KR100941036B1 - Three-step Differentiation protocol for generation of spinal cord oligodendrocytes from human embryonic stem cells - Google Patents

Abstract

The present invention is to increase the ventral spinal nervous system cells by culturing human embryonic stem cells in a medium containing Sonic Hedgehog (Shh), Retinoic acid (RA), noggin and vitronectin The present invention relates to a method of inducing differentiation from human embryonic stem cells to spinal cord oligodendrocyte glial cells, characterized by inducing isolated embryoid bodies.

According to the present invention, human embryonic stem cells can be effectively induced to differentiate oligodendrocyte glial cells from human embryonic stem cells without artificially introducing external genes by forming an optimal environment for epigenetic cell differentiation, which is very similar to the normal development process. Can be. In addition, it is possible to develop a cell therapy composition for treating spinal cord disease and spinal cord injury by using the differentiation-derived oligodendrocytes or their progenitor cells.

Description

Three-step Differentiation protocol for generation of spinal cord oligodendrocytes from human embryonic stem cells

The present invention relates to a method of inducing differentiation into spinal cord oligodendrocytes that can be used in the treatment of spinal cord injury patients with human embryonic stem cells, and more specifically, 1) from embryonic stem cells from human embryonic stem cells. Treatment of Noggin, Retinoic acid (RA) and Sonic hedgehog (Shh) together in the formation of neural embryoderm, more specifically spinal nerve ectodermal embryos (dorsal spinal cord) Neuronal cells), 2) treatment with bFGF and PDGF to promote progenitor cell proliferation of oligodendrocytes, and 3) cyclic adenosine monophosphate (cAMP) and insulin-like growth factor (IGF) on proliferated progenitor cells. -1), and a method for treating neurotrophic factor (NT-3) to differentiate into final oligodendrocytes.

Methods for obtaining cells that can be used for the purpose of treating spinal cord injury include culturing cells obtained from human spinal cord tissue and differentiating stem cells, but those who have spinal cord injuries due to lack of donor cells. In order to obtain a large amount of tissue that can treat the disease, research using stem cells has shown the greatest potential at this time. Recently published studies show that cell therapy compositions can treat spinal cord damage caused by genetic causes or accidents by treating the stem cells with various factors related to the differentiation of oligodendrocytes during normal human embryonic development. It is expected to be able to obtain.

In particular, human embryonic stem cells possessing infinite self-proliferative capacity and capable of differentiating into more than 200 kinds of cells constituting the human body are self-proliferating to infinity and constituting the body when placed under culture conditions including various differentiation-related factors. It has the potential to differentiate into specific cells. Therefore, attempts are being made worldwide to use the cells obtained through this purpose for various cell therapies including the treatment of neurological diseases.

As a method of differentiating oligodendrocyte glial cells using embryonic stem cells, it is possible to treat oligodendrocyte glial cells using a complex treatment of appropriate growth factors or hormones and control of physical and chemical culture conditions, and using viral vectors and specific compounds. A method of facilitating differentiation by introducing genes known to play an important role in differentiation into cells and inducing overexpression of the genes may be mentioned. In the former case, attempts have been made to differentiate high-purity oligodendrocytes using rodent-derived mouse embryonic stem cells, but the differences in mouse and human development, and morphological differences between human embryonic stem cells and mouse embryonic stem cells. Due to the differences in expression of markers and differences in culture conditions, the development of oligodendrocyte glial cells is very limited.

In addition, the latter study using genetic engineering techniques requires the process of artificially introducing foreign genes into cells to modify the traits of the cells. Therefore, due to the safety of genetically changed cells, it is difficult to apply clinically, which requires the injection of cells into the body for the treatment of diseases, and it is only experimental to find genetic factors that play an important role in differentiation into oligodendrocytes. It is used as a research method.

In view of this fact, the theory is that the induction of differentiation that can be used in practice should be carried out through epigenetic differentiation process that does not cause genetic transformation of cells. However, while there have been many studies on the technique of inducing differentiation from human embryonic stem cells to neurons, the development record on the differentiation technique to spinal cord oligodendrocytes, another important cell constituting the nervous system, has been developed. Is a very poor situation. Moreover, epigenetic differentiation from human embryonic stem cells to oligodendrocyte glial cells is mostly focused on the final differentiation of oligodendrocytes, rather than early stages of differentiation, and from spinal cord from human embryonic stem cells. The development of epigenetic differentiation methods by stages of development into oligodendrocytes is essential for the cellular processes that require quality control.

