JP2012075380A - Method for producing nerve cell and nerve cell differentiation promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a nerve cell having excellent safety including the rapid differentiation of a stem cell into a nerve cell using a medicinal agent.SOLUTION: There is disclosed a method for producing a nerve cell, wherein the differentiation of an embryoid into a nerve cell is promoted by bringing the embryoid into contact with a sulfation inhibitor and inhibiting the sulfation of sugar chain in the embryoid.

Description

  The present invention relates to a method for producing nerve cells from embryoid bodies. The present invention also relates to a nerve cell differentiation promoting agent that can be used in the production method.

  Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) can be subcultured over a long period of time with the ability to differentiate into various cells existing in the living body (differentiation pluripotency). It has the property of Therefore, it is expected to use these stem cells for the treatment of leukemia, Parkinson's disease and the like for which cell transplantation is expected as an effective treatment method.

  In cell transplantation therapy using pluripotent stem cells, it is necessary to induce the differentiation of pluripotent stem cells into target cells or precursor cells thereof before transplantation. For example, in the treatment of Parkinson's disease, pluripotent stem cells are induced to differentiate into nerve cells or their progenitor cells and transplanted into patients. Until now, molecular biological techniques such as gene transfer and gene modification have been reported mainly for the induction of differentiation of pluripotent stem cells (for example, Non-Patent Document 1), but there is concern about the safety of the cells induced to differentiate. However, it has not been put into practical use. In addition, the molecular biological technique requires skilled techniques and expensive reagents, while the differentiation induction rate of pluripotent stem cells is relatively slow.

Plos ONE December 2009 Vol. 4 Issue 12 e8262

If the present inventors can induce differentiation of pluripotent stem cells using a drug without using conventional gene transfer and gene modification techniques, differentiation can be induced more easily and inexpensively. We thought it was possible to create highly practical cells with excellent safety.
An object of the present invention is to provide a method for producing a nerve cell excellent in safety, including rapidly differentiating pluripotent stem cells into nerve cells using a drug.
Another object of the present invention is to provide a drug capable of rapidly inducing differentiation of pluripotent stem cells into nerve cells excellent in safety.

  The present inventors have conducted intensive studies in view of the above problems. As a result, when an embryoid body (EB) derived from a pluripotent stem cell such as an ES cell or iPS cell is brought into contact with a sulfation inhibitor, the embryoid body is rapidly induced to differentiate into a neuron. I found what I could do. The present invention has been made based on this finding.

The object of the present invention has been achieved by the following means.
[1] A nerve cell comprising bringing an embryoid body into contact with a sulfation inhibitor and suppressing sulfation of a sugar chain in the embryoid body to promote differentiation of the embryoid body into a nerve cell. Manufacturing method,
[2] The method according to [1], wherein the sulfation inhibitor is chlorate.
[3] The method according to [1] or [2], wherein the embryoid body is contacted with the sulfation inhibitor by culturing the embryoid body in a medium containing the sulfation inhibitor.
[4] The method according to any one of [1] to [3], further comprising a step of bringing the embryoid body into contact with retinoic acid.
[5] The method according to any one of [1] to [4], wherein the embryoid body is derived from a mammalian ES cell or a mammalian induced pluripotent stem cell.
[6] A neuronal cell differentiation promoter containing a sulfation inhibitor as an active ingredient,
[7] The nerve cell differentiation promoter according to [6], wherein the sulfation inhibitor is chlorate,
[8] The nerve cell differentiation promoting agent according to [6] or [7], further containing retinoic acid.

  According to the production method of the present invention, neurons can be easily and rapidly obtained from embryoid bodies derived from ES cells or iPS cells. Moreover, the nerve cell differentiation promoting agent of the present invention contains a sulfation inhibitor as an active ingredient, and does not have the property of being incorporated into cells and replicated with cell division like genes. Therefore, the nerve cell differentiation promoting agent can be easily removed from the cells, and a highly safe nerve cell can be provided.

