KR101385509B1 - Method for Differentiating of Embryo Stem Cell into Nurons Using Substrate having Nano-Patterened Surface - Google Patents

Abstract

The present invention relates to a method of differentiating embryonic stem cells into neurons, characterized in that a support having a patterned surface is used. More particularly, the present invention relates to a method of differentiating embryonic stem cells into neural cells, The present invention relates to a method for differentiating human embryonic stem cells into neurons in a short time by culturing human embryonic stem cells.
According to the present invention, it is possible to efficiently differentiate embryonic stem cells into desired nerve cells on the surface of a nano unit pattern. Especially, since the increase in the length of nerve cells is elongated with a certain direction along the pattern, it is effective as a cell therapy agent.

Description

TECHNICAL FIELD The present invention relates to a method of differentiating embryonic stem cells into nerve cells using a support having a patterned surface having a nano unit,

The present invention relates to a method of differentiating embryonic stem cells into neurons, characterized in that a support having a patterned surface is used. More particularly, the present invention relates to a method of differentiating embryonic stem cells into neural cells, The present invention relates to a method for differentiating human embryonic stem cells into neurons in a short time by culturing human embryonic stem cells.

It has long been known that pluripotent cells such as embryonic stem (ES) cells can differentiate into neurons in vitro. In principle, working with ES cells leads to the possibility of isolating cells and characterizing neuronal precursors at selected differentiation stages. ES cells have stimulated the study of molecular and gene-based pathways in vitro and are a potential source of cells for transplantation into the brain to treat neurological diseases.

However, these and other applications are hampered by heterogeneous, fissionable and often non-renewable properties of neuronal development in culture. Cell heterogeneity is a major problem in using ES cells to generate neurons.

Typically, neuron cultures derived from ES cells contain non-neuronal cells, including glial cells, as well as a variety of different neuronal subtypes. The lack of a sufficient number of cells with a defined and uniform phenotype is a major difficulty in neurobiology

There is no way to generate neurons from ES cells, leading to a certain number of neurons or limited populations of neurons, and thus a homogeneous population of cells can be obtained in sufficient quantities to characterize brain neurons using a biochemical approach none. In addition, the systematic relationship between neurons and nerve precursors remains unclear.

In view of the use of ES cell-derived neurons for transplantation, contrary to the mixture of cells comprising some cells which can continually isolate and form tumors, it is desirable to obtain defined primitive cells which induce known offspring cells . The xenogeneic cells may also interfere with nutritional and / or instructional signals from the host tissue that facilitate incorporation of the implanted tissue into the brain.

The transplanted cell type is functionally important and dopaminergic neurons may be required to treat diseases such as, in particular, Parkinson's disease, and thus for such medical applications, the precursor and neuronal cell type An increase in the control for the above is desirable. By increasing the cell population with the desired neuronal lineage, a reduction in cell heterogeneity is required to reduce undesirable side effects, lower the risk of tumors and improve the therapeutic potential.

Recently, advances have been made through the use of inductive signals and transcription factors, in order to substantially enrich the subtype of neurons, including dopaminergic neurons and motor neurons, in particular. In other words, while transcription factors such as Nurr1 or other types of co-culture with stem cells significantly increase the development of dopaminergic neurons, the addition of external factors, including sonic hedgehog, , Which has been found to integrate into post-transplant host tissues. However, despite these advances, there is still little known about the in vitro development of defined neuronal precursors that can cause a particular neuronal phenotype.

There are a number of different protocols that describe the development of neurons and glial cells. As previously recognized for embryonic carcinoma cell line P19, treatment of pluripotent cell aggregates with retinoic acid leads to neuronal differentiation. Subsequently, treatment of ES cell-derived somata (EBs) with RA was observed to promote neurons and inhibit mesodermal gene expression. EBs are 3-dimensional assemblies caused by aggregation and proliferation of ES cells. EBs can be produced by culturing ES cells on a bacterial culture dish, typically on a substrate on which they can not be assimilated.

Usually, early ES cell cultures are performed on nutrient cell layer supports (inactivated fibroblasts) to maintain ES cells in the form of colonies of pluripotent undifferentiated ES cells. Fibroblasts are thought to support the undifferentiated state of ES cells. Leukemia inhibitory factor (LIF) may be included in the culture medium to inhibit differentiation. However, even in the presence of LIF, it has been observed that some ES cells tend to differentiate and that different lines of cells can be observed during EB formation

Growth factors, etc., and to differentiate neural cells from stem cells through an induction medium containing antioxidants such as DMSO and BHA, but the results are still insufficient. In addition, the conventional techniques have a problem that the differentiation induction time is as long as 24 hours, and that the mark of differentiation into neural cells is not expressed to a sufficient degree as it is differentiated into mature neurons.

Accordingly, the present inventors have found that, when human embryonic stem cells are cultured on a surface having a pattern of a certain specific size, the embryonic stem cells can be efficiently and rapidly administered to non-neuron cells including glial cells And the present invention has been completed.

It is an object of the present invention to provide a method of culturing embryonic stem cells on a support having a patterned surface in a nanosized form without neuronal differentiation inducing factor to differentiate into neurons.

It is another object of the present invention to provide a support having a nano-patterned surface for neural cell differentiation of embryonic stem cells.

It is still another object of the present invention to provide a cell therapy agent containing neurons differentiated from embryonic stem cells obtained using the above method and extending in the direction of a pattern.

In order to achieve the above object, the present invention provides a method of differentiating embryonic stem cells into neurons using a support having a surface patterned in a nano unit.

The embryonic stem cell is most preferably derived from a mammalian tissue, especially a human tissue.

