KR20080023078A - Methods for inducing differentiation from human embryonic stem cells to dopaminergic neurons by using vascular endothelial growth factor - Google Patents

Abstract

Methods for inducing differentiation from human embryonic stem cells to dopaminergic neurons are provided to block possibility of differentiation into various cells including myocardial cells, muscle cells, liver cells and hematopoietic cells, so that differentiation efficiency into high purity nerve cells for cell therapy is increased. A method for inducing differentiation from human embryonic stem cells to dopaminergic neurons comprises the steps of: treating embryoid bodies formed from human embryonic stem cells with 40-60 ng/ml of a vascular endothelial growth factor(VEGF) for 7-9 days to induce the neuroectoderm-increased embryoid bodies; culturing the neuroectoderm-increased embryoid bodies attached to a tissue culture dish to induce neural precursor cells; detaching the induced neural precursor cells from the tissue culture dish and re-attaching and culturing them in the dish to increase purity and maintain density uniformly; and finally differentiating the high purity of neural precursor cells to dopaminergic neurons. Further, a 20-30 ug/ml of fibronectin is added into the neuroectoderm-increased embryoid bodies.

Description

Methods for promoting differentiation from human embryonic stem cells to dopaminergic neurons by using vascular endothelial growth factor}

1 is a photograph of a typical embryonic stem cell that maintains an undifferentiated state through several passages and expresses markers of representative undifferentiated embryonic stem cells such as Oct4, Nanog, SSEA4, Alkaline phosphatase, and forms a colony. Scale bar is 100 μm.

2 is an immunostaining photograph and a polymerase chain demonstrating that the undifferentiated embryonic stem cells used in the present invention can differentiate into ectoderm, mesoderm, and endodermal when differentiated into germ cells by forming embryos and inducing early differentiation. The result is a reaction. Scale bar is 100 μm.

Figure 3 shows that the expression of Pax6 and Nestin markers of neuroectodermal cells capable of differentiating into neurons, which are the final target cells of differentiation, are increased in the embryonic body by treating vascular endothelial growth factors in the embryonic formation. Shown are the results of immunostaining and polymerase chain reaction. Scale bar is 100 μm.

Figure 4 shows the formation of neural rosettes, which are typical morphological characteristics of neuroepithelial cells, when the embryos are disassembled and cultured. The neural rosettes express Pax6 and Nestin, which are markers of neuroepithelial cells, and vascular endothelial growth. This figure shows that when the factor is processed in embryonic state, the number increases. Scale bars are 100 μm (A, B, C, D), 50 μm (F).

5 shows that the process of overall differentiation and thus the number of differentiated neurons increased by treatment of vascular endothelial growth factor at the embryonic stage, most of which are dopamine neurons in the midbrain and other GABA neurons Non-neuronal cells, such as astrocytes and endothelial cells, are rarely expressed. Scale bar is 50 μm.

The present invention relates to a method for promoting differentiation from human embryonic stem cells to dopamine neurons, and more specifically, by treating the embryonic body formed from human embryonic stem cells with vascular endothelial growth factor (VEGF), A method for promoting differentiation into dopamine neurons by inducing increased embryonic bodies.

The present invention relates to a method for differentiating embryonic stem cells into neurons with high efficiency, and to form an embryonic body with embryonic stem cells with pluripotent capacity to induce early differentiation, blood vessels involved in neuroprotection and neurogenesis. The present invention relates to a method of finally increasing neuronal differentiation efficiency by increasing endoderm cells destined to differentiate into neurons by treating endothelial cells.

Stem cells are cells that divide indefinitely, remain undifferentiated, and have the ability to differentiate into all the cells of an organism when subjected to the right conditions and signals. That is, stem cells refer to cells capable of differentiating into adult cells that have morphological characteristics of cardiomyocyte epidermal neurons and perform specialized functions. Stem cells are considered to be the next generation of cell therapy and developmental biological research because of their self-renewal and differentiation capacity. Conventional cell transplantation has been spotlighted in that stem cells of the present invention, which are not easily secured for treatment cells and are inadequate to maintain and proliferate even after securing, provide insufficient amounts for treatment. In the same vein, as a good biological research data, the differentiation process reflects the developmental process in the human body.