It is a figure which shows the result of having analyzed the change of the sulfation degree of HS and CS in EB derived from mESC 1 day after contact with sodium chlorate (3 days after EB formation) by FACS. In FIG. 1, a shows the FACS analysis result of EB not contacted with sodium chlorate, and b shows the FACS analysis result of EB contacted with sodium chlorate. Plots other than a and b are results when using IgM isotype control as the primary antibody. In EB from mESCs, is a graph showing the results, a marker gene of mesodermal differentiation T and Goosecoid, and the amount of mRNA, a marker of ectodermal differentiation Pax6 and Mash1 was quantified by real-time PCR. The vertical axis represents the relative amount of β-actin mRNA, and the unit is [1000 / β-actin mRNA amount]. Numbers 1 to 6 on the horizontal axis are as follows: 1: EB after 0 days from EB formation, 2: EB after 2 days from EB formation, 3: EB after 3 days from EB formation without contact with sodium chlorate, 4: EB 3 days after EB formation in contact with sodium chlorate, 5: EB 4 days after EB formation not in contact with sodium chlorate, 6: EB 4 days after EB formation in contact with sodium chlorate, Indicates. In EB from mESC after 3 days of contact with the sodium chlorate (5 days after EB formation), a marker gene for neuronal differentiation Nestin, Musashi-1, Mash1, Math1, NeuroD1 and NeuroD2 mRNA quantity real time PCR of It is a graph which shows the result quantified by. A vertical axis | shaft shows a relative value when the mRNA amount of each gene in EB which is not made to contact any of sodium chlorate and retinoic acid is set to 1. Three bars showing the mRNA amount of each gene in the graph are, in order from the left, EB not contacted with either sodium chlorate or retinoic acid, EB contacted with only retinoic acid, sodium chlorate and retinoic acid EB in contact with both. In hiPSC-derived EB 3 days after contact with sodium chlorate (5 days after EB formation), Nestin , Musashi-1 , Sox1 and NCAM1 which are marker genes for neuronal differentiation, and Oct3 / which is a marker for undifferentiated cells It is a graph which shows the result of having quantified the amount of 4 mRNA by real-time PCR. The vertical axis represents relative values when the mRNA amount of each gene on day 0 after EB formation is taken as 1. The four bars showing the amount of mRNA of each gene in the graph are, in order from the left, EB on the 0th day after EB formation, EB after 5 days from EB formation not contacted with either sodium chlorate or retinoic acid, EB after 5 days from EB formation contacted with only retinoic acid, and EB after 5 days from EB formation contacted with both sodium chlorate and retinoic acid. It is a microscope picture which shows the result of having carried out the immuno-staining using the anti- βIII-tubulin antibody in EB derived from mESC 3 days after contact with sodium chlorate (5 days after EB formation). In FIG. 5, a is an EB that is not in contact with either sodium chlorate or retinoic acid, b is an EB that is in contact with only retinoic acid, and c is an EB that is in contact with both sodium chlorate and retinoic acid. Show. The white fibrous material in the photograph is βIII-tubulin. It is a microscope picture which shows the result of having carried out the immuno-staining using the anti-beta III-tubulin antibody in EB derived from hiPSC 3 days after contact with sodium chlorate (5 days after EB formation). In FIG. 6, a is an EB that is not in contact with either sodium chlorate or retinoic acid, b is an EB that is in contact with only retinoic acid, and c is an EB that is in contact with both sodium chlorate and retinoic acid. Show. The white fibrous material in the photograph is βIII-tubulin. It is a figure which shows the result of having performed the immunoblot using the anti-betaIII-tubulin antibody in EB derived from mESC 3 days after contact with sodium chlorate (5 days after EB formation). The vertical axis shows the relative amount of βIII-tubulin relative to β-actin (βIII-tubulin amount / β-actin amount). Numbers 1 to 3 on the horizontal axis are 1: EB not in contact with either sodium chlorate or retinoic acid, 2: EB in contact with only retinoic acid, 3: both sodium chlorate and retinoic acid EB brought into contact is shown. It is a figure which shows the result of having performed the immunoblot using the anti-beta III-tubulin antibody in EB derived from hiPSC 3 days after contact with sodium chlorate (5 days after EB formation). The vertical axis shows the relative amount of βIII-tubulin relative to β-actin (βIII-tubulin amount / β-actin amount). Numbers 1 to 3 on the horizontal axis are 1: EB not in contact with either sodium chlorate or retinoic acid, 2: EB in contact with only retinoic acid, 3: both sodium chlorate and retinoic acid The result of contacted EB is shown. The amount of β-catenin and p-Smad1 protein for mESC-derived EBs 1 day after contact with sodium chlorate (3 days after EB formation) and 3 days after contact with sodium chlorate (5 days after EB formation) It is a figure which shows the result analyzed by the immunoblot. The vertical axis of the graph represents the relative amount of β-catenin relative to lamin B1 and the relative amount of p-Smad1 relative to β-actin. Numbers 1 to 5 on the horizontal axis are: 1: EB after 3 days from EB formation not in contact with either sodium chlorate or retinoic acid, 2: EB after 3 days from EB formation in contact with sodium chlorate, 3: EB after 5 days from EB formation not contacted with either sodium chlorate or retinoic acid, 4: EB after 5 days after EB formation contacted with retinoic acid alone, 5: sodium chlorate and retinoic acid The result of EB 5 days after EB formation which contacted both is shown.