The patterning is characterized by having a plurality of longitudinal groove / groove structures. In particular, the patterning preferably has a size of 350 nm, 600 nm, 800 nm, and most preferably a size of 350 nm.

The differentiation method of the present invention is characterized by not using conventional neurogenic differentiation factors such as retinoic acid and laminin.

In addition, the support having the patterned surface may be any material that has no resistance to the implantation of a human body, can easily produce a desired shape or pattern, and is excellent in biocompatibility. For example, a polyurethane acrylate , PUA) can be used.

At this time, it is preferable that gelatin is coated on the surface of the sheet such as the patterned PUA in order to attach the embryonic stem cells. In particular, in order to enhance the adhesion ability of embryonic stem cells, It is more preferable to carry out the oxygen plasma treatment.

The present invention also provides a cell therapy agent for treating neurological diseases, which is obtained by the above method and is fixed to a support having a patterned surface and contains neurons extending in the longitudinal direction of the pattern.

According to the method of the present invention, the neurons differentiated from embryonic stem cells elongate in the longitudinal direction of the pattern, and the neurons are mature neurons (nerve cells) rather than glial cells such as astrocytes.

Examples of the neurological diseases include degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, Mad Cow disease, Prion disease, Huntington disease, Amyotrophic lateral sclerosis, and Pick's disease.

Further, the present invention provides a support for neuron differentiation of embryonic stem cells comprising:

A polyurethane acrylate (PUA) sheet patterned to a size of 300 nm to 800 nm;

An oxygen plasma layer treated on the sheet; And

A gelatin layer coated on the oxygen plasma layer.

In addition, the present invention provides a method for producing a support for neuron differentiation of embryonic stem cells, comprising:

(a) curing the PUA prepolymer with ultraviolet light to the silicon main feature;

(b) peeling the cured PUA mold;

(c) patterning the PUA mold to a size of 300 nm to 800 nm and contacting the PUA precursor on a treated glass cover slip;

(d) curing with ultraviolet light and then stripping;

(e) coating the nano-patterned surface of the peeled sheet with a gelatin solution.

At this time, it is preferable to further perform oxygen plasma treatment on the nano-patterned surface of the sheet prior to the step of coating with the gelatin solution to improve the ability to adhere embryonic stem cells.

In the present invention, since embryonic stem cells can be differentiated into nerve cells on a nano-unit pattern support in a short period of time without specific neuronal differentiation inducing factors, and since the increase in the length of differentiated neurons has a certain directionality, (Such as spinal cord injury or nerve injury).

In other words, when stem cells are differentiated into neurons and are differentiated in a short time, they are advantageous for treatment as well as the length of differentiated neurons is sufficiently increased to regenerate broken nerves. It is more effective to use the nano unit pattern support of the present invention.

FIG. 1 is a schematic diagram and a scanning electron microcopy (SEM) image of a patterned array fabricated in the nano unit of the present invention.
Figure 2 is an SEM image of a cover slip patterned with a conventional dry-coating method and coated with gelatin.
FIG. 3 is a photograph of cultured hESCs on a patterned support, which is a comparative photograph of cases (a and b) in which only gelatin coating is performed and cases (c and d) in which gelatin coating is performed after oxygen plasma treatment.
FIG. 4 is a comparative graph (p < 0.001) for hESC adherence with or without oxygen plasma treatment.
Figure 5 is a SEM image and fluorescence image observing the differentiation state of hESCs on a flat surface and a 350 nm-patterned surface.
Figure 6 shows gene expression analysis results for hESCs, undifferentiated hESCs, and differentiated hEB5 differentiated on a flat surface and a 350 nm-patterned surface.
Figure 7 is an immunostained photograph showing the results for neuronal differentiation on a flat surface and a 350 nm-patterned surface over time.
FIG. 8 is a photograph showing immunofluorescent staining of hESCs differentiated by the method of the present invention with Nerual cells and glial cell markers.
FIG. 9 is a photograph of immunofluorescent staining with various nerve cell markers.

The terms used in the present invention are defined as follows.

The term "differentiation" refers to a phenomenon in which cells and tissues specialize in structure or function, while cells grow and proliferate, that is, the form or function changes to carry out the task given to each of them . In general, a relatively simple system is separated into two or more qualitatively different systems. For example, there may be qualitative or quantitative differences between parts of a biological system that were initially homogeneous, such as head or trunk distinction between eggs that were initially homogeneous in origin, or distinctions of cells such as muscle cells or neurons The state in which there is a difference or as a result is divided into qualitative or inferior parts or partial systems is called differentiation.

"Undifferentiated" refers to a property that indicates undifferentiated state which can be confirmed by expression of at least one undifferentiated marker in pluripotent stem cells, for example, expression of ALP activity or 0ct-3/4 gene (product) . The undifferentiated state of pluripotent stem cells is a state in which pluripotent stem cells are capable of proliferating almost permanently or for a long time, exhibiting a normal nucleus (chromosome) type, capable of differentiating into all lineage cells of the third leaf under appropriate conditions .

"Pluripotent stem cells" are cells that can be almost permanently or prolonged cell proliferation while retaining their undifferentiated state by in vitro culture and exhibit normal nucleus (chromosome) type, and under the proper conditions, they have three trimesters (ectoderm, mesoderm, Quot; means cells capable of differentiating into all the lineages of the lineage. "Pluripotent stem cells" are ES cells and their like cells isolated from early embryos.

Hereinafter, the present invention will be described in detail.

The present inventors have found that when human embryonic stem cells are cultured on a surface having a pattern of a certain specific size, the embryonic stem cells are effectively differentiated into neurons in a short time without adding any neural differentiation inducer, The present invention relates to a method for differentiating new embryonic stem cells into neurons based thereon.

