EA044339B1 - METHOD FOR CULTURING CELLS - Google Patents

Description

Field of technology

The present invention relates to a method for producing neural crest cells, a method for allowing neural crest cells to proliferate, a medium, a frozen stock culture, and a method for producing various types of cells from neural crest cells. More specifically, the present invention relates to a method for producing neural crest cells, a method for allowing neural crest cells to proliferate, a medium for use in these methods, a frozen stock culture containing neural crest cells, and a method for producing various types of cells differentiated into which can be induced from neural crest cells.

State of the art

Neural crest cells (NCC) are cells that form between the neuroectoderm and epidermal ectoderm when the neural plate forms the neural tube during early development. These cells have the multipotency to differentiate into many cell types such as neural cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells and pigment cells, and the ability to self-proliferate. This multipotency and ability to self-proliferate indicate the suitability of neural crest cells as a cell therapy for regenerative medicine. Thus, there remains a need for a method for effectively maintaining or proliferating neural crest cells.

Non-Patent Document 1 describes that neural crest cells are induced from human induced pluripotent stem cells (iPSCs), and further, mesenchymal stromal cells and the like are induced from neural crest cells. In Non-Patent Document 1, neural crest cells are maintained in an adherent culture using a medium supplemented with TGFe inhibitor, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF (also referred to as FGF2)).

Non-Patent Document 2 describes that neural crest cells are induced from the neural tube of a chick embryo, and further, glial cells and the like are induced from neural crest cells. In Non-Patent Document 2, neural crest cells are maintained in suspension culture using a medium supplemented with chicken embryo extracts, insulin-like growth factor (IGF), bFGF and retinoic acid (RA).

Non-Patent Document 3 describes that neural crest cells are induced from human embryonic stem cells (ESCs) or human iPSCs, and further, smooth muscle cells and the like are induced from neural crest cells. In Non-Patent Document 3, neural crest cells are maintained in an adherent culture using a medium supplemented with a GSK3e inhibitor and a TGFe inhibitor.

Non-Patent Document 4 describes that neural crest cells are induced from human iPSCs and maintained by adherent culture or suspension culture using medium supplemented with a GSK3e inhibitor, a TGFe inhibitor, an EGF inhibitor, and a bFGF inhibitor. Non-Patent Document 4 describes that the number and proportion of neural crest cells in the cultured cell population in maintenance culture carried out by culturing adherent cells decreased in a concentration-dependent manner over the bFGF concentration range from 0 pg/ml to 10 ng/ml (see FIG. 5A-5C). In maintenance culture carried out by suspension culture, the presence of Sox10-positive cells was confirmed by culture for 7 days using 1 μM CHIR99021 as a GSK3μ inhibitor. However, it was not determined whether the cells were multipotent.

None of the patent documents 1-4 describe that neural crest cells are maintained and allowed to proliferate through suspension culture using medium supplemented with CHIR99021 at a concentration greater than 1 μM and bFGF.

Bibliography

Non-patent documents

Non-patent document 1: Fukuta M. et al.: Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media., PLoS One, 2014, 9(12):e112291.

Non-Patent Document 2: Kerosuo L. et al.: Crestospheres: Long-Term Maintenance of Multipotent, Premigratory Neural Crest Stem Cells., Stem Cell Reports, 2015, 5(4):499-507.

Non-patent document 3: Menendez L. et al.: Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells., Proc. Natl. Acad. Sci., 2011, 108(48): 19240-5.

Non-Patent Document 4: Horikiri T. et al.: SOX10-Nano-Lantern Reporter Human iPS Cells; A Versatile Tool for Neural Crest Research., PLoS One, 2017, 12(1):e0170342.

Essence of the invention Technical problem

Neural crest cells are initially multipotent to differentiate into many cell types, such as neural cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells, and pigment cells. However, it is known that in neural crest cell culture, multipotency gradually decreases and the number of cell types into which neural crest cells can differentiate decreases.

The main object of the present invention is to provide a method for culturing and proliferating neural crest cells that maintain multipotency over an extended period of time.

Solution

To accomplish the object, the present invention relates to the following [1]-[21b].

[1] A method for producing neural crest cells, comprising the steps of:

(1) obtaining neural crest cells; and (2) culturing using a suspension culture of neural crest cells in a medium containing a GSK3e inhibitor and basic fibroblast growth factor (bFGF), wherein the medium contains a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration greater than 1 μM .

[1a] The preparation method according to [1], wherein the concentration of the GSK3e inhibitor is a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration above 1 μM and below 5 μM.

[1b] The production method according to [1], where the effect is evaluated based on the GSK3e inhibitor activity of the GSK3e inhibitor, where the GSK3e inhibitor activity of the GSK3e inhibitor is determined by methods:

(i) culturing cells whose reporter gene expression is suppressed under the control of GSK3e, in the presence and absence of a GSK3e inhibitor;

(ii) determining the level of reporter gene expression in the presence and absence of a GSK3e inhibitor; and (iii) determining the GSK3e inhibitory activity of the GSK3e inhibitor based on the amount of increase in the level of reporter gene expression in the presence of the GSK3e inhibitor relative to the level of expression of the reporter gene in the absence of the GSK3e inhibitor.

[2] The production method according to [1], where the medium additionally contains a TGFe inhibitor.

[3] The production method according to [1], where the medium is a CDM medium.

[4] The production method according to [1], where the medium additionally contains epidermal growth factor (EGF).

[5] The production method according to [1], wherein the GSK3e inhibitor is at least one member selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, kenpaullone, AR-AO144-18, TDZD-8, SB216763, BIO , TWS-119 and SB415286.

[6] The production method according to [5], wherein the GSK3e inhibitor is CHIR99021.

[6a] The production method according to [6], where CHIR99021 has a concentration of above 1 μM and below 5 μM.

[6b] The production method according to [6a], wherein CHIR99021 has a concentration of 2 or higher and 4.5 μM or lower.

[6c] The production method according to [5], wherein the GSK3e inhibitor is CP21R7.

[6d] The production method according to [6c], wherein CP21R7 has a concentration of 0.5 or higher and 1 μM or lower.

[7] The production method according to [2], wherein the TGFe inhibitor is at least one member selected from the group consisting of SB431542, A83-01, LDN193189, Wnt3a/BIO, BMP4, GW788388, SM16, IN-1130, GW6604 and SB505124.

[7a] The production method according to any one of [1] to [7], wherein bFGF has a concentration of 20 to 40 ng/ml.

[8] The production method according to [1], where at stage (2) neural crest cells are passaged every 5-8 days after inoculation.

[9] The production method according to [1], wherein step (1) is a step of inducing differentiation of stem cells into neural crest cells.

[10] A method for promoting proliferation of neural crest cells, comprising the step of:

(I) culturing using a suspension culture of neural crest cells in a medium containing a GSK3e inhibitor and basic fibroblast growth factor (bFGF), wherein the medium contains a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration greater than 1 μM.

[10a] The proliferation method according to [10], wherein the concentration of the GSK3e inhibitor is a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration above 1 μM and below 5 μM.

[10b] The proliferation method according to [10], where the medium additionally contains a TGFe inhibitor.

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[10c] The proliferation method according to [10], where the medium is CDM medium.

[10d] The proliferation method according to [10], wherein the medium further contains epidermal growth factor (EGF).

[10e] The proliferation method according to [10], wherein the GSK3e inhibitor is at least one member selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, kenpaullone, AR-AO144-18, TDZD-8, SB216763, BIO, TWS-119 and SB415286.

[10f] The proliferation method according to [10e], wherein the GSK3e inhibitor is CHIR99021.

[10g] The proliferation method according to [10f], where CHIR99021 has a concentration of above 1 μM and below 5 μM.

[10h] The proliferation method according to [10g], where CHIR99021 has a concentration of 2 or higher and 4.5 μM or lower.

[10i] The proliferation method according to [10e], wherein the GSK3e inhibitor is CP21R7.

[10j] The proliferation method according to [10i], wherein CP21R7 has a concentration of 0.5 or higher and 1 μM or lower.

[10k] The proliferation method according to [10b], wherein the TGFe inhibitor is at least one member selected from the group consisting of SB431542, A83-01, LDN193189, Wnt3a/BIO, BMP4, GW788388, SM16, IN-1130, GW6604 and SB505124.

[10l] Proliferation method according to [10], where at stage (I), neural crest cells are passaged every 5-8 days after inoculation.

[11] A medium containing a GSK3e inhibitor, basic fibroblast growth factor (bFGF) and neural crest cells, wherein the medium contains a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration greater than 1 μM.

[11a] Medium according to [11], where the concentration of GSK3e inhibitor is the concentration that shows an effect equivalent to that shown by CHIR99021 at a concentration above 1 μM and below 5 μM.

[12] Medium according to [11], additionally containing a TGFe inhibitor.

[13] Environment according to [11], where the environment is a CDM environment.

[14] Medium according to [11], additionally containing epidermal growth factor (EGF).

[15] The medium according to [11], wherein the GSK3e inhibitor is at least one member selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, kenpaullone, ARAO144-18, TDZD-8, SB216763, BIO, TWS-119 and SB415286.

[16] Environment according to [15], where the GSK3e inhibitor is CHIR99021.

[16a] Medium according to [16], where CHIR99021 has a concentration above 1 µM and below 5 µM.

[16b] Medium according to [16a], where CHIR99021 has a concentration of 2 or higher and 4.5 µM or lower.

[16c] Medium according to [15], where the GSK3e inhibitor is CP21R7.

[16d] Medium according to [16c], where CP21R7 has a concentration of 0.5 or higher and 1 µM or lower.