In the present invention, in order to overcome the problems of the prior art and more efficiently develop differentiation protocols similar to the normal embryonic development process, neuroectodermal formation, spinal nervous system differentiation, progenitor cell differentiation of oligodendrocytes, proliferation of these precursor cells and The present invention has been completed, which systematically includes a step of final differentiation into oligodendrocytes.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior art, while inhibiting the differentiation of human embryonic stem cells having pluripotency into unwanted cells, spinal cord neuropathy and spinal cord injury, etc. In case of differentiating spinal cord oligodendrocytes, which are potentially used for cell therapy, considering the infinite proliferation ability of embryonic stem cells, it is easy to supply a large amount of spinal cord injured tissue, which is the biggest problem of cell therapy. In addition, since the differentiation process of the present invention is very similar to the development process of the normal spinal nerve, the laboratory model system that enables the discovery of various genes and unknowns involved in the spinal cord development process (in It was confirmed that it can be used as an in vitro model system, and the present invention was completed.

During normal early embryonic development, spinal oligodendrocytes begin to differentiate in the ventral part of the neural tube tail (the caudal part), and various differentiation factors for neural tube formation and the differentiation of ventral spinal nerve cells. Acts alone or in combination to induce differentiation of oligodendrocytes.

Therefore, the main object of the present invention is to create an optimal environment of cell differentiation very similar to the normal development process step by step, the optimal differentiation technique that can effectively induce the differentiation of rare progenitor cells from human embryonic stem cells without the artificial introduction of external genes Is in developing.

To this end, we investigated the effects of various factors involved in the development, differentiation, proliferation, maturation and survival of spinal cord oligodendrocytes during the normal developmental stages from human embryonic stem cells to final oligodendrocyte differentiation. We have developed an effective differentiation technique.

According to one aspect of the invention, the present invention is cultured in human embryonic stem cells in a medium containing Sonic Hedgehog (Shh), Retinoic acid (RA), and noggin (abdominal spinal nerve ectoderm) A first step of forming the increased embryoid body; A second step of incubating the formed embryoid body in a medium containing basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) to induce proliferation of progenitor cells of oligodendrocytes; And the proliferated progenitor cells are cyclic adenosine monophosphate (cAMP), insulin-like growth factor 1 (IGF-1), and neurotrophin 3 (neurotrophin 3, NT-3). It provides a method for inducing differentiation from human embryonic stem cells to spinal cord oligodendrocyte glial cells comprising a third step of inducing differentiation of oligodendrocytes by culturing in a medium containing a).

The ventral spinal cord nervous system cells of the present invention can be differentiated into neurons, astrocytes, and oligodendrocytes that form the brain and spinal cord, and the oligodendrocytes are in turn the cranial nerve or the spinal cord nervous system. It is divided into cells that make up and differentiated. Nogin of the present invention is effective in inducing the antagonist and neuronal differentiation of BMPs that inhibit the formation of neural tube ventral cells during early development, and retinoic acid (RA) is a mouse embryonic stem cell. It is effective in inducing neuronal differentiation.

In the method of inducing differentiation into spinal cord oligodendrocyte glial cells of the present invention, it is preferable to further treat 3-7 μm / ml of vitronectin in the first step, and 5 μm / ml of vitronectin is most preferred. desirable. Vitronectin of the present invention is a constitutive protein of the extra cellular matrix ECM, which is known to enhance the activity of SHH after binding to sonic hezog (SHH) during normal development. Treatment with vitronectin in the present invention was found to significantly increase the rate of differentiation into oligodendrocytes (FIG. 7C).

In the method of inducing differentiation into spinal cord oligodendrocyte glial cells of the present invention, in the first step, initial fibroblast growth of 1.5 to 2.5 ng / ml in order to adapt human embryonic stem cells to differentiation culture medium in the first 1 to 3 days. Adaptation to differentiation cultures in the presence of a factor (bFGF) is preferred and differentiation, most preferably after adaptation to differentiation cultures in the presence of 2 ng / ml basic fibroblast growth factor (bFGF) for the first 2 days It is good to let. Although various cytokines and growth factors may promote differentiation of human embryonic stem cells, sometimes cells may die due to cytotoxicity of cytokines or growth factors. In this case, there is an advantage of reducing the toxicity to stabilize the embryoid body formation and to facilitate the differentiation.

