KR101204894B1 - Method of differentiating stem cells into ectodermal cells - Google Patents

Abstract

The present invention relates to a method of differentiating stem cells into ectoderm cells, and more particularly, to a method of differentiating stem cells into ectoderm cells, comprising culturing globular bodies formed by aggregation of adult stem cells. will be. The present invention does not use existing biochemical differentiation inducing substances, and differentiates them into ectoderm cells by increasing the intercellular interactions caused by the aggregation of stem cells, and therefore, the risk of contamination by other substances is much lower and simpler. Adult stem cells can be differentiated into ectoderm cells.

Stem cells, cell aggregation, ectoderm, differentiation, spheroid

Description

Method of differentiating stem cells into ectodermal cells

The present invention relates to a method of ectoderm differentiation of stem cells. More specifically, the present invention relates to a method of differentiating stem cells into ectoderm cells, which induces differentiation into ectoderm cells by increasing intercellular interactions due to cell aggregation of adult stem cells.

Biotechnology in the 21st century presents the possibility of new solutions to food, environment and health problems as the final goal of human welfare. Recently, the use of stem cells has emerged as a new chapter in the treatment of intractable diseases.

Stem cells are cells that have the ability of self-replication and differentiate into two or more cells. Totipotent stem cells, pluripotent stem cells, and multipotent stem cells can be classified as a multipotent stem cell.

The ultimate goal of stem cell research is to create cells and tissues of the desired type for use in cell therapy and tissue engineering. In order to practically utilize stem cells, a challenge to be solved is the development of a technology that can induce differentiation into desired cells.

The most time consuming step in stem cell differentiation research is to analyze the effects of specific culture conditions on differentiation induction. In particular, in order to increase the usefulness of stem cells as cell therapeutics, a technique for efficiently differentiating stem cells into specific cells is required.

Among them, cerebral nervous system disease is considered to be the most appropriate target for cell transplantation treatment than any other disease. Unlike other tissues, cerebral nervous system has almost no immune rejection reactions. This is because long-term survival can be expected.

Therefore, there is a need for an effective method for differentiating stem cells into ectoderm cells such as neurons.

WO 2005/003320 discloses a method for inducing differentiation of stem cells into neurons, and more specifically, (a) culturing stem cells with basic fibroblast growth factor; (b) culturing the cells of step (a) with fibroblast growth factor 8 and Sonic Hedgehog; (c) culturing the cells of step (b) with brain-derived neurotrophic factors; And (d) co-culturing the cells of step (c) with glial astrocytic cells. WO 2004/093812 discloses that compounds with specific chemical formulas can act as inducers for the differentiation of embryonic stem cells into neurons.

However, these conventional methods have been difficult to apply clinically due to the low differentiation rate. Due to the long time of differentiation, a large number of cells die during the differentiation process, and the survival rate of the cells has become a big problem. There was also a risk of contamination by the material.

Thus, the present inventors can induce differentiation of adult stem cells into ectoderm cells, cells having a differentiation rate that can be clinically applied by simply changing the culture conditions without adding any known biochemical differentiation inducing substance. It was confirmed that the present invention was completed.

An object of the present invention is to provide a method for differentiating adult stem cells into ectoderm cells.

In order to achieve the above object, it provides a method for differentiation of stem cells into ectoderm cells, comprising culturing a globular body formed by the aggregation of adult stem cells.

In particular, the aggregation of the adult stem cells does not contain a biochemical for ectoderm differentiation, characterized in that it is carried out in a culture vessel coated with polyethyleneimine or poly-D-lysine.

Moreover, the said spherical body is 0.2x10 <^5> - It can be formed by inoculating 6.0 × 10 ⁵ stem cells / cm 2 into the culture vessel and aggregation, preferably 1.0 × 10 ^5- It can be inoculated at 1.5 × 10 ⁵ cells / cm 2. It is preferable that the formed spherical body has a diameter of 50 to 400 µm, particularly 100 to 200 µm.