Parkinson's disease, Alzheimer's disease, and spinal cord injury are disorders caused by the degeneration or damage of certain nerve cells in the nervous system. The cure is dominant. The stem cell differentiation and transplantation technique replaces the degenerated tissue and forms a new synapse with the existing neural tissue to carry out a function of transmitting a neural signal, thereby providing a therapeutic measure that is the basis of various neurodegenerative diseases. present.

The present invention is a neurodegenerative disease in terms of the smooth supply of cells for cell replacement therapy as a technology for differentiating neurons necessary for cell replacement therapy through stem cells having the advantage of differentiation into specific cells under infinite proliferation and appropriate stimulation. This is a technical field that will shape a new paradigm in the treatment of medicine.

Cell replacement therapy has already been performed to treat neurodegenerative diseases. For example, the implantation of midterm brain tissue from a stillborn human fetus showed improvement in the patient, but this technique caused ethical controversy and proliferated to the level required for securing cerebral center tissue and cell therapy of a stillborn human fetus. It is almost impossible to treat many patients with neurodegenerative diseases because it is almost impossible to do so. The stem cell-derived neurons that can proliferate in vitro and supply cells as desired can be said to be the technology underlying the cell replacement therapy. In this context, a technique for differentiating several stem cells into neurons has already been proposed, but the differentiation efficiency is low and differentiation into other cells besides neurons, which can be called impurities in cell transplantation, has not been an alternative to previous studies. The present invention induces embryos with increased ectoderm through vascular endothelial growth factors, thereby blocking the possibility of differentiation into cells derived from various mesoderm and endoderm such as cardiomyocytes, muscle cells, hepatocytes, and hematopoietic cells. It is characterized by differentiating high-purity neuronal cell populations for cell therapy.

Accordingly, the present inventors have made intensive studies to overcome the problems of the prior art, when the embryonic body formed from human embryonic stem cells is treated with vascular endothelial growth factor (VEGF), neuroectoderm is increased. It was confirmed that it can be induced into an embryo and thus promote the final differentiation into dopamine neurons, thus completing the present invention.

Therefore, the main object of the present invention is to provide a method capable of differentiating human embryonic stem cells into neurons with high efficiency for the supply of stable neurons for cell therapy for neurodegenerative diseases.

According to one aspect of the present invention, the present invention provides a method for promoting differentiation into neurons by treating embryos formed from human embryonic stem cells with vascular endothelial growth factor (VEGF) and inducing them into increased embryonic embryos. do.

In the present invention, the method for promoting differentiation is a step 1 to induce an embryonic body with an increase in neuroectoderm by treating an embryonic body formed from human embryonic stem cells with vascular endothelial growth factor (VEGF). Two steps of attaching to the culture culture dish to induce neutrophil progenitor cells, three steps to remove induced neuroprogenitor cells from the tissue culture dish and reattach to culture to increase the purity and maintain the density uniformly, and It characterized in that it comprises a four steps to finally differentiate the neural progenitor cells into dopamine neurons.

In the present invention, the embryonic body in the first step is preferably treated with vascular endothelial growth factor VEGF for 6 to 10 days, preferably, it is characterized in that it is treated with VEGF for 7 to 9 days.

In the present invention, the treatment concentration (VEGF) of the vascular endothelial growth factor in the first step is preferably 10 ~ 100 ng / ml, preferably characterized in that the treatment using VEGF of 40 ~ 60 ng / ml. . VEGF 50 ng / ml used in the embodiment of the present invention is to dissolve the vascular endothelial growth factor in a mixed solution of PBS and 0.1% BSA so that the final concentration in the culture medium to 50 ng / ml.

In the present invention, it is preferable to culture the embryos with serum-free during the VEGF treatment period. Recent studies have reported that there is a major factor in the differentiation of embryonic stem cells into blood vessels or hematopoietic cells in serum. In the present invention, serum-free cultures are used to reduce the effects of VEGF on angiogenesis and to reduce the effects of neurogenesis. Maximized.

