KR20200105461A - Composition and kit for differentiating stem cells to neural crest stem cell comprising TGF-β I receptor inhibitor and BMP inhibitor, and method using the same - Google Patents

Abstract

The present invention relates to a composition for differentiation to a neural crest stem cell (NCSC) of a stem cell including a transforming growth factor beta (TGF-β) I receptor inhibitor and a bone morphogenetic protein (BMP) inhibitor of 0.001 to 2.5 μM, a kit, and a method using the same. By using a low-cost composition having a simple component, the composition is differentiated from the stem cell to the NCSC.

Description

Composition and kit for differentiating stem cells to neural crest stem cell comprising TGF-β I receptor for differentiating stem cells into neural crest cells, including TGF-beta I receptor inhibitors and BMP inhibitors inhibitor and BMP inhibitor, and method using the same}

It relates to a composition for differentiation of stem cells into neural crest stem cells (NCSC), a kit, and a method using the same.

Nervous system cells can be divided into two types, the central nervous system cells constituting the brain and spinal cord, and the peripheral nervous system cells constituting the motor, sensory and autonomic nerves. Neurons, astrocytes, and oligodendrocytes that make up the central nervous system (brain and spinal cord) are neural stem cells or neural progenitor cells differentiated from pluripotent stem cells: NPC ), whereas the peripheral nerve cells (motor, autonomic, and sensory nerves) and Schwann cells that make up the peripheral nervous system are neural crest stem cells differentiated from pluripotent stem cells. cell: NCSC). Therefore, central nervous system cells and peripheral nervous system cells are made from pluripotent stem cells along different differentiation pathways, respectively, through neural progenitor cells and neural crest cells, and these different pathways depend on the surrounding environment and intracellular signaling systems. It is known to be.

There are about 360,000 people suffering from various peripheral nervous system diseases caused by the death of peripheral nervous system cells in the United States alone, costing about 150 trillion dollars annually. Therefore, since it places a great social/economic burden on society, efforts to treat them are urgent.

Therefore, in order to develop a cell therapy method that is considered to be the most fundamental treatment for peripheral nervous system diseases, which are mostly intractable and progressive diseases, it is necessary to establish a method of efficiently differentiating stem cells into neural crest cells, which are the parent cells of peripheral nervous system cells. Essential and very important.

It provides a composition for differentiation of stem cells into neural crest cells.

It provides a kit for differentiation of stem cells into neural crest cells.

It provides a method of differentiating stem cells into neural crest cells.

One aspect is a neural crest cell of stem cells containing an inhibitor of transforming growth factor beta (TGF-β) I receptor and an inhibitor of 0.001 μM to 2.5 μM bone morphogenetic protein (BMP) ( It provides a composition for differentiation into neural crest stem cell: NCSC).

The term "TGF-β I receptor (Transforming growth factor beta I receptor)" refers to a serine/threonine receptor belonging to the TGF-β receptor family. The TGF-β I receptor may also be called TGFBR1, AAT5, ACVRLK4, ALK-5, ALK5, ESS1, LDS1, LDS1A, LDS2A, MSSE, SKR4, TGFR-1, tbetaR-I, TBRI, or TBR-i.

The inhibitor of the TGF-β I receptor may be a small molecule compound or a polypeptide that inhibits cellular signaling of the TGF-β I receptor. For example, the inhibitor of the TGF-β I receptor is SB431542(4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2- Yl]benzamide); SB525334(6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinozaline); SB505124(2-(4-(benzo[d][1,3]dioxol-5-yl)-2-tert-butyl-1H-imidazol-5-yl)-6-methylpyridine); Galunisertib (LY2157299) (Galunisertib, 4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl] Quinoline-6-carboxamide); GW788388(4-(4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide ); LY2109761(4-(2-((4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-7 -Yl)oxy)ethyl)morpholine); RepSox(2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine); A 77-01(4-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), and SD-208 (2-(5-Chloro-2-fluorophenyl) pteridin-4-yl]pyridin-4-yl-amine) may be one or more selected from the group consisting of.