The present invention does not use any biochemical differentiation-inducing substance and differentiates into ectoderm cells using increased intercellular interactions due to the aggregation of stem cells, thereby simplifying the differentiation method and significantly shortening the time required for differentiation. This can increase the viability of differentiated cells and eliminate the risk of contamination by other substances by eliminating differentiation-inducing material processing or gene conversion processes. Therefore, as compared with the existing method, it is possible to differentiate stem cells into ectoderm cells by a simpler and safer method, which will be useful for developing cell therapeutics for neurological diseases.

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

'Stem cell' refers to cells that can differentiate into each cell constituting the tissue (differentiation), and refers to a cell that can be differentiated to a specific cell by a specific differentiation stimulation (environment). Stem cells can be classified into 'embryonic stem cells' derived from embryos and 'adult stem cells' derived from organs present in the adult depending on their cytological origins.

The term 'precursor cells' refers to cells that are at a stage before they have the shape and function of a particular cell.The term 'neural precursor cells' refers to neurons, astroglia, and oligodendrocytes that form the central nervous system. It refers to progenitor cells that can differentiate into neurons such as cells.

The term 'tissue' refers to a collection of cells having substantially the same function and / or shape in a multicellular organ. It can also be an aggregate of cells with the same origina- tion, or a collection of cells of the same origin with the same function and / or morphology.

'Differentiation' refers to a phenomenon in which structures or functions are specialized during the division and proliferation of cells, that is, the shape or function of a living cell or tissue is changed to perform a given task. Generally, a relatively simple system is divided into two or more qualitatively different sub systems. For example, qualitatively between parts of a living organism that were almost homogeneous in the first place, such as head or torso distinctions between eggs that were initially homogenous in the development, or cells such as muscle cells or neurons. Phosphorus difference, or as a result, is a state divided into subclasses or subclasses that can be distinguished qualitatively.

'Biochemical differentiation inducing substance' means any biological or chemical agent such as a protein, lipid or carbohydrate, salt, mineral, or organic substance that enhances the differentiation ability of stem cells into specific cells in vivo or ex vivo. Refers to all differentiation inducing agents known to induce differentiation into differentiated cells. The differentiation inducing substances may be used alone or in combination as necessary.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention relates to a method of differentiating "stem cells", in particular "adult stem cells" into ectoderm cells. Preferably, adult stem cells of primates, more preferably human tissues, are used.

Stem cells, unlike differentiated cells that have ceased cell division, are capable of self-renewal by cell division and thus have proliferation (expansion) characteristics. Differentiation into cells can be differentiated into other cells by different environment or differentiation stimulus, so it has plasticity in differentiation.

These stem cells can be divided into embryonic and adult stem cells according to their origin, and both of them can be used in the present invention, but preferably adult stem cells that have little biological, ethical or legal problems.

Adult stem cells, like embryonic stem cells, are capable of self proliferation in culture and have differentiation ability to develop into cells having characteristic shapes and specialized functions according to changes in culture conditions and culture conditions.

The adult stem cells can differentiate into tissue-specific progenitor cells inherent in the human body. Adult stem cells can be virtually any differentiated cell in the body and have the potential to generate replacement cells for a wide range of tissues and organs such as the heart, pancreas, nerve tissue, muscle, cartilage, and the like.

Such adult stem cells can be obtained from most tissues such as primates, preferably human bone marrow, cord blood, blood, fat, liver, skin, gastrointestinal tract, placenta, uterus, brain, pancreas, liver, eyes and fetal tissue. have. In one preferred embodiment, it is possible to use placental stem cells which have differentiation ability to various tissues, are easily manipulated in vitro, and have immunomodulatory ability.

As a method for separating adult stem cells from various tissues of humans, methods conventional in the art, which are appropriate for each tissue, can be used. For example, the obtained specific tissues are separated into single cell bodies by treatment with trypsin solution and / or collagenase, and then cultured in a suitable medium in which growth factors such as bFGF and EGF are added in an appropriate amount. Then, it can be separated by FACS or the like can be used to isolate adult stem cells according to the growth rate.

