

selection/dissociation of ES cells in 2 days after seeding, and, optionally, re-seeding.

ES cells can be isolated by cleavage (dissociation) 2 days is to after sowing. Normal cultivation procedures (passage) should be at least 2-3 times to selection of cells with high proliferative activity for the formation of the Mat.

For example, about 2×106cells can be seeded into the Cup for the cultivation area of 10 cm2. The above-described method allows normal to select from 10×106up to 35×106cells with the area of 10 cm2after 2 days, for example, 10-20×106.

The stage of proliferation can be measured in units of time of doubling of ES cells. The methods of the present invention can include a time dimension doubling of ES cells and selection of cells with high proliferative activity. For example, cells with high proliferative activity can have a doubling time of 8 hours or less 16 hours or less, or 24 h or less, usually 8-24 hours

Morphological features

ES cells used for the formation of the Mat, preferably are morphologically homogeneous, all or almost all ES cells have the same or similar morphological properties.

Preferably the methods of the present invention include the selection of morphologically homogeneous ES cells for the formation of methadone treatment and seeding of these cells for the formation of the Mat. Preferably all or nearly all (for example, at least 80%, at least 90%, at least 98% or at least 99%) ES CL the weave selected for the formation of the ST, have one or more, and most preferably all, of the following morphological characteristics in culture without feeder cells): growth in the form of a flat monolayer, the cells are not in direct contact with neighboring cells (but, nevertheless, firmly Packed), large nuclei, many of the nuclei, the cells do not grow on top of each other or in the form of structures, reminiscent of the colony. Preferably the cells are tightly Packed, for example, the density of the cells is approximately 20×106cells on the Cup area of 10 cm2(2×106cells/cm2), preferably about 10-30×106cells, for example, 15-25×106cells on the Cup area of 10 cm2. The present method may include monitoring one or more preferred morphological properties of ES cells and/or selection of cells having one or more of these signs.

The morphology of the cells can also be used as an indicator of the stage of proliferation. Cells with high proliferative activity preferably have one or more, preferably all, of the above morphological features.

Preferably all cultured ES cells are the offspring of one ES cells, for example, at an early stage of the method may be the selection of one colony of ES cells and cultivation is the use of ES cells from the colony. Due to this, in the culture of uniformity and homogeneity of ES cells, including morphology, can be raised.

The density of seeding in cups

For the formation of the Mat corresponding having a high proliferative activity of the cells and/or morphologically homogeneous cells normally should be sown using approximately 0.5×106and 5×106cells in 15 ml of culture medium for the formation of the Mat, preferably of 2.5 to 2.5×106cells, for example, 3×106cells in 15 ml of medium. Normal should be sown about 0.3 to 3.5×105cells·ml-1preferably 1,6-2,5×105cells·ml-1most preferably 2×105cells·ml-1. Volume environment 15 ml is preferred, although 10 ml or 10-15 ml of normal can be used for planting in the Cup with a diameter of 10 cm

The density of cells seeded for the formation of methadone treatment should be adjusted depending on the stage of proliferation applied ES cells. Therefore, if the culture of ES cells is more dense, will be sown more cells, if the culture is less dense, will be planted fewer cells. In the present work it was found that the best results are achieved when using the most rapidly proliferating ES cells.

As an example, ES cells with homogeneous morphology, with a doubling time of about 12-16 h, mo the ut to be selected and planted with a density of about 0.5×10 5cells/cm2.

Dissociation of cells

In the methods of the present invention mainly dissociation of cells preferably lies in the separation of cells (ES cells or ST) to form a suspension of individual cells, mainly leaving aggregates of more than 2-3 cells. Preferably, the suspension consists of a whole separate dissociated cells (i.e. suspension does not contain aggregates of cells). Preferably more than 90%, 95%, 98% or 99% of cells in suspension dissociative into individual cells. Preferably less than 5% of the cells in suspension forms aggregates of 4 or more cells.

Trypsin (for example, 0.05%) and/or grinding can be used for dissociation of cells, using the methods described in the present invention.

ES cells should be well dissociate before sowing for education program. Therefore, preferably the methods of the present invention include the dissociation of ES cells to form a suspension of individual cells, containing no units more than 2-3 cells. Preferably the suspension is composed entirely of single dissociated cells (i.e. suspension does not contain aggregates of cells). Preferably more than 90%, 95%, 98% or 99% of cells in suspension dissociative into individual cells. Preferably less than 5% of the cells in suspension forms and the regatta of 4 or more cells.

The methods of the present invention may include the identification or assessment of the level of dissociation of ES cells. Preferably the methods include the dissociation of ES cells and selection of suspension of dissociated cells according to the present invention. Microscopy or conventional counters cells can be used to determine or assess dissociation occurred. For example, the use of counter cells Casy®1 allows to identify peaks corresponding to the larger diameter of the cells in the presence of aggregates.

Direct dissociation of cells methadone

Embryonic body (ST) grown in suspension culture, and the cells are then ST is subjected to dissociation, resulting in a suspension of dissociated cells methadone. OK ST is subjected to dissociation after 8 days, i.e. on the 8th day after seeding cells for the formation of methadone or 4 days after adding the Republic of Kazakhstan. Dissociation may be performed earlier or later than this period, but usually within 3-5 days after adding the Republic of Kazakhstan. The person skilled in the art can determine experimentally the optimum time for dissociation.

Preferably methadone is not plated on adherently substrate prior to dissociation, and instead support in leatherete culture to the dissociation of cells. Thus, the Mat preferably should be subjected to a dissociation prior to sowing, and not be sown directly is directly.

Dissociation ST in norm involves incubation of methadone treatment with trypsin (at the rate of 0.05% or 0.01%to 0.5%). Preferably the methods of the present invention include filtering suspensions of dissociated ST to remove the agglutinated cells, for example, cells can be filtered through a sieve or filter, usually nylon mesh fabric or filter. OK applies the sieve or filter cell 40 microns. In embodiments implementing the present invention, the pore size is preferably 20, 30 or 40 microns, preferably 100, 80, 60, or 50 microns or less.

Storage cells methadone

The methods of the present invention may include storage cells dissociated ST, for example, by freezing the cells in liquid nitrogen. For example, the storage may include centrifugation of the cells, re-suspension of cells after centrifugation for ST+10% DMSO and freezing the cells in liquid nitrogen. Thus, in some embodiments, implementation of the present invention the method includes the dissociation of methadone treatment and storage of cells dissociated ST. Thus, can be easily obtained suitable precursor cells nerve cells.

Frozen original cell suspension can unfreeze as necessary, for example, seeding and cultivation for receiving neurons. Earlier in this technical field no publications about who is agnosti storage of such precursor cells for later use. In normal cells is thawed and immediately after that resuspending in the environment, usually in 10 ml of medium N2, centrifuged (usually within 5 min at 1000 rpm and room temperature) and resuspended (usually N2).

The seeding density of cells dissociated ST

When the sowing of ST cells was determined that the density of seeded cells methadone is an important indicator for the survival and differentiation of cells. Too sparsely seeded cells lead to a decrease in their survival, and too dense crops adversely affect the rate of differentiation. The density of the seed also influences the purity of the culture, i.e. the ratio of neuronal cells and cells of a different type. Preferably it should be sown about 0.5×105up to 2.5×105cells dissociated ST on cm2for example, approximately 1-2×105most preferably about 1-1,5×105cells / cm2.

The methods of the present invention may include measuring, evaluating or determining the number or density of inoculated cells ST, using the methods described in this invention.

Replacement of culture medium

It was found that a marked significant increase in the survival rate of cells is achieved if the culture medium replaced after approximately 2 h after seeding cells dissociated ST. These discoveries create and the possibility of obtaining long-lasting neuronal cultures, not previously common in this area.

In this context, the replacement of the culture medium means updating or replacement of the culture medium. New medium preferably has the same composition as the medium in which the cells are dissociated ST were originally or previously sown, i.e. on the medium of the same type as used in the environment. Can be applied to the environment, are similar in composition, but the preferred composition is the same as that of the previously used medium. For example, as the environment can be used in the N2 environment.

Thus, the methods described in the present invention include replacement of culture medium with subsequent dissociation of methadone treatment and seeding of cells dissociated ST into the culture medium. Preferably the culture medium is replaced after about 1-6 h after seeding.

The culture medium may be replaced within 6 h after seeding, preferably within 5, 4, 3, or 2.5 h after seeding. The culture medium may be replaced, at least approximately 1, 1.5 or 2 h after seeding.

Most preferably, the culture medium replaced after about 1-3 hours after seeding, more preferably approximately 1.5-2.5 hours, and most preferably approximately 2 hours

Culture inoculated cells dissociated ST

Cells dissociating prefer ST is Ino plated on Wednesday N2.

After 2 days the medium is preferably replaced with a suitable environment for neuronal differentiation, for example, "complete medium" (see examples). Selection and composition of the medium may depend on what line of differentiation of neuronal cells want to get. For example, the basis of complete medium used in the present invention, is the environment Brever, created to enable the development of pyramidal neurons. Other environment or factors can be selected to support different lines of differentiation of neuronal cells, for example, Shh (signaling protein Sonic hedgehog) to obtain the cholinergic motor neurons.

It was found that precursor cells obtained according to the present invention, can be differentiated in a number of different specific line of differentiation of neuronal cells, including motor neurons, with subsequent implantation in chicken embryos.

In some embodiments, implementation of the present invention it is preferable that the culture medium did not contain TK (triiodothyronine). Used in the present invention cultural environment based on the environment Brever, but TK in the composition of the medium is not included. It is possible that TK detected in fetal calf serum (FCS), can inhibit neuronal differentiation.