[17] The medium according to [12], wherein the TGFe inhibitor is at least one member selected from the group consisting of SB431542, A83-01, LDN193189, Wnt3a/BIO, BMP4, GW788388, SM16, IN-1130, GW6604 and SB505124 .

[18] Frozen stock culture containing neural crest cells obtained by the production method according to [1].

[18a] Frozen stock culture according to [18], wherein the frozen stock culture is obtained through the steps of:

separating the neural crest cells obtained through the steps of the production method according to [1]; and suspending the separated neural crest cells in a cell preservation solution followed by freezing.

[19] A method for producing nerve cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells or pigment cells, comprising the steps of:

(i) culturing using a suspension culture of neural crest cells in a medium containing a GSK3e inhibitor and basic fibroblast growth factor (bFGF), wherein the medium contains a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration greater than 1 μM; and (ii) differentiating the neural crest cells obtained in step (i) into cells of at least one lineage selected from the group consisting of neural cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells and pigment cells .

[19a] The production method according to [19], wherein the concentration of the GSK3e inhibitor is a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration above 1 μM and below 5 μM.

[19b] The production method according to [19], where the medium additionally contains a TGFe inhibitor.

[19c] The production method according to [19], where the medium is a CDM medium.

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[19d] The production method according to [19], where the medium additionally contains epidermal growth factor (EGF).

[19e] The production method according to [19], wherein the GSK3e inhibitor is at least one member selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, kenpaullone, AR-AO144-18, TDZD-8, SB216763, BIO, TWS-119 and SB415286.

[19f] The preparation method according to [19e], wherein the GSK3e inhibitor is CHIR99021.

[19g] The preparation method according to [19f], where CHIR99021 has a concentration of above 1 μM and below 5 μM.

[19h] Media according to [19g], where CHIR99021 has a concentration of 2 or higher and 4.5 µM or lower.

[19i] Environment according to [19e], where the GSK3e inhibitor is CP21R7.

[19j] The production method according to [19i], wherein CP21R7 has a concentration of 0.5 or higher and 1 μM or lower.

[19k] The production method according to [19b], wherein the TGFe inhibitor is at least one member selected from the group consisting of SB431542, A83-01, LDN193189, Wnt3a/BIO, BMP4, GW788388, SM16, IN-1130, GW6604 and SB505124.

[19l] Method of production according to [19], where at stage (I), neural crest cells are passaged every 5-8 days after inoculation.

[20] A method for culturing neural crest cells with multipotency for a long period of time, including the steps of:

(1) obtaining neural crest cells; and (2) culturing using a suspension culture of neural crest cells in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, wherein the medium contains a GSK3e inhibitor at a concentration that demonstrates an effect equivalent to that demonstrated by CHIR99021 in concentrations above 1 µM.

[20a] The culture method according to [20], wherein the concentration of the GSK3e inhibitor is a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration above 1 μM and below 5 μM.

[20b] The cultivation method according to [20], where the medium additionally contains a TGFe inhibitor.

[20c] The culture method according to [20], where the medium is CDM medium.

[20d] The culture method according to [20], wherein the medium further contains epidermal growth factor (EGF).

[20e] The culture method according to [20], wherein the GSK3e inhibitor is at least one member selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, kenpaullone, AR-AO144-18, TDZD-8, SB216763, BIO, TWS-119 and SB415286.

[20f] The culture method according to [20e], wherein the GSK3e inhibitor is CHIR99021.

[20g] The culture method according to [20f], where CHIR99021 has a concentration above 1 μM and below 5 μM.

[20h] The culture method according to [20g], where CHIR99021 has a concentration of 2 or higher and 4.5 μM or lower.

[20i] The culture method according to [20e], wherein the GSK3e inhibitor is CP21R7.

[20j] The culture method according to [20i], wherein CP21R7 has a concentration of 0.5 or higher and 1 μM or lower.

[20k] The cultivation method according to [20b], wherein the TGFe inhibitor is at least one member selected from the group consisting of SB431542, A83-01, LDN193189, Wnt3a/BIO, BMP4, GW788388, SM16, IN-1130, GW6604 and SB505124.

[20l] Cultivation method according to [20], where at stage (I) neural crest cells are passaged every 5-8 days after inoculation.

[21] Using a medium containing basic fibroblast growth factor and a GSK3e inhibitor at a concentration that shows an effect equivalent to that shown by CHIR99021 at a concentration above 1 μM to culture multipotent neural crest cells for an extended period of time.

[21a] Application of basic fibroblast growth factor and GSK3e inhibitor at a concentration that shows an effect equivalent to that shown by CHIR99021 at a concentration above 1 μM to culture neural crest cells.

[21b] Use of basic fibroblast growth factor and GSK3e inhibitor at a concentration that shows an effect equivalent to that shown by CHIR99021 at a concentration above 1 μM to produce media for neural crest cells.

As used herein, pluripotency means the ability to differentiate into tissues and cells having different forms and functions, and the ability to differentiate into cells of any lineage of the 3 germ layers. Pluripotency differs from totipotency, which is the ability to differentiate into any tissue of a living organism, including the placenta, in that pluripotent cells cannot differentiate into the placenta and, thus, do not have the ability to form an individual.

Multipotency refers to the ability to differentiate into significant but limited numbers of cell lineages. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent, but not pluripotent. Neural crest cells have the multipotency to differentiate into cells such as neural cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells, and pigment cells.

As used herein, culture refers to the maintenance, proliferation (growth) and/or differentiation of cells under in vitro conditions. Culturing means maintaining cells and/or allowing the cell to proliferate (grow) and/or differentiate from a tissue or organism, for example, in a cell culture dish or flask.

Attached culture means a culture in a state where the cells are attached to a container, for example, in a state where the cells are attached to a cell culture dish or flask made of sterilized plastic (or coated plastic) in the presence of a suitable medium.

Suspension culture means a culture in a state where cells are dispersed as single cells or as cell spheres, each consisting of two or more cells, in a suitable medium without attachment to a container.

Expansion culture means culture for the purpose of allowing the proliferation of desired cells.

A GSK3e inhibitor is a substance that has GSK3e (glycogen synthase kinase 3β) inhibitory activity. GSK3 (glycogen synthase kinase 3) is a serine/threonine protein kinase and is involved in many signaling pathways associated with glycogen production, apoptosis, stem cell maintenance, etc. GSK3 has 2 isoforms: α and β. The GSK3e inhibitor used in the present invention is not particularly limited as long as the GSK3e inhibitor has GSK3e inhibitory activity. The GSK3e inhibitor may be a substance having both GSK3e inhibitory activity and GSK3a inhibitory activity.

A TGFe inhibitor is a substance having TGFe (transforming growth factor β) inhibitory activity. TGFe is a cytokine that binds to two types of serine/threonine protein kinase receptors and controls cell proliferation, cell differentiation, cell death, etc. via signal transduction, mainly to activate Smads (R-Smads). Examples of a substance having TGFe inhibitory activity include substances that inhibit the binding of TGFe to its receptor, and substances that inhibit downstream signals after binding of TGFe to its receptor. Examples of the following signals include phosphorylation of the TGFeI receptor by the TGFeII receptor and phosphorylation of Smad by the phosphorylated TGFpI receptor. The TGFe inhibitor used in the present invention is not particularly limited as long as the TGFe inhibitor has TGFe inhibitory activity.

As used herein, a marker is a marker protein or a marker gene and means a protein that is specifically expressed on the cell surface, cytosol and/or nucleus of a given cell type, or a gene thereof. The marker may be a positive selection marker and a negative selection marker. Preferably the marker is a cell surface marker. In particular, the cell surface marker for positive selection allows the concentration, isolation and/or detection of living cells.

Detection of the marker protein can be accomplished using an immunoassay, such as ELISA, immunostaining, or flow cytometry, using an antibody specific for the marker protein. As the marker protein-specific antibody, an antibody that binds to a specific amino acid sequence of the marker protein or a specific sugar chain associated with the marker protein, etc. can be used. In the case of an intracellularly expressed marker protein that does not appear on the surface of cells (eg, a transcription factor or a subunit thereof), detection of the marker protein of interest can be accomplished by expressing the marker protein with a reporter protein and detecting the reporter protein (eg, Non-Patent Document 4). This method can preferably be used when a suitable cell surface marker cannot be found. Detection of the marker gene can be accomplished using a nucleic acid amplification and/or detection method known in the art, such as RT-PCR, microarrays, bioarrays, or RNA sequencing.

As used herein, expression is defined as the transcription and/or translation of a specific nucleotide sequence driven by an intracellular promoter.

As used herein, the term contain(s) or containing refers to

- 5 044339 inclusion of element(s) after this word without its restrictions. Thus, it indicates the inclusion of the element(s) after that word, but does not indicate the exclusion of any other element.

As used herein, the term approximately or approximately refers to a value that may deviate by up to plus or minus 30, 25, 20, 15, 10, 8, 6, 5, 4, 3, 2 or 1% from the reference value. Preferably, the term approximately or approximately refers to a range of minus to plus 15, 10, 5 or 1% of the reference value.

Advantageous effects of the invention

The present invention relates to a method for culturing and proliferating neural crest cells that maintain multipotency over an extended period.

Brief description of drawings

In fig. 1 is a graph showing the change in the total number of cells after the start of cultivation using expansion culture of neural crest cells (Example 1). The y-axis represents the total number of cells, and 1.E+ corresponds to a multiplier of 10. For example, 1.E+04 means 10,000 cells. The abscissa represents the number of days of cultivation.

In fig. Figure 2 is a graph showing the change in the percentage of cells positive for SOX10 expression after the start of cultivation using expansion culture of neural crest cells (Example 1). The y-axis represents the percentage of cells positive for SOX10 expression (%). The abscissa represents the number of days of cultivation.