The present invention relates to a method for "differentiating" stem cells, in particular adult stem cells.

In general, in order to differentiate adult stem cells into endoderm, mesoderm, or ectoderm cells, a method using a biochemical that induces differentiation in each germline is used.

In other words, conventional methods for differentiation into ectoderm cells include retinoic acid, lithium chloride, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). ), Betacellulin, activin, and sonic hedgehog (shh) are used for differentiation into endoderm cells, and decks are used for differentiation into mesodermal cells. As it uses dexamethasone, dimethyl sulfoxide, fibroblast growth factor, and vascular endothelial growth factor (VEGF), it uses a wide variety of drugs depending on the direction of differentiation. Various combinations of these differentiation-inducing biochemicals are also used. .

However, as mentioned above, there has been a problem of low differentiation rate despite the use of these various biochemicals. Currently, 100% differentiation has not been reported in the literature. In addition, in the conventional method of differentiation, since the differentiation process proceeds for a long time, a large amount of cells die during the differentiation process, and the survival rate of the cells also becomes a big problem. In addition, since most differentiated cells, including neurons, do not divide anymore, there is a limitation that large-scale culturing is necessary at the time of differentiation of stem cells. In addition, the bFGF, FGF8, SHH, BDNF, etc. are very expensive, there is no economic advantage.

In particular, in relation to the differentiation rate, even in the differentiation of neurons known to have the highest differentiation rate among conventional methods, the rate of differentiation is only about 70%. This is not only a poor efficiency for clinical applications, but also a problem because the remaining 30% of the cells that may be in an undifferentiated state are not precisely identified. That is, from a clinical point of view, in clinical application or in vivo transplantation, there is a dangerous result of transplanting the cells together which are not precisely identified. This is because even after the differentiation process is completed, it does not differentiate into a specific target cell, but also includes the possibility of remaining undifferentiated or differentiating into other cells at all unexpected.

However, the present invention solves the above problems and differentiates adult stem cells into ectoderm cells with an efficiency of about 100%.

That is, the present invention relates to a method for differentiating stem cells to "high efficiency" into "ectoderm cells".

Specifically, the present invention relates to a method for agglomerating stem cells, preferably adult stem cells, to form a globular body without using a biochemical derivative for ectoderm tissue induction, and to obtain ectoderm cells by culturing the globular body. will be. That is, according to the method of the present invention, the aggregation of the stem cells increases the cell interaction between the aggregated stem cells to differentiate into ectoderm cells without other differentiation inducing substances.

As described above, existing methods for differentiation into ectoderm cells have been performed by culturing in a medium containing biochemical differentiation inducers such as soluble factors and growth factors.

On the contrary, in the present invention, the method of aggregating adult stem cells increases cell interactions between stem cells, and thus, adult stem cells are differentiated into ectoderm cells without a specific ectoderm differentiation inducing substance.

At this time, adult stem cells can be aggregated in a culture vessel (plate) coated with polyethyleneimine or poly-D-lysine to form a globular body.

The polyethyleneimine is preferably diluted with a general buffer solution such as water or PBS at a concentration of 0.01-1% (w / v) and coated on a culture dish, and the concentration of 0.1% is most preferred. The poly D-lysine is preferably coated at a concentration of 0.1 to 100 µg / ml, most preferably at a concentration of 1 µg / ml.

In general, the polyethyleneimine or poly-D-lysine has a positive charge on the surface of the culture medium to enhance the ability of the cells to adhere to the surface, but when the stem cells are cultured at high concentrations, the cells do not adhere to the surface of the culture plate and Aggregate to form spherical body. It is preferable that the spherical body formed by aggregation of stem cells has a diameter of 50 to 400 μm, and more preferably, a diameter of 100 to 200 μm.

Stem cells inoculated at high concentration in the culture vessel to form such a globular form is 0.2 x 10 ⁵ ~ 6.0 x 10 ⁵ cells / ㎠ is appropriate, preferably 1.0 x 10 ⁵ ~ 3.0 x 10 ⁵ cells / ㎠, Most preferably 1.0 x 10 ⁵ to 1.5 x 10 ⁵ cells / cm 2. In the case of culturing more stem cells than 6.0 x 10 ⁵ cells / cm 2, the spherical body formed is too large to maintain its shape, and in the case of culturing less than 0.2 x 10 ⁵ stem cells, It will adhere to the culture dish surface.