Preferably the medium Neurobasal not premastersecret Neurobasal + B27 Supplement (both products can be purchased in the company GIBCO) was commonly used in the prior art for neuronal cultures. However, it is established that the Neurobasal medium can activate the development of glial cells rather than the development of neuronal cells. Consequently, the use of Neurobasal medium can lead to undesirable presence of glial cells among derived neuronal cells. Conversely, it was found that complete environment used in the present invention, suppresses the development of glial cells and thereby contribute to neuronal development.

Preferably planted in cups cells (cells dissociated ST, neuronal precursor cells/progenitor cells) are cultured in the absence of serum. (Serum can be used to inaktivirovanie trypsin after dissociation of the cells, but then it should be removed, for example, by centrifugation to precipitate cells and almost complete removal of the supernatant).

Preferred growth factors (especially EGF, FGF/bFGF and PDGF) is not contained in the culture media and precursor cells or progenitor cells not cultured in the presence of these or other growth factors.

The methods can include culturing neurons, and neurons also preferably not cultivated in the presence of serum and preferably not cultivated in the presence of growth factors, particularly EGF, FGF/bFGF or PDGF.

In addition, the methods of the present image is etenia not require positive or negative selection, and preferably do not contain these stages, for example, genetic selection for Sox-2, for the enrichment of neural cells, or neurons, although if desired such stage selection can be made. The methods of the present invention allow to obtain a substantially homogeneous population of neural cells even without screening stage. Preferably the methods of the present invention do not include the stage of culling cell types that are not related to neural or neuronal (e.g., dividing cells). Preferably the methods of the present invention do not contain a stage of positive selection for the enrichment of nerve cells or neurons. Known methods of selection include genetic selection for Sox-2, i.e. to reduce not containing Sox-2 cells, and cells negative selective agent for suppression and/or destruction of cells that are not neural or neuronal, for example, treating the cells with anti-mitogenic agent, for example, AraC or FRDU, for the inhibition and/or destruction of dividing cells.

Embryonic stem cells (ES cells)

Embryonic stem cells are poly potent stem cells isolated from the inner cell mass of the mammalian blastocyst. Embryonic stem cells used in the present invention can be taken from any mammal, human or animal, for example, the Orsk mumps, rats, mice or other rodents, cats, dogs, pigs, sheep, goats, cows, horses, or primates, for example, monkeys. Usually use ES cells mouse.

In the present invention, ES cells normally are pluripotential cells, but not totipotent cells and can not form germ cells. ES cells used in the examples of the present invention are pluripotential. Optional can be used totipotent ES cells.

Several lines of ES cells, known in the field, can be obtained according to the present invention (for example, J1, E14).

Can be applied to ES cells, designed to avoid the stages of selection, for example, selection of Sox2.

In the present invention are described that used ES cells can be targeted cell lines or genetically modified lines containing the introduced gene, or mutant gene, or sverkhekspressiya endogenous gene.

You can apply a line of ES cells, comprising a reporter gene operatively associated with a promoter (e.g., promoter neuron-specific expression). The present invention describes the use of a line of cells Tau-GFP. To the properties of the Tau locus include relevant levels of expression insertionindex of cDNA molecules, the high efficiency of recombination, expression only in neurons, and so is e the fact, the removal of the Tau do not have a pronounced phenotype. In the present invention is applied to the tau locus for the insertion of the analyzed cDNA. Tau can be easily replaced by a different cDNA molecules or cDNA molecules can be insertion in the Tau locus (so that their expression of the operatively linked to a promoter Tau) in order to quickly establish a high-level stable expression specifically in neurons (reference 42).

Nerve cells

In the present invention nerve cell is called a cell of the nervous system, including neural stem cell, neuronal cell, the precursor or progenitor cell and neuron (neural cell), unless the context indicates otherwise. The term "neuron" and "neuronal cell" are used interchangeably.

The term "stem cell" means any cell type that can simulatenously and if it prepotency or is a neural stem cell that can give rise to cells of the nervous systems of all types, including neurons, astrocytes and oligodendrocytes. A stem cell may Express one or more of the following markers: Oct-4, Sox1-3, embryonic antigens that are specific to certain stages (SSEA-1, -3 and -4) (Tropepe and other Neuron, 30, 2001, cc.65-78). Neural stem cell may Express one or more of the following markers: nestin, neurotrophic receptor P75, Noth 1, SSEA-1 (Capela and Temple, Neuron 35, 2002, cc.865-875).

The concept of "neural progenitor cell" means a subsidiary of a cell or a cell of the subsequent divisions of the nervous stem cells with more differentiated phenotype and/or more reduced differentiation potential compared to the stem cell. The term "cell-precursor" refers to any cell, regardless of whether it is in direct communication with neurons during development, but under certain environmental conditions can be induced in transdifferentiation, or re-graded, or acquired neuronal phenotype.

The concept of "line of cell differentiation" refers to the progeny cells of a particular cell type. The concept of "subline cell differentiation" refers to the subtype specific line of differentiation of the cells.

Identification of markers and identification of cell types

By using the methods of the present invention preferably produces a population of cells in which at least 80%, at least 85%, at least 90% or at least 95% neuronal precursor cells/progenitor cells, for example, radial glial cells, or neurons, for example, pyramidal neurons. The methods preferably include the identity of at least 80%, at least 85%, at least 90%, at least 95%, m is Nisha least 98% or at least 99% of the cells are neuronal cells-precursor/progenitor cells, for example, radial glial cells, or neurons, for example, pyramidal neurons. Methods culture of neuronal cells according to the present invention preferably allow to obtain a population of cells containing less than 5% of astrocytes, for example, less than 4%, 3%, 2% or 1%.

The methods of the present invention described above, preferably are ways that reach these proportions. The present invention provides methods to achieve, obtain or generate these proportions of cells using one or more from among the above-described stages, and properties.

The methods of the present invention can enable the identification of cells dissociated ST as neuronal precursor cells or (later inoculated culture) as neurons. The method may include determining, monitoring or confirmation that at least 80%, at least 85%, at least 90% or at least 95% of the cells, and identification of at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the cells are neuronal cells-precursor/progenitor cells, for example, radial glial cells, or it is areas, for example, pyramidal neurons. Usually less than 5% of the cells obtained in culture of neuronal cells by the methods described in the present invention are astrocytes, for example, less than 4%, 3%, 2% or 1%.

Line of cell differentiation and/or cell types can be identified according to morphological characteristics, for example, by the method of the microscope. The present method may include the study of the morphology of neuronal precursor cells/progenitor cells or neuronal cells, at least in the described proportions of the resulting cells. Neuronal precursor cells/progenitor cells may be elongated and/or bipolar spindle forms. The line of differentiation of neuronal cells can be determined by morphological characteristics of neuronal cells, for example, pyramidal neurons have a triangular shape and branched processes of neurites, and cholinergic neurons are bipolar form.

Obtained according to the methods of the present invention, the cells can be identified in another way or additionally be identified by identifying markers, usually token the cell surface, as determined using antibodies. The method may include determining whether one or more markers whose presence indicates that the cell belongs to a certain whether the AI or as compared to cells or to a particular type or subtype of cells. Specialist known markers that can be identified and applicable as an indicator belonging to the line of differentiation or cell type.

For example, the method can include identifying marker RH on the surface of cells and identification of cells, which are neuronal precursors, for example, radial glial cells. Other detectable markers include nestin, RC2 and BLBP, which are present on the surface of the radial glial cells, and P75, GluR1, synaptophysin, Trks (e.g., TrkA, TrkB, TrkC) and the RDA, which are present on the surface of certain neuronal cells.

The present method may include determining a high percentage of cells expressing markers of neuronal precursor cells, for example, at least 80%, at least 85%, at least 90% or at least 95% of the cells, and identification of at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of neuronal precursor cells.

The present method can include identifying a high percentage of cells expressing markers of neuronal cells, for example, at least 80%, at least 85%, at least 90% or at least 95% of the cells, and identification, at IU is e, 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the cells as neurons, preferably of neurons in certain lines of cell differentiation, for example, pyramidal neurons and dopaminergic neurons.

Thus, the method allows to obtain an almost completely homogeneous population of neural precursor cells or neurons. At least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the cells may be of the same type/line of differentiation or subtype/line differentiation, for example, neuronal precursor cells of the same type, for example, radial glial cells, or neurons in the same line of differentiation, for example, pyramidal neurons.

In the present description, the examples show, in what terms is the expression of different markers and morphological what stage of development in which the terms occur. The methods of the present invention may include the identification of markers and/or monitoring specific morphology at a certain time after dissociation ST (as noted in the examples), for example, neuronal morphology is observed in less than two days after the dissociation ST and/or manifested expression of Trk receptors example is about 7 days. For example, in the present invention shows that approximately 99% of the cells obtained in the described manner, were radial glial cells, as detected by detecting expression RC2+ 99% of the cells dissociated ST. The present invention also shows that at least 80% of the neurons can stably be obtained by the methods of the present invention, which is confirmed by measuring the expression or calculation of vGLUT1 and GFP approximately 7 days after the dissociation ST.

The percentage can be calculated as the percentage of viable cells or the percentage of cells expressing nuclear marker, for example, after processing by DAPI or Hoechst.

The formation of methadone treatment and handling of Kazakhstan

In the methods of the present invention methadone cultivate and form in the culture medium. During the formation of methadone treatment and culturing the culture medium is usually replaced every two days.