Accordingly, the present invention relates to a method of differentiating embryonic stem cells into neurons, characterized in that a support having a surface patterned in units of nanometers is used.

The undifferentiated cells used in the present invention are embryonic stem cells.

Embryonic stem cells are cell lines obtained from early embryos and are normal non-transformed cells capable of cell proliferation in an undifferentiated state. It is also pluripotent that can differentiate into various tissues and germ cells when re-implanted in blastocyst. Therefore, it is possible to supply endogenous embryonic stem cells through cell culture, and theoretically, it is recognized as the best source of cell therapy because it can differentiate into cells of all tissues. In addition, embryonic stem cells can be used for gene targeting by homologous recombination, so that they can produce better cells through gene manipulation and make their own embryonic stem cells by nuclear replacement, The immune rejection reaction, which is a problem of the therapeutic method, can be fundamentally solved. In the present invention, mammalian embryonic stem cells can be used, preferably human embryonic stem cells.

The human embryonic stem cell refers to an omnipotent cell derived from an inner cell mass of a human cell. Human embryonic stem cells include, for example, CHA-hES3 (Ahn SE, Kim S, Park KH, Moon SH, Lee HJ, Kim GJ, Lee YJ, Park KH, Cha KY, Chung HM. Biochem Biophys Res Commun. (2006) 10; 340 (2): 403-408), but the present invention is not limited to these examples.

The embryonic stem cells are obtained by transferring a cell mass (cell mass) called an inner cell mass inside an embryo of a blastocyst stage to an in vitro culture and repeating dissociation and passage of the cell mass, Can be isolated as a stem cell population. The cells can also be used as feeder cells prepared by using interstitial (stromal line) cells such as mouse embryonic fibroblasts (hereinafter, AEF cells) or ST0 cells cells can be established as a cell line having the properties of undifferentiated stem cells by maintaining the optimal cell density and repeating the subculture while frequently changing the culture medium.

The term "feeder cell" is also referred to as a support cell, and refers to a cell which is cultured in advance when culturing cells that can not survive or cultivate alone, and plays a role of supplying nutrients or growth factors lacking in the medium. "Feeder cell" includes, but is not limited to, MEF cells, and stromal line cells such as STO cells. As the supporting cells of human embryonic stem cells, usual supporting cells can be used. For example, mouse fibroblasts, mouse embryonic fibroblasts, human embryonic fibroblasts, human bone marrow cells, adult epithelial cells and the like can be used. Mouse fibroblasts can be preferably used since double mouse fibroblasts can more stably maintain undifferentiated state and growth of human embryonic stem cells.

In addition, as the culture medium for culturing human embryonic stem cells, any well-known culture medium for culturing human embryonic stem cells can be used, for example, serum replacement (SR), mercaptoethanol, non-essential amino acid, and bFGF KO-DMEM (KNOCKOUT Dulbecco's modified Eagle's medium, KO-DMEM).

In order to use embryonic stem cells for cell therapy, it is necessary to differentiate into cells of a desired tissue. When the MEF is removed under culturing conditions and the basic fibroblast growth factor (bFGF) is subtracted, the embryonic stem cells naturally begin to differentiate. At this time, when grown on a hydrophobic culture dish such as a bacterial culture dish, the embryonic stem cells A mass of cells less than 1 mm in size is called an embryoid body (EB).

As a method of differentiation of embryonic stem cells, gene manipulation method, inducer treatment method, coculture method, cell separation method and the like have been used so far. Each differentiation method has advantages and disadvantages and various attempts have been made to achieve perfect differentiation by properly mixing various methods.

In addition, a review of several references, such as Guide to Techniques in Mouse Development (Wasserman et al., Academic Press, 1993), as a review of ES cell characteristics, culture methods, and in vivo / in vitro differentiation potential; Embryonic Stem Cell Differentiation in vitro (MV Wiles, Meth. Enzymol. 225: 900, 1993); Manipulating the Mouse Embryo: A laboratory manual (Hogan et al., Cold Spring / Harbor Laboratory Press, 1994); Embryonic Stem Cells (Turksen, Humana Press, 2002).

The method of the present invention is characterized in that embryonic stem cells are cultured on a supporter having a nano-sized patterned surface in differentiating the embryonic stem cells into neurons.

In this case, the 'patterned surface' means having a plurality of longitudinal groove / groove structures, and the 'pattern size' means a PUA support manufactured by a silicon mold as shown in FIG. 1, Which corresponds to the width of the portion darkened in FIG. 1, which means the gap size of the concave groove.

The present invention has a pattern having a size of 300 nm to 800 nm, which is suitable for differentiating a specific undifferentiated cell called embryonic stem cell into a specific cell called a neuron, more preferably a pattern having a size of 300 nm to 350 nm. In one embodiment of the present invention, a pattern having a size of 350 nm was used.

The size of this pattern is a decisive factor in differentiating embryonic stem cells into specific types of cells called neurons.

In other words, when the pattern size according to the present invention is applied, the differentiation of human embryonic stem cells into neurons is effectively induced. This is because, compared to the case where the differentiation is induced in the absence of the pattern, Is remarkably large. It can be inferred that this specific size of the pattern creates the most suitable physical environment for the neurite to grow longitudinally along the pattern.

In particular, the increase in the length of the nerve cell that grows with a certain direction is more likely to occur when the nerve cell is disconnected during clinical application, It can be said that it is significant.

When the size of the pattern is different from the above range when the neural cell is differentiated, that is, when a pattern having a large size in units of micro or a pattern smaller in size than the present invention is used, the differentiation rate into a neuron is lowered .