In fig. 3 is a graph showing the results of measuring the change in the total number of neural crest cells cultured using expansion culture by using bFGF and EGF concentrations of 20 ng/ml (A) or 40 ng/ml (B) and using CHIR99021 concentrations 1, 2. 3 or 5 µM (indicated as 1x, 2x, 3x and 5x, respectively) (Example 2). The y-axis represents the total number of cells and 1.E+ corresponds to a multiplier of 10. For example, 1.E+04 means 10,000 cells. The abscissa represents the number of days of cultivation.

In fig. 4 is a graph showing the results of measuring changes in the total number of neural crest cells cultured using expansion culture using bFGF and EGF concentrations of 40 ng/ml and using CHIR99021 concentrations of 3, 3.5, 4, 4.5 or 5 μM (example 2). The y-axis represents the total number of cells and 1.E+ corresponds to a factor of 10.

In fig. 5 is a graph showing the results of measuring the change in the percentage of neural crest cells positive for SOX10 expression that were cultured using expansion culture using bFGF and EGF concentrations of 20 ng/ml (A) or 40 ng/ml (B) and using CHIR99021 concentrations of 1, 2, 3, or 5 μM (indicated as 1x, 2x, 3x, and 5x, respectively) (Example 2). The y-axis represents the percentage of cells positive for SOX10 expression (%). The abscissa represents the number of days of cultivation.

In fig. 6 is a graph showing the results of measuring the change in the percentage of neural crest cells positive for SOX10 expression that were cultured using expansion culture using bFGF and EGF concentrations of 40 ng/ml and using CHIR99021 concentrations 3, 3.5, 4, 4 .5 or 5 µM (example 2). The y-axis represents the percentage of cells positive for SOX10 expression (%). The abscissa represents the number of days of cultivation.

In fig. 7 is a graph showing the change in the percentage of cells with positive SOX10 expression after the start of expansion culture of neural crest cells using CP21R7 as a GSK3e inhibitor (Example 3). The y-axis represents the percentage of cells positive for SOX10 expression (%). The abscissa represents the number of days of cultivation.

In fig. 8 is a graph showing the results of measuring the change in the total number of neural crest cells subjected to expansion culture using bFGF concentrations of 10, 12.5, 15.0 or 17.5 ng/ml (Example 8). The y-axis represents the total number of cells and 1.E+ corresponds to a factor of 10.

In fig. 9 is a graph showing the results of measuring the change in the percentage of total neural crest cells positive for SOX10 expression that were cultured using expansion culture using bFGF concentrations of 10, 12.5, 15.0, or 17.5 ng/ml (Example 8). The y-axis represents the percentage of cells positive for SOX10 expression (%). The abscissa represents the number of days of cultivation.

Description of Embodiments

Suitable methods for carrying out the present invention are described below. The embodiments described below are provided only to illustrate typical embodiments of the present invention. The scope of the present invention should not be interpreted as being limited to these embodiments.

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Method for obtaining neural crest cells and method for ensuring proliferation of neural crest cells

The method for producing neural crest cells in accordance with the present invention includes the steps below. Of these steps, step (2) is particularly a method for allowing neural crest cells to proliferate in accordance with the present invention.

(1) Obtaining neural crest cells; and (2) culturing using a suspension culture of neural crest cells in a medium containing a GSK3e inhibitor and basic fibroblast growth factor (bFGF), wherein the medium contains a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration greater than 1 μM .

Step (1) obtaining neural crest cells.

Step (1) of obtaining neural crest cells is a step of obtaining neural crest cells for carrying out step (2). Examples of the method for producing neural crest cells in step (1) include, but are not specifically limited to, a method for inducing differentiation of stem cells into neural crest cells, a method for acquiring commercially available neural crest cells, and a method for collecting naturally occurring neural crest cells.

In one embodiment of the present invention, to obtain neural crest cells for step (2), stem cells can be induced to differentiate into neural crest cells.

Examples of stem cells that can be used within the scope of the present invention include pluripotent stem cells. Pluripotent stem cells that can be used in the present invention refer to stem cells that can differentiate into various tissues and cells having different forms and functions, and have the ability to differentiate into cells of any size from the 3 germ layers (endoderm, mesoderm and ectoderm ). Examples thereof include, but are not specifically limited to, embryonic stem cells (ESC), embryonic stem cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells, embryonic germ cells and induced pluripotent stem cells (hereinafter also referred to as iPSCs) . Multipotent stem cells that can be used within the scope of the present invention refer to stem cells that have the ability to differentiate into significant but limited numbers of cell lineages. Examples of multipotent stem cells that can be used in the present invention include dental pulp stem cells, oral mucosal stem cells, hair follicle stem cells, and somatic stem cells derived from cultured fibroblasts and bone marrow stem cells. Pluripotent stem cells are preferably ESCs and iPSCs.

Available ESCs include mouse ESCs, such as the various mouse ESC lines developed at the inGenious Targeting Laboratory, Riken (Institute of Physical and Chemical Research), etc., and human ESCs, such as the various human ESC lines developed by the University of Wisconsin, NIH, Riken, Kyoto University, National Center for Child Health and Development, Cellartis, etc. For example, the human ESC lines that can be used are the START-1 to START-12 lines, the RUES1 line, the RUES2 line, and the HUES1 to HUES28 lines developed by ESI Bio, the H1 line and the H9 line developed by WiCell Research, and the KhES-1 line , KhES-2 line, KhES-3 line, KhES-4 line, KhES-5 line, SSES1 line, SSES2 line and SSES3 line developed by Riken.

Induced pluripotent stem cells refer to cells that are obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various induced pluripotent stem cells and iPSCs developed by Yamanaka, et al. by introducing 4 factors: Oct3/4, Sox2, Klf4 c-Myc, into mouse fibroblasts (Takahashi K., Yamanaka S., Cell, (2006) 126: 663-676); iPSCs, derived from human cells, developed by introducing similar 4 factors into human fibroblasts (Takahashi K., Yamanaka S., et al. Cell, (2007) 131: 861-872.); Nanog-iPSCs developed by cell sorting using Nanog expression as an indicator after the introduction of 4 factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.); iPSCs obtained by a method without using c-Myc (Nakagawa M., Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106); iPSCs developed by introducing 6 factors in a virus-free manner (Okita K et al. Nat. Methods 2011 May; 8(5): 409-12, Okita K et al. Stem Cells. 31 (3) 458-66), etc. .P. Induced pluripotent stem cells developed by Thomson et al can also be used. through the introduction of 4 factors: OCT3/4, SOX2, NANOG and LIN28 (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920); induced pluripotent stem cells obtained by Daley et al. (Park IH, Daley GQ.et al., Nature (2007) 451: 141-146); induced pluripotent stem cells obtained by Sakurada et al. (Japan Unexamined Patent Application Publication No. 2008-307007), etc.

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In addition, any of the known induced pluripotent stem cells known in the art, which are described in all published articles, can be used (for example, Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol 3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; HuangfU D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No. 7, 795-797) or patents (for example, Japanese Unexamined Patent Application Publication No. 2008-307007, Japanese Unexamined Patent Application Publication No. 2008-283972, US 2008-2336610, US 2009-047263, WO 2007069666, WO 2008-11822 0 , WO 2008-124133, WO 2008-151058, WO 2009-006930, WO 2009-006997, WO 2009-007852).

Available induced pluripotent cell lines include various iPSC lines developed by NIH, Riken, Kyoto University, etc. Examples of such human iPSC lines include the HiPS-RIKEN-1A line, the HiPS-RIKEN-2A line, the HiPS-RIKEN-12A line, and the Nips-B2 line from Riken, and the 253G1 line, 253G4 line, 1201C1 line, 1205D1 line, 1210B2 line, line 1383D2, line 1383D6, line 201B7, line 409B2, line 454E2, line 606A1, line 610B1, line 648A1, line 1231A3 and line FfI-01s04 from Kyoto University. Line 1231A3 is preferred.

Induction of differentiation of stem cells into neural crest cells can be carried out by a known method described in the literature (eg, Non-Patent Document 1). In the case of using, for example, human iPSCs, the iPSCs are inoculated onto a plate or the like, subjected to adherent culture, and then subjected to adherent culture in a medium containing a TGFe inhibitor and a GSK3e inhibitor, and thereby can be allowed to differentiate into neural crest cells.

In this regard, the medium used is not particularly limited and is suitable, for example, TeSR1 medium and chemically defined medium (CDM). In addition, you can use BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Enhanced MEM medium (IMEM), Advanced MDM medium (IMDM medium), Medium 199 medium, Eagle MEM medium, aMEM medium, DMEM medium (high glucose). or low glucose), DMEM/F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium and their mixed medium, etc.

The CDM medium is not particularly limited, and, for example, medium obtained from Iscove's modified Dulbecco's medium (manufactured by GE Healthcare Japan Corp.) can be used. As a more specific example, the CDM medium described in Non-Patent Document 1 is used. The CDM medium may contain apotransferrin, monothioglycerol, bovine serum albumin (BSA), insulin and/or an antibiotic.

The culture period before adding the TGFe inhibitor and GSK3e inhibitor may be the period during which the number of cells of interest is reached. This cultivation period is not particularly limited and is, for example, from 2 to 6 days.