Normally in the methods of the present invention methadone cultivated in the presence of RK for one or more days, usually two, preferably three or four days, or up to five, six, seven or eight days. Originally methadone may be cultured in the absence of RK for one day or more, usually from two to six days, usually within two, preferably three or four days, or up to five or six days is it before processing the Republic of Kazakhstan. Methodology 4 days/4 nights + used Bain and others, and Li and others

The expert can select a suitable concentration of the Republic of Kazakhstan. For example, the concentration may be at least 0.25 mcmole at least 0.5 mcmole, or at least 1 micromole. The concentration may be, for example, 10 mcmole or less, 7.5 mcmole or less, or 5 mcmole or less. Preferably the concentration is from 0.5 to 5 mcmole inclusive. For example, the concentration may be 1 µm or 5 mcmole.

The study of nerve cells

Other objects of the present invention are methods of studying cells performed with neuronal cells precursor or progenitor cells, or neuronal cells, which are normally formed in vitro (these neurons are not the primary), and preferably the cells formed by the method of the present invention. The ways of learning can be described in the present invention is a method of obtaining neuronal precursor cells, or progenitor cells, or neurons. The methods of the present invention can provide a method of the present invention for neuronal differentiation (formation of neuronal precursor cells, or progenitor cells, or neurons), and additionally enable stage of the method of the study of cells described in this izaberete the AI.

Thus, the above methods of neural differentiation can be applied in a research context.

In addition, because the present invention were first derived largely homogeneous culture/population of neuronal progenitor cells, or progenitor, or neuronal cells, the present invention also provides methods study performed with a high degree of homogeneous cultures/populations of neuronal precursor cells, or progenitor cells, or neuronal cells that can be obtained or cannot be obtained) ways of differentiation of nerve cells of the present invention, but in normal they get by in vitro methods.

Methods of study the present invention may include detecting, counting, monitoring, or determining one or more characteristics of neuronal precursor cells, or progenitor cells, or neurons ("neuronal characteristics"), for example, of neurite growth or elongation/degeneration, neuronal form, neuronal cell death, neurogenesis, neuronal differentiation, the electrical activity of the cells, synaptogenesis and/or markers of neuronal cells.

In some embodiments, implementation of the present invention the methods of the study this izobreteny may include the described method of differentiation of nerve cells to obtain neuronal precursor cells, or progenitor cells, or neuronal cells, and this method also includes the cultivation of ES cells and/or methadone in the study condition, and identifying, counting, observation or determination of one or more neuronal characteristics of neuronal precursor cells, or progenitor cells, or neuronal cells.

According to other variants of implementation of the present invention, provided for them in the ways of learning may include the cultivation of neuronal precursor cells, or progenitor cells, or neuronal cells in the test condition, and identifying, counting, observation or determination of one or more neuronal cells. Optional cells can be obtained and/or produced according to the methods of differentiation of nerve cells described in the present invention.

Methods of studying may not necessarily include a comparison of neuronal properties in the test condition (test culture) with the neuronal properties of cells cultured in the second state ("control culture"), not necessarily with the established data about the course of the process of culturing cells of a second, control, culture. The methods can include culturing the cells in the second state.

Thus, the methods and the teachings can include a method of differentiation of nerve cells, described in the present invention, to obtain neuronal precursor cells, or progenitor cells, or neuronal cells, comprising culturing ES cells or ST in the first and second conditions, and mapping one or more properties of neuronal precursor cells, or progenitor cells, or neuronal cells, cultured in the initial conditions, with similar neuronal properties or properties of neuronal precursor cells, or progenitor cells, or neuronal cells, cultivated in their second terms, respectively.

Cultivation in the test culture, i.e. in the first embodiment of cultivation, may include treating the cells with the test compound or culturing cells in the presence of the compounds that can be added to the culture medium or included in its composition. In the second variant of cultivation, i.e. under cultivation in the control cells can be grown in the absence of the compounds, or does not come into contact with the test compound, or not subject to its effects.

The investigated compound may be any molecule, for example, from the library of the studied compounds. In some embodiments, implementation of the present invention investigated the connection is a molecule which voicewas RNA (dnrc) and the first version of the cultivation, i.e. with the test compound, is in the handling of ES cells or cells ST molecules of double strand RNA and, as a result of such processing, in the inhibition of the gene in the cells by the mechanism of RNA interference (Rnci).

It was found that dnrc to suppress gene expression even more effective than individual threads of sense or antisense RNA (Fire, A. and others, Nature, 391, 1998). Suppression of gene expression using dnrc is genespecific and it is often called RNA interference (Rnci) (see also Fire in Trends Genet., 15, 1999, cc.358-363, Sharp, Genes Dev., 15, 2001, cc.485-490, Hammond and others in the Nature Rev. Genes, 2, 2001, cc.1110-1119, Tuschl in Chem. Biochem. 2, 2001, cc.239-245).

RNA interference is a two-step process. First, molecules dnrc broken down inside the cell with the formation of short interfering RNA molecules (kirk), a length of about 21-23 nucleotides from the 5'-terminal phosphate and a short 3'-protruding end (from about 2 nucleotides). Molecules kirk focus specifically on the corresponding sequence of the mRNA for destruction (Zamore P.D. in Nature Structural Biology, 8, 2001, cc.746-750).

Rnci can be effectively induced by using chemically synthesized duplexes kirk the same structure with a 3'-protruding ends (Zamore in the Cell, 101, 2000, cc.25-33). It is established that synthetic duplexes kirk specifically suppress expression of endogenous and foreign genes in widely the range of lines of mammalian cells (Elbashir, etc. in Nature, 411, 2001, cc.494-498). Can be used duplexes kirk containing 20-25 base pairs, more preferably 21 to 23 base pairs, inhibiting sequence.

Another way kirk can be obtained by using a vector, in vitro (selection and use) or in vivo.

In other embodiments, implementation of the present invention investigated the connection can be a nucleic acid (DNA, cDNA or RNA), optional encoding gene, for example, cDNA. Thus, the analyzed connection can be a vector encoding a gene, and the exposure time of the cells of the nucleic acid or vector leads to the expression of the gene in the cells. In one of the embodiments of the present invention, the vector can include a nucleic acid sequence, which according to the present invention may be semantic, and antisense orientation, in the case of expression of RNA sense and antisense fragments associated with the formation of double-RNA. Such RNA, for example, can be a long Dunaeva RNA (for example, consisting of more than 23 nucleotides), which result from processing in the cell can lead to the formation of molecules kirk for Rnci (see, for example, Myers in Nature Biotechnology 21, 2003, cc.324-328).

In another embodiment of the present invention investigated the connection can be an antibody.

JV is the event study can also identify compounds or condition, which increase or reduce the required property.

Typically correlated with neuronal cells, for example, one week after seeding cells dissociated ST.

Study and control culture are usually two different crops grown in the same conditions for all the other parameters. If cultivated together with a test compound, especially with nucleic acid, this cultivation, which is the cultivation of the first type may involve the processing of cells (usually ES cells or cells dissociating ST) investigated the connection and then in the cultivation of cells.

Neuronal properties (e.g., increase or elongation of neurites) can be detected by induction or assumptions the expression of neuron-specific reporter gene, and detection or quantitative evaluation of the expression of the reporter gene. Reporter gene can encode fluorescent protein such as green fluorescent protein (GFP). Reporter gene can be targeted or operatively associated with the neuron-specific locus or promoter, for example, tau locus or promoter for neuron-specific expression. We described the expression of neuron-specific reporter gene with locus tau (Tucker and others, reference 42). The reporter gene expression is activated immediately after differentiation of the cells and in the neuron, and only in neurons and not in precursor cells or cells of other types of the nervous system. In the methods of the present invention, including in studies of neuronal cells can be used line cells (ES cells)containing the reporter gene, with neuron-specific expression and reporter gene operatively linked to a promoter or locus, expressed only in neurons (for example, line Tau-GFP described in this invention).

The present invention provides methods of research designed to identify an agent that inhibits or reduces the growth of neuronal characteristics caused by the condition, which is known that it increases the characteristic of or associated with the growth of this characteristic (for example, in some embodiments, implementation of the present invention, according to which the condition lies in the cultivation with amyloid beta-peptide), i.e. the identification of an agent that reduces or inhibits the effects associated with this condition.

Such research may include:

cultivation of neuronal precursor cells, or progenitor cells, or neuronal cells in the presence of an investigational agent, provided, of which it is known that it increases or is associated with an increased neuronal properties is a,

cultivation of neuronal precursor cells, or progenitor cells, or neuronal cells in the absence of an investigational agent, provided, of which it is known that it increases neuronal property,

the measurement or determination of the levels of neuronal properties and

comparison of the levels of neuronal properties in the presence of an investigational agent with levels of neuronal properties in the absence of an investigational agent,

at this lowered level of neuronal properties in the presence of an investigational agent compared to the level in the absence of an investigational agent indicates that the agent inhibits or reduces the gain of the manifestations of neuronal properties, caused by or associated with the condition of the study.

The condition, which is known that it increases neuronal characteristic or related to its increase may be a condition identified by the examination method of the present invention as a condition that increases the neuronal response.

For example, if the target compound is a nucleic acid (e.g., dnrc), cultivation, subject to the condition, which is known that it increases neuronal characteristics may include exposure of cells nucleic acid and subsequent cultivation cleto is.

Cultivation with a test agent and cultivation subject to certain conditions may be performed simultaneously or cultivation with a test agent can be carried out before cultivation subject to certain conditions, or cultivation subject to certain conditions can be performed prior to culturing with the investigational agent. The specialist may choose the order, and in some embodiments, implementation of the present invention, one sequence may be preferable to another. For example, preferably the cells are exposed to the nucleic acid and then cultured in the presence of an investigational agent.