The term "bone morphogenetic protein (BMP)" refers to a growth factor that induces the formation of bone and cartilage. The BMP may be selected from the group consisting of BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, and BMP15.

The BMP inhibitor may be a small molecule compound or a polypeptide that inhibits cell signaling of BMP. The BMP inhibitor may be an inhibitor of the BMP receptor, for example, an inhibitor of BMP receptor type 1 or an inhibitor of BMP receptor type 2. The BMP inhibitor may be a small molecule inhibitor or a polypeptide inhibitor. The BMP inhibitor is Dorsomorphin (Dorsomorphin, (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine); Dorso Dorsomorphin homolog 1: DMH1, 4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline); K 02288(3-[ (6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol); LDN 212854(5-(6-(4-(1-Piperazinyl)phenyl)pyrazolo[1,5-a] pyrimidin-3-yl) quinolone); and Noggin polypeptide The dorsomorphine may also be referred to as compound C or BML-275.

The composition is Dulbecco Modified Eagle's Medium (DMEM), DMEM/F12 (Dulbecco's Modified Eagle's Medium), F-10 nutrient medium (Nutrient M), minimum essential nutrient medium (Minimum Essential Media: MEM), RPMI Medium 1640, Opti-MEM I Depleted Serum Medium (Reduced Serum Medium), IMDM (Iscove's Modified Dulbecco's Medium), Alpha-MEM, and Neurobasal (Neurobasal) may further include a cell culture medium selected from the group consisting of medium. The composition comprises Ham's F12 nutrient mixture, B27 supplement, F-10 nutrient mixture, F-12 nutrient mixture, N2 supplement, HT supplement, G-5 supplement, lipid supplement, serum Substitutes (serum replacement), non-essential amino acids, β-mercaptoethanol, and ITS (insulin, transferrin, selenium) may further include those selected from the group consisting of supplements.

The concentration of the inhibitor of the TGF-β I receptor in the composition is about 0.1 μM to about 100 μM, about 0.5 μM to about 90 μM, about 1 μM to about 80 μM, about 2 μM to about 70 μM, about 3 μM to about 60 μM, about 4 μM to about 50 μM, about 5 μM to about 40 μM, about 6 μM to about 30 μM, about 7 μM to about 20 μM, about 8 μM to about 10 μM, or about 9 μM to about 10 may be μM.

The concentration of the BMP inhibitor in the composition is about 0.001 μM to about 2.5 μM, about 0.001 μM to about 2 μM, about 0.001 μM to about 1.5 μM, about 0.001 μM to about 1 μM, about 0.001 μM to less than about 1 μM, about 0.01 μM to about 2.5 μM, about 0.01 μM to about 2.5 μM, about 0.01 μM to about 2 μM, about 0.01 μM to about 1.5 μM, about 0.01 μM to about 1 μM, about 0.01 μM to less than about 1 μM, about 0.1 μM to about 2.5 μM, about 0.1 μM to about 2.4 μM, about 0.1 μM to about 2.3 μM, about 0.1 μM to about 2.2 μM, about 0.1 μM to about 2.1 μM, about 0.1 μM to about 2.0 μM, about 0.1 μM to About 1.9 μM, about 0.1 μM to about 1.8 μM, about 0.1 μM to about 1.7 μM, about 0.1 μM to about 1.6 μM, about 0.1 μM to about 1.5 μM, about 0.1 μM to about 1.4 μM, about 0.1 μM to about 1.3 μM, about 0.1 μM to about 1.2 μM, about 0.1 μM to about 1.1 μM, about 0.1 μM to about 1.0 μM, about 0.1 μM to less than about 1.0 μM, about 0.1 μM to about 0.9 μM, about 0.1 μM to about 0.8 μM , About 0.1 μM to about 0.7 μM, about 0.1 μM to about 0.6 μM, about 0.1 μM to about 0.5 μM, about 0.1 μM to about 1 μM, about 0.2 μM to about 1 μM, about 0.3 μM to about 1 μM, about 0.4 μM to about 1 μM, about 0.5 μM to about 1 μM, or about 0.1 μM to less than about 1 μM.