Hereinafter, the present invention will be described in detail based on preferred embodiments thereof.
[Production method of the present invention]
The embryoid body used in the present invention is a spherical cell cluster having a structure resembling an early embryo that can be formed when pluripotent stem cells are cultured in suspension. There are no particular limitations on the type of pluripotent stem cells that form the embryoid body used in the present invention, but ES cells or iPS cells are preferred. The pluripotent stem cells are not particularly limited as long as they are derived from tridermal animals. For example, pluripotent stem cells derived from mammals, birds and the like can be used, but pluripotent stem cells derived from mammals can be preferably used. Examples of the mammal include humans, monkeys, cows, goats, sheep, pigs, horses, rabbits, rats, mice, guinea pigs and the like.
The pluripotent stem cell can be prepared by a usual method. It can also be obtained by selling cell lines deposited with depositary institutions.

The embryoid body used in the present invention can be prepared by a usual method. For example, mouse ES cells can be obtained by suspension culture in an ESC medium or the like without leukemia inhibitory factor (LIF) in a culture dish with low cell adsorption ability. Moreover, if it is a human iPS cell, it can obtain by carrying out suspension culture in the iPSellon culture medium etc. without bFGF with a culture dish with low cell adsorption ability. These specific culturing methods will be described in detail in Examples below.
Normal conditions such as the temperature and CO ₂ concentration for forming an embryoid body can be adopted. For example, an embryoid body can be formed from pluripotent stem cells under conditions of 37 ° C. and 5% CO _2. Can do.
The method for preparing the embryoid body used in the present invention is not particularly limited, and a method prepared by any method may be used as long as it can form an embryoid body.

The sulfation inhibitor used in the present invention is a substance that can be taken into cells and suppress at least sulfation of sugar chains in the cells. In the present invention, “suppressing sulfation of sugar chains” is a concept including not only completely inhibiting sulfation of sugar chains but also partially inhibiting sulfation of sugar chains. “Partially inhibit sulfation of a sugar chain” means that a sulfate group is bound to a sugar chain, but the number of sulfate groups is less than usual. The number of sulfate groups of sulfated glycans such as heparan sulfate (HS) and chondroitin sulfate (CS) present in cell membranes and cells can be reduced by the sulfation inhibitor used in the present invention.
The sulfation inhibitor used in the present invention is not particularly limited as long as it has an action of inhibiting sulfation of sugar chains in cells, but chlorate can be preferably used. There is no restriction | limiting in particular in the kind of chlorate, For example, sodium chlorate, potassium chlorate, lithium chlorate, ammonium chlorate, calcium chlorate etc. are mentioned. The sulfation inhibitor used in the present invention can be obtained by synthesis by a usual method, or a commercially available product may be used.

In the present invention, the method for bringing the embryoid body into contact with the sulfation inhibitor is not particularly limited. By culturing an embryoid body in a medium containing a sulfation inhibitor, the embryoid body and the sulfation inhibitor can be easily brought into contact with each other. The medium is preferably a liquid medium. There are no particular limitations on the type of medium as long as the embryoid body can survive. For example, in the case of an embryoid body derived from mouse ES cells, the above-mentioned ESC medium without LIF or the like is used as an embryoid body derived from human iPS cells. If it is a body, the above-mentioned iPSellon medium without bFGF can be suitably used.
From the viewpoint of promptly promoting differentiation induction, the initiation time of the contact between the embryoid body and the sulfation inhibitor is preferably 0 to 5 days after the formation of the embryoid body, and 0 to 4 days later. More preferably, it is more preferably after 0 to 3 days, further preferably after 1 to 2 days, and particularly preferably after 2 days.
There is no particular limitation on the contact time between the embryoid body and the sulfation inhibitor, but it is preferable to contact the embryoid body for at least 1 day in order to incorporate the sulfation inhibitor into the cell and exert physiological action. More preferably, the contact is performed for 1 day, more preferably 1 to 4 days, more preferably 1 to 3 days, and particularly preferably 2 to 3 days.
The amount of the sulfation inhibitor that is brought into contact with the embryoid body is not particularly limited, but when the embryoid body is cultured in a medium containing the sulfation inhibitor, the concentration of the sulfation inhibitor in the medium is 0.1. It is preferably ˜500 mM, more preferably 1 to 500 mM, further preferably 1 to 300 mM, further preferably 1 to 200 mM, and particularly preferably 10 to 100 mM.