As described above, when the spherical body formed by the aggregation of stem cells is transferred to a culture medium, cultured cells are differentiated into ectoderm cells, for example, neural tissues or progenitor cells for neural tissue generation.

Genes specifically expressed in ectoderm and ectoderm tissues include Nestin, Beta Tubulin III, MAP-2, Neurofilament Heavy Chain, Dopamine Beta hydroxylase, Neural Neural cell adhesion molecules, S-100, Pax-6, neural tubulin, and choline acetyltransferase, and the like, and other known genes can be used depending on the type of cell. For example, in the case of neural progenitor cells, expression of genes such as Nestin, Dcx, Sox1, and HuD can be confirmed, and in the case of fully differentiated neurons, MAP2, NeuN, NF200, and NSE can be identified. In one embodiment of the present invention it was confirmed whether the expression of Nestin.

Nestin is a cell marker (specific expression gene) that is specific to neural progenitor cells, and the fact that nestin is expressed in all cells targeted for differentiation enhances the interaction between cells aggregated into a globular form. It suggests itself as a strong signal for ectoderm differentiation.

Meanwhile, the ectoderm progenitor cells obtained by aggregating stem cells according to the present invention are characterized by expressing at least one of genes such as Dcx, Sox1, and HuD as well as nestin. In particular, DCX, Sox1 and HuD genes are known as genes that are characteristically expressed in early neuronal progenitor cells.

Conventionally, studies to differentiate embryonic stem cells and the like into ectoderm cells have been partially successful, but the differentiation rate was not satisfactory enough for the clinical application, and thus there is still a risk of teratoma formation after transplantation. This was a difficult situation.

However, the method of the present invention has the effect of showing a differentiation rate of 100%, in particular, all stem cells of interest, as compared to the previously known method. In addition, the process is very simple, which significantly reduces the time required for differentiation, thereby increasing the viability of differentiated cells and further reducing the risk of contamination by other substances by eliminating differentiation-inducing drugs or gene conversion processes. There is this.

Thus, by methods as disclosed herein, stem cells can be more efficiently differentiated into ectoderm cells such as neural progenitor cells or neurons, and the like, particularly for transplantation treatment for various neurodegenerative disorders and neuropathies. Clinical usefulness is great.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples. That is, the following steps are described as an example, and the scope of the present invention is not limited thereto.

Example 1 Isolation of Adult Stem Cells from Human Placenta

First, stem cells were prepared in order to implement the ectoderm differentiation method of stem cells according to the present invention.

After embryo implantation from Maria Fertility Hospital (Seoul, Korea), the embryo was successfully implanted. However, the placenta was removed in sterile state after 5-7 weeks, which was confirmed to be cardiac. It was washed with Dulbecco's Phosphate Buffered Saline (DPBS), chopped and separated into single cell bodies by treatment with trypsin solution (0/25% trypsin / 0.5 nM EDTA) at 37 ° C. for 5-20 minutes. The isolated cells were recovered using 100 μm nylon mesh and then washed with DPBS.

Alternatively, place the finely cut placenta tissue in a culture dish to induce cells to seep out and grow in the placenta tissue, remove the remaining placental tissue fragments after 3-7 days and allow the cells to grow on the culture dish. Induced to form cell lines.

After centrifugation of the cells obtained by the above methods, 10% fetal bovine serum (FBS), 4ng / ml fibroblast growth factor, 0.1 mM beta-mercaptoethanol (β- mercaptoethanol) , Dulbecco's Modified Eagle's Media (DMEM) containing 1% non-essential amino acids and antibiotics was suspended in a humidified chamber at 37 ℃ and 5% CO ₂ .