Examples of TGFe inhibitor include SB431542 (4-(5-benzene[1,3]dioxol-5-yl-4-pyridin-2-yl-1Nimidazol-2-yl)benzamide, 4-[4-(1,3-benzodioxole -5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide, 4[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2- yl]benzamide), A83-01 (3-(6-methylpyridin-2yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), LDN193189 (4-[6-[4-(1-piperazinyl)phenyl]pyrazolo [1,5-a]pyrimidin-3-yl]-quinoline), Wnt3a/BIO (Wnt family member 3A/(2'Z,3'E)-6bromoindirubin-3'-oxime), BMP4 (bone morphogenetic protein 4 ), GW788388 (4-[4-[3-(pyridin-2yl)-1H-pyrazol-4-yl]pyridin-2-yl]-N-(tetrahydro-2H-pyran-4-yl)benzamide), SM16 (4-[4-(1,3benzodioxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]-bicyclo[2.2.2]octane-1-carboxamide), IN-1130 (3-[[5-(6-methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]benzamide), GW6604 (2-phenyl-4-[ 3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine) and SB505124 (2-(5-benzo[1,3]dioxol-5-yl-2tert-butyl-3H-imidazol-4- yl)-6-methylpyridine). Two or more of these substances may be used in combination.

The concentration of TGFe inhibitor to be added at this stage is suitably adjusted depending on the type of TGFe inhibitor added, and is, for example, 1 to 40 μM, preferably 5 to 20 μM.

In the case of using SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2yl]benzamide), the concentration of TGFe inhibitor added may in particular be 10 µM.

Examples of GSK3e inhibitor include CHIR98014 (2-[[2-[(5-nitro-6-aminopyridin-2yl)amino]ethyl]amino]-4-(2,4-dichlorophenyl)-5-(1H-imidazol-1- yl)pyrimidine), CHIR99021 (6-[[2-[[4-(2,4dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile ), CP21R7 (3-(3-aminophenyl)-4-(1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione), LY2090314 (3-[9-fluoro-1,2, 3,4tetrahydro-2-(1-piperidinylcarbonyl)pyrrolo[3.2.1 -jk] [1,4]benzodiazepin-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1 h- pyrrole-2,5-dione), TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4- (1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), TWS-119 (3-[6-(3-aminophenyl)7H-pyrrolo[2,3-d ]pyrimidin-4-yloxy]phenol), kenpaullone, 1-azakenpaullone, SB415286 (3-[(3-chloro-4hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), AR-AO144-18 (1-[(4-methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)urea), ST99021, ST20026, BIO ((2'Z,3Έ) -6-bromoindirubin-3'- 8 044339 oxime), BJ-acetoxime, pyridocarbazole-ruthenium cyclopentadienyl complex, OTDZT, alpha-4dibromoacetophenone and lithium. Two or more of these substances may be used in combination.

The GSK3e inhibitor is not limited to these substances, and also antisense oligonucleotides and siRNAs against GSK3e mRNA, GSK3e binding antibodies, dominant negative mutants of GSK3e, and the like can be used as the GSK3e inhibitor. These GSK3e inhibitors are commercially available or can be synthesized in a known manner.

The concentration of the GSK3e inhibitor added in this step is suitably adjusted depending on the type of GSK3e inhibitor added, and is, for example, 0.01 to 20 μM, preferably 0.1 to 10 μM.

In the case of using CHIR99021, the concentration of the added GSK3e inhibitor is not particularly limited and can be, for example, 0.1 to 1 μM, preferably 0.5 to 1 μM, in particular 1 μM.

The culture period after the addition of the TGFe inhibitor and the GSK3e inhibitor may be the period during which the cell number of interest is reached. This culture period is not particularly limited and is, for example, 6 to 14 days, 8 to 12 days, 9 to 11 days or 10 days.

For adherent culture, use a culture container such as a dish, vial, microplate or cell culture sheet such as OptiCell (product name) (Nunc). The culture container preferably has a coated surface to enhance cell adhesiveness (hydrophilicity), or is coated with a cell adhesion substrate such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel (e.g., Matrigel BD (Nippon Becton Dickinson Company, Ltd)) or vitronectin.

Matrigel is a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, enriched with extracellular matrix protein. A Matrigel-coated culture container is commercially available. Matrigel consists mainly of laminin, collagen IV, heparan sulfate proteoglycan and entactin/nidogen-1,2. Matrigel contains, in addition to these main components, TGFe, epithelial cell growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogen activator 3,4 and other growth factors naturally produced in the EHS tumor.

The cultivation temperature is not particularly limited and is from 30 to 40°C (for example, 37°C). The carbon dioxide concentration in the culture container is of the order of, for example, 5%.

In one embodiment of the present invention, commercially available neural crest cells can be purchased to obtain neural crest cells for step (2). Examples of commercially available neural crest cells include human hair follicle outer root sheath cells (manufactured by Cosmo Bio Co., Ltd.) and the mouse cranial neural crest cell line O9-1 (manufactured by Merck Millipore).

In one embodiment of the present invention, naturally occurring neural crest cells can be collected to obtain neural crest cells for step (2). Neural crest cells are reported to be present in living mammals, for example, in the human embryonic neural tube at approximately 30 days postfertilization, in the mouse embryonic neural tube at approximately 9 days postfertilization, and in the adult skin of humans, pigs, and rodents. (Betters et al., Developmental biology, 2010, 344 (2): 578-592; Jiang et al., Development, 2000, 127 (8): 1607-1616; Dupin et al., Developmental biology, 2012, 366 ( 1): 83-95; and Nagoshi et al., Cell Stem Cell 2, April 2008, 392-403). Such neural crest cells can be collected using a known method (eg, Motohashi et al., Biology open, 2016, 5: 311-322; and Pfaltzgraff et al., Journal of Visualized Experiments, 2012, 64: 4134) and subjected to the stage ( 2).

Cultivation step (2) using expansion culture of neural crest cells

The neural crest cell expansion culture step (2) is a suspension culture step of neural crest cells in a medium containing a GSK3e inhibitor and basic fibroblast growth factor (bFGF).

In this regard, the medium used is not particularly limited, and, for example, a CDM medium is suitable. You can also use TeSR1, BME, BGJb, CMRL 1066, Glasgow MEM, Enhanced MEM (IMEM), Enhanced MDM (IMDM), Medium 199, Needle MEM, aMEM, DMEM ( high glucose or low glucose), DMEM/F12 medium, Ham's medium, RPMI 1640 medium, Fisher's medium and their mixed medium, etc.

The GSK3e inhibitor mentioned above can be used without specific restrictions.

The GSK3e inhibitor is preferably at least one selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, kenpaullone, AR-AO144-18,

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TDZD-8, SB216763, BIO, TWS-119 and SB415286. Preferably, the GSK3e inhibitor is

CHIR99021 or CP21R7.

The concentration of GSK3e inhibitor added at this stage is the concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration above 1 μM (or at a concentration above 1 μM and below 5 μM). CHIR99021 alone can be used as a GSK3e inhibitor. In the case of using CHIR99021 itself as a GSK3e inhibitor, as mentioned below, a suitable concentration of the added GSK3e inhibitor is a concentration above 1 μM, preferably 2 μM or above and below 5 μM, more preferably above 2 and 4.5 μM or below, in particular preferably 3 µM or higher and 4.5 µM or lower. Thus, in the case of using a GSK3e inhibitor other than CHIR99021, at this stage, the concentration of the GSK3e inhibitor added is a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration above 1 μM, preferably a concentration that exhibits an effect equivalent to that of which exhibits CHIR99021 at a concentration of 2 µM or higher and below 5 µM, more preferably a concentration that exhibits an effect equivalent to that which exhibits CHIR99021 at a concentration above 2 and 4.5 µM or lower CHIR99021, especially preferably a concentration that exhibits an effect equivalent effect exhibited by CHIR99021 at concentrations of 3 μM or higher and 4.5 μM or lower.

Neural crest cells that remain multipotent can be cultured and allowed to proliferate over a culture period of more than 9 weeks (63 days) by suspension culture of neural crest cells in the presence of an appropriate concentration of GSK3e inhibitor and bFGF. Neural crest cells that remain multipotent can be cultured and allowed to proliferate over a culture period of more than 9 weeks (63 days) by suspension culture of neural crest cells in the presence of an appropriate concentration of GSK3e inhibitor and bFGF.

In one embodiment of the present invention, neural crest cells that maintain multipotency have the ability to differentiate into or differentiate into neural cells, glial cells, and mesenchymal stromal cells, as well as bone cells, chondrocytes, corneal cells, and pigment cells. .

Neural crest cells that maintain multipotency can be assessed in a variety of ways. Examples of methods include, but are not specifically limited to, a method of inducing differentiation of neural crest cells to be assessed into each lineage, such as neural cells, glial cells and mesenchymal stromal cells. Assuming that neural crest cells can actually differentiate into neural cells, glial cells and mesenchymal stromal cells, etc., the neural crest cells to be evaluated can be determined to be neural crest cells that maintain multipotency. Another example of a method includes a method for determining the expression of a marker protein or gene. Provided that the SOX10 transcription factor is expressed in the neural crest cells being evaluated, the neural crest cells being evaluated can be defined as neural crest cells that maintain multipotency. Detection of SOX10 can be performed using an immunoassay, such as ELISA, immunostaining or flow cytometry, using an antibody specific for the marker protein. Detection of the marker gene can be performed using a nucleic acid amplification and/or detection method known in the art, such as RT-PCR, microarrays or bioarrays. When cells have an insertion of a nucleotide sequence encoding a reporter protein (for example, Nano-Lantern (Saito K. et al., Luminescent proteins for high-speed single-cell and wholebody imaging. Nat. Commun., 2012; 3: 1262)) below gene SOX10, a marker gene for NCC, and express a fusion protein of SOX10 and a reporter protein under the control of the SOX10 promoter, a method for detecting the reporter protein (eg, detecting fluorescence intensity) can be used.