The elongation or degeneration of neurites

The methods of the present invention may include a quantitative assessment of neurite growth, elongation of neurites or degeneration of neurites. Quantitative assessment may involve determining the levels of expression of Nairit-specific protein, with increased expression indicating a higher level of neurite growth, and/or elongation of neurites, and/or low level degeneration of neurites, and reduced expression level indicates a low level of neurite growth, and/or elongation of neurites, and/or an increased level of degeneration of neurites. Quantitative OC the NCA may include the induction or the assumption of the expression of neuron-specific reporter gene and measuring the levels of expression of the reporter gene, thus quantified neurite outgrowth, elongation of neurites or degeneration of neurites. For example, if the reporter gene encodes a fluorescent protein, for example, GFP, measurement of expression levels is to measure fluorescence. The methods of the present invention may include a quantitative assessment of neurite growth, elongation of neurites or degeneration of neurites by treating neurons with an antibody to marker Narita (e.g. tubulin, neurofilament, synaptophysin), determining or calculating the binding of an antibody to a marker and, thus, determining or calculating the formation of outgrowths or elongation of neurites.

For treatment of neuronal antibody can be used in cell extracts obtained after lysis of the cells (for example, when Western-blotting). According to another variant, the whole neurons can be contacted with the antibody. Methods of research may include the cultivation of neuronal precursor cells, or progenitor cells, or neuronal cells in observance of the first or second conditions, respectively, and comparing the levels of neurite growth, elongation or degeneration of neuronal precursor cells, or progenitor cells, or neuronal cells in compliance with the first condition with the level of neurite growth, elongation or degeneration of a neuron is lnyh precursor cells, or progenitor cells, or neuronal cells in compliance with the second condition, respectively. For example, if the levels of neurite growth, elongation or degeneration above (for example, according to high/low level of expression of Nairit-specific protein, see above) in cells cultured in compliance with the first condition, compared with cells cultured in compliance with the second condition, we conclude that, in compliance with the first condition (compared to the second) increases growth, elongation or degeneration of neurites, respectively.

In a preferred embodiment of the present invention cultivation in compliance with the first condition involves culturing cells in the presence of the tested compound, which is preferably of amyloid β peptide (β) (which is derived from the amyloid precursor protein, APP).

The present invention provides methods of study, allowing to identify an agent that inhibits or reduces growth of the degeneration of neurites arising under the influence of the state of which it is known that it increases the degeneration of neurites (for example, when culturing in the presence of amyloid beta-peptide), i.e., to identify an agent that reduces or inhibits the effects associated with this condition is. Such study may include:

cultivation of neuronal precursor cells, or progenitor cells, or neuronal cells in the presence of an investigational agent, subject to the condition, which is known that it increases the degeneration of neurites,

cultivation of neuronal precursor cells, or progenitor cells, or neuronal cells in the absence of an investigational agent, subject to the condition, which is known that it increases the degeneration of neurites,

the calculation or determination of the levels of degeneration of neurites in the presence of an investigational agent, or in his absence, and

comparison of levels of degeneration of neurites in the presence of an investigational agent with levels degeneration of neurites in the absence of an investigational agent,

at this lowered level of degeneration of neurites in the presence of an investigational agent compared to the level of degeneration of neurites in the absence of an investigational agent indicates that the agent inhibits or reduces the gain degeneration of neurites, caused by or associated with this condition.

According to the above, the comparison of the levels of degeneration of neurites may include comparing the level of expression of Nairit-specific protein, with increased expression (lower level of degradation) in prisutstvie and the investigational agent compared to expression level in the absence of an investigational agent shows that the investigational agent inhibits or reduces the gain degeneration of neurites induced by this condition of the study.

The condition is likely to be in the presence of compounds, which can be a compound identified using the method of the study the present invention due to its ability to enhance the degeneration of neurites, namely β-peptide.

Neuronal cell death

There is a need to research the death of neuronal cells in this area and such studies described in this invention.

Research the death of neuronal cells can be used to determine or assess the sensitivity of a population of neurons or neuronal cells to a specified condition, for example, to the presence of one or more compounds, for example, to identify conditions (e.g., connections), which increases or reduces neuronal cell death.

For example, the study in accordance with the present invention may include:

the cultivation of neurons in compliance with the first condition ("learning culture"),

the cultivation of neurons in compliance with the second condition ("control culture"),

quantifying or determining the death of neuronal cells in observance of the first and second conditions and

cf is giving levels the death of neuronal cells in compliance with the first condition with the levels of death of neuronal cells in compliance with the second condition,

this increased level of loss of neuronal cells in compliance with the first condition compared to the level of the death of neuronal cells in compliance with the second condition indicates that the subject of the first conditions of cell death increases, and/or

the lowered level of the death of neuronal cells in compliance with the first condition compared to the level of the death of neuronal cells in compliance with the second condition indicates that the subject of the first condition cell death is reduced.

When learning of the death of neuronal cells, particularly in identifying state, which reduces neuronal cell death, neurons are preferably genetically predisposed to apoptosis. For example, neurons can Express the receptor of neurotrophin P75 and/or to Express the apoptotic protein (e.g., caspase), operatively associated with a neuron-specific promoter (e.g., locus Tau). Thus, ES cells used in the present invention for receiving neurons to study the death of neuronal cells, can Express the apoptotic protein (e.g., caspase), operatively associated with a neuron-specific promoter (e.g., locus Tau).

The study of the death of neuronal cells can be used to identify an agent that inhibits or reduces the increase in g is Beli neuronal cells, received subject to certain conditions, which it is known that it increases neuronal cell death, i.e. an agent that reduces or inhibits the effect of such conditions. Such research may include:

culturing neurons in the presence of an investigational agent, subject to the condition, which is known that it increases neuronal cell death,

culturing neurons in the absence of an investigational agent, subject to the condition, which is known that it increases neuronal cell death,

quantifying or determining levels of death of neuronal cells in the presence of an investigational agent, or in his absence, and

comparison of the levels of the death of neuronal cells in the presence of an investigational agent with levels of death of neuronal cells in the absence of an investigational agent,

at this lowered level of the death of neuronal cells in the presence of an investigational agent compared to the level of the death of neuronal cells in the absence of an investigational agent indicates that the agent inhibits or reduces the amplification of the death of neuronal cells caused by this condition.

Cell death can be determined by methods known in the art, for example, by identifying the mechanisms of induction of apoptosis in neurons. Signs of the death of the notches, which can be determined include the induction of apoptotic proteins (e.g., caspases, especially caspase-3, see reference 43), the color of the iodide of propecia and/or DNA fragmentation and/or destruction of the nucleosome (detected, for example, binding of an antibody to DNA or histone, see reference 44),

Neurogenesis and neuronal differentiation

The methods of the present invention may include studies of neurogenesis or neuronal differentiation, and the production or generation of neurons or differentiation of ES cells and/or neuronal precursor cells, or progenitor cells identify and/or assess quantitatively. The method may include detection and/or quantification of one or more neuron-specific markers. The methods of the present invention can include monitoring levels of neurogenesis for one or more specific neuronal subtypes or lines of cell differentiation, or the levels of neurons in General, depending on the selected markers. Generation of neurons in certain lines of differentiation of the cells can be studied by identifying and/or quantifying markers specific to lines of cell differentiation. The methods may include treating the cells with an antibody to the cell marker and a binding definition, and the presence of the marker (and the binding of an antibody according to the government) shows what cells belong to a certain type, subtype, line differentiation of cells or sub-line of cell differentiation. The methods can include determining or calculating levels of antibody binding sites and, thus, the determination or calculation of the levels of differentiation, stage of cell differentiation and/or percentage of cells of a particular type, subtype, line differentiation of cells or sub-line differentiation of cells, or a particular stage of differentiation. In more detail, the identification of markers for the identification of the cell types represented in the present invention, and suitable markers known to specialists in this field.

Methods study neuronal differentiation according to the present invention can be used to identify markers that can be used to identify ES cells and/or nerve cells at certain stages of differentiation, or to identify the type or subtype of cells and, thus, to establish the stage of differentiation of cells, or cell type, or subtype. For example, methods of study may include the induction or admission differentiation of ES cells to obtain neuronal precursor cells, or progenitor cells, and/or culturing the neural precursor cells or progenitor cells for receiving neurons (preferably using the methods ner the Neu differentiation, described in the present invention), comparison of expression levels of proteins in the cells at the same stage of differentiation with levels of protein expression in cells at the second stage, differentiation, and identification of proteins, the expression level of which is different in the cells on the first and second stages of differentiation. The difference in levels of expression shows that protein can be used as a marker to identify the stage of differentiation, type or subtype of cells and/or for differentiating the cells in the first and second stages of differentiation. The levels of expression can be mapped using a suitable method, which can be selected by the person skilled in the art. Preferably mapped protein expression on the cell surface, for example, by examining the interaction of cells or cellular extracts with the library expressed on the cell surface antibody and determining binding. For example, the method may include mapping protein expression in neuronal cells-precursor/progenitor cells (e.g., radial glial cells) protein expression in ES cells. The difference in levels of expression may be at least 1.2 times, at least 1.5, at least 1.6-fold, at least 1.8-fold, at least 2 times, at least 3 times, at least once, at least 10 times or more. Expression can be detected in the cells at the first stage of differentiation and not detected in the cells at the second stage of differentiation.

The electrical activity

The levels of electrical activity in neurons, for example, electrical activity, indicating about the discovery of specific channels (e.g., ion channels), can be observed, detected, determined or calculated.