In order to efficiently induce differentiation of the embryoid body into nerve cells, it is preferable to further contact the embryoid body with retinoic acid. Retinoic acid is a substance conventionally used for inducing differentiation of pluripotent stem cells into neurons. Although there is no restriction | limiting in particular in the method to which an embryoid body and a retinoic acid are made to contact, An embryoid body and a retinoic acid can be made to contact easily by culturing an embryoid body in the medium containing a retinoic acid. The medium is preferably a liquid medium. There are no particular limitations on the type of medium as long as the embryoid body can survive. For example, in the case of an embryoid body derived from mouse ES cells, the above-mentioned ESC medium without LIF or the like is used as an embryoid body derived from human iPS cells. If it is a body, the above-mentioned iPSellon medium without bFGF can be suitably used. The onset of contact between the embryoid body and retinoic acid may be before or after initiating contact between the embryoid body and the sulfation inhibitor. Simultaneously with the initiation of contact, contact between the embryoid body and retinoic acid may be initiated. The start time of the contact between the embryoid body and retinoic acid is preferably 0 to 5 days after the formation of the embryoid body, more preferably 1 to 5 days, and further preferably 1 to 4 days later. Preferably, after 2 to 4 days, more preferably after 3 to 4 days.
There is no particular limitation on the contact time between the embryoid body and retinoic acid, but it is preferably contacted for at least 5 hours and preferably contacted for 5 to 72 hours in order for retinoic acid to be taken into cells and exert physiological functions. Is more preferable, contact for 12 to 48 hours is further preferable, and contact for 20 to 30 hours is particularly preferable.
The amount of retinoic acid to be brought into contact with the embryoid body is not particularly limited, but when the embryoid body is cultured in a medium containing retinoic acid, the concentration of retinoic acid in the medium may be 0.1 to 10 μM. Preferably, it is 0.5-5 μM, more preferably 0.5-2 μM.
For retinoic acid, a trans form (all-trans) is usually used to exert physiological functions.

The embryoid body may continue to induce differentiation into nerve cells in the presence of a sulfation inhibitor and / or retinoic acid. Usually, the preferred contact time sulfation inhibitor and / or retinoic acid described above is used. After the contact, the contact with the sulfation inhibitor substance and / or retinoic acid is terminated.
In order to terminate the contact between the embryoid body and the sulfation inhibitor and / or the embryoid body and retinoic acid, the sulfation inhibitor and retinoic acid may be removed from the culture environment of the embryoid body. For example, the embryoid body may be transferred to a medium containing no sulfation inhibitor or retinoic acid and cultured to terminate the contact between the embryoid body and the sulfation inhibitor and / or the embryoid body and retinoic acid. it can.
Even after the contact with the sulfation inhibitor and retinoic acid is terminated, the embryoid body is differentiated into nerve cells by culturing under an appropriate culture condition. Although there is no restriction | limiting in particular in the culture medium used for the said culture | cultivation, For example, DMEM-F12 etc. which contain N2 supplement are mentioned. Moreover, it is preferable to change a culture medium regularly.

Ordinary conditions can be employed for the temperature at which the embryoid body is cultured, the CO ₂ concentration, and the like. For example, the embryoid body can be cultured under conditions of 37 ° C. and 5% CO ₂ .

It is known that signal transduction via Wnt, BMP, FGF, etc. is involved in the differentiation of embryoid bodies. Wnt and BMP inhibit differentiation into nerve cells and induce differentiation into mesoderm. Prompting has also been suggested (see, for example, Plos ONE December 2009 2009 Vol. 4 Issue 12 e8262). It has also been reported that sulfated glycans present in cell membranes can be involved in the above signal transduction.
The sulfation inhibitor used in the present invention has an action of inhibiting the sulfation of sugar chains in cells. Therefore, the number of sulfate groups of sulfated glycans existing in cell membranes and / or cells such as heparan sulfate and chondroitin sulfate is decreased, thereby inhibiting Wnt and BMP signaling and differentiation of embryoid bodies into neurons. It is thought to promote.
Until now, there has been a report that differentiation of embryoid bodies into nerve cells has been induced by suppressing the action of specific genes involved in sulfation of sugar chains (Plos ONE December 2009 Vol. 4 Issue 12). e8262), there is no known example in which differentiation of embryoid bodies into nerve cells was successful only by contacting the embryoid body with a sulfation inhibitor.
According to the present invention, it becomes possible to obtain a nerve cell by rapidly differentiating an embryoid body into a nerve cell by a simple method unprecedented. Moreover, the sulfation inhibitor used in the present invention can remove the sulfation inhibitor from the differentiated nerve cells by removing the sulfation inhibitor from the medium. Therefore, the nerve cells induced to differentiate by the method of the present invention are excellent in safety and expected to be put to practical use for cell transplantation therapy.
In the present invention, the “nerve cell” includes both a nerve cell and a precursor cell of the nerve cell.