An effect equivalent to that exhibited by CHIR99021 at a concentration above 1 μM (or at a concentration above 1 μM and below 5 μM) as a GSK3e inhibitor can be assessed based on GSK3e inhibitory activity. The GSK3e inhibitory activity of a GSK3e inhibitor can be determined using a method per se known and can be measured, for example, by the method described in Patsch et al., Nature cell biology, 2015, 17 (8): 994-1003, or Uno et al. al., Brain Res., 2009, 1296: 148-163. In particular, the inhibitory activity of GSK3e can be determined using the gene expression regulation function (in particular, the phosphorylation function of βcatenin) of GSK3e in the Wnt/β-catenin cascade as an indicator. More specifically, this approach involves: (i) culturing cells in which expression of a reporter gene is suppressed under the control of GSK3e, in the presence and absence of a GSK3e inhibitor; (ii) determining the level of reporter gene expression in the presence and absence of a GSK3e inhibitor; and (iii) determining the GSK3e inhibitory activity of the GSK3e inhibitor based on the magnitude of the increase in reporter gene expression in the presence of the GSK3e inhibitor relative to the reporter gene expression in the absence of the GSK3e inhibitor (see Example 9).

When a GSK3e inhibitor exhibits a GSK3e inhibitory activity (% inhibition) equivalent to that exhibited by CHIR99021 at a concentration greater than 1 μM, the measurement results determine that the GSK3e inhibitor exhibits an effect equivalent to that exhibited by CHIR99021 (GSK3e inhibitory activity equivalent to the effect which exhibits CHIR99021) at concentrations above 1 µM. In this context, equivalent value (% inhibition) refers to a value that may differ by up to plus or minus 30, 25, 20, 15, 10, 8, 6, 5, 4, 3, 2 or 1% from the reference value. Preferably the value is in the range of minus to plus 15, 10, 5 or 1% of the reference value.

An effect equivalent to that demonstrated by CHIR99021 at a concentration above 1 μM (or at a concentration above 1 μM and below 5 μM) as a GSK3e inhibitor could more appropriately be assessed based on the period over which the neural crest cells that retain multipotency and are culturable when neural crest cells are cultured in the manner described in the examples herein. When neural crest cells that remain multipotent can be cultured in increasing numbers relative to the start of culture by culturing the neural crest cells in a medium containing a GSK3e inhibitor for a period of time equivalent to the period of time culturing the neural crest cells in a medium containing a higher concentration of CHIR99021 1 μM, the GSK3e inhibitor is determined to exhibit an effect equivalent to that demonstrated by CHIR99021 (neural crest cell proliferation activity equivalent to the effect demonstrated by CHIR99021) at a concentration greater than 1 μM. In this context, an equivalent value (period) refers to a value that can differ by up to plus or minus 30, 25, 20, 15, 10, 8, 6, 5, 4, 3, 2 or 1% from the reference value. Preferably, the value is in the range of minus to plus 15, 10, 5 or 1% of the reference value.

In the case of using CHIR99021 itself as a GSK3e inhibitor, the concentration of the added GSK3e inhibitor is a concentration higher than 1 μM, preferably 2 μM or higher or lower than 5 μM, more preferably higher than 2 μM and 4.5 μM or lower, especially preferably 3 μM or higher and 4.5 µM or lower. The effect of allowing neural crest cells to proliferate while maintaining differentiation ability over a long period of time was found to be high for 2 μM or above and below 5 μM CHIR99021, and highest for 3 μM or above and 4.5 μM or below CHIR99021. Specifically, CHIR99021 at concentrations of 3 to 4.5 μM allowed the cultivation and proliferation of neural crest cells that maintained multipotency for 112 days (16 weeks) or more. Also, CHIR99021 at a concentration of 2 μM maintained multipotency for 9 weeks (63 days) or more. On the other hand, 1 μM or 5 μM CHIR99021 decreased the number of cells that remained multipotent after culture for 3 weeks (21 days) and 6 weeks (42 days), respectively.

In the case of using CP21R7 as a GSK3e inhibitor, the concentration of the GSK3e inhibitor added is a concentration higher than 0.1 μM, preferably 0.5 μM or higher, more preferably 1 μM or higher. The effect of allowing neural crest cells to proliferate while maintaining differentiation ability over a long period of time was found to be high for 0.5 μM or higher and 1 μM or lower of CP21R7. In particular, CP21R7 at concentrations of 0.5 to 1 μM allowed the cultivation and proliferation of neural crest cells that maintained multipotency for 84 days (12 weeks) or more. On the other hand, 0.1 μM CP21R7 reduced the number of cells that remained multipotent after cultivation for 3 weeks (21 days).

The concentration of bFGF added is not particularly limited and is, for example, 10 to 200 ng/ml, preferably 20 to 40 ng/ml.

The medium can be supplemented with a TGFe inhibitor and/or epidermal growth factor (EGF), in addition to the GSK3e and bFGF inhibitor. The TGFe inhibitor mentioned above can be used without specific restrictions.

The TGFe inhibitor is preferably at least one selected from the group consisting of SB431542, A83-01, LDN193189, Wnt3A/BIO, BMP4, GW788388, SM16, IN-1130, GW6604 and SB505124. Particularly preferably, the TGFe inhibitor is SB431542.

The concentration of the TGFe inhibitor added is suitably adjusted depending on the type of TGF3e inhibitor added, and is, for example, 1 to 50 μM, preferably 5 to 20 μM.

In the case of using SB431542, the added concentration of TGFe inhibitor is not particularly limited and can be, for example, 1 to 40 μM, preferably 5 to 20 μM, in particular 10 μM.

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The concentration of EGF added is not particularly limited and is, for example, 5 to 100 ng/ml, preferably 20 to 40 ng/ml.

In suspension culture, the neural crest cells obtained in step (1) are detached from the culture container and then dispersed in the medium to form an aggregated cell mass, while the components of the medium and the internal oxygen concentration of the medium are uniformized by stirring or shaking. The appropriate stirring speed is appropriately selected depending on the cell density and the size of the culture container. Excessive mixing or shaking provides physical stress to the cells and inhibits the formation of aggregated cell mass. Thus, the speed of stirring or shaking is controlled to ensure uniformity of the media components and the internal oxygen concentration of the media, and not to inhibit the formation of aggregated cell mass. Suspension cultivation can be done by simply letting the culture stand without stirring or shaking.

The cultivation temperature is not particularly limited and is from 30 to 40°C (for example, 37°C). The carbon dioxide concentration in the culture container is on the order of, for example, 5%.

The culture period at this stage may be the period during which the number of cells of interest is achieved. This stage allows the cultivation and proliferation of neural crest cells, which remain multipotent for an extended period. The proportion of neural crest cells that remain multipotent in the cultured cell population is at least 20% or more, 30% or more, or 40% or more, and can be maintained at preferably 50% or more, 60% or more, or 70% or more, more preferably 80% or more, 90% or more, or 95% or more.

During this culture, the cells are properly passaged. Passaging is carried out every 5-8 days after inoculation, for example. Preferably, the interval between passagings is a sufficient period for expansion of the aggregated cell mass, and this period is shorter than the period during which the aggregated cell mass prevents oxygen or nutrients from reaching the cells in the aggregated cell mass.

The period during which this stage allows the cultivation and proliferation of neural crest cells that retain multipotency is not particularly limited and can be, for example, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77. 84, 91, 98, 105 or 112 days or more. Preferably, this period is 35 days or more, more preferably 42 days or more, even more preferably 63 days or more, particularly preferably 84 days or more, most preferably 112 days or more.

Wednesday

The present invention also relates to a medium for use in a method for producing neural crest cells and a method for causing proliferation of neural crest cells mentioned above, including neural crest cells. The preferred composition of the medium is as stated above.

Initial cell culture

The present invention also relates to a frozen stock culture containing neural crest cells obtained by the method for producing neural crest cells and the method for causing proliferation of neural crest cells mentioned above. Neural crest cells are positive for the expression of SOX10 and positive for the expression of p75, an antigenic marker of the cell surface of NCC.

A frozen stock culture can be obtained by separating the neural crest cells obtained by the method of producing neural crest cells and the method of allowing neural crest cells to proliferate from the medium and suspending the neural crest cells in a cryopreservation solution for freezing. Separation of neural crest cells from the medium can be accomplished using a cell sieve or centrifugation. Cells separated in this way can be washed if necessary. The frozen stock culture may contain an additional cell population in addition to the neural crest cells and preferably contains purified neural crest cells. Purification of neural crest cells can be accomplished, for example, by separating neural crest cells from other cell populations through cell sorting using the expression of the markers described above as an indicator. As the cell preservation solution, a conventional reagent for use in cell cryopreservation can be used. For example, Cryostem Freezing Medium and CELLBANKER(R) are commercially available.

The frozen stock culture can be used as starting material to induce differentiation of neural crest cells to produce neural cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells and pigment cells. Also, frozen stock culture can be used to generate tissue models having neural crest cells as a component.

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Method for obtaining various cells from neural crest cells

The method for producing nerve cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells or pigment cells in accordance with the present invention includes the steps below. Of these steps, step (i) is the same as step (2) of the method for producing neural crest cells according to the present invention or the method for causing proliferation of neural crest cells according to the present invention.

(i) A suspension culture of neural crest cells in a medium containing a GSK3e inhibitor and basic fibroblast growth factor (bFGF), wherein the medium contains a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration greater than 1 μM; and (ii) allowing the neural crest cells obtained in step (i) to differentiate into cells of at least one lineage selected from the group consisting of neural cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells and pigment cells.

Induction stage into various cells

According to the method for producing neural crest cells and the method for causing proliferation of neural crest cells in accordance with the present invention, it is possible to obtain neural crest cells that retain multipotency to differentiate into neural cells, glial cells and mesenchymal stromal cells, or multipotency to differentiate into these cells , as well as bone cells, chondrocytes, corneal cells and pigment cells.