How research can be used to identify compounds capable of modulating the electrical activity of neurons. The methods may include the cultivation of neurons in compliance with the first condition, the cultivation of neurons in compliance with the second condition and the comparison of the electrical activity of neurons cultured in compliance with the first condition, with the electrical activity of neurons cultured in compliance with the second condition, respectively. The difference in electrical activity shows that the condition of cultivation modulates electrical activity.

Synaptogenic

Methods of study can include the identification or quantification of synaptogenesis in neuronal cells. Detection or quantification may include measuring electrophysiological activity of the cells and/or detecting or measuring Express and one or more markers synaptogenesis, for example, synaptophysin.

Comparison of genetically different neurons

The present invention describes methods for comparison of standard (usually cells wild type) neuronal precursor cells, or progenitor cells, or neurons with mutant neuronal precursor cells, or progenitor cells, or neurons, neurons with other genotypes. The method of the present invention may include the above-described method of obtaining nerve cells.

Thus, the present invention describes a method including:

receiving the first and second cultures of neuronal cells or neuronal precursor cells, or progenitor cells, which in the first culture have the genotype that is different from the genotype of the cells in the second culture, and

comparison of neuronal precursor cells, or progenitor cells, or neurons, in the first culture of neuronal cells precursor or progenitor cells, or neurons in the second culture.

Can be compared neuronal precursor cells, or progenitor cells, or neuronal cells, or not containing the mutation in the gene of interest. The mutation can be, for example, a deletion of all or part of the gene, a deletion of all or part of gene promoters and/or enhancers, or substitution of one or several the x nucleotides in the coding region of the promoter or enhancer. Normal mutation leads to a change (decrease or increase) the level of expression of a gene or expression of the mutated protein (eg, shortening or content of one or more deletions in amino acid sequence). In another embodiment, the neuronal precursor cells, or progenitor cells, or neuronal cells in the first culture may have introduced gene (for example, insertional genome or insertional cDNA) or overexpression of an endogenous gene, but neuronal precursor cells, or progenitor cells, or neuronal cells in the second culture they do not possess.

ES cells can be genetically modified, mutations can be induced in ES cells or ES cells can be isolated from animal cells which carry the mutation, for example, ES cells from mice carrying a mutation in the gene of interest, to obtain nerve cells, such as neurons or neuronal progenitor cells/precursor cells possessing or not possessing the required mutation, respectively. Thus, using the present invention can be obtained mutant nerve cells and nerve cells of the wild type, for example, neurons or neuronal progenitor cells/precursor cells. Comparison of neural cells derived from ES cells of different types (from a to the x alone are interesting mutation, and others not), could, for example, be performed to identify the mechanism responsible for the loss of the type of neural cells in neurodegenerative disease, or affecting this loss, and to identify appropriate targets associated with disease phenotypes.

In some embodiments, implementation of the present invention, the method may include receiving neuronal precursor cells/progenitor cells or neurons of the first and second cultures of ES cells, respectively, and ES cells in the first and second cultures have different genotypes. Optional neuronal precursor cells/progenitor cells or neurons can be derived from ES cells by the methods described in the present invention. ES cells in the first culture may contain a mutation of the gene of interest, and ES cells in the second culture do not contain this mutation (e.g., cells of the wild type). In another embodiment, the ES cells in the first culture may contain the introduced gene or sverkhekspressiya endogenous gene, and ES cells in the second culture is not.

In another embodiment, for use genetically different ES cell genetic manipulation may be subjected to ES cells. The methods may include the first transfection cell culture dissociated ST, or neuronal precursor cells, or progenic the situations of the cell structure of nucleic acids and, as a result, the change in the genotype of the cells in the first culture compared to cells in the second culture. For example, the nucleic acid can encode an endogenous gene, or to encode the gene of interest containing the mutation. Such methods of the present invention allow normal expression (usually transient expression of approximately 2, 3 or 4 days) to design nucleic acid. The present method can include culturing the cells to obtain neuronal cells. Cells in the first culture should be compared with neuronal cells precursor, progenitor cells or neuronal cells in the second culture, and cells in the second culture does not contain the design of nucleic acid, the introduced gene and/or mutation.

Comparison of neuronal precursor cells, or progenitor cells, or neurons can be compared (usually in the determination or calculation) of one or more characteristics, for example, of neurite growth or elongation of neurites, neuronal form, the death of neuronal cells, neurogenesis, neuronal differentiation, electrical activity, synaptogenesis and/or markers of neuronal cells. In other embodiments, implementation of the present invention, the comparison may involve the capture of the gene of interest, e.g. the measures introduced, or mutated, or sverkhekspressiya gene, or the manifestations of this gene. The nature of the reading depends on the gene and can be determined by a specialist for this gene. Thus, the transfer mechanisms of neuronal signal can be identified, blocked and/or affected.

Comparison of neuronal precursor cells, or progenitor cells, or neuronal cells may include comparing one or more characteristics of the cells at a specific target condition, and can be used ways of comparing genetically distinct neuronal precursor cells, or progenitor cells, or neuronal cells in the context of the research methods described in this invention. Thus, in preferred embodiments, the implementation of the present invention, the first and second cell culture is cultivated in the studied condition, and neuronal characteristics of the cells are compared. Other methods and variations of methods of the study of cells described above. For example, the cultivation when the monitored condition may lie in the cultivation of presence β-peptide.

Antibodies

In the context of the present invention the term "antibody" or "antibodies" means any substance which can specifically bind or link the and, having a binding domain with the required specificity. Thus, this concept refers to the fragments of the antibodies, derivatives, functional equivalents and homologues of antibodies, including any polypeptide containing the immunoglobulin binding domain, whether natural or synthetic. This term also includes chimeric molecules comprising immunoglobulin-binding domain, or equivalent, linked to another polypeptide. Cloning and expression of chimeric antibodies are described in applications EP-A-0120694 and EP-A-0125023.

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL domains, VH, CL and CN; (ii) the Fd fragment consisting of the VH domains and SN; (iii) the Fv fragment consisting of the domains V1 and VH a single antibody; (iv) the dAb fragment (Ward, E.S. and others, Nature 341, 1989, cc.544-546), consisting of a VH domain; (v) selection of CDR; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked the Fab fragment, and (vii) single-chain Fv molecules (scFv), in which the VH domain and a VL domain are linked by peptide linker which allows the two domains to associate with the formation of the antigen-binding site (Bird and others, Science, 242, 1988, cc.423-426; Huston and others, PNAS USA, 85, 1988, cc.5879-5883; (viii) bespecifically single-chain Fv dimers (PCT/US92/09965) and (ix) "dimers", polyvalent or polyspecific fra the cops, constructed by fusion of genes (WO94/13804; Holliger P, and others, Proc. Natl. Acad. Sci. USA 90, 1993, cc.6444-6448).

The dimers are multimarine polypeptides, each polypeptide comprising a first domain containing the binding region of a light chain immunoglobulin and a second domain comprising a binding region of a heavy chain immunoglobulin, and the two domains are linked (for example, a peptide linker), but can't associate with each other for the formation of the antigen-binding site: the antigen-binding sites are formed by the Association of the first domain of the polypeptide in the composition of multimer with the second domain of another polypeptide in the composition of multimer (WO 94/13804).

Antibodies can be modified in a number of ways, for example, they can be marked, for example, a fluorescent dye, allowing to quantify the bound antibodies by measuring the levels of fluorescence.

Various additional objects and embodiments of the present invention described in the present invention as understood by the experts in this field.

All documents referred to in the present description, is included by reference in their entity.

Certain objects and embodiments of the present invention are illustrated below by example and by reference to the drawing, which shows the percentage of Pax-6-positive cells defined in the TES time after seeding cells dissociated ST. First, Pax-6 is expressed by most cells, but the expression quickly disappears. The results represent the average of 4 experiments performed with two different lines of ES cells. They are expressed as percentage ± standard deviation of the number of DAPI-positive nuclei taken for 100%.

Examples

Culture of ES cells

The way leading to the generation of neurons from ES cells, includes the following stages, summarized as follows:

1. Cells grown on the feeding layer, grow as colonies, although after deprivation supply layer they grow in the form of a flat monolayer of cells.

2. ES cells in readerenter bacteriological cups form cell aggregates (embryonic body, GTO), which grow in suspension.

3. After 4 days, during which the formed Mat, add the Republic of Kazakhstan for the next 4 days.

5. ST subjected to dissociation in total in 8 days and sown in the Cup with a coating of PEP/laminin in medium with N2.

6. Wednesday N2 replaced after 2 h and again after 12-24 hours At this stage the majority of progenitor cells has a fusiform shape. Environment for neuronal differentiation through add 30-48 hours

This technique was developed using ES cells, which Express GFP from the tau locus (reference 13). Expression of GFP from the endogenous promoter allows the VI is to oilservice neurons and their processes in UV light, what was used to maximize the generation of fluorescent cells.

After thawing ES cells are first cultured on feeding the cells for 2-3 passages and then gradually remove the power cell. A certain number of (3×106cells are then used for the formation of aggregates (embryonic bodies, ST), inkubiruemykh in the non-adhesive bacteriological cups (a Cup with a diameter of 10 cm, 15 ml of medium) for 8 days. Retinoic acid (RK, 5 mcmole) contribute over 4 days and leave for the next 4 days. An important step is the selection of ES cells without feeder cells with homogeneous flat morphology and high proliferative activity (see materials and methods).