[Neuronal differentiation promoting agent of the present invention]
The nerve cell differentiation promoting agent of the present invention is a drug for promoting differentiation of embryoid bodies into nerve cells, and contains the above-mentioned sulfation inhibitor as an active ingredient. The nerve cell differentiation promoting agent of the present invention can be used as a supply source of a sulfation inhibitor in the production method of the present invention described above. That is, the embryonic somatic cells can be rapidly differentiated into neural cells by contacting the neuronal differentiation promoting agent of the present invention with the embryoid bodies obtained by suspension culture of pluripotent stem cells. Can do.

  The nerve cell differentiation promoting agent of the present invention may contain retinoic acid in addition to the sulfation inhibitor. Retinoic acid is usually an all-trans form in order to exert physiological functions. Moreover, the nerve cell differentiation promoting agent of the present invention may further contain other components such as nutrients, pH adjusters, preservatives, stabilizers, and emulsifiers.

  There is no restriction | limiting in particular in the dosage form of the nerve cell differentiation promoter of this invention, The liquid which each component melt | dissolved or disperse | distributed may be sufficient, and a powder form may be sufficient. Moreover, the shape of tablets, such as a tablet, may be sufficient.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these, unless the summary is exceeded.

[Maintain cell line]
As mouse ES cells (mESC), the R1 cell line (described in Proc. Natl. Acad. Sci. USA 90 (1993) 8424-8428, provided by the National Institute of Physiological Sciences, Institute for Physiological Sciences, Molecular Neurophysiology Research Division) and the E14TG2a cell line (Dev. Biol. 121 (1987) 1-9, provided by the University of Tokyo Graduate School of Science). Each cell was treated with ESC medium (15% by mass FBS (manufactured by Hyclone)), 1% by mass penicillin / streptomycin (manufactured by Gibco), 0.1 mM at a concentration of 1000 U / mL. Of mouse fibroblasts inactivated with 10 μg / mL mitomycin C (Sigma) in 2-Mercaptoethanol (Gibco) and 0.1 mM non-essential amino acid (Gibco) in DMEM). Maintained on (MEFs).

  Further, as human iPS cells (hiPSC), MRC-hiPS_Fetch (cell number: NIHS0604) and MRC-hiPS_Tic (cell number: JCRB1331) (both described in Exp. Cell Res. 315 (2009) 2727-2740, National Growth Medicine) Obtained from the center). On each MEFs inactivated with 10 μg / mL mitomycin C (manufactured by Sigma) in iPSellon (manufactured by Cell-Sight) with bFGF (manufactured by Wako Pure Chemical Industries) at a concentration of 10 ng / mL. Maintained at.

[Formation of embryoid body]
The above mESC and hiPSC were transferred to Low Cell Binding 60 mm dishes (manufactured by Nunc), and mESC was cultured in the above ESC medium without LIF, and hiPSC was suspended in iPSellon without bFGF to give embryoid bodies ( EB) was formed. The hiPSC was transferred to a gelatin-coated dish before EB formation to remove feeder cells.
In the present invention, the time point when the above suspension culture is started is defined as the time of EB formation. Therefore, for example, “one day after EB formation” means one day after the start of the above suspension culture for pluripotent stem cells.

[Induction of differentiation of embryoid bodies into neurons]
Two days after EB formation, sodium chlorate (manufactured by Sigma) was added as a sulfation inhibitor so that the final concentration in the medium was 50 mM. Further, 2 days later (4 days after EB formation), all-trans-retinoic acid (hereinafter sometimes referred to as RA; Sigma) was added so that the final concentration in the medium was 1 μM. One day after the addition of RA (5 days after EB formation), EB was transferred to PDL / laminin-coated 60 mm dishes (Becton Dickinson) and cultured in DMEM-F12 containing N2 supplement (Gibco). did. By this operation, sulfation inhibitor and RA were removed from the culture solution.