That is, when the pattern is larger than the size of the cell, the cell recognizes it as a general plane, and the meaning of the uneven pattern disappears. In the smaller unit pattern, the cell attachment portion does not recognize it as the attachment point, . That is, it is judged that the pattern of irregularities of 300 nm to 800 nm promotes adhesion, elongation and migration of embryonic stem cells, and this is an essential condition for induction of neural differentiation

The method of the present invention for differentiating embryonic stem cells into neurons is characterized in that no known neuronal differentiation factor is used.

In order to differentiate stem cells into ectodermal cells such as neurons, they were conventionally cultured in a medium containing a biochemical differentiation inducing substance such as a soluble factor and a growth factor

As used herein, the term "growth factor" means a protein that binds to a receptor on the cell surface as a primary result of cell proliferation and differentiation activity. Examples of the soluble factor include neurotrophins, mitogens, , TGF-beta phase and agonist, neurotrophic factor, oxidant, neurotransmitter and survival factor. Many soluble factors stimulate cell division in a number of different cell types, while others are specific to certain cell types. Examples of typical differentiation agents that specifically promote neuronal cell type differentiation include progesterone, putrescine, laminin, insulin, sodium selenite, transferrin, neururin, sonic hedgehog (SHH), nogin, FGF-8, basic fibroblast growth factor (bFGF), growth and differentiation factor 5 (GDF-5), neurotrophin 3 (NT-3), fibroblast growth factor ), Transforming Growth Factor alpha (TGF-alpha), Transforming Growth Factor Beta-3 (TGF beta 3), Platelet Inducing Growth Factor (BDNF), Neurotrophin 4 (BMP-2, BMP-4), glial cell-derived neurotrophic factor (GDNF), retinoic acid (RA), midkine, ascorbic acid (E.g., LIF, CNTF, SCF, IL-11, and IL-6) for receptors that complex with an acid, dibutyryl cAMP, dopamine and gp130. In particular, it is important to treat retinoic acid or to treat neural stem cells with embryonic stem cells in a stepwise fashion, such as basic fibroblast growth factor (FGF2), epidermal growth factor (EGF), and platelet-derived growth factor It is known.

In this way, the differentiation of embryonic stem cells using a neuronal differentiation inducer is the most common method. The inducer added to the culture medium expresses a specific gene of embryonic stem cells, thereby changing growth and differentiation conditions to promote differentiation into specific cells .

However, when such differentiation inducing factors are used stepwise or in combination, there is a disadvantage in that not only the differentiation rate is high but also the differentiation time is long and the cost is high. In addition, there are limitations in the mechanism studies that can identify which factors are essential for the induction of neuronal differentiation due to the combined action of various factors.

Therefore, the method of the present invention, which does not use a neuronal differentiation inducing factor, overcomes the above-mentioned problems and exposes a remarkable effect that can shorten neuron differentiation time to 10 days or less at low cost, It can also be used as a model system for the mechanism research.

That is, the present invention is a method capable of efficiently differentiating embryonic stem cells into neural cells without inducing other neuronal differentiation factors.

In another aspect, the present invention relates to a support which can be used to differentiate embryonic stem cells into neurons as described above, and a method for producing the same.

In one embodiment, the present invention provides a method for producing the supporter for differentiating neuron cells, comprising the following steps.

(a) curing the PUA prepolymer with ultraviolet light to the silicon main feature;

(b) peeling the cured PUA mold;

(c) patterning the PUA mold to a size of 300 nm to 800 nm and contacting the PUA precursor on a treated glass cover slip;

(d) curing with ultraviolet light and then stripping;

(e) coating the nano-patterned surface of the peeled sheet with a gelatin solution.

In principle, any substance which can be used as a main component of the support in the present invention can be easily produced in a desired shape or pattern and has no biocompatibility when implanted in a human body. For example, PLGA poly (lactic-co-glycolic acid) and PEG poly (ethylene glycol) may be used, and polyurethane acrylate (PUA) is preferably used. In one embodiment of the present invention, the PUA was used.

Urethane acrylate is one of commercially widely used UV-curable oligomers. UV cured resins containing urethane acrylate usually have excellent friction resistance, rigidity, and flexibility. Therefore, UV curing type urethane acrylate is used as a binder of magnetic material, a continuous phase of ink, and a coating agent of vinyl tile, optical fiber, paper, wood, metal, plastic and the like because of such excellent physical properties. And UV coating is a process that has been attracting attention due to short reaction time, high energy efficiency, low curing temperature, proper space requirement, low operating cost.

Various methods have been proposed for the synthesis of urethane acrylate series in the reactive oligomer, but they are synthesized primarily by reacting a polyol with a diisocyanate to obtain an isocyanate at the terminal, is prepared by reacting an acrylate containing a hydroxy group with an isocyanate.

In the present invention, a 300 nm to 800 nm nano-sized ridge / groove pattern array can be fabricated on a glass cover slip by UV-assisted capillary force lithography using the PUA.

In the method for producing the support, photo-curing, in particular photo-curing by ultraviolet light, can be used as a method for curing urethane acrylate.

Photocuring using ultraviolet (UV), visible ray or electron beam (EB). Generally, free radicals or cations generated from the photoinitiaor by light irradiation. ) Initiates the initiation reaction and the monomer or oligomer having reactivity is cured through the continuous reaction.

UV curing is most widely used in the industry for most of the photocuring industry. Advantages of such ultraviolet curing are excellent cost performance, low energy consumption, very little emission of VOCs, easy curing facilities, It is possible to make materials having excellent chemical resistance and mechanical properties after curing.