Induction of differentiation of neural crest cells into cells of each lineage selected from neural cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells and pigment cells can be carried out in a known manner described in the literature (for example, in non-patent documents 1-4) .

In particular, the induction of differentiation into nerve cells can be carried out based on the method described in Non-Patent Document 1 or 4.

For example, neural crest cells are inoculated onto a fibronectin-coated plate and the medium is replaced with DMEM/F12 supplemented with N-2 Supplement (17502-048, Gibco), BDNF (028-16451, Wako Pure Chemical Industries, Ltd.), GDNF (074-06264, Wako Pure Chemical Industries, Ltd.), NT-3 (14106643, Wako Pure Chemical Industries, Ltd.) and NGF (141-07601, Wako Pure Chemical Industries, Ltd.), and then cultured at 37 °C for 14 days in 5% CO ₂ .

Alternatively, neural crest cells are inoculated onto a plate and cultured for 1 day in CDM medium containing 10 μM SB431542 and 1 μM CHIR99021, and then the medium is replaced with Neurobasal Medium (21103-049, Gibco) supplemented with B-27 Supplement (17504-044 , Gibco), N-2 Supplement, L-glutamine (073-05391, Wako Pure Chemical Industries, Ltd.), penicillin/streptomycin (15140122, Gibco), BDNF, GDNF, NT-3 and NGF, and then cultured at 37°C for 35 days in 5% CO2.

Cells thus cultured were fixed in 4% paraformaldehyde, and differentiation into neural cells expressing TUBB3 protein was confirmed by immunostaining.

Induction of differentiation into glial cells can be carried out using a similar approach to inducing the onset of differentiation into neural cells in the manner described in Non-Patent Document 1 or 4. After completion of the differentiation induction period, the onset of differentiation into glial cells is confirmed based on the expression of GFAP protein.

Induction of differentiation into mesenchymal stromal cells can be carried out based on the method described in Non-Patent Document 1. Specifically, for example, neural crest cells are inoculated at a density of 6.5 x ^{10 4} cells/cm ² per dish and cultured for 1 day in CDM medium containing 10 µM SB431542 and 1 µM CHIR99021. After one day, the medium is replaced with aMEM (Nacalai Tesque, Inc.) containing 10% fetal bovine serum (FBS, Nichirei Corp.). After approximately 4 days, a morphological change in the cells is observed. Cells are passaged by cell detachment using 0.25% trypsin-EDTA (Gibco) and inoculation at a density of 1.0 x 10 ⁴ cells/cm ² . 14 days after the start of induction of differentiation, the expression of surface antigenic markers CD73, CD44, CD45 and CD105 of human mesenchymal stromal cells is analyzed using FACS to confirm differentiation into mesenchymal stromal cells.

The induction of differentiation into bone cells can be carried out based on the method described in Non-Patent Document 1. Specifically, for example, the mesenchymal stromal cells described above are inoculated at 2.5 x 105 cells per fibronectin-coated dish and cultured for 2 weeks in aMEM , containing 10% FBS, 0.1 μM dexamethasone, 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate. The medium is replaced once every two days only during the first 1 week. The calcified nodule is identified by alizarin red staining to confirm differentiation into bone cells.

Induction of differentiation into chondrocytes can be carried out based on the method described in

- 13 044339 patent document 1. Specifically, for example, the mesenchymal stromal cells described above are suspended at a concentration of 1.5 x 105 cells/5 μl in DMEM:F12 (Invitrogen Corp.) containing 1% (v/v) pre ITS+ mixture (BD), 0.17 mM AA2P, 0.35 mM proline (Sigma-Aldrich Co. LLC), 0.1 mM dexamethasone (Sigma-Aldrich Co. LLC), 0.15% (v/v). ) glucose (Sigma-Aldrich Co. LLC), 1 mM Napyruvate (Invitrogen Corp.), 2 mM GlutaMax, 0.05 mM MTG, 40 ng/ml PDGF-BB, and 1% (v/v) FBS (Nichirei Corp.). 5 μl of cell suspension is spotted onto a fibronectin-coated dish and cultured for 1 hour. After 1 hour, 1 ml of differentiation induction medium described above is added to them. 3-5 days after the start of differentiation induction, 10 ng/ml TGFe3 (R&D Systems, Inc.) is added to them. On day 10 after the start of differentiation induction, 50 ng/ml BMP4 is added to them. The cells are cultured for 16 days and the onset of differentiation into chondrocytes is confirmed by Alcian blue staining.

Induction of differentiation into corneal cells can be carried out based on the method described in Non-Patent Document 1. Specifically, for example, neural crest cells are inoculated onto a fibronectin-coated dish and cultured for 1 day in CDM medium containing 10 μM SB431542 and 1 μM CHIR99021 . After one day, the medium is replaced with conditioned medium obtained by culturing human corneal endothelial cells in CDM medium. The medium is replaced once every two days. On day 12 after the start of differentiation induction, the expression of ZO-1, a marker molecule of corneal cells, was confirmed by immunostaining, and the expression of COL4A1 and COL8A1 was confirmed by qPCR.

Induction of differentiation into pigment cells can be carried out based on the method described in Non-Patent Document 1. Specifically, for example, neural crest cells are inoculated onto a fibronectin-coated dish and cultured for 1 day in CDM medium containing 10 μM SB and 1 μM CHIR . After one day, the medium is replaced with CDM medium containing 1 μM CHIR, 25 ng/ml BMP4 and 100 nM endothelin-3 (American Peptide Company). The medium is replaced once every two days. On day 7 after the start of induction of differentiation, differentiation into pigment cells is confirmed by the expression of the MITF and c-KIT genes.

All of the resulting nerve cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells and pigment cells can be used to produce cells for regenerative medicine.

Neural crest cells are cells that have the multipotency to differentiate into many cell types, such as neural cells, glial cells, mesenchymal stromal cells, bone cells, chondrocytes, corneal cells and pigment cells, and the ability to self-proliferate. Based on this ability found for neural crest cells, it is possible to use neural crest cells as cellular agents for regenerative medicine. If neural crest cells that remain multipotent could be efficiently maintained or allowed to proliferate, their large-scale production would be possible. If it were possible to obtain a stock culture of neural crest cells that retain multipotency, the stock culture would be suitable as starting material for cell-based agents. For example, the use of cells (eg, neural crest cells) midway through the differentiation of stem cells into cells of interest (eg, neural cells) as starting material has been explored for the purpose of, for example, simplifying production methods, shortening production times, or reducing cost. production during the production of cellular agents. The present invention is believed to provide a useful method for this purpose. This may be true not only for the production of cellular agents, but also for the construction of screening systems using various cells, etc.

Examples

Example 1 Cultivation Using Neural Crest Cell Expansion Culture

Induction of iPSC differentiation into NCC

Human iPSC cells were allowed to differentiate into neural crest cells (NCC) in accordance with the method described in Non-Patent Document 1. The iPSCs used were SOX10-Nano-Lantern Reporter Human iPSC (line 201B7) described in Non-Patent Document 4. This cell the line has an insertion of a nucleotide sequence encoding the fluorescent protein Nano-Lantern (Saito K. et al., Luminescent proteins for high-speed single-cell and wholebody imaging. Nat. Commun., 2012; 3: 1262) downstream of the SOX10 gene, which is a marker NCC genome, and expresses a fusion protein of SOX10 and Nano-Lantern under the control of the SOX10 promoter.

iPSCs were inoculated onto a Matrigel-coated dish, adherent cultured for 4 days in TeSR1 medium, and then adherent cultured for 10 days in CDM medium containing 10 μM SB431542 (TGFe inhibitor) and 1 μM CHIR99021 ( GSK3e inhibitor), and thereby allowed differentiation into NCC.

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NCC expansion culture

NCCs were detached from the plate and cultured using suspension culture in CdM medium containing 10 μM SB431542, 3 μM CHIR99021, 40 ng/ml bFGF, and 40 ng/ml EGF.

Cells were passaged every 7 days.

The change in the total number of cells after the start of cultivation using expansion culture is presented in Fig. 1, and the change in the percentage of cells positive for SOX10 expression is presented in FIG. 2. To determine the percentage of cells positive for SOX10 expression, aggregated cell masses were dissociated using StemPro Accutase Cell Dissociation Reagent (Invitrogen Corp.) to obtain single cell suspensions. Single cell suspensions in FACS buffer (2% BSA HBSS) supplemented with 1 μg/mL PI (propidium iodide, Wako Pure Chemical Industries, Ltd.) were filtered through a 35 μm nylon mesh tube (BD Falcon) and then analyzed using a flow cytometer (FACS Aria, BD Biosciences) to determine the proportion of GFP-positive cells in living cells (PI-negative cells). The total number of cells increased throughout the entire period and a high percentage of cells positive for SOX10 expression was confirmed even on day 121 of culture. Expressed SOX10 serves as a marker for multipotent NCCs. These results indicate that NCCs proliferate while maintaining differentiation capacity over long periods of culture.

Example 2. Study of the concentration of the additive in the medium

GSK3e inhibitor, bFGF and EGF were examined for the effect of their concentrations on the ability of NCC to self-proliferate and their ability to differentiate in NCC expansion culture. Next, cultivation was carried out using expansion culture under the same conditions as in example 1, unless otherwise indicated.

In fig. 3 shows the change in total cell number when bFGF and EGF concentrations of 20 ng/ml (A) or 40 ng/ml (B) were used and CHIR99021 concentrations of 1, 2, 3, or 5 μM were used. The level of cell proliferation within 1 week using 1 or 2 μM CHIR99021 was 8- to 30-fold (also see Example 8 below). In fig. Figure 4 shows the change in total cell number when bFGF and EGF concentrations of 40 ng/ml were used and CHIR99021 concentrations of 3, 3.5, 4, 4.5, or 5 μM were used. The concentrations of bFGF, EGF and CHIR99021 did not affect the change in the total number of cells.