[FACS analysis]
In EB derived from mESC R1 cell line, anti-HS antibody or anti-CS antibody (both mouse-derived IgM) was used as a primary antibody in order to examine the change in the degree of glycan sulfation by addition of a sulfation inhibitor. FACS analysis was performed. 10E4 or HepSS-1 (both trade names, manufactured by Seikagaku Corporation) was used as the anti-HS antibody, and 2H6 (trade name, manufactured by Seikagaku Corporation) was used as the anti-CS antibody. In addition, a case using mouse IgM isotype (Chemicon) instead of the above antibody was used as a control. The specific experimental method is as follows.
After treatment of the cell mass one day after contact with the sulfation inhibitor (3 days after EB formation) with EDTA, this cell suspension was treated with FACS buffer (0.5% by weight bovine serum albumin (BSA) and Incubated in the primary antibody solution diluted with PBS containing 0.1% by mass sodium azide). After washing, the cell suspension was incubated in a FITC-labeled anti-mouse IgM antibody (Sigma) solution diluted with the FACS buffer. After washing, FACS analysis was performed using a FACSAria Cell Sorter (Becton Dickinson).
The results are shown in FIG.

  HS and CS are sulfated glycans that play an important role in cell differentiation, and mESC is known to be expressed until at least 8 days after EB formation. From the results shown in FIG. 1, EB (a in FIG. 1) in which a sulfation inhibitor was added to the medium was compared with EB in which sulfation inhibitor was not added (b in FIG. 1). It can be seen that the amount of CS antibody bound is small (the fluorescence intensity is small), and the amount of sulfate groups bound to the sugar chain is decreased. The result of FIG. 1 shows that the sulfation of the sugar chain was remarkably suppressed in only one day after the contact between the EB and the sulfation inhibitor.

[Real-time PCR analysis]
In EBs derived from the R1 cell line, which is an mESC, it was examined by using the expression level of mRNA of a specific gene as an index that a sulfation inhibitor induces differentiation of EBs into ectoderm-like (nerve cells). Specifically, T and Goosecoid markers of mesoderm differentiation, and for each gene of a marker of ectodermal differentiation Mash1 and Pax6, the expression level of their mRNA ABI PRISM (registered trademark) and 7700 sequence detection system Quantified by the real-time PCR used. J. et al. Biol. Chem. 283 (2008), page 3597, right column, lines 3 to 15 were used for quantification using primer sets and probes having the following base sequences.
Primer set Probe
T : SEQ ID NO: 1 and SEQ ID NO: 2 SEQ ID NO: 31
Googlesecid : SEQ ID NO: 3 and SEQ ID NO: 4 SEQ ID NO: 32
Mash1 : SEQ ID NO: 5 and SEQ ID NO: 6 SEQ ID NO: 33
Pax6 : SEQ ID NO: 7 and SEQ ID NO: 8 SEQ ID NO: 34
β-actin: SEQ ID NO: 19 and SEQ ID NO: 20 SEQ ID NO: 40
The probe was purchased from Applied Biosystems with the 5 ′ end labeled with 3FAM of the reporter dye and the 3 ′ end labeled with the quencher dye TAMRA.
The results are shown in FIG.

From the results shown in FIG. 2, the EBs brought into contact with the sulfation inhibitor were suppressed in the levels of T and Goseoside , which are mesoderm markers, compared to the EB not in contact with the sulfation inhibitor. mRNA levels of a marker Mash1 and Pax6 are seen to be increased significantly (in FIG. 2, compare the number 3 on the horizontal axis and compared with 4, and No. 5 and 6). The change in the mRNA level of each gene occurs only 1 day after contact between EB and the sulfation inhibitor (comparison between numbers 3 and 4 on the horizontal axis), and the differentiation-inducing action of the sulfation inhibitor is extremely rapid. It is shown that.

Similarly, in the EB derived from the R1 cell line, which is mESC, the fact that the sulfation inhibitor induces the differentiation of EBs into neurons is confirmed after 3 days from contact with the sulfation inhibitor (5 days after EB formation). ). Said Specifically, Nestin, a marker of neuronal differentiation, the Musashi, Mashl, Math1, NeuroD1 and each gene NeuroD2, the expression level of their mRNA, using the primer set and probe of the following nucleotide sequence In the same manner as described above, real-time PCR was used.
Primer set Probe
Nestin : SEQ ID NO: 9 and SEQ ID NO: 10 SEQ ID NO: 35
Musashi-1 : SEQ ID NO: 11 and SEQ ID NO: 12 SEQ ID NO: 36
Mash1 : SEQ ID NO: 5 and SEQ ID NO: 6 SEQ ID NO: 33
Math1 : SEQ ID NO: 13 and SEQ ID NO: 14 SEQ ID NO: 37
NeuroD1 : SEQ ID NO: 15 and SEQ ID NO: 16 SEQ ID NO: 38
NeuroD2 : SEQ ID NO: 17 and SEQ ID NO: 18 SEQ ID NO: 39
The probe was purchased from Applied Biosystems with the 5 ′ end labeled with 3FAM of the reporter dye and the 3 ′ end labeled with the quencher dye TAMRA.
The results are shown in FIG.