The composition may comprise a cell culture medium, a serum substitute, an inhibitor of the TGF-β I receptor, and 0.001 μM to 1 μM of DMH1. The composition may comprise a cell culture medium, a serum substitute, an inhibitor of the TGF-β I receptor, and 0.001 μM to less than 1 μM dorsomorphine.

* The concentration of the constituents in the composition may be the final concentration. The composition may be concentrated, dried, or diluted. For example, when the composition is concentrated 50 times, the composition may be added to a medium containing stem cells at a final concentration diluted 1/50.

The composition may be a composition for cell culture.

The term "stem cell" refers to a totipotent cell capable of differentiating into all types of cells or a pluripotent cell capable of differentiating into various types of cells, and stem cells are undifferentiated. As a cell, it can differentiate into cells of a specific tissue. The stem cells are embryonic stem cells (ESC), adult stem cells (adult stem cells), induced pluripotent stem cells (iPSCs), or nuclear transfer embryonic stem cells (Somatic cell nuclear transfer embryonic stem). cell). The embryonic stem cell refers to an in vitro culture obtained by extracting an inner cell mass from a blastocyst embryo, just before the fertilized egg implants in the mother's uterus. The adult stem cells are undifferentiated cells that exist only in a small amount in each tissue of the body and refer to cells that replace dead cells or damaged tissues. The induced pluripotent stem cell (iPSC) refers to a cell that induces pluripotency like embryonic stem cells by injecting genes related to cell dedifferentiation into somatic cells that have undergone differentiation to return to the stage of pluripotent stem cells in an early stage. The induced pluripotent stem cells are, for example, induced pluripotent stem cells derived from human skin cells (human dermal fibroblast-iPSC: hDF-iPSC), induced pluripotent stem cells derived from blood cells (blood cell-iPSC), or induced pluripotent stem cells derived from urine cells. It may be a cell (urine-iPSC). The nuclear-replaced embryonic stem cell is an omnipotent cell cultured in vitro by extracting the inner cell mass from the blastocyst embryo formed during the initial development process from the cell after removing the nucleus of the egg and replacing it with the nucleus of the somatic cell. Say.

The stem cell may be a cell derived from a mammal, for example, a human, mouse, rat, ape, cow, horse, pig, dog, sheep, goat or cat.

The term "neural crest" refers to a population of ectodermal cells in a cuticle derived from the ectoderm in the abdomen of a vertebrate and located along the midline below the dorsal epidermis, just above the neural tube. The neural crest is often tubular in cross section, visible above the neural tube, and is caused by deepithelial cell populations at the outer edge of the neural fold. As the neural ridge develops, it splits left and right, passes through the edge of the neural tube, descends, and moves widely in the vesicle, so that it can differentiate into ganglion, ganchungjik, head, sagittal skeletal system, and pigment cells. Neural crest stem cells (NCSC) may be cells that appear inside the abdomen at the binding site when two nerve wrinkles on both sides of the left and right are bound to each other and de-epithelial. According to their location, neural crest cells are largely divided into cranial neural crest, cardiac neural crest, trunk neural crest, and vagal and sacral neural crest. cell).

The neural crest cells may express P75 protein, HNK1, or a combination thereof. The neural crest cells may further express SOX10, AP2, FoxD3, NOTCH1, SNAIL, vinculin, bone morphogenetic protein (BMP)-4, BMP-7, or a combination thereof. The neural crest cells may not express SOX1. SOX1 can be expressed in neural precursor cells (NPC), which is the parent of central nervous system cells.