In the present invention, the embryonic body in which the neuroectodermal increased in the second step is preferably added to 5 ~ 50 μg / ml of the extracellular matrix fibronectin (fibronectin) to enhance the adhesion, preferably 20 to 30 μg / ml of fibronectin is added. When the embryos are attached and cultured, a Falcon 3002 tissue culture dish is used. When culturing cells in the tissue culture dish, the embryos can be attached and cultured without a separate coating. Just add fibronectin to ensure a support base.

In the present invention, it is preferable to use the ITS culture medium to increase the cells expressing Nestin in the second step. Here, the ITS culture medium includes insulin, transferrin, and selenium, and refers to a culture medium used to propagate nestin-expressing cells, ie, neural progenitor cells.

In the present invention, it is preferable that the density of the cells to be reattached in the three steps is maintained at 1 × 10 ⁴ to 10 ⁶ cells / cm ² . The range of cell density is the concentration of the most active differentiation of nerve cells when tested at various densities, and outside of these ranges, differentiation or death occurs. When the cell is attached to the culture plate and propagated, differentiation may occur or die in an undesired direction by intercellular signal transmission depending on the degree of saturation of the cell.

In the present invention, it is preferable to coat the tissue culture plate to be reattached in step 3 with poly-l-ornithine. Poly-l-ornithine of the present invention serves to induce a signal that affects differentiation into neurons by attaching cells as an extracellular matrix.

In the present invention, it is preferable to propagate the cells reattached in step 3 in N2 culture medium to which 5 to 50 ng / ml stromal fibroblast growth factor (bFGF) is added, and more preferably, the cells reattached to 10 to 10 It is characterized in that the proliferation for 5 to 7 days in N2 culture medium added 30 ng / ml bFGF.

In the present invention, the final differentiation into neurons in the fourth step is preferably characterized in that the nerve precursor cells are cultured in bFGF-free N2 culture for 5 to 7 days. In bFGF-added N2 culture embryos are differentiated and proliferated into neural progenitor cells. In order to differentiate these neural progenitor cells into neural cells, the final object of the present invention, the embryonic bodies should be cultured in N2 culture medium with bGFG removed for 5-7 days. .

According to another aspect of the present invention, there is provided a composition for promoting neuronal differentiation containing vascular endothelial growth factor (VEGF) as an active ingredient.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example  1. Culture of Human Embryonic Stem Cells

Human embryonic stem cell lines Miz-hES4 and Miz-hES were cultured for stable supply of embryonic stem cells for neuronal differentiation. The medium for culture included 20% knockout serum replacement in Dulbecco's modified Eagle's medium (DMEM) and undifferentiated embryonic stem cells in 0.1 mM nonessential amino acid, 0.1 mM mercaptoethanol, 0.5 U / ml penicillin- streptomycin, and 0.1 mM β-mercaptoethanol. An important 4 ng / ml basic fibroblast growth factor (bFGF) was added. The culture medium was changed once a day, and then passaged using collagenase type IV (Invitrogen, Carlsbad, CA) once every 5 to 7 days to provide a new feeder layer and to maintain a constant colony size of embryonic stem cells in the culture dish. Embryonic stem cells were maintained at a constant level. In consideration of the possibility that collagenase type IV may have a negative effect on the maintenance of undifferentiated cells, some embryonic stem cells were maintained in the subdifferentiated state by subculture using micro-dissecting technique.

It was confirmed that the stem cells cultured in this way remain undifferentiated even after several passages. First, in the morphological aspect, embryonic stem cell colony in undifferentiated state is formed by forming round circular colony by the feeder layer supporting the stem cells physically and nutritionally and clearly distinguishing the marginal area where the feeder layer and colony meet. The chemical properties are well represented. In addition, the individual stem cells are well represented the morphological characteristics of the typical stem cells with a relatively large nucleus (round A). In addition, it can be confirmed that the transcription factors SSEA4, Nanog, and Oct4, which are markers for pluripotency, are well expressed in the nucleus of all cells in the embryonic stem cell colony (FIG. 1C). As a result of investigating the activity of alkaline phosphatase, another marker used for the omnipotence of such embryonic stem cells, it can be seen that the activity appeared in almost all embryonic stem cells (FIG. 1B).