After 5 days, the adherent tissue debris was removed while changing medium. When cells proliferated and became dense (passfluent ¹ ) on the culture surface (75 cm ² ). Through this process, a total of 11 human placenta stem cells (hPLCs) were established.

(1) Fluorescence activated cell sorting (FACS) analysis

To analyze the surface antigen of hPLC, cells were harvested with PBS with 2 mM EDTA / 5% FBS and washed twice with PBS. Subsequently, FITC or PE (phycoerythrin) -bound antibody diluted in an appropriate ratio in 2% FBS / PBS buffer was added to the cell suspension, and treated at 4 ° C. for 30 minutes, followed by FACS Vantage SE (Becton & Dickson). It was. 5 μg / ml of propidium iodide was added to remove dead cells and debris prior to analysis. The results obtained were analyzed using the CellQuest (Becton & Dickinson) program.

FACS analysis showed positive for CD13, CD9, CD29, CD44, CD90, CD105 and HLA-ABC, as shown in FIG. 1; Negatives were shown for CD117, CD10, CD14, CD24, CD34, CD45, CD50, CD38, and HLA-DR.

These results are similar to those expressed in mesenchymal stem cells (MSCs) of cord blood, which are known in the art, and that the expression of CD13, CD9, CD90 and CD105 and the lack of CD45, CD10 and HLA-DR are Although it was shown that both cells have in common, the expression of CD34, which is an identification marker of MSCs, did not appear in the hPLCs of the present invention.

(2) Karyotype analysis

HPLC having about 70% confluency was exposed to 0.1 µg / ml of colcemid in a 100 mm culture dish for 2 hours, collected, washed and treated in 75 mM potassium chloride solution at 37 ° C. for 20 minutes. It was. After incubation, the stock solution was washed by centrifugation, and then fixed with 5 ml of a fixed solution containing 3: 1 of methanol and acetic acid.

The fixed cells were harvested and resuspended in 0.5 ml of fixed solution. One drop of suspension was dropped on a frozen slide at a height of 30 cm. After drying the slides were exposed to 0.025% trypsin for 30 seconds and washed with PBS. The chromosomes were stained with 10% Giemsa solution and the karyotypes of normal human chromosomes were analyzed by Cytovision of Applied Imaging.

The results are shown in FIG. without noticeable karyotypes or morphological changes in vitro Proliferation continued for 90-150 days.

Example 2: Induction of differentiation into ectoderm progenitor cells

The hPLCs established in Example 1 were incubated in a culture dish coated with 0.1% polyethylenelenimine at about 1.0 × 10 ⁵ cells per cm 2. Meanwhile, in another embodiment, about 1.0 × 10 ⁵ cells per cm 2 were cultured in a culture dish coated with poly-D-lysine (10 μg / ml). On day 2, the stem cells in each culture dish aggregated with each other without adhering to the culture dish surface to form spheroids.

(1) Immunostaining Analysis

First, immunostaining analysis was performed on nestin antibodies on the stem cell globules.

The cultured globulars were transferred onto a cover slip, incubated for about 3 days, fixed with 4% paraformaldehyde solution at room temperature for 15 minutes, and washed with PBS. Fixed cells were diluted in PBS solution with 5% Triton X-100 and 5-10% normal goat serum at the rate indicated by the producer, added to the sample, and then at room temperature. The reaction was reacted overnight for 1 hour or in the cold.

After washing with PBS solution added with 2% Triton X-100 and 3% normal goat serum, the secondary antibody of tetrarhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) conjugate at the room temperature or in the cold state It was processed by the method. After control staining, the cell nuclei were stained with Hoechst 33342 (10 µg / ml, Sigma) and observed by fluorescence microscopy.

The results are shown in Fig. Undifferentiated human placental cells were indicated by NV, Sph when spherical bodies were formed, Adh when the spherical bodies were cultured in a general culture dish, and P5 when the fifth passages of the globular bodies were cultured.