The PUA mold produced using the photo-curing was patterned at a size of 300 nm to 800 nm, adhered to a glass cover slip treated with a PUA precursor, and then cured by ultraviolet rays to form a nano-sized patterned sheet .

And the PUA sheet is coated with gelatin for attachment of embryonic stem cells.

In one embodiment of the present invention, the adhesion of the hESCs is enhanced by coating the surface of the nano / groove pattern with 0.1% gelatin. Since there is a report that fibronectin and laminin promote the differentiation of hESCs and neural progenitor cells, gelatin was used for attachment of undifferentiated hESCs to prevent this and accurately examine the differentiation inducing effect of the pattern.

In order to improve the adhesion of the embryonic stem cells, it is preferable to perform the oxygen plasma treatment before the gelatin coating.

Plasma is a generic term for substances that are negatively charged in the system as a whole, with negative (+) and positive (+) charged particles moving freely. Plasma is the element or molecule excited and in the ionized state. Plasma chemistry is referred to as the chemical of the gas discharge or the chemistry of the excited state, which is a highly excited state of instability relative to normal gas.

The high temperature and active chemical properties of the plasma can be used to provide different physical and chemical properties to the body by changing the properties of the synthesis of new materials and the surface properties of metals or polymers by providing extreme environments that are difficult to obtain by conventional methods.

In addition, when the surface of the polymer is treated with nitrogen or an oxygen plasma, the surface of the polymer may have a dimensional or hydrophobic property, or may improve the antistatic property, dyeability, By applying a bias, a nitriding or carburizing layer is formed on the surface to improve the hardness, abrasion resistance and corrosion resistance of the metal. Some of the effects obtained by the surface coating and modification technique using plasma can be obtained by the conventional wet method, but considering the environmental problem, the plasma drying method has many advantages.

In the present invention, an oxygen plasma treatment can be additionally performed so as to take advantage of such plasma treatment. The treatment can be carried out by known methods.

Therefore, based on the above description, the present invention relates to a support for neuron differentiation of embryonic stem cells, which comprises, from another aspect, the following constituent elements.

A polyurethane acrylate (PUA) sheet patterned to a size of 300 nm to 800 nm;

An oxygen plasma layer treated on the sheet; And

A gelatin layer coated on the oxygen plasma layer.

Specific details of each component are as described above.

The present invention also relates to the differentiation of embryonic stem cells into "neural cells ". That is, it provides an improved method of inducing and / or promoting the generation and / or differentiation into neurons (neurons) or neuronal precursors or primitive cells.

As used herein, nerve cells are cells of the nervous system and include neural stem cells, neuronal precursors or primitive cells, and neurons (neuronal cells) unless otherwise indicated herein. The terms "neuron" and "neuron cell"

"Neural stem cells" refers to cell types capable of self-replicating and capable of producing all cell types in the nervous system, including pluripotent, i.e., neurons, astrocytes, and rare dendritic glial cells. Stem cells can express one or more of the following markers: Oct-4; Sox1-3; Stage specific embryonic antigens (SSEA-1, -3 and -4) (Tropepe et al., 2001, Neuron 30, 65-78). Neural stem cells can express one or more of the following markers: nestin; p75 neurotrophin receptor; Notch 1, SSEA-1 (Capela and Temple, 2002, Neuron 35, 865-875).

"Neuronal primate" refers to a progeny or descendant of neural stem cells and has a more differentiated phenotype and / or a further reduced differentiation potential than stem cells. A precursor cell is any cell that is not in direct lineage with neurons during development, but transfected or regenerated under defined environmental conditions, or other cells that can be induced to obtain a neuronal phenotype.

"Lineage" means a cell or a descendent of a cell derived from one defined cell type. "A-line" means the sub-type of a particular line.

In particular, the neurons obtained by the present invention are free from problems such as heterogeneity, fission and often non-renewable properties of neuronal development.

In general, neuron cultures derived from ES cells have had a problem of containing non-neuronal cells including glial cells as well as various different neuron-types.

Glial cells are the cells that support the tissues of the central nervous system and are the cells that function to supply the necessary substances to the neurons inside the brain and spinal cord and to create a chemical environment suitable for the activity of neurons. If neurons are responsible for the essential functions of neural tissue, they are located between blood vessels and nerve cells to support nerve cells, supply nutrients, remove waste, and phagocytosis. Astrocytes, astrocytes, small cells, Schwann cells, and satellite cells.

However, the neuronal cells differentiated by the method of the present invention do not contain glial cells (non-neuronal cells) such as astrocytes. Thus, the present invention solves the conventional problem that a neuron culture derived from ES cells is present.

On the other hand, the present invention, on the other hand, also provides the use of differentiated neural cells in embryonic stem cells to restore neural function. In particular, it provides therapeutic uses for neurological diseases caused by damage to nerve cells.

That is, the present invention relates to a cell therapy agent for treating a neurological disease, which is obtained by the above-described method in one aspect and which is fixed on a support having a patterned surface and contains neurons extending in the longitudinal direction of the pattern.

The cell therapy agent of the present invention replenishes (reconstructs) or restores (restores) damaged neurons.

"Regeneration" is a phenomenon in which a portion of a formed organs or an individual is lost when it is lost, and "restoration" is sometimes referred to as "reconstitution" It refers to reconstituting tissues or organs from dissociated cells or tissues.

This can be advantageously carried out by direct implantation into the lesion site in the form of a neuron or the composition containing it (cell therapy agent). Methods of neural transplantation and cell culture can be performed using known methods well known to those skilled in the art.