In the method of inducing differentiation into spinal cord oligodendrocyte glial cells of the present invention, Sonic Hedgehog (Shh) of 450 to 550 ng / ml, Retinoic acid (RA) of 8 to 12 μM in the first step, And 6 to 10 days of incubation in a culture solution containing 0.8 to 1.2 μg / ml of noggin, most preferably 500 ng / ml of Sonic Hedgehog (Shh), 10 μM of retinol acid ( Retinoic acid (RA) and 1 μg / ml of noggin (noggin) culture medium containing 8 days is recommended. In the present invention, when cultured in the medium of the above conditions it can be seen that there is an ability to induce to retain the properties of spinal cord neurons (Fig. 4, 5).

In the method of inducing differentiation into spinal cord oligodendrocyte glial cells of the present invention, in the second step, 8 to 12 ng / ml basic fibroblast growth factor (bFGF) and 8 to 12 ng / ml platelet derived growth factor (PDGF) It is preferable to incubate for 6 to 10 days in a culture medium containing), and most preferably in a culture medium containing 10 ng / ml basic fibroblast growth factor (bFGF) and 10 ng / ml platelet derived growth factor (PDGF). It is good to culture one. In the present invention, when cultured in the medium under the above conditions, the proliferation of progenitor cells proceeded rapidly and the proliferation rate was found to be the highest (FIG. 6I).

In the method of inducing differentiation into spinal cord oligodendrocyte glial cells of the present invention, 0.5-1.5 μg / ml cyclic adenosine monophosphate (cAMP), and 80-120 ng / ml insulin-like growth factor in the third step It is preferable to incubate for 6 to 10 days in a culture medium containing -1 (insulin-like growth factor 1, IGF-1) and 3 to 7 ng / ml of neurotrophin 3 (NT-3). For example, 1 μg / ml cyclic adenosine monophosphate (cAMP), 100 ng / ml insulin-like growth factor 1 (IGF-1) and 5 ng / ml neurotrophic factor 3 ( Incubate for 8 days in culture containing neurotrophin 3, NT-3. In the present invention, it can be seen that the differentiation efficiency is significantly increased when cultured in the medium of the above conditions, and that the factors play an important role in the survival of differentiated oligodendrocytes during final differentiation into oligodendrocytes. Could be (FIG. 7E).

According to one aspect of the present invention, the present invention provides a neural basal medium (DMEM / F-12 medium) of 23 to 27 μg / ml insulin, 45 to 55 μg / ml apo-transferrin, 5 to 7 ng / ml progesterone, 8-12 μg / ml Putrescine, 45-55 ng / ml Sodium selenite, 36-44 ng / ml triiode tyrodine (tri Sonic Hedgehog (Shh) in addition to 450-550 ng / ml neural restricred media containing -iodo-thyroidin T3), 36-44 ng / ml thyroxine T4 and B27 adjuvant It provides a media composition for the formation of embryoid body with increased ventral spinal nerve ectodermal comprising 8 to 12 μM of retinoic acid (RA) and 0.8 to 1.2 μg / ml of noggin. DMEM (Dulbecco's modified Eagle's medium) used as a neural basal medium of the present invention is a basic medium used for cell culture. In an embodiment of the present invention, specifically DMEM / F-12 (Gibco) was used. Neural restricred media were treated with insulin, apo-transferrin, progesterone, Putrescine, sodium selenite, It is a medium for inducing differentiation into neuronal cells, further comprising triiodine tyrodine, thyroxine, and B27. The B27 is a medium composition generally added to nerve cell culture, and is a growth enhancer similar to serum, and is generally used for long-term survival of neurons of the central nervous system. In the present invention, Gibco 50 times stock B27 reinforcement was purchased, diluted (1ml B27 reinforcement / 50ml) was used. In the medium composition for embryonic body formation in which the ventral spinal neural extradermal embryo of the present invention is increased, the medium composition is a medium composition for embryonic body formation, further comprising 3 to 7 μm / ml of vitronectin. desirable.

According to one aspect of the present invention, the present invention further comprises a basic fibroblast growth factor (bFGF) of 8-12 ng / ml and platelet-derived growth of 8-12 ng / ml in addition to the neural restricred media described above. Provided is a composition for inducing progenitor glial progenitor cell proliferation comprising a factor (PDGF).

According to one aspect of the present invention, the present invention further comprises cyclic adenosine monophosphate (cAMP), insulin-like growth factor-1, in addition to the neural restricred media described above. Provided is a medium composition for inducing oligodendrocyte glial differentiation comprising IGF-1) and neurotrophin 3 (NT-3).