After 8 days ST subjected to dissociation using a freshly prepared suspension of trypsin and plated on the substrate containing poly-D-lysine (PEP) and laminin. It is established that the density of seeded cells (1.5 x 105cells/cm2) is critical, because at lower densities the cells are prone to rapid death. Dissociatively cells plated on medium without serum, which replace the 2 h after seeding to remove cell fragments and dead cells. Through the day (approximately 24 h) environment replace again. After 48 h the medium is replaced by medium without serum, enriched with additives (reference 12). In addition to ES cells, EC is pressious GFP with both tau alleles, also used by more than 7 other lines of ES cells with results that do not differ from the results described in this study. These include ES cells wild-type J1 and E14, and J1 with GFP in one or both tau alleles. Also contains 4 different lines of ES cells from blastocysts mixed BL6/SV129 genetic environment and used in the differentiation Protocol with similar results.

Neuronal precursor cell

ES cells differentiate into a homogeneous population of radial glial cells.

Cells differentiated from ST, take a clear elongated fusiform shape that resembles the shape of the radial glial cells (see reference 16). Phase-contrast microscopy illustrates bipolar fusiform shape after 2 h the process of differentiation.

These cells are identified as neuronal precursor cells staining with antibody to intermediate filament protein nestin (reference 9). After 2 h after seeding is established that in the vast majority of cells are nestin-positive, which follows from the comparative quantitative analysis of the total number of inoculated cells, after staining of the nuclei (see table).

Then apply RC2 - marker, which Express all radial glial cells, and almost all cells are positive (see table). krasivaya antibodies to the protein, communicating with the lipids of the brain (BLBP), - antigen, which is expressed by radial glial cells in the developing Central nervous system (reference 18), further confirms the identity of the cells, their in the dissociation of methadone (see table). Homeodomains the transcription factor Pax-6 Express all cortical radial cells (reference 19), and found that almost all cells in the ST Express it before dissociation, therefore, at this stage they are already cells predecessors.

Counting the 2 h after seeding shows that in the vast majority of cells still positive for Pax-6 (see drawing), and that its expression is rapidly declining for the next day until complete disappearance after 7 days (see drawing).

The percentage of neuronal precursor cells after 2 h after seeding
NestinRC2BLBPRaj
88±2,599±2,597±2,684±12,2

Nestin, RC2 and BLBP, Pax-6 analyze the method of immunocytochemistry after 2 h after seeding dissociated ST. Percentage (± standard the disturbances is giving) positive cells determined from the total number of cells, painted the nuclear marker DAPI.

Neuronal differentiation

Cells with signs of neuronal morphology begin to appear no later than 2 days after the dissociation ST. All differentiated cells Express GFP, which means that they belong to the neurons. This conclusion is confirmed by experiments on staining using an antibody that recognizes the neuron-specific form of tubulin.

After 4 days approximately 85% of the cells are GFP-positive and tubulin-positive. Using phase-contrast and fluorescence microscopy have shown the emergence of a remarkably homogeneous neuronal cell phone With time in culture, they are more and more taking a pyramidal shape, celebrated for cells isolated from the hippocampus of rodents (reference 20). After staining with antibodies to synaptophysin see the process of building numerous clusters of GFP-positive cells, indicating the possibility of synaptic contact in these cultures.

To install, do these neurons glutamate as a neurotransmitter, cells stained with antibody to vesicular the glutamine Transporter (vGlut1) is a membrane protein expressed by the majority of pyramidal neurons in the cerebral cortex and in the hippocampus (reference 21). After 7 suto is 93±4.7% of cells in culture stained with antibodies against vGlut1. The results obtained with the use of vGlut1 antibodies confirmed the identity of the neurons as pyramidal cells. By the end of the first week after dissociation ST less than 0.1% of the cells stained positive Is1-1, tirosingidroksilazy and cholineacetyltransferase. After 3 weeks less than 5% of the cells are GABA-positive.

To identify proteins expressed during the transition from radial glial cells to neurons, carry out Western blotting using differentiated in vitro neurons obtained at different time intervals.

Although AMPA-receptor subunit GluR1, for example, synaptophysin not detected in lysates of radial glial cells, after several days of growth is clearly detected in the culture. The levels of GluR1 protein and synaptophysin increase after the beginning of differentiation of neurons.

Because pyramidal neurons Express high levels of Trk receptors in the cortex and in the hippocampus (reference 22), also analyze their expression using anticigarette to the intracellular domain of these neurotrophin receptors. Although the Trk receptors hardly detected at 5 days, their level increases dramatically in the next day. Significant expression of Trk receptors observed after about 7 days in vitro, after which it increases sharply. And, in contrast, found that the levels of neuro who eroticheskoe receptor P75 fall during neuronal maturation, that is largely similar to the process in vivo (reference 23).

In conclusion, investigating the expression of protein - amyloid precursor (APP). Found that this membrane protein Express radial glial cells (reference 24), as well as a number of cells, including neurons at later stages of development. It is established that, in contrast to other studied membrane proteins, the RDA is clearly detected in the lysates of radial glial cells. The levels of expression of a later increase, probably due to maturation of neurons, which includes a pronounced growth of neuronal processes.

The in vivo differentiation of implanted cells predecessors

The potential development of neuronal precursor cells according to the present invention examined by implantation of these cells in chicken embryos, in which they can be differentiated in various specific line of differentiation of neuronal cells, including motor neurons.

Electrophysiology

Electrophysiological experiments show that neurons form synapses show action potentials and are very uniform in electrophysiological characteristics. These neurons are predominantly glutamatergic (as shown by blocking synaptic transmission NBQX, vGAT staining) with some GABAergic input (blocks which has been created by bicucullin, otherwise, the culture cannot survive). Using electrophysiology clearly established that under these conditions there are no other types of neuronal cells.

To describe the electrophysiological properties obtained from the EC cells of the neurons carry the recognition of the patch-clamav on the surface of individual cells in culture for 10 to 22 days. All investigated cells (n=22) show spontaneous or induced depolarization of action potentials, and in all cases they can be blocked by application of tetrodotoxin. In addition, the electrophysiological properties of the investigated cells show that they are highly homogeneous functional properties similar to the previously described properties of pyramidal neurons. You may experience spontaneous synaptic transmission (STD), which can be completely blocked by the addition of NBQX/AP-5 and bicuculline, or only one NBQX/AP-5. These results show that obtained from ES cells neurons form functional synapses that use glutamate as a neurotransmitter. Because these experiments also reveal the presence of functional GABA synapses in the long-supported crops, count the number of GABA-neurons after 3 weeks. Simultaneously with the loss of staining for neurotransmitte is s, not related to glutamate and GABA system does not detect any of synaptic activity, which would not be associated with glutamate or GABA.

Discussion

The use of mouse ES cells has allowed to establish the conditions that lead to the generation of essentially pure populations of neural progenitor cells, which are called radial glial cells. Then these cells continue to develop in a homogeneous population of neurons with properties of pyramidal cells.

It was found that if vysokopoligonalnye undifferentiated stem cells are selected for transformation ST, processing RK converts entire population of cells in neuronal precursor cells of a particular type. The selection of undifferentiated ES cells is important because it was found that even in the presence of LIF some ES cells tend to differentiate, and that during the formation of the ST often see the cells of different lines of differentiation (see reviews, links 3, 34).

For selection of ES cells with high proliferative activity after removal of feeder cells monitor the division rate by counting cells using phase-contrast microscopy, and determination of the attained degree of merge cells before the formation of the Mat with a specific number of cells.

Earlier it was reported about the presence of radial glial cells in medicationabana ST established using either ES cells or cells of embryonal carcinoma P19 (reference 35). If methadone plated on polylysines substrate, can be observed radial migration of elongated cells in the direction from methadone treatment, with subsequent transformation into astrocytes (reference 35). Identification of the cells is carried out by dissociation ST, based on cell morphology and number after staining with antibodies to RC2 and BLBP and Pax-6. Previously it was found that this set of markers Express the radial glial cells in the cerebral cortex (references 11, 16). It is known that not all radial glial cells Express Pax-6. In particular cells, localized in the ganglion tubercle, does not Express Pax-6 and are not neurogenic (reference 11). Previously made an interesting observation, namely, that add RK to methadone treatment leads to the induction of Wnt signaling antagonist sFRP2 (reference 36). It is possible that suppression of Wnt signaling antagonist molecules present in the development of forebrain induces the cells of the phenotype of radial glial cells. Spatial and temporal expression patterns sFRP1 combined with this observation (reference 37).

In the conditions of the present invention in vitro, the key is EXT is the relation RK (reference 38). If after 4 days of processing RK virtually all cells in the ST Express Pax-6 in the absence of RK Pax-6-positive cells can be detected, and after dissociation raw Mat neurons were not found. Although it seems unlikely that the ROK has a physiological effect, which lies in the induction of Pax-6 in the developing cerebral cortex, it can happen in other parts of the developing CNS. In fact, although Pax-6 has a restricted expression pattern in the CNS, including the cerebral cortex, the expression of Pax-6 in most parts of the ventral portion of the neural tube during development, and the results indicate that the segment is a derivative of RK has physiological significance in the development of certain patterns the ventral portion of the neural tube (REFs 39, 40). In the case of methadone-treated RK, Renoncourt and others (reference 28) and Wichterle and others (reference 7) also observed that some cells in the ST are Pax-6-positive after treatment of the RK. However, Pax-7-positive cells were also observed under similar exposure (reference 7), which confirms the heterogeneity of cells after treatment methadone retinoic acid (RK).