For example, when a stem cell treatment agent containing stem cells immobilized on a nano-patterned support according to the present invention is used for injecting stem cells to connect neurons broken from the nervous system damage, a sufficient number of cells It is possible to maximize the effect of the stem cells. In addition, due to the large number of neuronal differentiation and prolonged nerve cells, a definite cross-linking function is performed at the nerve ending portion, so that a stem cell-based histological engineering method is fused to treat the nervous system damage, The effect can be doubled.

The term " treating "or" treating "as used herein is a therapeutic treatment and prevention or prevention measure. Thus, those in need of treatment already include those with neurodegenerative disorders or neuropathy, as well as those who seek to prevent neurodegenerative disorders or neuropathy. The methods of the invention can be used to treat any mammal in need of treatment, including, but not limited to, humans, primates and livestock, raising, pet or sports animals such as dogs, horses, cats, sheep, pigs, Can be used. "Disorder" is any condition that is beneficial in treating two types of neuronal cells, differentiated neurons, or cells of the invention.

As used herein, a "therapeutically effective amount" of a cell is an amount sufficient to stop or alleviate the physiological effect of the patient caused by loss, damage or denaturation of the differentiated neuron.

The therapeutically effective amount of the employed cell will depend on the needs of the patient, the age, physiological condition and health of the patient, the desired therapeutic effect, the size and area of the tissue to be targeted for treatment, the severity of the lesion and the selected delivery route. For example, treatment of a disorder affecting a larger area of the brain may require more cells to achieve a therapeutic effect when compared to a smaller target area. In addition, the cells can be administered at one or more sites within a given target tissue with multiple sub-doses of a low cell dose. The cells of the invention can be completely isolated prior to transplantation, for example, to form a suspension of single cells, or to be almost completely isolated prior to transplantation, e.g., to form small aggregates of cells. The cells may be administered in a manner such that they are transplanted or transferred to a predetermined tissue site and the functionally deficient region is reconstituted or regenerated.

The appropriate range of cells that can be administered therapeutically effective can be suitably used for the patient within the ordinary knowledge of those skilled in the art.

It should be understood, however, that the actual dosage should be determined in light of various relevant factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease, And are not intended to limit the scope of the invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One : PUA  Nano Units / Home ( ridge / groove ) Pattern Array Fabrication

1.1 PUA  Mold making

A polyurethane acrylate (PUA) mold was prepared by curing a PUA pre-polymer (311RM, Minuta) on a silicon mold produced by photolithography for nano-sized patterning.

UV-curable PUA mold materials consist of acrylate groups for crosslinking, monomeric modulators, photoinitiators and functionalized precursors with radiation-curable release agents for surface activity.

In order to produce a sheet-type mold, a liquid precursor was dropped for a silicon mold, and then a flexible and transparent polyethylene terephthalate (PET) film was brought into contact with the precursor surface.

Thereafter, the mold was exposed to ultraviolet light (? = 200-400 nm) for 20 seconds through a transparent rear plate (dose = 100 mJ / cm2). After UV curing, the mold was stripped from the mold and the remaining active acrylate groups were terminated by further curing overnight before use as the first model. The PUA mold used in the experiment is a thin sheet type with a thickness of about 50 μm.

1.2 Fabrication of nano / groove-patterned surface

PUA micro- and nano-ridge / groove pattern arrays were fabricated on glass cover slips using UV-assisted capillary force lithography. The glass cover slip was rinsed with ethanol in an ultrasonic bath for 30 minutes, washed with distilled water, and then dried in a drying oven. To increase the adhesion between the PUA nanostructure and the glass interface, an adhesion promoter (phosphonic acrylate: isopropyl alcohol = 1: 10, volume ratio) was coated on the glass substrate.

A small amount of the PUA precursor (about 0.1-0.5 mL) was spread on the substrate and spread out, placing the first-replicated PUA mold (no identical material or active acrylate group) directly on the surface.

The PUA precursor material is cured by itself by capillary action into the hole of the mold and by exposure to ultraviolet light (? = 250-400 nm) for about 30 seconds through a transparent back plate (dose = 100 mJ cm-2). After curing, the mold was stripped from the substrate using a sharp tweezer.

Thus, a 350-nm (spacing of 350 nm, height of 500 nm) / groove-patterned surface was fabricated and a flat surface was created as a control.

The surface topography of the PUA nano / groove pattern structures was investigated using scanning electron microscopy (SEM).

The nano-groove / groove pattern array fabricated by the above method is illustrated in FIG. 1.

The capillary force filled the liquid PUA precursor into the hole of the PUA mold and cured by exposure to ultraviolet light.

The surface topography of the PUA nano / groove pattern structure was observed using SEM, and the results are shown in FIGS. 1C and 1D. 1C is a top-down bird's-eye view of a SEM image of a 350 nm / groove pattern array. FIG. 1D is a cross section of a SEM image of a 350 nm / groove pattern array, It shows that the size of the home pattern array is very constant. It was also confirmed that the sidewall angle of the land / groove pattern array was almost 90 degrees.

In addition, the nano-ridge / groove patterns were neatly formed over a large area with almost no defects (the diameter of the cover slip was 25 mm). This can be confirmed by the diffraction color of FIG. 1B

Example  2: human embryonic stem cells ( hESCs ) Culture

2.1 hESCs Maintenance of maintencance )

The hESCs (H9) were maintained following the research protocol of the WiCell Research Institute. As previously reported (28, 29), the hESC line H9 was plated as a colony and the feeder layer of MEF (mouse embryonic fibroblasts) inactivated with 10 μg / ml mitomycin C (seeded at 2 x 105 per 35 mm dish) Ml of recombinant human FGF (Sigma) supplemented with DMEM (Dulbecco's modified Eagle's medium) / F12, 20% serum replacement, 1 mM glutamine, 0.1% nonessential amino acid, 0.1% penicillin / streptomycin, 0.1 mM beta- -2 (Invitrogen Corporation, USA) were cultured in 5% CO2 overnight with alternating. For passaging, the hESC colonies were removed mechanically with a glass pipette during transport to reduce the number of differentiated cells. The passage was performed at a split ratio of 1: 2 or 1: 3.