According to an aspect of the present invention, the present invention provides a therapeutic agent for spinal cord disease and spinal cord injury comprising spinal cord oligodendrocytes or their progenitor cells differentiated by the method of inducing differentiation into spinal cord oligodendrocytes as an active ingredient. To provide a composition. In the cell therapy composition of the present invention, the composition containing the spinal cord oligodendrocyte or its progenitor cells as an active ingredient may be prepared in a general formulation in the art, for example, in the form of an injection, and spinal injury site by surgical operation. It may be directly implanted in or moved to a spinal injury site after administration to a vein, but it is preferable to move to a spinal injury site after administration to a vein. Differentiation-induced oligodendrocyte progenitor and oligodendrocyte glial cells in the composition of the present invention may vary depending on the type of disease, the route of administration, the age, sex, and severity of the patient, but preferably in the case of the average adult. Administer 10 ⁴ to 10 ⁸ cells.

Hereinafter, the differentiation technique for the production of spinal nerve oligodendricular glial cells from human embryonic stem cells of the present invention will be described in more detail step by step. The present invention is similar to the normal development process 1) Induction of differentiation of neuroectodermal (ventral spinal nerve ectodermal) by treating Noggin, Retinoic acid, Sonic hedgehog from human embryonic stem cells, 2) Induction of proliferation of oligodendrocyte progenitor cells by treatment of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), 3) Neurotrophic factor-3 ( neurotrophin-3 (NT-3), insulin-like growth factor (IGF-I) and cyclic adenosine monophosphate (cAMP) to induce differentiation into mature oligodendrocytes Includes three steps in total. In addition , in step 1) , Vitronectin is added to the culture medium to maximize the efficiency of differentiation induction. The present invention is characterized by remarkably increasing the differentiation yield due to the specific differentiation of spinal cord oligodendrocytes and the use of survival factors, and further, Noggin, Retinoic acid, and Sonic hedgehog. It is characterized by undergoing the process of differentiation of neuroectodermal (ventral spinal neuroectodermal) by treatment.

In the present invention, in order to differentiate human embryonic stem cells into spinal cord oligodendrocytes, neuronal cell-derived culture medium was used, and DMEM / F-12 (neural basal medium) was used as a basal medium, 25 ug / ml insulin, 50 ug / Neural restricted media with the addition of ml apo-transferrin, 6.3 ng / ml progesterone, 10 ug / ml putrescine and 50 ng / ml sodium selenite ), 40 ng / ml triiodine tyrodine, 40 ng / ml thyroxine and B27 adjuvant were additionally used. Based on this neuronal cell-derived culture, in order to specifically induce spinal cord oligodendrocyte differentiation, cytokines, growth factors, and hormones are treated alone or in combination in the order of the differentiation stages. Produced.

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. Embryonic Stem Cells Embryonic body  In the process of formation Into spinal nerve cells  Differentiation Promotion

One). Human Embryonic Stem Cell Culture

Human embryonic stem cells contained 20% KnockOut SR (Gibco), 0.1 mM nonessential amino acids (Gibco), 0.1 mM beta-mercaptoethanol (β-mercaptoethanol) (Sigma) in DMEM / F-12. Cultured daily using culture medium containing 100 unit / ml penicilly / streptomycin (Gibco Co.) and 4 ng / ml hbFGF (Peprotech), and maintained with subculture once every 7 days. Cell lines were used for differentiation of oligodendrocytes. In order to confirm that the characteristics of stem cells are maintained, immunostaining with Oct4, Nanog, SSEA-4, and Alkaline phosphatase, which are undifferentiated markers of human embryonic stem cells, was performed. Expression was confirmed through polymerase chain reaction. In FIG. 1, (B) alkaline phosphatase, a marker of undifferentiation, is related to whether human embryonic stem cells, which are raw materials of the present experiment, properly maintain their characteristics as undifferentiated human embryonic stem cells before initiation of differentiation into oligodendrocytes. phosphatase), (C) SSEA-4, (D) Nanog, (E) Oct-4, and (G) Rex-1. Figure 1A is the form of a typical human embryonic stem cell colony and Figure 1F shows the synthesis of Figures 1D and E. Figure 1G shows the expression of human embryonic stem cell-specific genes by polymerase chain reaction, it can be seen that they are not expressed for differentiation marker factors. Pax6 to HNF3beta are genes that appear when differentiated. Human embryonic stem cells were used Miz-hES4 and Miz-hES6 cell line.