  From the results shown in FIG. 3, the EB brought into contact with the sulfation inhibitor was EB 3 days after the addition of the sulfation inhibitor (5 days after EB formation) compared to the EB not in contact with the sulfation inhibitor. , It was found that the mRNA level of the marker gene for neuronal differentiation was significantly increased.

Subsequently, for the EBs derived from Fetch and Tic, which are hiPSCs , Nestin and Musashi , which are markers of neuronal differentiation, are used for cells 3 days after contact with a sulfation inhibitor (5 days after EB formation). -1 , NCAM1 and Sox1 and the mRNA level of each gene of Oct3 / 4 which is a marker of non-differentiated cells were examined by real-time PCR. In addition, for the real-time PCR analysis of hiPSC-derived EB, quantitative analysis using FastStart Universal SYBR GreenMaster (manufactured by Roche) was performed without using a probe. The base sequence of the primer set used is as follows.
Primer set
Nestin : SEQ ID NO: 21 and SEQ ID NO: 22
Musashi-1 : SEQ ID NO: 23 and SEQ ID NO: 24
Sox1 : SEQ ID NO: 25 and SEQ ID NO: 26
NCAM1 : SEQ ID NO: 27 and SEQ ID NO: 28
Oct3 / 4 : SEQ ID NO: 29 and SEQ ID NO: 30
The results are shown in FIG.

  From the results shown in FIG. 4, it is found that the expression level of the marker gene for neuronal differentiation is significantly increased in the EB brought into contact with the sulfation inhibitor as compared with the EB not in contact with the sulfation inhibitor. It was. That is, it can be seen that the sulfation-inhibiting substance shows excellent nerve cell differentiation-inducing ability even for hiPSC-derived EB.

[Immunostaining analysis]
As described above, EB contacted with a sulfation inhibitor was transferred to a PLL / laminin-coated glass chamber slide (Iwaki) 3 days after contact with the sulfation inhibitor (5 days after EB formation). The cells after 2 days were analyzed for differentiation into neurons by immunostaining. Specifically, the cells on the glass chamber slide were fixed with 4% paraformaldehyde and permeable with 0.1% saponin. This was washed and subjected to blocking treatment, and reacted with an anti-βIII-tubulin antibody (mouse IgG, manufactured by Chemicon). After washing, FITC-labeled anti-mouse IgG antibody (Chemicon) was reacted, followed by counterstaining with propidium iodide (PI). Immunofluorescence images were obtained using LSM5 Pascal confocal laser scanning microscope (Carl Zeiss). FIG. 5 shows the results of cells differentiated from EBs derived from mESC cells (R1 cell line), and FIG. 6 shows the results of cells differentiated from hiPSCs (Fetch and Tic).

From the results of FIGS. 5 and 6, it can be seen that the amount of βIII-tubulin, which is an indicator of neuronal differentiation, is markedly increased at the protein level in cells differentiated from EBs contacted with a sulfation inhibitor ( That is, it can be seen that EB is structurally greatly changed into nerve cells.) This structural change is observed only 2 days after transfer of EB onto a PLL / laminin-coated glass chamber slide (manufactured by Iwaki) (7 days after EB formation). It can be said that the structural change of EBs to nerve cells by contacting them is extremely rapid.
In addition, Plos ONE December 2009 Vol. 4 Issue 12 e8262 describes that an embryoid body was differentiated into a neuron by knocking out a gene involved in sulfation. According to this, even in the cells 14 days after EB formation, expression of βIII-tubulin in an amount comparable to or less than the amount shown in FIGS. 5 and 6 can only be confirmed.