The term "differentiation" refers to a phenomenon in which a cell's structure or function is specialized during growth by division and proliferation. Pluripotent or pluripotent stem cells can be completely differentiated into specific cells after passing through certain types of progenitor cells. The embryonic stem cells, induced pluripotent stem cells, or nuclear-substituted embryonic stem cells may be differentiated into the neural crest cells. The neural crest cells may differentiate into peripheral nerve cells, Schwann cells, melanocytes, bones, cartilage, muscle cells, and the like.

The composition may be a single composition or a separate composition.

Another aspect provides a kit for differentiation of stem cells into neural crest cells, including a composition according to one aspect, and a cell culture dish.

The composition, stem cells, neural crest cells, and differentiation are as described above.

The cell culture dish refers to a cell culture container, and includes a cell culture container regardless of the material, size, and shape of the culture dish.

The cell culture dish may be a culture dish for suspension culture or a culture dish for adherent culture. The culture dish for attachment culture may be coated with a polypeptide. The polypeptide may be a polypeptide for attaching or culturing stem cells. The polypeptide is, for example, vitronectin (VTN), laminine, fibronectin, poly ornithine, or Matrigel™.

Another aspect is the step of obtaining an embryoid body (EB) by culturing stem cells in suspension in a cell culture medium containing an inhibitor of TGF-β I receptor and a BMP inhibitor of 0.001 μM to 2.5 μM; And it provides a method for differentiating stem cells into neural crest cells, comprising the step of inducing differentiation into neural crest cells by attaching and culturing the cells obtained by crushing the embryonic body.

The stem cells, TGF-β I receptor, TGF-β I receptor inhibitor, BMP, BMP inhibitor, cell culture medium, neural crest cells, and differentiation are as described above.

The method may further include inoculating the stem cells in the cell culture dish. The stem cells can be inoculated in the presence of a basal culture medium.

The method includes the step of culturing stem cells in suspension in a cell culture medium containing an inhibitor of TGF-β I receptor and a BMP inhibitor of 0.001 μM to 2.5 μM to obtain an embryoid body.

The suspension culture may be cultured without attaching the stem cells to the bottom of the culture dish. In the case of suspension culture of stem cells, an embryoid body (EB), which is a cell mass formed by lumping stem cells together in a ball shape at the beginning of cell division, can be formed.

In the above method, the culture time of the stem cells may vary depending on the culture conditions. For example, stem cells from about 1 day to about 10 days, about 1 day to about 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about It may be carried out by culturing for 1 to about 5 days, about 1 to about 4 days, about 2 to about 4 days, about 3 to about 4 days, or about 4 days. In the above method, stem cells may be cultured at about 30°C to about 40°C, about 30°C to about 37°C, or about 37°C.

The method includes the steps of inducing differentiation into neural crest cells by attaching and culturing cells obtained by crushing the embryonic body.

The adherent culture may be cultured by attaching the cells to the bottom of a culture dish.

The adherent culture is about 1 to about 15 days, about 1 to about 14 days, about 1 to about 13 days, about 1 to about 12 days, about 1 to about 11 days, about 1 to about 10 Day, about 1 day to about 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, or about 2 days to about 5 days It can be carried out by culturing during.

The proportion of neural crest cells among the cells cultured by the above method is about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97 % Or more, about 98% or more, or about 99% or more.

According to a composition for differentiation of stem cells into neural crest cells (NCSC) comprising an inhibitor of TGF-β I receptor and a BMP inhibitor of 0.001 μM to 2.5 μM according to an aspect, a kit and a method of using the same, the component is simple and low cost The phosphorus composition can be efficiently differentiated from stem cells into neural crest cells.