Example  2. Owning both inside, outside and mesoderm Tricotyledonous Embryonic  Differentiation Capacity Survey

To confirm the omnipotence of embryonic stem cells, we examined whether embryos can be differentiated into trioderm. All the tissues of the adult are derived from ectoderm, mesoderm, and endoderm, which are produced during the formation of the embryonic stage, which is a stage of embryonic formation. At the same time, it is the first step in inducing differentiation into specific cells.

In the present invention, using embryo collagen type IV (2mg / ml) (Invitrogen, Carlsbad, CA), a colony grown to an appropriate size in order to form an embryoid body from embryonic stem cells whose omnipotence and self-proliferation have been confirmed. Separate from layer. This separated colony is then broken into a cell aggregate consisting of about 50 to 100 cells using a pipette. The cell aggregates broken down to the appropriate size are embryonic stem cell cultures except for basic fibroblast growth factor (bFGF) (R & D Systems, Minneapolis, MN). To form. Embryos formed were fixed with 4% Paraformaldehyde solution and frozen sectioned to analyze their initial differentiation.

Cell aggregates cultured for 8 days formed spherical embryos with typical morphological characteristics of embryos. As a result of fragmentation and immunofluorescence staining, it was confirmed that the embryonic stem cells were initially differentiated into individual cells exhibiting the properties of ectoderm, mesoderm, and endoderm. In addition, induction of differentiation into a specific type of cells further confirmed that the induction of differentiation through embryonic bodies has various possibilities. GATA4, a representative marker of endoderm, is concentrated in some regions of the embryonic body (Fig. 2 B, C). Another endoderm markers, AFP and Sox17, also expressed similarly (B, C in Figure 2). Brachyury, a marker of mesoderm, can also be seen in the embryo (FIG. 2D). As the differentiation progresses, ectoderm cells to be differentiated into neurons can be identified as markers of neuroectoderm such as Pax6 and nestin. These ectoderm cells are mainly distributed just below the surface of the embryoid body and can be seen to show the distribution of the morphological characteristics of neural rosettes throughout the embryonic body (FIG. 2E).

Example  3. The ectoderm cells are induced by vascular endothelial growth factor. Increased Embryonic  Formation and Enhancement of Neuroprogenitor Cell Differentiation Efficiency

  At the stage of organogenesis, the nervous system and the vascular system are the first organs to develop from the germ tissue, and these organs are known to mutually participate in differentiation during the process of embryogenic development. From the interrelationship between neurogenesis and angiogenesis, it was hypothesized that vascular endothelial growth factors related to angiogenesis would affect differentiation of neural stem cells into neural cells during embryonic formation. Embryos formed from human embryonic stem cells were cultured in the presence of vascular endothelial growth factor for 8 days. As a result, it was confirmed that the expression areas of Pax6 and Nestin, which are markers of neuroectodermal growth, were increased compared to the control group that did not process vascular endothelial growth factor (FIGS. 3A and 3B). However, it was confirmed that a small amount of CD34, a marker of hematopoietic progenitor cells, did not increase, and thus the treatment of vascular endothelial growth factor during embryonic formation was related to neurogenesis, not angiogenesis (FIG. 3C). This result was once again confirmed by RT-PCR analysis. Sox1, another marker of neuroectodermal growth, was increased by vascular endothelial growth factor, and the markers of endoderm were decreased (FIG. 3D).

To determine how the endothelial treated embryos differentiated continuously, embryos cultured for 8 days were attached to tissue culture dishes and cultured in ITS medium containing fibronectin (Sigma, St. Louis, MO). After 8 days of incubation, cells migrated from the endothelium-treated embryonic bodies formed a much larger number of neural rosettes compared to cells from the control embryos without the vascular endothelial factor treatment (Fig. 4). C), the formed rosettes express Pax6 and nestin well and were clearly distinguished morphologically (A, B, D of FIG. 4). In addition, the expression of Pax2, a transcription factor involved in the formation of midbrain and hindbrain, is well expressed in neural rosettes, suggesting that most of the neural precursor cells migrated from embryonic bodies will differentiate into dopamine neurons in the midbrain (Fig. 4). E).

