JP2011152111A - Culture medium for culturing pluripotent stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a serum-free culture medium and free from mammal-derived components, and allowing culturing and proliferation of pluripotent stem cells while maintaining an undifferentiating property and pluripotency of the pluripotent stem cells. <P>SOLUTION: The culture medium for culturing the pluripotent stem cells includes sericin as a serum substitute. The culture medium for culturing the pluripotent stem cells further includes a differentiation repressor. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a culture medium for pluripotent stem cell culture. More specifically, the present invention relates to a medium that does not contain a component derived from a mammal and enables culture of pluripotent stem cells.

  A pluripotent stem cell is a self-replicating stem cell capable of differentiating into differentiated cells belonging to at least one type of three germ layers (ectoderm, mesoderm, endoderm) (multipotent, also called pluripotent) For example, embryonic germ cell (Embryonic Germ Cell: EG cell), embryonic cancer cell (Embryonal Carcinoma Cell: EC cell), embryonic stem cell (Embryonic Stem Cell: ES cell), induced pluripotent stem cell (Induced Pluripotent Stem Cell: iPS cell), adult pluripotent Stem Cell (APS cell) and the like.

  Among these, ES cells are established by culturing cells derived from the inner cell mass of blastocysts under special conditions. ES cells have the ability to differentiate into all three germ layers, including germ cells, and can continue to renew themselves by culturing under special conditions. In addition, chimeric animals can be created by injecting ES cells into embryos at the blastocyst stage or morula stage. In mice, mice that have a specific gene disrupted by mating chimeric mice (knockout mice) ) Has been established. In addition, ES cells can be differentiated into specific cells by inducing differentiation in vitro. Therefore, in the future, ES cells will be a source of cells in regenerative medicine that regenerates lost functions. Expected.

  In recent years, a technique for artificially introducing genes into adult-derived cells to produce induced pluripotent stem cells (iPS cells) having properties similar to those of ES cells has been invented (Patent No. 4183742 (Patent No. 4183742)). Literature 1)). iPS cells, like ES cells, have the ability to differentiate into all three germ layers including germ cells, and chimeric mice can be created from mouse iPS cells. iPS cells differ from ES cells in several respects, such as the cells from which they are derived, the method of establishment and the potential for canceration, and the significance of regenerative medicine. However, maintenance culture methods, differentiation induction methods, chimera formation abilities, etc. The nature of the cells involved is considered to be equivalent to ES cells in many ways.

  A technique for culturing ES cells while maintaining “undifferentiated” and “pluripotency” (hereinafter sometimes referred to as “maintenance culture”) has already been established. That is, in order to maintain undifferentiation and pluripotency, ES cells are usually cultured in a medium in which (1) basal medium is added (2) serum and (3) differentiation inhibitory factor, and further if necessary. (4) Use feeder cells (supporting cells). In addition, L-glutamine, a non-essential amino acid for assisting growth, β-mercaptoethanol and the like as a reducing agent are added as necessary.

Of the above,
(1) The basal medium is a minimum medium containing inorganic salts, saccharides, amino acids, vitamins, and the like, and is used with additives as necessary.
(2) As the serum, fetal bovine serum (FBS) or the like is usually used to supply a cell growth factor. Serum is thought to have a function to suppress differentiation and promote establishment of ES cells in addition to proliferation, and in fact, ES cells may lose their pluripotency if serum is removed from the medium. (Wiles and Johansson, Exp. Cell Research, 1999 (247) 241-248 (Non-patent Document 1)).
(3) Differentiation-inhibiting factors vary depending on animal species. For example, leukemia inhibitory factor (LIF) is used in mouse ES cells, and basic fibroblast growth factor (basic fibroblast growth factor in monkey and human ES cells). Factor: bFGF) can be used.
(4) Feeder cells are thought to provide a scaffold (matrix) for ES cells to adhere to and supply various humoral factors that suppress differentiation and promote proliferation. Mouse or human primary fibroblasts or their cell lines are used as feeder cells, and the proliferation is stopped by mitomycin C treatment or X-ray irradiation. Feeder cells can be replaced by coating the surface of the culture vessel with gelatin using an ES cell line.

  As described above, it has been conventionally known that a combination of serum and a differentiation inhibitory factor is used for culturing ES cells. However, when ES cells are used for medical purposes, animal-derived components can be an infection source or a heterologous antigen. Therefore, it is preferably removed from the culture. Among these, regarding LIF and bFGF which are differentiation inhibitory factors, recombinants not derived from animals are commercially available and can be easily obtained. On the other hand, regarding the serum, several reports have already been made that it was possible to culture while maintaining the undifferentiation and pluripotency of ES cells by using a specific serum substitute.

  For example, International Publication No. WO 98/30679 (Japanese Patent Publication No. 2001-508302) (Patent Document 2) describes albumin or albumin substitute, amino acid, vitamin, transferrin or transferrin substitute, antioxidant, insulin instead of serum. Alternatively, it is disclosed that ES cells can be established and cultured with an alternative composition containing an insulin substitute, a collagen precursor, trace elements, and the like. This composition generally used as KSR (Knockout Serum Replacement) is widely used for serum-free culture of mouse, monkey and human ES cells.

  In addition, International Publication WO2006 / 036925 (Japanese Patent Publication No. 2008-514230) (Patent Document 3) describes the growth of primate ES cells using a serum-free medium supplemented with several cytokines including bFGF and albumin. A supporting culture method is disclosed.