In fig. Figure 5 shows the change in the percentage of cells positive for SOX10 expression when a concentration of bFGF and EGF of 20 ng/ml (A) or 40 ng/ml (B) was used and a CHIR99021 concentration of 1, 2, 3 or 5 μM was used. In fig. Figure 6 shows the change in the percentage of cells positive for SOX10 expression when bFGF and EGF concentrations of 40 ng/ml were used and CHIR99021 concentrations of 3, 3.5, 4, 4.5 or 5 μM were used. When the concentration of CHIR99021 was 3-4.5 μM, 90% or more cells were confirmed to be positive for SOX10 expression during the long culture period (at 121 days of culture). Even when the concentration of CHIR99021 was 2 μM, approximately 55% or more cells were confirmed to be positive for SOX10 expression during a relatively long culture period (at day 63 of culture). On the other hand, when the concentration of CHIR99021 was 5 μM, approximately 55% or more of SOX10 positive cells were maintained during culture for up to 42 days, while the percentage of SOX10 positive cells decreased to approximately 5% or less at 63 days of cultivation. When the concentration of CHIR99021 was 1 μM, a decrease in the percentage of SOX10 positive cells was observed during a short culture period (at 21 days of culture), and the percentage of SOX10 positive cells was approximately 25% or less at 63 days of culture. The concentrations of bFGF and EGF1 did not affect the change in the percentage of cells positive for SOX10 expression.

Example 3 GSK3e Inhibitor Study

NCC was subjected to expansion culture in the same manner as in Example 1, except that the GSK3e inhibitor used in NCC expansion culture was changed from CHIR99021 to CP21R7. The concentration of CP21R7 used was 0.1, 0.5, or 1 μM.

The change in the percentage of cells positive for SOX10 expression is presented in Fig. 7. At a CP21R7 concentration of 0.5 or 1 μM, a high percentage of cells positive for SOX10 expression was confirmed even on the 84th day of cultivation. On the other hand, at a CP21R7 concentration of 0.1 μM, the percentage of cells positive for SOX10 expression began to decrease within a short period of time (on day 21 of culture).

Example 4 Induction of differentiation of neural crest cells into nerve cells

The differentiation ability of NCCs cultured using expansion culture according to example 1 into nerve cells was confirmed.

Induction of differentiation into nerve cells was carried out based on the method described in non-patent document 4.

NCCs subjected to expansion culture by maintenance culture for 30 days were inoculated at 5x105 cells per plate and cultured for 1 day in CDM medium containing 10 μM SB431542 and 1 μM CHIR99021. After one day, the medium was replaced with neurobasal medium (21103-049, Gibco), supplemented with B-27 Supplement (17504-044, Gibco), N-2 Supplement, L-glutamine (073-05391, Wako Pure Chemical Industries, Ltd.) , penicillin/streptomycin (15140-122, Gibco), BDNF (028-16451, Wako Pure Chemical Industries, Ltd.), GDNF (074-06264, Wako Pure Chemical Industries, Ltd.), NT-3 (141-06643, Wako Pure Chemical Industries, Ltd.) and NGF (141-07601, Wako Pure Chemical Industries, Ltd.), followed by culture at 37°C for 35 days with 5% CO2. During this culture period, the medium was replaced every 3-4 days.

Cells were fixed by adding 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.) and incubating at 4°C for 1 hour. Cells were reacted with anti-TUBB3 antibody (845502, BioLegend, Inc.) and anti-GFAP antibody (ab7260, Abcam PLC ) as primary antibodies, then sequentially reacted with 488-labeled secondary antibody (Invitrogen Corp.) and Alexa 568-labeled secondary antibody as secondary antibodies compatible with the species of animals that were immunized with primary antibodies, and then observed under a fluorescence microscope.

The onset of differentiation into neural cells expressing TUBB protein and glial cells expressing GFAP protein was confirmed for NCCs subjected to maintenance culture for 30 days.

The ability to differentiate into neural cells was further confirmed in the same manner as described above using NCCs cultured using expansion culture based on maintenance culture for 84 days. As a result, differentiation into neural cells (peripherin-positive cells) and glial cells (GFAP-positive cells) was confirmed.

Example 5 Induction of differentiation of neural crest cells into pigment cells

The differentiation ability of NCCs cultured using expansion culture according to example 1 into nerve cells was confirmed.

Induction of differentiation into melanocytes was carried out based on the method described in Non-Patent Document 1.

NCCs subjected to expansion culture via maintenance culture for 84 days were inoculated into a 6-well fibronectin-coated plate and cultured for 1 day in CDM medium containing 10 μM SB431542 and 1 μM CHIR99021. The next day, the medium was replaced with CDM medium supplemented with BMP4 and endothelin-3 (Wako Pure Chemical Industries, Ltd.), followed by cultivation at 37°C for 7 days in 5% CO ₂ . During this culture period, the medium was replaced every 2 days.

Cells were fixed by adding 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.) and incubating at 4°C for 1 hour. Cells were reacted with anti-MITF antibody (Sigma-Aldrich Co. LLC) as the primary antibody, and then reacted with 568-labeled secondary antibody (Invitrogen Corp.) as the secondary antibody, compatible with the species of animals that were immunized with primary antibodies and then observed under a fluorescence microscope. The onset of differentiation into MITF protein-expressing melanocytes from NCCs subjected to maintenance culture for 84 days was confirmed.

Example 6 Induction of Differentiation of Neural Crest Cells into Mesenchymal Stromal Cells The ability to differentiate into mesenchymal stromal cells was confirmed in NCCs cultured using expansion culture according to Example 1.

Induction of differentiation into mesenchymal stromal cells was carried out based on the method described in Non-Patent Document 1.

NCCs subjected to expansion culture based on maintenance culture for 84 days were inoculated onto a 6 cm cell culture dish coated with fibronectin and cultured for 1 day in CDM containing 10 μM SB431542 and 1 μM CHIR99021. The next day, the medium was replaced with CTS StemPro MSC SFM (Gibco, A1033201). Cells were passaged by cell dissociation with StemPro Accutase Cell Dissociation Reagent (Invitrogen Corp.) and inoculation at a density of 1.0-2.0 x 106 cells/dish (for a 10 cm dish).

14 days after the onset of induction of differentiation into mesenchymal stromal cells, NCCs were immunostained with anti-CD73 (BD), anti-CD44 (BD), anti-CD45 (BD), and anti-CD105 (eBioscience, Inc.), i.e. . antibodies against surface antigenic markers of human mesenchymal stromal cells, and then FACS analysis was performed. Onset of differentiation into mesenchymal stromal cells expressing each of CD44, CD73 and CD0105 proteins, and not expressing CD4 protein, was confirmed for NCC treated

- 16 044339 maintenance cultivation for 84 days.

Example 7 Induction of Differentiation of Neural Crest Cells into Bone Cells, Chondrocytes or Adipocytes

The ability of NCCs cultured using expansion culture according to Example 1 to differentiate into bone cells, chondrocytes or adipocytes was confirmed.

Induction of differentiation into bone cells, chondrocytes or adipocytes was carried out based on the method described in Non-Patent Document 1.

NCC cells cultured using expansion culture based on maintenance culture for 84 days were allowed to differentiate into mesenchymal stromal cells in the manner described in Example 6. Mesenchymal stromal cells were inoculated at 4.0 x 10 ⁴ cells/well in a 12-well plate, coated with fibronectin and cultured for 4 weeks in aMEM containing 10% FBS, 0.1 μM dexamethasone, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate to induce differentiation into bone cells. The medium was replaced once every two to three days. The calcified nodule was identified by alizarin red staining to confirm differentiation into bone cells.

Induction of differentiation into chondrocytes was carried out as follows: mesenchymal stromal cells, which were induced to differentiate from NCC, subjected to expansion culture through maintenance culture for 84 days, were carried out by suspending the cells at a concentration of 1.5x105 cells/5 μl in DMEM:F12 (Invitrogen Corp.) containing 1% (v/v) ITS+ premix (BD), 0.17 mM AA2P, 0.35 mM proline (Sigma-Aldrich Co. LLC), 0.1 mM dexamethasone (Sigma -Aldrich Co. LLC), 0.15% (v/v) glucose (SigmaAldrich Co. LLC), 1 mM Na-pyruvate (Invitrogen Corp.), 2 mM GlutaMax, 0.05 mM MTG, 40 ng/ ml PDGF-BB and 1% (v/v) FBS (Nichirei Corp.). The cell suspension was spotted in an amount of 5 μl/well onto a 12-well plate coated with fibronectin and cultured for 1 hour. After 1 hour, the medium was further supplemented with 10 ng/ml TGFe3 (R&D Systems, Inc.) and 100 ng/ml was added to it. BMP7 (Wako Pure Chemical Industries, Ltd.) at a concentration of 1 ml/well. The cells were cultured for 10 days and the onset of differentiation into chondrocytes was confirmed by Alcian blue staining.

Induction of differentiation into adipocytes was carried out as follows: mesenchymal stromal cells, into which NCCs were induced to differentiate, subjected to expansion culture based on maintenance culture for 84 days, were carried out by inoculation in an amount of 4.0x104 cells/well per 24-well plate, coated with fibronectin and cultured for 4 weeks in hMSC-adherent media using Human Mesenchymal Stem Cell Adipogenic Differentiation Medium Bullet Kit (Lonza Japan Ltd.). The medium was replaced once every two to three days. Oil droplet detection was performed in cells stained with Oil Red O staining to confirm differentiation into adipocytes.