2). Nogin Noggin Induction of Nervous System Cells by Treatment

For differentiation of spinal cord oligodendrocytes from human embryonic stem cells, the differentiation of neural cells should be promoted first, and these cells are induced to differentiate into spinal nerve cells, specifically ventral spinal nerve cells, located in the rear of neural tube. Promoting them is a prerequisite. Nogin is known to be effective in inducing antagonists and neuronal differentiation of BMPs that inhibit neural tube ventral cell formation during early development. In addition, retinoic acid (RA) is used to induce neuronal differentiation of mouse embryonic stem cells.

In view of this fact, in the present invention, the differentiation efficiency into neural cells was compared and analyzed by RA or nogin for 8 days during the formation of embryoid bodies which appear as an early differentiation form after the start of differentiation of human embryonic stem cells. As a result, as shown in A, B, and C of FIG. 2, the embryonic bodies treated with noggin (1 μg / ml) were significantly larger in size than the control group (A) and group (B) treated with RA (10 μM). Large and healthy form appeared.

As a result of analyzing the differentiation efficiency into neuroectoderm by the expression level of genes specifically expressed in neuroectoderm, when treated with nogin, as shown in FIGS. 2D and E, compared with the control and RA-treated groups, endoderm Markers of differentiation (HNF3beta, Sox 17, AFP (alpha-fetoprotein) and mesoderm (differentiation into cells that form muscle, bone, blood, etc. after differentiation) Gene expression (Mixl1, GATA4, T) expression is reduced effectively and expression of neuroectoderm markers (Pax6, Sox1, Nestin) that can be differentiated into oligodendrocytes increases, so as to neuroectoderm Differentiation factor was determined by nogin. 2E quantitatively shows the gene expression results of FIG. 2D. Figure 3 is a test result re-tested at the protein level using the immunofluorescent staining technique shown in the genetic level of 2D and E, endoderm expressing HNFβ, Sox17, AFP, GATA4, Bry, etc. due to nogin treatment Differentiation into mesodermal cells was effectively blocked, and differentiation induction was promoted into progenitor cells (neural extracellular germ cells (expressing Pax6 and Nestin together)) of oligodendrocyte glial cells, the ultimate target cells of the present technology.

3). RA  And Sonic Hezog  ( Sonic hedgehog By treatment Ventral Spinal nervous system  Induction of Cell Differentiation.

The human central nervous system is composed of the brain and spinal cord. During the initial formation of the central nervous system, the brain is formed in front of the neural tube and the spinal cord is generated in the rear. Spinal oligodendrocytes, along with motor neurons, derive from the pMN domain, whose developmental origin is at the ventral side of the spinal cord, so that neural cells formed from embryonic stem cells become neuroid glial cells. Differentiation should be made so that the identity of the person retains the properties of the spinal nervous system, especially the ventral spinal nervous system. Markers that express genes from the head of the early embryo to the tail, i.e., forebrain-midbrain-hindbrain-spinal cord, and from ventral to ventral The differentiation pattern of neuronal cells was observed.

Differentiation efficiency into spinal cord nervous system cells by various combination treatment of differentiation factors during 8 days of embryoid body formation was investigated using genes expressed in sections from the whole brain to the spinal cord during normal fetal development. At this time, during the first two days of the embryonic body formation process, in order to adapt human embryonic stem cells to differentiation cultures, the embryos were differentiated after adapting to differentiation cultures in the presence of basic fibroblast growth factor (bFGF).

As a result of the polymerase chain reaction, the experimental group (S / R / N) treated with retinol acid and nogin (R / N), or sonic hexog (500 ng / ml), retinol acid and nogin was treated with control, noginman only. The expression of spinal cord genes (Hoxb5, Hoxc5, Hoxb6) was higher than that of Noggin and Sonic Hezog and Noggin treated groups (S / N). As a result of the present invention, retinol acid has no great effect on the formation of neuronal cell itself, but has been shown to have the ability to induce the formed nervous system cells to retain the properties of spinal cord neurons.

4B to E are the results of re-verification by immunofluorescence staining at the protein level that the fate of the cells was converted to the spinal nerve cells by the treatment of S / R / N, which is expressed in the brain by S / R / N treatment. The expression of Otx2 is decreased and the expression of HB9 expressed in the spinal cord is increased. The green color of E is Tuj1, a marker of neuronal cells, indicating that some cells also differentiate into spinal motor neurons.