1A is a schematic diagram showing the process of inducing differentiation of human embryonic stem cells, and FIG. 1B is a microscopic image of an embryoid body in which human embryonic stem cells are suspended and cultured for about 4 days (top: 10 μM SB431542 and various concentrations of Dorso Morphine, bottom: 10 μM SB431542 and various concentrations of DMH1), Figure 1c is an image of cells obtained after about 4 days of suspension culture and about 5 days of adherent culture (top: 10 μM SB431542 and various concentrations of dorsomorphine , Bottom: 10 μM SB431542 and various concentrations of DMH1, black arrow: rosette-formed cells, white arrow: embryonic body), Figure 1d is a graph showing the number per unit area of embryonic bodies according to the concentration of DMH1 (pcs/cm2) Is (***: p <0.001).
Figure 2a is a graph showing the results of flow cytometry for detecting P75 on cultured cells, and Figure 2b is a graph showing the results of flow cytometric analysis for detecting SOX1 on cultured cells (top: SB431542 and dorsomorphine, bottom: SB431542 and DMH1).
3A is a microscopic image of an embryoid body in which human embryonic stem cells are suspended in the presence of 10 μM SB431542 and 0.5 μM to 2.5 μM DMH1, and FIG. 3B is a human embryonic stem cell 10 μM SB431542 and 0.5 μM to 2.5 Microscopic images of embryoid bodies suspended for about 4 days in the presence of μM dorsomorphine, and FIG. 3C is an image of cells obtained after suspension culture and adhesion culture in the presence of 10 μM SB431542 and 0.5 μM to 2.5 μM DMH1 And (black arrow: rosette-formed cells, white arrow: embryoid body), FIG. 3D is an image of cells obtained after suspension culture and adhesion culture in the presence of 10 μM SB431542 and 0.5 μM to 2.5 μM dorsomorphine. , Figure 3e is a graph showing the results of flow cytometry for detecting P75 for cultured cells (top: SB431542 and DMH1, bottom: SB431542 and dorsomorphine).

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Differentiation of human pluripotent stem cells into neural crest cells

1. Induction of differentiation into neural crest cells

Colonies obtained by culturing H9 human embryonic stem cells (hESC) (WiCell Research Institute, Inc. Madison, WI, U.S.A.) in a culture medium were made into single cells using accutase (Life Technologies).

Single-celled cells were treated with vitronectin (VTN) (vitronectin XF, STEMCELL Technologies), laminine (rhLaminin-521, Thermo Fisher Scientific Inc., Waltham, MA, USA), fibronectin (Thermo Fisher). Scientific Inc.), or Matrigel™ (StemCell Technologies, Inc.) was inoculated into a culture vessel coated with a protein. Undifferentiated culture was performed by adding an ESC (embryonic stem cell) culture solution to the inoculated cells. As the ESC culture medium, Essential 8 (E8) medium (StemCell Technologies Inc.), TeSR2 medium (StemCell Technologies Inc), or StemMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) were used.

The obtained colonies were crushed finely, and SB431542 (GlaxoSmithKline: GSK) as a 10 μM TGF-βI receptor inhibitor and 0.5 μM to 1 μM DMH1 (dorsomorphin homolog 1) as a BMP inhibitor under conditions of 37° C. and 5% CO ₂ ( Tocris Bioscience, USA) or dorsomorphine (Tocris Bioscience, USA) in EB medium containing about 4 days suspension culture (suspension culture) to prepare embryos (embryoid body (EB)). EB medium was DMEM/F12 (Life Technologies), 20% (v/v) Knockout Serum Replacement (KSR, Invitrogen), 1x non-essential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen) , And 1x penicillin/streptomycin (Invitrogen). As a negative control, dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was used instead of the drug.

After crushing the embryoid body (EB), which is a round cell mass produced by floating culture for about 4 days, adhered to the bottom of the culture vessel and cultured for about 5 days (DMEM-F12, 1x N2 supplement, insulin 25 ㎍/㎖, bFGF 20 ng/ml, using a matrigel-coated culture vessel).