On poly substrate coated with laminin, radial glial cells quickly lose their typical fusiform shape. When using lines EGP-ES the number fluoresce is their cells increases rapidly and that the size and shape of neurons look highly homogeneous. To 4 days after the dissociation of almost all cells have neuronal characteristics. In the subsequent period, almost all neurons receive a pyramidal shape and are positive for the presence of vesicular Transporter glutamine. All cells stained at different times, regardless of their identity. In contrast, less than 0.1% of the cells clearly positive, which follows from staining after 1 week in culture with antibodies to Is1-1, tyrosinekinase or polytransfused. Three weeks less than 5% of the cells are vGAT-positive. The lack of staining of GABA and Is1-1 excludes the number of interneurons and neurons great length, including, in particular, motor neurons, many of which are also derived Pax-6-positive cells in vivo. Presumably, inductive signals, for example, sonic hedgehog, must be present for the movement of the offspring of Pax-6-positive radial glial cells on a particular path of differentiation (reference 7). Glutamatergic phenotype of neurons in the present invention corresponds to their identity as cortical pyramidal neurons, which follows from their form. Most importantly, this characteristic is consistent with the observation that these neurons originate from radial glial cells. Indeed, earlier Malatesta and others (reference 11) found that the offspring of cortical what's radial glial cells is pyramidal neurons located in all layers of the cortex and in the hippocampus. Thus, provided in the present invention, the culturing conditions can be described as "permissive (permissive), allowing the internal program of differentiation of glial cells to turn in vitro. In accordance with that used in the present invention, the environment was initially created to support the survival and differentiation of pyramidal neurons isolated from the hippocampus of rodent embryos (reference 12). The property of this environment is the ability to prevent or to repress reproduction of such cells as astrocytes. We should expect that these cells are present in the cultures obtained according to the present invention, because they also belong to the progeny of radial glial cells.

Using GFAP antibodies see the development of several branched astrocytes in culture. However, their number is too small (in the range of 1-2% of the total number of cells after 3 weeks).

The relative homogeneity derived neuronal cultures stimulated to investigate the expression of membrane proteins, known as they are expressed in specific terms of development. In accordance with the obtained in vivo results (reference 23) found that the expression of P75 is closely correlated with the appeared who eat neurons in these cultures and later regulation is reduced. On the contrary, although the expression of Trk receptor is not detected at early stages, it increases sharply after a few days, confirming that the development of neurons occurs synchronously. High levels of expression of the Trk receptor are characteristic of pyramidal neurons in vivo (reference 22). Experiments using the method of PCR confirm that and TrkB, and TrkC affect the signal obtained with the use of pan-Trk antibody, although in a few the first day in vitro only detect the expression of TrkA. Unlike receptors P75 and Trk, APP clearly detect after a 2 h after dissociation ST and their levels increase during the course of neuronal differentiation. This is in line with the results of the immunohistochemical experiments, showing that APP specifically marks radial glial cells in the developing cerebral cortex of rodents (reference 24).

Materials and methods

Materials

The ingredients for the environment for cell culture were obtained company Gibco, LIF obtained from the company Chemicon, PEP and original solutions for N2 and environment complete environment - from the company Sigma. Powder fraction V BSA manufactured by Gibco. Laminin was isolated from the cells of sarcoma Engelbreth-Holm-Swann (firm Roche). PK purchased the company Sigma, and the results did not differ when using different doses.

Antibodies

Primary antibodies for immunocytochemistry reveal the Xia mouse monoclonal antibody Intenstinal (rat401, IgG1; 1:10; firm DSHB - Bank hybrid for scientific research), mouse monoclonal antibody RC2 (IgM; 1:4; firm DSHB), rabbit polyclonal antibody anti-BLBP (1:2000; courtesy of N. Heintz for M. Goetz, Rockefeller University, new York), mouse monoclonal antibody anti-RH (IgG1; 1:100; firm DSHB), mouse monoclonal antibody anti-β III (IgG2b; 1:100; firm Sigma) and rabbit polyclonal antibody anti-vGlut1 (1:5000; firm SYSY). Subclass-specific Cy2 - or Cy-3-related antisera used as secondary antibodies. For Western blotting is used mouse monoclonal antibody anti - synaptophysin (IgG1; 1:1000; firm Sigma), rabbit polyclonal antibody anti-GluR1 (1:1000; firm Upstate), rabbit polyclonal antibody anti-Trk (C-14, sc-11; 1:1000; firm Santa Cruz), rabbit polyclonal antibody anti-APP (1:3000; courtesy of P. Paganetti, Novartis, Basel), rabbit polyclonal antibody anti-P75 (1:2000; firm Promega).

Environment

Medium for ES cells (500 ml):

DMEM410 ml
FTS (fetal calf serum)75 ml

(inactivate by heating at 55°C, 30 min)

LIF 5 ml
Glutamine5 ml
Amino acids that are not related to irreplaceable5 ml
β-Meon5 ál

Environment for methadone treatment (500 ml):

DMEM440 ml
FTS50 ml
Glutamine5 ml
Amino acids that are not related to irreplaceable5 ml
β-Meon5 ál

Wednesday N2:

DMEM125 ml
Glutamine1.25 ml
F-12, Gibco, catalog number 21765029)125 ml
Insulin1.25 ml 25 mg/ml
Transferrin6,25 ml 50 mg/ml
Progesterone0.25 ml 6 ng/ml
Putrescine 0.25 ml 16 ág/ml
Sodium Selenite25 μl of 30 nm
BSA (bovine serum albumin)1.25 ml 50 mg/ml
P/S2.5 ml 1%

P/S denotes antibiotics, such as penicillin/streptomycin. Antibiotics may not necessarily be excluded from the environment and replaced with an equal volume of DMEM.

Original solutions for N2 environment:

BSA company Gibco, catalog number A-9418, powder fraction V 100 g

Aliquots of 10 mg/ml stored at -20°C

The final concentration of 50 μg/ml

Insulin firm Sigma, catalog number I-6634 100 mg

The original solution of 5 mg/ml in water (acidified with a drop of concentrated HCl to pH 2 for dissolving insulin)

Stored at -80°C

Transferrin company Sigma, catalog number I-1147, apotransferrin person 100 mg

The original solution of 2 mg/ml in water

Stored at -80°C

Progesterone company Sigma, catalog number P-8783 5 g

The original solution of 2 mm in ethanol stored at -80°C.

The working solution of 20 μm obtained by dilution of the original solution in water and stored at -80°C.

Putrescine company Sigma, catalog number P-5780

The original aqueous solution 100 μm stored at -80°C.

Sodium Selenite company Sigma, catalog number S-5261 25 g

The original aqueous solution of concentratie is 300 μm keep at 4°C.

Complete environment:

Aqueous solutions:

L-alanine (Sigma, catalog number A-7627) [original solution of 2 mg/ml] 2 mg/ml

Biotin (Sigma, catalog number B-4501) [original solution of 0.1 mg/ml] 0.1 mg/ml

L-carnitine (Sigma, catalog number C-0283) [2 mg/ml] 2 mg/ml

Ethanolamine (Sigma, catalog number E-9508) [1 mg/ml) 1 mg/ml

D - galactose (Sigma, catalog number G-0625) [15 mg/ml] 15 mg/ml

L-Proline (Sigma, catalog number P-0380) [7,76 mg/ml] 7,76 mg/ml

Putrescine (Sigma, catalog number P-7505) [16,1 mg/ml] 16,1 mg/ml

The sodium pyruvate (Sigma, catalog number P-5280) [25 mg/ml] 25 mg/ml

Sodium Selenite (Sigma, catalog number S-1382) [0,016 mg/ml] 0,016 mg/ml

Vitamin B12 (Sigma, catalog number V-2876) [0,34 mg/ml] 0,34 mg/ml

Zinc sulfate (Sigma, catalog number Z-4750) [0,194 mg/ml] 0,194 mg/ml

Catalase (Sigma, catalog number C-40) [16 mg/ml] 16 mcg/ml

Glutathione (Sigma, catalog number G-6013) [1 mg/ml] 1 mg/ml

SOD (Sigma, catalog number S-2515) [2.5 mg/ml] 2.5 µg/ml

Ethanol solutions:

Linoleic acid (Sigma, catalog number L-1376) [100 mg/ml] 1 mg/ml

Linolenic acid (Sigma, catalog number L-2376) [100 mg/ml] 1 mg/ml

Progesterone (Sigma, catalog number P-8783) [0,63 mg/ml] of 6.3 ng/ml

Retinol (TRANS-form) Retinol (Sigma, catalog number R-7632) [10 mg/ml] 100 ng/ml

Reinjected (firms is Sigma, catalog number R-7882) [10 mg/ml] 100 ng/ml

Tocopherol (Sigma, catalog number T-3251) [100 mg/ml] 1 mg/ml

Tocopherol acetate (Sigma, catalog number T-3001) [100 mg/ml] 1 mg/ml

Dissolve to:

BSA1 g
Transferrin2 mg
Insulin1.6 mg
Glutamine2 mm
P/S (optional)1%

400 ml DMEM and add the above solutions.

Culture of ES cells

Originally ES cells cultured on feeding the cells representing inactivated by mitomycin fibroblasts mouse embryos, at least for two passages after thawing. Subsequent passages of ES cells cultured without feeder cells, and differentiation may begin either immediately through at least two passage without feeder cells, or biomass frozen ES cells without feeder cells. The biomass of cells used for differentiation, passedout at least twice prior to the procedure. After culturing ES cells on power cells are very important is the first passage on the environment b is C feeder cells. Successful differentiation depends on the density of ES cells used for the first passage. ES cells must occupy at least one third Cup after 1 day after splitting. The base medium for ES cells is DMEM containing 15% FTS (fetal calf serum specially studied for the culture of ES cells, subsequently undergoing neuronal differentiation), LIF (1000 U/ml), non-essential amino acids and β-mercaptoethanol. In the Cup for cell cultures always put a solution of 2% gelatin for at least 10 minutes found that the incubation temperature is an important factor and at a temperature above 37°C, successful differentiation does not occur. Each environment is pre-heated to 37°C.