To obtain more pure neuronal progenitor cells, dissociate the neural rosette using trypsin and attach to Poly-l-ornithine (PLO) (15 μg / Sigma) -coated culture dish and bFGF (20 ng / ml) in N2 culture. ) Was added and cultured. For about 6 days after the start of the culture, the attached cells multiplied rapidly, resulting in most cells expressing Nestin, a marker of neural progenitor cells (FIG. 4F).

Example  4. Final differentiation into neurons

In order to finally differentiate the increased neural progenitor cells into neurons, the neural progenitor cells were incubated for about 6 days with bFGF removed in N2 culture (FIG. 5A). After termination of differentiation, it was confirmed by Tuj1, which is a marker of neurons, compared with the control group (43.9 ± 5.2%, n = 5) (Fig. 5B), and neurons differentiated from ectodermal-derived embryos with vascular endothelial growth factor. It can be seen that the amount of is increased (64.9 ± 3.4%, n = 5) (Fig. 5C). On the other hand, it was confirmed that a very small amount of glial cells or endothelial cells, etc., which are not neurons (FIG. In particular, recent studies have reported that there is an important factor inducing embryonic stem cells into serum to mesodermal cells that will differentiate into vascular cells or hematopoietic cells. In the present invention, the vascular endothelial growth factor of vascular endothelial growth factor using serum-free culture. Reduced production and maximized the effects of neuronal formation.

Analysis to define differentiated neurons from embryonic bodies induced by vascular endothelial growth factor revealed that 57.14 ± 3.0% of all differentiated neurons were dopamine neurons and less than 1% of other types of neurons. It turns out to exist. RT-PCR analysis results also show that the genes involved in dopamine neuron formation in the midbrain are expressed in differentiated neurons.

As described above, according to the present invention, by using the vascular endothelial growth factor, it is possible to form an embryonic body in which neuroectodermal cells are increased from human embryonic stem cells, and by increasing the differentiation efficiency from such embryonic bodies to neurons, In addition, it is expected to facilitate the supply of cells for cell therapy of neurodegenerative diseases such as Parkinson's disease caused by the destruction of midbrain dopaminergic neurons.

Claims (11)

A method for promoting differentiation into neurons by treating embryos formed from human embryonic stem cells with vascular endothelial growth factor (VEGF) and inducing them into increased embryonic extracellular germ layers. The method of claim 1, wherein the embryonic body formed from human embryonic stem cells is treated with vascular endothelial growth factor (VEGF) to induce an embryonic embryo with increased neuroectodermal lobe, and the embryonic body with increased neuroectodermal embryo is placed in a tissue culture dish. Attaching and culturing two steps to induce neural progenitor cells, detaching the induced neural progenitor cells from a tissue culture dish and reattaching them to culture to increase the purity and maintain the density uniformly, and the high purity neural progenitor cells And four stages of final differentiation into dopamine neurons. The method of claim 2, wherein in the first step the embryoid body is treated with vascular endothelial growth factor (VEGF) for 7 to 9 days. The method of claim 2, wherein the concentration of vascular endothelial growth factor (VEGF) in the first step is characterized in that 40 ~ 60 ng / ml. 4. The method of claim 3, wherein said embryos are cultured in serum free during said period. The method of claim 2, wherein the embryonic embryos with increased neuroectodermal at step 2 is characterized in that to enhance adhesion by adding 20 ~ 30 μg / ml of the extracellular matrix fibronectin (fibronectin). The method of claim 2, wherein the ITS culture is used to increase the cells expressing Nestin in the second step. The method of claim 2, wherein the density of the cells to be reattached in the step 3 is characterized in that to maintain 1 × 10 ⁴ ~ 10 ⁶ cells / cm ² . The method of claim 2, wherein the tissue culture plate reattached in the step 3 is characterized in that the coating with poly-l-ornithine. The method of claim 2, wherein the cells reattached in step 3 are grown in N2 culture medium containing 10-30 ng / ml stromal fibroblast growth factor (bFGF) for 5-7 days. The method according to claim 2, wherein the final differentiation into neurons in step 4 is characterized in that the neural progenitor cells are cultured in bFGF-free N2 culture for 5-7 days.

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