A microscopic image of an embryoid body suspended for about 4 days is shown in FIG. 1B, and a microscopic image of cells in which a rosette was formed by suspension culture for about 4 days and adherent culture for 5 days is shown in FIG. 1C (top: 10 μM SB431542 and several concentrations of dorsomorphine, bottom: 10 μM SB431542 and several concentrations of DMH1, black arrow: rosette-forming cells, white arrow: embryoid body). The number of spherical embryos was counted, and the number of embryos per unit area (pcs/cm2) according to the concentration of DMH1 is shown in FIG. 1D (***: p <0.001). As shown in FIG. 1D, the number of embryos per unit area significantly decreased as the concentration of DMH1 in the EB medium increased in the suspension culture.

2. Identification of differentiated cells

Flow cytometry was performed to confirm whether the cultured cells were neural crest cells as described in Example 1.1.

2% (v/v) paraformaldehyde (Sigma-Aldrich) was added to the cultured cells for a total of 9 days (4 days of floating culture + 5 days of adherent culture), and the cells were fixed by reacting for about 10 minutes at room temperature. . 2% (v/v) normal serum/1xPBS (Vector Laboratories, Inc., Burlingame, CA) containing 0.1% (v/v) TRITON™ X-100 (Sigma-Aldrich) was added to the cells and at room temperature. It was reacted at for 30 minutes to block fixation. To analyze the proportion of cells expressing P75 protein, a marker of neural crest stem cells (NCSC) or SOX1, a marker of neural precursor cells (NPC), phycoerythrin (PE) Labeled anti-P75 monoclonal antibody (1:50 dilution) (Miltenyi Biotec), and phycoerythrin (PE)-labeled anti-Sox1 monoclonal antibody (1:100 dilution) (BD Biosciences) Using immunostaining. The immunostained cells were subjected to flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences, Sparks, MD, USA). The flow cytometry results are shown in FIGS. 2A and 2B (FIG. 2A: P75 flow cytometry graph, FIG. 2B: SOX1 flow cytometry graph).

2A and 2B, when H9-hESC is cultured in EB medium containing 10 μM SB431542 and dorsomorphine or DMH1, the proportion of P75-expressing cells in 0.5 μM dorsomorphine or DMH1 is about The proportion of SOX1 expressing cells was about 86% or more in 5 μM dorsomorphine or DMH1, whereas 61% or more. Therefore, the lower the concentration of a BMP inhibitor such as dorsomorphine or DMH1, the more differentiated into P75-expressing cells (i.e., neural crest cells), and the higher the concentration of dorsomorphine or DMH1, the higher the concentration of SOX1-expressing cells (ie, neural progenitor cells). It was confirmed that it was differentiated into.

3. Culture of embryonic stem cells in the presence of 10 μM SB431542 and 0.5 μM to 2.5 μM BMP inhibitor

Since human embryonic stem cells differentiated into neural crest cells as the concentration of the BMP inhibitor dorsomorphine or DMH1 was lower, the concentration of dorsomorphine or DMH1 was set in the range of 0.5 μM to 2.5 μM, as described in Example 1.1. Embryonic stem cells were cultured.

Microscopic images of cells suspended in H9-hESC in the presence of 10 μM SB431542 and 0.5 μM to 2.5 M DMH1 or dorsomorphine are shown in FIGS. 3A and 3B. In addition, microscopic images of embryoid bodies that were suspended and cultured for about 4 days thereafter are shown in FIGS. 3C and 3D (black arrow: rosette-formed cells, white arrow: embryonic body).

Cells obtained after suspension culture for about 3 days and adherent culture for about 4 days were subjected to P75 flow cytometry as described in Example 1.2, and the flow cytometric analysis results are shown in Figure 3e (top: SB431542 and DMH1, bottom: SB431542 and dorsomorphine).