2.2 On the nano / groove-patterned surface hESCs  culture

First, only the gelatin-coated surface was used to determine whether the nano / groove pattern table leads directly to the differentiation of hESCs.

When coating the cover slip patterned with the conventional dry-coating method with gelatin, the nano-groove / groove pattern array was covered with gelatin as shown in FIG. The patterned cover slip is immersed in 0.1% gelatin solution for 12 hours and rinsed with deionized water.

On the other hand, the nano-patterned surface was treated with an oxygen plasma for 60 seconds (60 W, PDC-32G, Harrick Scientific, Ossining, NY) in 0.1% gelatin solution After soaking for 12 hours, rinse with deionized water.

Human embryonic stem cells were plated in DMEM medium containing 15% FBS until the hESCs adhered to the substrate. After 24 hours, the attached hESC colonies were counted under a microscope.

Data under each condition was obtained from separate culture wells. Then, the adhered hESCs were cultured in hESCs medium without bFGF for 5-10 days to induce differentiation.

HESC colonies cultured on the surface of PUA nano / groove pattern structures were examined using scanning electron microscopy (SEM).

The sample was washed and fixed in sodium chocodilate buffer with 2.5% glutaraldehyde at 4 캜 for 5 hours. After fixation, the cells were washed with chocodilate buffer and allowed to stand in a series of ethanol dehydrations by concentration and air-dried. The samples were then sputter-coated with gold and analyzed at 20 kV under a scanning electron microscope of Japan Electron Optics Laboratory model (JSM 5600, Tokyo, Japan).

The results are shown in Fig.

The gelatin-coated pattern was almost the same as the pattern before gelatin coating as shown in FIG. 3 (a). And hESC adhesion on the surface was also poor. The white circles in Figure 3b represent hESC desorption from the surface.

This is believed to be due to the hydrophobicity of the nano / groove-patterned surface, which was sparsely coated with gelatin.

on the contrary. After oxygen plasma treatment, hESC adhesion on the surface was significantly enhanced as can be seen in Fig. 4 (p < 0.001). And, as shown in FIG. 3C, the patterns were almost the same as those observed before gelatin coating.

In addition, the hESC fraction attached to the 350-nm-grooved / grooved-patterned surface treated with oxygen plasma prior to gelatin coating was significantly less than that of hESC on the 350-nm / groove-patterned surface coated with gelatin alone (Fig. 3 (c) and (d)).

Example  3: on the nano-groove / groove pattern array hESCs Of neuronal differentiation

Human embryonic stem cells were seeded on a plasma-treated 0.1% gelatin-coated surface and cultured in DMEM / FBS medium until hESCs adhered to the surface. The hESCs were further cultured for 5 days in DMEM / F12-SR medium without any additional differentiation inducers, such as retinoic acid or laminin.

As a result, human embryonic stem cells seeded on the flat surface of the control group differentiated while spreading in an arbitrary direction as shown in Figs. 5A, 5C and 5E. However, the hESCsp seeded on the 350-nm well / groove pattern array was elongated in the direction of the ridge / groove pattern as shown in Figs. 5 (b), 5 (d) and 5 (f).

3.1 DAPI , Oct4  And nestin Immunostaining for

To investigate the differentiation of hESCs, cells were immunostained for DAPI, Oct4 and nestin and gene expression was analyzed by RT-PCR.

(1) Immunohistochemical analysis Immunocytochemistry )

Cells were fixed with 4% (w / v) paraformaldehyde for 30 min and permeabilized with 0.1% (v / v) Triton X-100 in PBS for 5 min.

After treatment with a blocking solution of 10% (v / v) goat serum for 30 minutes, the cells were incubated with primary antibody overnight at 4 ° C. The commercial antibodies used for immunohistochemical analysis were as follows: mouse anti-Oct-4 (1: 500), mouse anti-Nestin (1: 1000), goat anti-PDX- (1: 500, Chemicon, USA), rabbit anti-Calbindin (1: 200), anti-Brachyury (1: 500, R and D, USA) , Rabbit anti-MAP2 (1: 500, Santa Cruz Biotechnology, USA).

After washing with PBS, stained cells were stained with FITC- and rhodamine-conjugated secondary antibody (Molecular Probe, USA). And visualized with a confocal microscope (LSM 510; Zeiss).

(2) RT - PCR

Total RNA from the cultured cells was extracted using a Trizol reagent (Invitrogen) according to the user's manual protocol. One microgram of DNase I -treated total RNA was reverse transcribed using any primers and Superscript II reverse transcriptase (Invitrogen).

The reverse transcription reaction was performed at 42 째 C in 20 ((5xRT buffer, 1.25 mM MgCl2, 5 mM DTT, 2.5 g random hexamer, 0.5 mM dNTP, and 50 units of Superscript II enzyme). For each PCR reaction, 1 μl of the single strand cDNA product was used as a template.