3A is a photograph showing the expression of Nestin about 30% of undifferentiated human placental cells, B is a photograph showing that the expression of Nestin is already increased when the globular body is formed, and C is a globular body Is a photograph showing that all cells express Nestin when incubated in a general culture dish, D is a photograph showing that the expression of Nestin is maintained when the globular body is passaged for more than one month. On the other hand, after subculture, as a result of counting about 10,000 cells, all cells expressed nestin (not shown).

In addition, referring to the middle photographs Aa to Da of FIG. 3, first, Aa is a photograph showing the nuclei of all cells by Hoechst staining of the cells shown in A of the upper photographs A to D of FIG. Hoechst staining of the cells shown in B of the photographs showing the nuclei of all cells, Ca is Hoechst staining of the cells shown in C of the top photo of Figure 3 is a photograph showing the nuclei of all the cells, Da is photograph D Hoechst staining of the cells shown in the photo shows the nuclei of all cells.

And, if you look at the lower pictures Ab to Db of Figure 3, Ab is a combination of A and Aa of Figure 3, Bb is a combination of B and Ba of Figure 3, Cb is a combination of C and Ca of Figure 3 It is a photograph and Db is the photograph which combined D and Da of FIG.

These results, in particular, based on the Db panel in Figure 3, it can be confirmed that 100% differentiation into ectoderm progenitor cells by enhanced cell interaction between hPLC.

(2) RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) Analysis

Total RNA was extracted using Trizol reagent (Invitrogen), one hour at 42 ° C using Superscript II (Invitrogen) and oligo-d (T) 20 primer, The reaction was carried out at 72 ° C. for 15 minutes to synthesize cDNA. Target sequences from cDNA were amplified using the I-taq premix kit (Invitron Biotechnology) under the following conditions: denatured at 95 ° C. for 30 minutes, then denatured at 95 ° C. for 30 seconds and then at 53-60 ° C. 45 The synthesized cDNA was amplified by annealing for 35 seconds and repeating the amplification for 45 seconds at 72 ° C. for 35 seconds. The amplified cDNA was identified by separation on 2% agarose gel and stained with ethidium bromide.

The results are shown in FIG. In Figure 4, undifferentiated human placental cells are NV, Sph when spherical bodies are formed, Ros when spheroids are cultured, and Ros, and cells of passage 1 of the spherical bodies. P1, three passages sefonpo is expressed as P3, five passages P5.

As shown in A and B of Figure 4, nestin induced by differentiation in stem cells was confirmed again by Western blotting (western immunoblotting) and confirmed the expression of DCX, Sox1 and HuD by RT-PCR It was confirmed that they differentiated into true neuroprogenitor cells.

Specifically, FIG. 4A is a photograph confirming the nestin expression by Western blotting for each generation, and FIG. 4B shows the expression of Sox1, Dcx and HuD increased according to differentiation by RT-PCR, and is characteristic only in stem cells. It is a photograph showing that the expression of Oct4, Nanog, Tbn is reduced. The expression, morphology, and progenitor growth rate of nested cells in these differentiated cells were almost unchanged during 4 weeks of incubation, and the phenotype was maintained even after cryopreservation for more than two months (not shown).

These results indicate that human placental stem cells differentiated (derived) into ectoderm cells by enhanced cell interaction.

Comparative Example 1 Comparison with Cases of Biochemical Inducer for Ectodermal Differentiation

Portmann-Lanz et al. (American J Obs Gyn 194; 664-673, 2006) isolated mesenchymal stem cells from different parts of the placenta, namely early chorion, late chorion and early villous, and differentiated them into retinoic acid. When 20-30% of the population was differentiated into ectoderm cells.

In addition, Yen et al. (Tissue Engineering 14; 9-17, 2008) differentiated cells from the placenta with bFGF, EGF, mercaptoethanol, dimethyl sulfoxide, and butylated hydroxyanisole for 6 days and then treated with retinoic acid for about 10%. Showed ectoderm differentiation, but when IBMX (1-methyl-3-isobutylxanthine) was treated, it was confirmed that about 40-60% of cells differentiated into ectoderm cells.