Bone cell staining using alizarin red S was performed as follows: cells were fixed by adding 100% ethanol and incubating at room temperature for 10 min. Cells were reacted with Alizarin Red staining solution (Merck Millipore), washed with water, dried, and then observed under a microscope.

Alcian blue staining of chondrocytes was performed as follows: cells fixed by adding 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.) and incubating at room temperature for 30 min were reacted with 1% Alcian blue staining solution (Muto Pure Chemicals Co. , Ltd.), washed with water and dried.

Staining of adipocytes with oil red O was carried out as follows: cells were fixed in 10% formaldehyde at room temperature for 1 hour, reacted with a 3:2 mixture of 0.5% solution of oil red O with isopropanol, and water at room temperature for 1 hour. and washed with water. The oil droplets were observed under a microscope.

Example 8. Study of the concentration of the additive in the medium - 2 bFGF was studied in relation to the effect of its concentration on the ability of NCC to self-proliferate and their ability to differentiate in the expansion culture of NCC. Next, cultivation was carried out using expansion culture under the same conditions as in example 1, unless otherwise indicated.

In fig. Figure 8 shows the change in total cell number when a CHIR99021 concentration of 1.5 μM and a bFGF concentration of 10, 12.5, 15.0, or 17.5 ng/ml were used. In fig. Figure 9 shows the change in the percentage of cells positive for SOX10 expression. The concentration of bFGF had no effect on the percentage of positivity for SOX10 expression.

The pattern of changes in total cell number was essentially the same over the range of bFGF concentrations described above, and the level of cell proliferation ranged from 6- to 13-fold over 1 week. The level of cell proliferation over 1 week in Example 2 was in the higher range of 8- to 30-fold at CHIR99021 concentrations of 1 or 2 μM and bFGF concentrations of 20 or 40 ng/ml (see Fig. 3), indicating that that bFGF contributes to the ability

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Claims (25)

NCC self-proliferates and has a suitable concentration range of 20 to 40 ng/ml. Example 9 Measurement of GSK3e Inhibitory Activity An experimental system was prepared to evaluate the activity of a GSK3e inhibitor in inhibiting GSK3e. In the Wnt/β-catenin cascade, GSK3e functions by phosphorylating β-catenin in the absence of Wnt ligand. Phosphorylated β-catenin is ubiquitinylated and degraded in the proteasome. Thus, gene expression downstream of the Wnt-to-catenin cascade is suppressed. If GSK3e in this cascade is inhibited, β-catenin translocates to the nucleus without degradation, inducing gene expression downstream of the Wnt-βcatenin cascade along with other transcription factors such as T cell factor (TCF)/lymphoid enhancer factor (LEF). The CellSensor LEF/TCF-bla HCT-116 Cell Line (Thermo Fisher Scientific Inc., K1676) stably incorporates LEF/TCF and includes a reporter gene (betalactamase reporter gene) for expression of the reporter gene under the control of LEF/TCF. Reporter gene expression in this cell line in the absence of Wnt ligand serves as an indicator of inhibition of function (β-catenin phosphorylation function) of GSK3e. The GSK3e inhibitory activity of the GSK3e inhibitor was determined by assay using this cell line. The assay was performed according to the Invitrogen Corp. protocol. (CellSensor(R) LEF/TCF-bla HCT 116 Cell-based Assay Protocol). Specifically, LEF/TCF-bla HCT-116 Cells were suspended in assay medium (OPTI-MEM, 0.5% dialyzed FBS, 0.1 mM NEAA, 1 mM sodium pyruvate, 100 U/ml/100 μg/ ml Pen/Strep) (312500 cells/ml). The cell suspension was inoculated into each well of the assay plate (10,000 cells/well) and cultured for 16 to 24 h. GSK3e inhibitor (CHIR99021 used here) was added to each well (concentration: 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 10000 nM) and cells were cultured for 5 hours Beta-lactamase substrate solution (LiveBLAzer-FRET B/G (CCF4-AM) Substrate Mixture) (8 μl/well) was added to each well and incubated for 2 h. The amount of fluorescence was determined using a plate reader. The measurement was carried out in two wells for each concentration. The results are presented in table. 1. The GSK3e inhibition activity exhibited by 1 μM CHIR99021 was 113.5 (average of two wells) in fluorescence magnitude values. The concentration that exhibits GSK3e inhibitory activity equivalent to the inhibition activity exhibited by 1 μM CHIR99021 (fluorescence value: 113.5) can also be determined for GSK3e inhibitors other than CHIR99021 by measuring fluorescence values at each concentration conditions using this experimental system and constructing calibration curves in accordance with the standard method. Table Compound concentration Fluorescence value (nM) Hole 1 Hole 2 10000 88 73 3160 99 136 1000 114 113 316 101 96 100 75 60 31.6 21 45 10.0 12 23 3.16 0 14 1.00 0 0 0.316 6 0 CLAIM 1. A method for ensuring proliferation of neural crest cells, comprising the step of: (i) culturing using a suspension culture of neural crest cells for a period of 35 days or more in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, wherein the medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor. 2. The method according to claim 1, where the medium additionally contains a TGFe inhibitor. 3. The method of claim 1, wherein the medium is a CDM medium. - 18 044339 4. The method according to claim 1, where the medium additionally contains epidermal growth factor. 5. The method according to claim 1, wherein in the specified medium containing (a), the GSK3e inhibitor is at least one representative selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, kenpaullona, AR-AO144-18, TDZD-8, SB216763, BIO, TWS-119 and SB415286. 6. The method of claim 5, wherein the GSK3e inhibitor is CHIR99021. 7. The method of claim 2, wherein the TGFe inhibitor is at least one member selected from the group consisting of SB431542, A83-01, LDN193189, Wnt3a/BIO, BMP4, GW788388, SM16, IN1130, GW6604 and SB505124. 8. The method according to claim 1, where at stage (2) the neural crest cells are passaged every 5-8 days after inoculation. 9. A medium for producing and allowing proliferation of neural crest cells over a period of 35 days or more, containing a GSK3e inhibitor, basic fibroblast growth factor and neural crest cells, wherein the medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor. 10. Medium according to claim 9, additionally containing a TGFe inhibitor. 11. The medium according to claim 9, where the medium is a CDM medium. 12. The medium according to claim 9, additionally containing epidermal growth factor. 13. The medium of claim 9, wherein said medium containing (a) the GSK3e inhibitor is at least one member selected from the group consisting of CHIR99021, CP21R7, CHIR98014, LY2090314, kenpaullone, AR-AO144-18, TDZD -8, SB216763, BIO, TWS-119 and SB415286. 14. The medium according to claim 13, wherein the GSK3e inhibitor is CHIR99021. 15. The medium of claim 10, wherein the TGFe inhibitor is at least one member selected from the group consisting of SB431542, A83-01, LDN193189, Wnt3a/BIO, BMP4, GW788388, SM16, IN1130, GW6604 and SB505124. 16. Frozen source culture containing neural crest cells obtained by the production method according to claim 1. 17. A method for obtaining nerve cells, including the stages: (i) culturing using a suspension culture of neural crest cells for a period of 35 days or more in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, wherein the medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor; and (ii) differentiating the neural crest cells obtained in step (i) into neural cells. 18. A method for culturing neural crest cells possessing multipotency for a period of 35 days or more, comprising the steps of: (1) obtaining neural crest cells; and (2) culturing using a suspension culture of neural crest cells in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, wherein the medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor. 19. The use of a medium for culturing multipotent neural crest cells for a period of 35 days or more, wherein said medium contains basic fibroblast growth factor and (a) a GSK3e inhibitor at a concentration that demonstrates an effect equivalent to that demonstrated by CHIR99021 in concentrations of 2 μM or higher and lower than 5 μM, where the effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor. 20. A method for obtaining glial cells, including the stages: (i) cultivation using a suspension culture of neural crest cells for - 19 044339 a period of 35 days or more in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, where the said medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor; and (ii) differentiating the neural crest cells obtained in step (i) into glial cells. 21. A method for obtaining mesenchymal stromal cells, including the stages: (i) culturing using a suspension culture of neural crest cells for a period of 35 days or more in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, wherein said medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor; and (ii) differentiating the neural crest cells obtained in step (i) into mesenchymal stromal cells. 22. A method for producing bone cells, including the stages: (i) culturing using a suspension culture of neural crest cells for a period of 35 days or more in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, wherein said medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor; and (ii) differentiating the neural crest cells obtained in step (i) into bone cells. 23. A method for producing chondrocytes, including the stages: (i) culturing using a suspension culture of neural crest cells for a period of 35 days or more in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, wherein said medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor; and (ii) differentiating the neural crest cells obtained in step (i) into chondrocytes. 24. A method for obtaining corneal cells, including the stages: (i) culturing using a suspension culture of neural crest cells for a period of 35 days or more in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, wherein said medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor; and (ii) differentiating the neural crest cells obtained in step (i) into corneal cells. 25. A method for producing pigment cells, including the stages: (i) culturing using a suspension culture of neural crest cells for a period of 35 days or more in a medium containing a GSK3e inhibitor and basic fibroblast growth factor, wherein said medium contains: (a) a GSK3e inhibitor at a concentration that exhibits an effect equivalent to that exhibited by CHIR99021 at a concentration of 2 μM or higher and lower than 5 μM, wherein said effect is assessed based on GSK3e inhibitory activity; or (b) CHIR99021 at a concentration of 2 μM or higher and below 5 μM as a GSK3e inhibitor; or (c) CP21R7 at a concentration of 0.5 μM or higher and 1 μM or lower as a GSK3e inhibitor; and (ii) differentiating the neural crest cells obtained in step (i) into pigment cells. -