Both the group treated with retinol acid and nogin (R / A) and the group treated with sonic hezog, retinol acid and nogin (S / R / N) had the effect of differentiating neurons into spinal cord neurons. The effects on the differentiation of the ventral spinal cord nervous system, a region where spinal cord oligodendrocytes differentiate, were compared and analyzed in two groups. To aid understanding, the expression range of genes expressed by regions from the dorsal to the ventral of the neural tube during the development process is modeled on the right side of FIG. 5A. As shown in FIG. 5A, the experimental group treated with sonic hezog, retinol acid, and nogin (S / R / N) specifically showed the pMN domain, which is an area where spinal cord oligodendrocytes differentiate during normal development. The expression of expressing Olig2 was significantly increased.

As a result of the immunofluorescence staining, the cells expressing Olig1 and Olig2 expressed in the pMN domain, which is a differentiation region of oligodendrocytes in the spinal cord, were treated with S / R / N in Figs. Increased, and expression of O4 expressed in oligodendrocytes was detected. Therefore, the final treatment of Sonic Hezog, RA and Noggin was finally determined as a pretreatment process for promoting differentiation of spinal cord oligodendricular glial cells from human embryonic stem cells.

Example  2. Differentiation-promoted spinal cord Ventral From neuronal cells Rare process  Proliferation and Differentiation of Glial Progenitor Cells

One of the requirements for cell therapy is that large quantities of raw materials must be obtained. As Sonic Hezog, RA and Noggin's combined treatment (S / R / N) promotes the differentiation of spinal cord ventral neuroectoderm, it is possible to induce proliferation of oligodendrocyte progenitor cells contained therein. Among the growth factors, bFGF (10 ng / ml) and PDGF (10 ng / ml) were compared and analyzed. To this end, the neural cells formed in the embryonic body were trypsinized, and then low density in a culture plate in which poly-L-ornithine and laminin were plated as extracellular matrix. Cells (1.25X104 cells / cm2) were attached and cultured for 8 days to examine the effect of growth factors. 6 shows the results of analysis of proliferation of progenitor cells of oligodendrocytes differentiated through neural ectoderm by S / R / N treatment. In order to analyze the proliferation rate of progenitor cells, BrdU (an analog of DNA component thymidine) was added to the culture medium to label the dividing cells, and then to NG2 specifically expressed in the progenitor cells of oligodendrocytes. Immunofluorescence was performed and the progenitor cells of dividing oligodendrocytes were counted and analyzed.

6A, C, E and G are the results of immunofluorescence staining using antibodies to NG2 and BrdU specifically expressed in progenitor cells of oligodendrocyte glial cells and treated with bFGF as shown in the photograph. The proliferation of progenitor cells proceeded rapidly in the group treated with bFGF and PDGF (platelet-derived growth factor) (Figs. 6B, D, F and H, respectively, showing phase morphology of left fluorescently stained cells). Photomicrograph). As a result of counting progenitor cells of proliferating oligodendrocyte glial cells, the proliferation rate of progenitor cells was highest in the group treated with bFGF and PDGF (FIG. 6I).

Example  3. Amplified Spinal Cord Ventral Rare process  From glial progenitor cells Rare process  Final differentiation into glial cells

In addition to the differentiation factors that regulate the development of specific tissues, extracellular matrix (ECM) that is external to the cell also plays an important role in cell differentiation. Therefore, after binding to sonic hezog (SHH) during normal development, 1) vitronectin (5 μg / ml), an extracellular matrix known to enhance the activity of SHH, was added to S / for 8 days of embryoid body formation. After additional treatment with R / R treatment, 2) treatment of bFGF and PDGF to amplify progenitor cells, and 3) spinal cord expressing O4 by final differentiation into oligodendrocytes in culture with survivors Differentiation rate of oligodendrocytes was measured.

O4-expressing oligodendrocytes were efficiently differentiated in the experimental group treated with S / R / N compared to the control group to which only vitronectin was added (FIG. 7A, VN) (FIG. 7B). When treated with N, the rate of differentiation into oligodendrocytes was significantly increased (FIG. 7C). 7D shows quantitatively counting oligodendrocyte glial cells for each treatment group.