Experimental Example 1 Cell Number for Maximum Differentiation Efficiency

The amount of inoculation of stem cells showing the most effective differentiation efficiency into ectoderm cells was examined. In other words, the cell density in the case where the interaction between stem cells is most effective for differentiation was investigated.

For this purpose, the experiment was divided into three groups.

Group 1: Inoculate at approximately 0.1 × 10 ⁵ cell / ㎠

Group 2: about 1.0 × 10 ⁵ cells / cm 2; 2.0 × 10 ⁵ cell / cm 2; Inoculate into the same medium at 3.0 × 10 ⁵ cell / ㎠

3 groups: inoculated at about 7.0 × 10 ⁵ cell / ㎠

The results are shown in FIG. In the case of the second group, the spherical body was best formed (B of FIG. 5), and the diameter of the spherical body was about 50 to 300 μm. However, as the inoculation concentration was lowered, the size and number of spherical bodies formed were smaller, and the first group inoculated with less than about 0.2 × 10 ⁵ cells / cm 2 could not form spherical bodies (FIG. 5A). On the other hand, even when inoculated at 3.0 x 10 ⁵ cell / cm 2 or more in the two groups, up to 6.0 x 10 ⁵ cell / cm 2, the number of spherical bodies formed gradually decreases, but the size of the spherical body gradually increased (not shown). When inoculated at 6.0 x 10 ⁵ cells / cm 2 or more, the shape of the globular body was not constant as in Group 3 (C of FIG. 5), and the cells were too large in size to die without differentiation.

Therefore, the globular culture of the present invention is 0.2 × 10 ⁵ ? It can be confirmed that it is preferable to form 6.0 × 10 ⁵ stem cells / cm 2 by inoculating the culture vessels and integrating them.

1 is a result of flow cytometry (FACS analysis) showing the surface expression of hPLC.

Figure 2 is the result of karyotype analysis by proliferating cell number and Kimja staining of hPLC.

Figure 3 is a micrograph confirming the induction of differentiation into ectoderm progenitor cells using immunostaining.

Figure 4 is a Western blot results (A) and RT-PCR results (B) pictures for differentiated ectoderm progenitor cells.

5 shows experimental results showing the differentiation efficiency into ectoderm cells according to the cell density.

Claims (13)

0.2 × 10 ⁵ ～ in culture vessel coated with polyethyleneimine or poly-D-lysine A method of differentiating mesenchymal adult stem cells into neural progenitor cells or neurons, comprising culturing spherical bodies formed by inoculating 6.0 × 10 ⁵ cells / cm 2 of mesenchymal adult stem cells into a culture vessel. The method of claim 1, wherein the aggregation of mesenchymal adult stem cells is a biochemical for inducing ectoderm differentiation, retinoic acid, lithium chloride, basic fibroblast growth factor (bFGF) or epithelium. A method characterized in that it is carried out in a culture vessel that does not contain Epidermal Growth Factor (EFG). delete The method of claim 1, wherein the polyethyleneimine is coated at a concentration of 0.01-1%. The method of claim 1, wherein the poly-D-lysine is coated at a concentration of 0.1-100 μg / ml. delete According to claim 1, wherein the mesenchymal adult stem cells are 1.0 × 10 ⁵ ~ Inoculated at 1.5 × 10 ⁵ cells / cm 2. The method of claim 1, wherein the size of the globular body is 50 ~ 400 ㎛ diameter mesenchymal stem cells, characterized in that differentiation into neuronal progenitor cells or neurons. The method of claim 8, wherein the size of the globular body is 100 ~ 200 ㎛ diameter mesenchymal stem cells, characterized in that differentiation into neuronal progenitor cells or neurons. The method of claim 1, wherein the mesenchymal adult stem cells are derived from humans. The method of claim 10, wherein the human-derived mesenchymal adult stem cells are derived from human tissue selected from the group consisting of bone marrow, umbilical cord blood, blood, liver, skin, fat, gastrointestinal tract, placenta, and uterus. delete The method of claim 1, wherein the neural progenitor cells express one or more genes selected from the group consisting of Nestin, DCX, Sox1 and HuD.

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