Standard PCR conditions were as follows: denaturation at 94 ° C for 10 minutes, denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 30 seconds. (30 cycles)

The primer sequence pairs are as follows:

GATA6 forward- 5'-aaaaagagggaattcaaacc-3 '(SEQ ID NO: 1),

GATA6 reverse-5'-cctatgtagagcccatcttg-3 '(SEQ ID NO: 2);

NeuroD1 forward-5'-attctaagacgcagaagctg-3 '(SEQ ID NO: 3),

NeuroD1 reverse-5'-actggtaggagtaggggtgt-3 '(SEQ ID NO: 4);

AFP forward-5-'agaacctgtcacaagctgtg-3 '(SEQ ID NO: 5),

AFP reverse- 5'-gacagcaagctgaggatgtc-3 '(SEQ ID NO: 6);

DCN forward-5'-gagctcaggaattgaaaatg-3 '(SEQ ID NO: 7),

DCN reverse-5'-aagcttgttgttgtccaagt-3 '(SEQ ID NO: 8);

Oct4 forward-5'-aactcgagcaatttgccaagctcc-3 '(SEQ ID NO: 9),

Oct4 reverse- 5'-aactcgagcaatttgccaagctcc-3 '(SEQ ID NO: 10); And

beta-Actin forward-5'-caggagatggccactgccgca-3 '(SEQ ID NO: 11),

beta-Actin reverse-5'-tccttctgcatcctgtcagca-3 '(SEQ ID NO: 12).

(3) Observation results

As a result, all of the hESCs seeded on the 350 nm ridge / groove-patterned surface and the flat surface of the control group showed nestin positive (early marker of differentiation) and Oct4 negative (undifferentiated marker and dominant hESCs).

Gene expression analysis showed a very different pattern of differentiation between the 350-nm ridge / groove-patterned surface and the flat surface of the control, as shown in Fig.

In addition, analysis of hESCs (hEB5) differentiated into undifferentiated hESCs and embryoid bodies for 5 days, as well as hESCs cultured on a flat surface control, showed that the neurogenic differentiation marker NeuroD1 had a 350-nm ridge / regulated in hESCs cultured in the groove pattern arrays, which is better understood when compared to the hESCs and hEB5 controls cultured on a flat surface.

And, the expression levels of the endoderm gene marker GATA6 and the mesodermal marker DCN were lower in the hESCs on 350-nm ridge / groove pattern arrays than on the flat surface hESCs and hEB5 controls (Fig. 6).

3.2 Tuj1 , brachyury , Pdx1  And nestin Immunostaining for

For further analysis of hESC differentiation, hESCs were immunostained for Tuj1, brachyury, Pdx1 and nestin. Culture conditions were the same as in the previous experiment.

As a result, hESCs on 350-nm ridge / groove pattern arrays were only Tuj1 (immature neuronal marker; d, g in Fig. 7) as compared to hESCs of a flat surface control group ) Positive.

Human embryonic stem cells on 350-nm ridge / groove pattern arrays were negative for brachyury (mesoderm marker, Figure 7, e, h) and Pdx1 (endoderm marker; Figure 7, f, I) When cultured for 10 days on ridge / groove pattern arrays, well-structured neurite elongation was observed along the direction of the ridge / groove pattern (Fig. 7 gi) and the cells were Tuj1-positive.

These results strongly suggest that the nano / groove pattern arrays induce the differentiation of hESCs into neuronal cells without any biochemical or biological differentiation inducer.

3.3 differentiation into different types of neurons

Samples were immunostained against other neuron and glial cell markers to confirm neural differentiation of hESCs on the 350-nm / groove pattern array: HuC / D (human neuronal protein: RNA-binding protein), MAP2 neuronal marker: microtubule-associated protein 2), and GFAP (intermediate filament proteins of mature astrocytes: glial fibrillary acidic protein).

As a result, as shown in Fig. 8, human embryonic stem cells immunostained for Tuj1 showed neurites elongated in the direction of 350 nm / groove pattern, as previously observed.

And the elongation of the neurites also increased with an increased incubation period of 5 to 10 days. The differentiated hESCs stained positively for HuC / D and MAP2 were not stained for GFAP.

These results suggest that hESCs differentiated into mature neurons without differentiation into astrocytes.

We further immunostained for calbindin (calcium binding protein), serotonin (monoamine neurotransmitter), γ-aminobutyric acid (GABA), and tyrosine hydroxylase (TH).

The results are shown in Fig.

Some of the hESCs were positive for calbindin, serotonin and GABA on 350-nm ridge / groove pattern arrays. However, it was not positive for TH.

Attach an electronic file to a sequence list

Claims (15)

Without using neurogenesis inducers,
Characterized in that a support comprising an oxygen plasma treated surface consisting of a plurality of longitudinal grooves / rims having a depth of 500 nm and an equal width of 350/350 nm to 800/800 nm is used. To differentiate into non-neuronal cells.
delete delete delete delete The method of claim 1, wherein the support is made of gelatin coated polyurethane acrylate (PUA).
delete 2. The method according to claim 1, wherein the embryonic stem cells are derived from a human tissue.
delete delete delete delete Supports for neural cell differentiation of embryonic stem cells, including:
A polyurethane acrylate (PUA) sheet having a plurality of longitudinal grooves / ridges having a depth of 500 nm and an equal width of 350/350 nm to 800/800 nm;
An oxygen plasma layer treated on the sheet; And
A gelatin layer coated on the oxygen plasma layer.
A method for producing a support for neuron differentiation of embryonic stem cells, comprising:
(a) curing the PUA prepolymer with ultraviolet light to the silicon main feature;
(b) peeling the cured PUA mold;
(c) forming a plurality of grooves / ridges in the PUA mold at a depth of 500 nm and an equal width of 350/350 nm to 800/800 nm and contacting a PUA precursor-treated glass cover slip;
(d) curing with ultraviolet rays to obtain a PUA sheet having grooves / grooves formed thereon, and peeling the PUA sheet;
(e) treating oxygen plasma on the surface of the peeled PUA sheet;
(f) coating the nano-patterned surface of the sheet with a gelatin solution. delete

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