Overall, as a result of analyzing the proliferation factor of progenitor cells and survival factors of the final differentiation process, when proliferating proliferation of cells with bFGF and PDGF, the proliferation factor was not treated as expected from the analysis results of FIG. 6. It was shown that oligodendrocyte differentiation was induced more efficiently than the control group (FIG. 7E). In addition, cyclic adenosine monophosphate (cyclic adenosine monophosphate, cAMP, 1 μg / ml), insulin-like growth factor-1 (insulin-like growth factor 1) in order to suppress the cell death occurring in the final differentiation induction for 8 days Treatment with IGF-1, 100 ng / ml) and neurotrophic factor 3 (neurotrophin 3, NT-3, 5 ng / ml) resulted in a statistically significant increase in differentiation efficiency. The final differentiation was analyzed to play an important role in the survival of differentiated oligodendrocytes (FIG. 7E). 8 is a schematic diagram illustrating the processing time and process for differentiation factors of the whole differentiation induction protocol.

Example  4. RNA  Extraction RT - PCR Wow Immunofluorescence Staining  Way.

Total amount of RNA was isolated using Trizol (Trizol, Invitrogen) and cDNA was prepared using SuperScript III (SuperScript III first-strand synthesis system, Invitrogen) to 1μg of extracted RNA. Since the primer and the PCR machine shown in Table 1 (AccuPower PCR-Premix, Bioneer, Daejeon, Korea) was performed using. PCR results were confirmed by 1% agarose gel electrophoresis. Relative band intensities were analyzed by an image analyzer (Image analyzer, AutoChemi Bioimaging system, UVP Inc., CA, USA). The expression level of mRNA was normalized compared to the expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA.

TABLE 1

Example  5. Immunofluorescence Staining  Way.

Cells were fixed with 4% paraformaldehyde in phosphate buffered saline PBS solution. Embryos saturated with 20% sugar were fixed with 4% paraformaldehyde and placed in an O.C.T compound (Tissue Tek, Sakura) and cut at 9 μm using Cryostat (LEICA CM 3050S). Immunofluorescence staining was performed by the general method and alkaline kinase activity staining was performed using Alkaline Phosphatase Substrate Kit III, Vector Laboratories. The list of antibodies is shown in Table 2 below. Appropriate fluorescently labeled secondary antibodies (Jackson ImmunoResearch Laboratories West Grove, PA) were used for visualization. Quantitative evaluation was performed using Axiovision (version 2.4 software, Zeiss).

TABLE 2

As described above, according to the present invention, spinal cord scarcity, which has the potential to be used for cell therapy such as spinal cord neuropathy and spinal cord injury, while inhibiting pluripotency-producing human embryonic stem cells into unwanted cells Dendritic glial cells can be differentiated.

Therefore, in consideration of the infinite proliferation ability, which is a characteristic of embryonic stem cells, the present invention can overcome the problem of securing raw materials, which is the biggest problem of cell therapy, by facilitating the supply of a large amount of spinal cord injury tissue.

In addition, since the differentiation process of the present invention is very similar to the development of normal spinal nerves, it is a laboratory model system (in vitro model system) that enables the discovery of various genes and discoveries involved in the spinal cord development process. Can be used.

1 is a diagram showing whether human embryonic stem cells, a raw material of the present experiment, properly maintain their characteristics as undifferentiated human embryonic stem cells before initiation of differentiation into oligodendrocytes.

2 is a diagram showing that Noggin, a type of cytokine, effectively induces differentiation into ectoderm cells, which are precursor cells of oligodendrocytes, in the early differentiation process of human embryonic stem cells.

Figure 3 is a diagram re-verification at the protein level of the experimental results shown in the gene level of 2D and E.

FIG. 4 is a diagram showing efficient cell differentiation from neuroectodermal cells with differentiation efficiency increased to oligodendrocytes from human embryonic stem cells shown in the results of FIGS. 2 and 3.

5 is a diagram illustrating the expression of genes that are expressed from the dorsal to the ventral of the neural tube during development.

FIG. 6 shows amplification of undifferentiated oligodendrocyte glial cells (i.e., progenitor cells of oligodendrocyte glial cells) differentiated via neuroectodermal by S / R / N treatment.

7 is a diagram measuring the differentiation yield of spinal cord oligodendrocytes expressing O4 obtained by further differentiating progenitor cells amplified by treating bFGF and PDGF together with oligodendrocytes.

8 is a diagram showing the processing time and process for each differentiation factor of the whole differentiation induction protocol.