[Immunoblot analysis]
In order to more quantitatively measure the increase in the expression level of βIII-tubulin confirmed by the immunostaining analysis, immunoblotting was performed. Cells transferred on a glass chamber slide (manufactured by Iwaki) coated with PLL / laminin as described above and lapsed 2 days were dissolved in lysis buffer (150 mM NaCl, 1% Triton X-100, 1 mM N ₃ VO ₄ , 10 mM NaF, protease inhibitor). It was dissolved in 50 mM Tris-HCl pH 7.4) containing (Sigma). The cell lysate was subjected to 10% SDS-PAGE and then transferred to a PVDF membrane (Millipore). After blocking this membrane, the above-mentioned anti-βIII-tubulin antibody (mouse IgG, manufactured by Chemicon) was reacted as a primary antibody, and a peroxidase-labeled anti-mouse IgG antibody (manufactured by Cell signaling) was reacted as a secondary antibody. After washing, the peroxidase on the membrane was developed using ECL Plus reagent (GE Healthcare). An anti-β-actin antibody (manufactured by Sigma) was used as a control antibody for analyzing changes in the expression level of βIII-tubulin. The results are shown in FIGS.

  From the results shown in FIG. 7 and FIG. 8, the amount of βIII-tubulin in EBs contacted with a sulfation inhibitor was about 10 times that derived from mESC, and the amount of βIII-tubulin in hiPSC-derived EBs. Was found to rise more than twice. The graphs in FIGS. 7 and 8 are the results of measuring the band intensity of an immunoblot with NIHimage.

Subsequently, the expression levels of proteins related to Wnt signal and BMP signal, which have been reported to be involved in mesoderm differentiation of cells, were also analyzed by immunoblotting.
A cell lysate was prepared by lysing R1-derived cells 1 day after contact with the sulfation inhibitor (3 days after EB formation) and 3 days (5 days after EB formation) with the lysis buffer. In addition, J.H. Biol. Chem. 283 (2008), page 3596, right column, lines 3 to 18 were used to prepare a nuclear extract. The cell lysate is used to analyze the expression level of phosphorylated Smad1 (p-Smad1) acting downstream of the BMP signaling pathway, and the nuclear extract promotes the expression of the target gene in the nucleus in the Wint signaling pathway It used for the analysis of the expression level of (beta) -catenin which plays a role.
The cell lysate or nuclear extract is transferred to a PVDF membrane (Millipore) after 10% SDS-PAGE. After blocking this membrane, the primary antibody, anti-p-Smad1 antibody (Ser463 / 465, rabbit IgG, cell) Signaling Technology) or anti-β-catenin antibody (rabbit IgG, manufactured by Cell Signaling Technology) was reacted, followed by reaction with a secondary antibody peroxidase-labeled anti-rabbit IgG antibody (Cell Signaling). Incidentally, the anti-β-actin antibody (mouse IgG, Sigma) for cell lysates with anti-lamin _{B 1} antibody (mouse IgG, manufactured by Zymed Corp.) for nuclear extracts, respectively p-Smad1 and β-catenin expression It was used as a control antibody for analyzing changes in the amount (in this case, a peroxidase-labeled anti-mouse IgG antibody was used as a secondary antibody). After washing, the peroxidase on the membrane was developed using ECL Plus reagent (GE Healthcare). The results are shown in FIG.

  From the results of FIG. 9, it can be seen that the expression levels of both β-catenin and p-Smad1 are reduced by contacting EB with a sulfation inhibitor. This result suggests that the sulfation inhibitor inhibits Wint signaling and BMP signaling that lead EBs to mesoderm differentiation, and as a result, the differentiation of EBs into neurons is promoted. is there.

  Of the above experimental results, it was confirmed that the experimental results using the mESC R1 cell line were also reproduced by another mESC cell line, E14TG2a.

Claims (8)

  A method for producing a nerve cell, comprising bringing an embryoid body into contact with a sulfation inhibitor and suppressing the sulfation of a sugar chain in the embryoid body to promote differentiation of the embryoid body into a nerve cell. .   The manufacturing method of Claim 1 whose sulfation inhibitory substance is a chlorate.   The production method according to claim 1 or 2, wherein the embryoid body is contacted with the sulfation inhibitor by culturing the embryoid body in a medium containing a sulfation inhibitor.   Furthermore, the manufacturing method of any one of Claims 1-3 including the process which an embryoid body and a retinoic acid are made to contact.   The production method according to any one of claims 1 to 4, wherein the embryoid body is derived from a mammalian ES cell or a mammalian induced pluripotent stem cell.   A neuronal differentiation promoter containing a sulfation inhibitor as an active ingredient.   The nerve cell differentiation promoting agent according to claim 6, wherein the sulfation inhibitor is chlorate.   The nerve cell differentiation promoting agent according to claim 6 or 7, further comprising retinoic acid.