As shown in Figure 3e, when H9-hESC is cultured in EB medium containing 10 μM SB431542 and DMH1, the proportion of P75-expressing cells is about 69.8% or more in 0.5 μM to 1 μM DMH1, whereas 2.5 μM of In DMH1, it was significantly reduced to about 28.9%. In addition, when H9-hESC was cultured in EB medium containing 10 μM SB431542 and dorsomorphine, the proportion of P75-expressing cells was about 83.2% or more in 0.5 μM, while about 58.7% in DMH1 of 1 μM to 2.5 μM. To about 40%. Therefore, it was confirmed that the lower the concentration of a BMP inhibitor such as dorsomorphine or DMH1, the differentiation into neural crest cells was induced.

Claims (13)

Transforming growth factor beta (TGF-β) I receptor inhibitor and 0.001 μM to 2.5 μM bone morphogenetic protein (BMP) inhibitor of stem cells neural crest stem cell: NCSC) as a composition for differentiation,
The inhibitor of the TGF-β I receptor is SB431542(4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide )ego,
The composition of the BMP inhibitor is a BMP receptor type 1 inhibitor. The method of claim 1, wherein the BMP receptor type 1 inhibitor
Dorsomorphin (6-[4-[2-(1-pipelinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine));
Dorsomorphin homolog 1: DMH1, 4-[6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline);
K 02288(3-[(6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol);
LDN 212854(5-(6-(4-(1-piperazinyl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone); And
A composition that is at least one selected from the group consisting of Noggin polypeptides. The method according to claim 1, Dulbecco Modified Eagle's Medium (DMEM), DMEM/F12 (Dulbecco's Modified Eagle's Medium), F-10 nutrient medium (Nutrient M), minimum essential nutrient medium (Minimum Essential Media: MEM) , RPMI medium 1640, Opti-MEM I depleted serum medium (Reduced Serum Medium), IMDM (Iscove's Modified Dulbecco's Medium), alpha-MEM, and neural basal (Neurobasal) the medium further comprising a cell culture medium selected from the group consisting of Phosphorus composition. The method of claim 3, Ham's F12 nutrient mixture, B27 supplement, F-10 nutrient mixture, F-12 nutrient mixture, N2 supplement, HT supplement, G-5 supplement, lipid supplement , Serum replacement (serum replacement), non-essential amino acids, β-mercaptoethanol, and ITS (insulin, transferrin, selenium) composition further comprising a selected from the group consisting of supplements. The composition of claim 1, wherein the concentration of the inhibitor of the TGF-β I receptor in the composition is 0.1 μM to 100 μM. The composition of claim 1, wherein the concentration of the BMP receptor type 1 inhibitor in the composition is 0.001 μM to 1 μM. The composition of claim 1 comprising a cell culture medium, a serum substitute, an inhibitor of the TGF-β I receptor, and 0.001 μM to 1 μM of DMH1. The composition of claim 1 comprising a cell culture medium, a serum substitute, an inhibitor of the TGF-β I receptor, and 0.001 μM to less than 1 μM dorsomorphine. The method according to claim 1, wherein the stem cell is an embryonic stem cell (ESC), an adult stem cell, an induced pluripotent stem cell (iPSC), or a nuclear replacement embryonic stem cell (Somatic cell nuclear transfer embryonic stem cell). A kit for differentiation of stem cells into neural crest cells comprising the composition of claim 1 and a cell culture dish. Stem cells are cultured in suspension in a cell culture medium containing an inhibitor of TGF-β I receptor and a BMP inhibitor of 0.001 μM to 2.5 μM to obtain an embryoid body (EB),
The inhibitor of the TGF-β I receptor is SB431542,
The BMP inhibitor is a BMP receptor type 1 inhibitor; And
Comprising the step of inducing differentiation into neural crest cells by attaching and culturing the cells obtained by breaking the embryonic body,
A method of differentiating stem cells into neural crest cells. The method of claim 11, wherein the suspension culture is cultured for 1 to 10 days. The method of claim 11, wherein the adherent culture is cultured for 1 to 15 days.

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