Claims (11)

The first step in cultured in human embryonic stem cells in a medium containing Sonic Hedgehog (Shh), Retinoic acid (RA), and noggin to form embryoid body with increased ventral spinal nerve ectoderm ; A second step of incubating the formed embryoid body in a medium containing basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) to induce proliferation of progenitor cells of oligodendrocytes; And the proliferated progenitor cells are cyclic adenosine monophosphate (cAMP), insulin-like growth factor 1 (IGF-1), and neurotrophin 3 (neurotrophin 3, NT-3). A method of inducing differentiation from human embryonic stem cells to spinal cord oligodendrocytes comprising a third step of culturing the oligodendrocytes in different cultures by culturing in a medium. The method of claim 1, wherein in the first step, differentiation from human embryonic stem cells to spinal cord oligodendrocytes, which is induced by differentiation into embryoid bodies with increased ventral ectoderm ectoderm by culturing in a medium further comprising vitronectin. Induction method. The method of claim 1, wherein in the first step, the first 1-3 days from the human embryonic stem cells, the embryonic stem cells are cultured in the presence of basic fibroblast growth factor (bFGF) to adapt to the culture medium for differentiation. Induction of differentiation into spinal cord oligodendrocytes. The method according to claim 1, wherein in the first step, 450 to 550 ng / ml of Sonic Hedgehog (Shh), 8 to 12 μM of Retinoic acid (RA), and 0.8 to 1.2 μg / ml of Noggin ( A method of inducing differentiation from human embryonic stem cells to spinal cord oligodendrocyte glial cells, which is cultured for 6 to 10 days in a culture medium containing noggin). The method of claim 1, wherein in the second step, 6 to 10 days in a culture medium containing 8 to 12 ng / ml basic fibroblast growth factor (bFGF) and 8 to 12 ng / ml platelet derived growth factor (PDGF) A method of inducing differentiation from human embryonic stem cells to spinal cord oligodendrocytes, comprising culturing. According to claim 1, wherein in the third step 0.5 to 1.5μg / ml of cyclic adenosine monophosphate (cyclic adenosine monophosphate, cAMP), 80 to 120ng / ml of insulin-like growth factor-1 (insulin-like growth factor 1, IGF-1) and human embryonic stem cells from spinal cord oligodendrocyte glial cells characterized by culturing for 6 to 10 days in a culture medium containing 3-7 ng / ml neurotrophin 3 (NT-3). Differentiation Induction Method. Neuronal basal medium (DMEM / F-12 medium) 23-27 μg / ml insulin, 45-55 μg / ml apo-transferrin, 5-7 ng / ml progesterone, 8-12 μg / ml putrescine, 45-55 ng / ml sodium selenite, 36-44 ng / ml tri-iodo-thyroidin T3, 36-44 ng / In addition to neural restricred media containing ml of Thyroxine T4 and B27 adjuvant, 450 to 550 ng / ml of Sonic Hedgehog (Shh), 8 to 12 μM of Retinoic acid (RA) ) And media composition for embryoid body formation, wherein the ventral spinal nerve ectoderm is increased, including noggin of 0.8 to 1.2 μg / ml. The method of claim 7, wherein the embryo composition forming medium composition is characterized in that it further comprises 3 to 7μm / ml of vitronectin (vitronectin). Neuronal basal medium (DMEM / F-12 medium) 23-27 μg / ml insulin, 45-55 μg / ml apo-transferrin, 5-7 ng / ml progesterone, 8-12 μg / ml putrescine, 45-55 ng / ml sodium selenite, 36-44 ng / ml tri-iodo-thyroidin T3, 36-44 ng / additional 8-12 ng / ml basic fibroblast growth factor (bFGF) and 8-12 ng / ml platelet derived growth in neural restricred media containing ml of Thyroxine T4 and B27 adjuvant A medium composition for inducing oligodendrocyte progenitor cell proliferation comprising a factor (PDGF). Neuronal basal medium (DMEM / F-12 medium) 23-27 μg / ml insulin, 45-55 μg / ml apo-transferrin, 5-7 ng / ml progesterone, 8-12 μg / ml putrescine, 45-55 ng / ml sodium selenite, 36-44 ng / ml tri-iodo-thyroidin T3, 36-44 ng / In addition to neural restricred media containing ml of Thyroxine T4 and B27 adjuvant, cyclic adenosine monophosphate (cAMP), insulin-like growth factor-1 (IGF) -1), and a media composition for inducing oligodendrocyte glial differentiation, including neurotrophin 3 (NT-3). delete

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