ES cells are split every 2 days and with a certain density component of 1.5×106-4×106cells were seeded into plates for cell cultures with a diameter of 10 cm (Corning). After 2 days 10-25×106cells can be selected, and their high proliferative activity is a necessary condition for a successful experiment. The cells are in a rapid growth phase and form a flat monolayer.

Splitting cells carry out the washing to twice the amount of PSF and incubation of cells in a thin layer of a solution of trypsin (once in the trypsin solution company Gibco - 0,05%, 02% EDTA) at 37°C, 7% CO2within 3 min, the Cup is shaken manually cells separated and resuspended in new net environment for ES cells by suction and blowing from a pipette (inactivation of trypsin). Then centrifuged 5 min at 1000 rpm at room temperature. The precipitate again resuspended in new net environment for ES cells using a pipette several times sucking and blowing suspension. Cells should be dissociativity to obtain a culture of individual cells, although there may be aggregates of 2-3 cells, larger clusters of cells should not be. The required number of cells re-seeded in the Cup, the surface of which is coated with gelatine.

For deprivation of ES cells from feeder cells can be cultivated after thawing approximately twice the supply cells and then at least 2 times passionate without feeder cells in such a way as to remove fibroblasts. ES cells, thus, change shape with colonizadores on the flat.

The thawing of ES cells enables rapid thawing of the original tubes with ES cells in an amount of 3×106, re-suspension of the cells in 10 ml medium for ES cells and centrifugation for 5 min at 1000 rpm at room temperature. Precipitated cells resuspended again in the medium for ES cells the number of cells seeded into the Cup for cell culture diameter, the rum 6 see Freezing of ES cells is carried out by resuspendable cells, split after treatment with trypsin and centrifugation in the medium for ES cells + 10% DMSO.

The Protocol neuronal differentiation

For the formation of the Mat 3×106ES cells are placed in leatherete bacteriological Cup (firm Greiner) in 15 ml of medium ES (environment ES without PPL and only 10% FTS) and incubated for 8 days.

Wednesday replaced every two days, which will remove the entire cell culture with bacteriological cups (in a test tube of 50 ml volume of the company's Falcon) and give methadone to settle (approximately 3-5 minutes). Then the supernatant carefully sucked off and to the Mat again add 15 ml of medium for methadone treatment. ST carefully resuspended in the environment using a pipette with a wide tip in order to avoid the decomposition or dissociation of methadone (for example, using a 10 ml plastic pipette).

RK (Sigma) in an amount of 5 mcmole add in 4 days directly into the Cup and distribute accurate rocking Cup. RK should not be left long in the light, because it is destroyed by exposure to light. ST then subjected to dissociation and cells were seeded into plates coated PEP/laminin as follows.

In cups for cell culture contribute to the solution of 10 μg/ml of PEP in borate buffer (150 mm, pH 8,4) and placed overnight in an incubator (37°C, 7% CO2 ). Poliorcetes also used at a concentration of 100 μg/ml with similar results. After triple washing of cups FSB (when using polymethine washed with H2O) laminin (approximately 0.5 μg/cm2) are added directly to the solution of the FSB and the Cup returned to the incubator for at least 2 hours

After 8 days of formation obtained methadone washed 2 times the FSB and treated with trypsin by incubation ST for 3 min in a water bath at 37°C in 0.05% solution of trypsin and 0.04% EDTA/FSB (freshly prepared solution with trypsin powder, TRNC-processed, firm Sigma). During the incubation tube company Falcon twice manually gently shaken, and then the disintegration of cells ST becomes noticeable. Dissociatively ST gently, but thoroughly, resuspended in 10 ml of medium Mat containing serum to inactivate the trypsin. Dissociation can be performed by approximately fivefold sucked into the pipette and blow out pipettes. The best way dissociation spend Pasteur pipette a small amount with drawn in the flame tip (approximately 1.5 ml), and then a plastic pipette with a volume of 5 ml. After grinding centrifuged 5 min at 1000 rpm at room temperature. The supernatant is completely removed, the residue resuspended under N2 and the cell suspension was filtered through nylon is iltr with a cell size of 40 μm (firm Falcon).

Laminin remove cups from the floor and immediately add cell suspension, preventing drying cups. Dissociatively cells were seeded with a density of 1.5×105cells/cm2. Wednesday N2 replaced after 2 h and again after 1 day. After 2 days Wednesday, replace enriched not containing serum medium described by Brewer and Cotman (reference 12), but in a modified version - without glutamate, HEPES, corticosterone, lipoic acid and T3.

Neuronal differentiation is complete and neuronal cultures can be maintained for several weeks.

Immunocytokine

Cover glasses prepared by washing in water and immersion in 65% nitric acid for 1-2 days. Later platinuum in H2O for several hours, washed with ethanol, dried in air and sterilized by UV light. Cells fixed with 4% paraformaldehyde (PFA) for 10 min, washed with FSB and blocked for 1 h in blocking buffer (0.03% of carageenan, 10% NGS and 0.3% Triton X-100). The drug is mounted in AquaPoly/Mount (company Polysciences).

Western blotting

Dissociatively ST seeded according to the above method, and samples for Western blotting gather at certain deadlines. Before selecting cells from cups washed them twice ice-cold phosphate-saline buffer (FSB). Extracts of whole cells prigoda is more in 750 μl of lytic buffer and placed in a Cup with a diameter of 6 cm (50 mm Tris pH 7,4, 150 mm NaCl, 10% glycerol, 1% Triton X-100), to which was added a mixture of protease inhibitors (firm Roche). After centrifugation for 30 min at 4200 rpm in a centrifuge company Eppendorf remove supernatant and the protein content determined using the method of DC Protein Assay, BioRad). The samples boiled in Laemmli buffer and 5 µg placed in polyacrylamide gels. Stain blocking with 5% milk solution, incubated overnight with the first antibody and 2 h with the secondary antibody. The determination is carried out with the help of the device ECL Plus, Amersham).

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1. Method induced the door is the embryonic stem (ES) cells into neuronal precursor cells, including
cultivation of ES cells,
the formation of cell aggregates (CA),
processing cell aggregates (CA) retinoic acid (RK) and
dissociation KA to obtain a culture of neuronal precursor cells,
in which the cultivation of ES cells includes a passage through seeding ES cells with a density of about 0.5×105- 2×105cells / cm2and dissociation of ES cells in 2 days after seeding, and the formation of the CA includes the selection of ES cells with high proliferative activity with a doubling time from 0 to 24 h and the seeding of these cells with a density of about 0.5×105- 5×105cells per ml for the formation of the AC.

2. The method according to claim 1, in which the formation of CA includes seeding ES cells with a density of about 2.5×105it is 3.5×105cells in ml.

3. The method according to claim 1 or 2, in which KA is supported in leatherete culture to the dissociation of cell aggregates.

4. The method according to claim 1 or 2, including the study of morphology of ES cells and selection of morphologically homogeneous ES cells to form CA.

5. The method according to claim 4, including the selection of ES cells with one or more of the following morphological characteristics: growth in the form of a flat monolayer, the lack of direct contact between neighboring cells, large nuclei, a large number of nucleoli, the absence of cell growth surface is px each other or in the form of, reminiscent of the colony.

6. The method according to claim 1 or 2, comprising determining the stage of proliferation of ES cells and selection of cells with high proliferative activity for the formation of the AC.

7. The method according to claim 1 or 2, in which the cultivation of ES cells involves the passage of ES cells in the absence of feeder cells.

8. The method according to claim 7, in which the passage is repeated at least twice in the absence of feeder cells.

9. The method according to claim 1 or 2, including the dissociation of ES cells to obtain a suspension of individual cells, in which less than approximately 5% of the cells form aggregates of 4 or more cells, and seeding cells for the formation of the AC.

10. The method according to claim 1 or 2, in which the dissociation KA is the dissociation of cells KA trypsin to form a suspension of dissociated cells KA, followed by filtering the suspension to remove accumulations of cells.

11. The method according to claim 10, in which the cells dissociated KA filtered through a mesh size of about 40 microns.

12. The method according to claim 1 or 2, providing an additional stage of storage cells dissociating KA by freezing the cells.

13. The method according to claim 1 or 2, further comprising seeding and culturing the dissociated cells KA to obtain neurons and optionally culturing neurons.

14. The method according to item 13, in which the cells dissociated KA sow splatnosti approximately 0.5×10 5is 2.5×105cells / cm2.

15. The method according to 14, in which the cells dissociated KA seeded with a density of approximately 1×105to 1.5×105cells / cm2.

16. The method according to item 13, including the replacement of the culture medium of cells dissociated KA in approximately 1-6 hours after seeding cells dissociated KA.

17. The method according to clause 16, which includes the replacement of culture medium after about 1-3 h after seeding.

18. The method according to item 13, in which the cells dissociated KA or neurons are not cultivated in the presence of serum.

19. The method according to item 13, in which neuronal precursor cells or neuronal cells not cultured in the presence of growth factors.

20. The method according to claim 19, in which neuronal precursor cells or neuronal cells not cultured in Neurobasal medium.

21. The method according to claim 1 or 2, in which there is no stage of culling cells.

22. The method according to claim 1 or 2, in which ES cells are ES cells human.

23. The method according to claim 1 or 2, including the identity of at least 80% dissociated cells KA as neuronal precursor cells.

24. The method according to claim 1 or 2, including the identity of at least 99% of the cells dissociated KA as neuronal precursor cells.

25. The method according to item 13, including the identity of at least 80% of the cells in which the number of neurons.

26. The method according A.25, including the identity of at least 90% of the cells as neurons.



 

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