  In addition, the literature (Yao S, et al., Proc. Natl. Acad. Sci. USA., 2006, 103: 6907-6912. (Non-patent Document 2)) does not use serum and has a clear composition. It has been reported that human ES cells could be continuously cultured in an undifferentiated state for a long time by combining commercially available additives.

  However, in any of these, although the use of serum itself is avoided, components that can be derived from mammals such as albumin are used, and the use of components derived from mammals is completely excluded. It was hard to say. In recent years, anxiety about using ingredients derived from mammals has increased due to concerns about various infectious diseases and diseases including mad cow disease. For this reason, even in the culture of pluripotent stem cells, which are expected to be applied to various medical uses, it is desired to use a culture medium that can completely eliminate the possibility of contamination with mammalian-derived components. I can say that.

  International Publication No. WO2004 / 039965 (Patent No. 4374419) (Patent Document 4) reports that an adenylate cyclase inhibitor is used in place of serum or support cells when culturing pluripotent stem cells. ing. However, albumin is also mentioned here as a serum substitute to be used, although avoidance of the use of serum components has been studied.

  For this reason, as far as the present inventors know, no medium composition for pluripotent stem cell culture has been reported that does not contain such a mammal-derived component at all.

  In recent years, it has become clear that sericin has excellent properties such as water retention, antioxidant, enzyme stabilization, cryoprotection and cell growth promotion, and its use as a cosmetic raw material, biochemical reagent or cell culture reagent It is expanding. For example, International Publication No. WO2002 / 086133 (Patent No. 4047176) (Patent Document 5) shows that sericin promotes the growth of animal cells and reduces the use of serum or enables serum-free culture. Yes. JP-A 2006-115837 (Patent Document 6) reports that cultured cells can be cryopreserved under serum-free conditions by using sericin.

On the other hand, as described above, it has been reported that when serum is removed from the medium, ES cells actually lose their pluripotency (Non-Patent Document 1). In addition, it is considered that there is an effect of maintaining its pluripotency while suppressing differentiation. Serum is a component derived from mammals and is a mixture containing unclear factors that have not been fully elucidated. Therefore, serum has the ability to suppress differentiation of pluripotent stem cells in addition to the effect of promoting cell proliferation. Even if it has the ability to maintain pluripotency, the known serum substitute is obtained mainly by paying attention to the effect of promoting cell proliferation. It is obvious that it cannot be said that the ability to suppress the differentiation of sexual stem cells and the ability to maintain pluripotency are simultaneously maintained.
For this reason, it is clear that the serum substitute of the conventional medium in which only the cell growth promoting effect is expected cannot immediately play the role as the serum substitute in the medium for pluripotent stem cell culture.

  As described above, sericin was only reported to be added to the medium as a serum substitute in the hope of promoting cell growth. As far as the present inventors know, it has not been known so far that sericin can be added to culture pluripotent stem cells under serum-free conditions while maintaining undifferentiation and pluripotency.

Japanese Patent No. 4183742 International Publication WO 98/30679 International Publication WO2006 / 036925 International Publication No. WO2004 / 039965 International Publication No. WO2002 / 086133 JP 2006-115837 A

Wiles and Johansson, Exp. Cell Research, 1999 (247) 241-248 Yao S, Chen S, Clark J, Hao E, Beattie GM, et al., "Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions", Proc. Natl. Acad. Sci. USA. , 2006, 103: 6907-6912

  The present inventors have recently replaced the serum component with sericin derived from silk thread among the serum component and differentiation inhibitory factor essential for maintaining the undifferentiated and pluripotent (pluripotent) pluripotent stem cells. As a result, the pluripotent stem cells were successfully cultured while maintaining the undifferentiation and pluripotency of the pluripotent stem cells under the condition that no mammalian-derived component was contained in the culture medium. In the culture of pluripotent stem cells, serum was thought to be essential for maintaining the undifferentiation and pluripotency of pluripotent stem cells in addition to its proliferative effect. It was surprising that this could be achieved with traditionally well known safe ingredients. Examination of serum substitutes in culture media for pluripotent stem cells is not simply the application of serum substitutes in conventional media, and various studies are necessary, which can be easily accomplished with difficulty However, the fact that sericin was revealed for the first time as a serum substitute in the culture, and the present inventors actually conducted such studies, although many studies of ES cells have been conducted so far. It will be clear to those skilled in the art when looking at the situation that had to be done. The present invention is based on these findings.

  Accordingly, the present invention provides a serum-free culture medium that does not contain a mammal-derived component and that can culture and proliferate pluripotent stem cells while maintaining the undifferentiation and pluripotency of pluripotent stem cells. Objective.

  The culture medium for pluripotent stem cell culture according to the present invention comprises sericin as a serum substitute.

  According to a preferred embodiment of the present invention, the culture medium according to the present invention further comprises a differentiation inhibitor. More preferably, the differentiation inhibitory factor is leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), adenylate cyclase activity inhibitor, MEK1 (MAPK / ERK kinase 1) inhibitor, GSK3 (glycogen synthase kinase 3) ) Selected from the group consisting of an inhibitor, an FGF receptor inhibitor, and a basic fibroblast growth inhibitory factor (bFGF).

  According to a more preferred embodiment of the present invention, the medium according to the present invention does not contain a mammal-derived component.

  According to one preferred embodiment of the present invention, the medium according to the present invention further comprises one or more auxiliary components selected from the group consisting of insulin, transferrin, ethanolamine and sodium selenite.

  According to another preferred embodiment of the present invention, the medium according to the present invention further comprises a medium basic component.

  The method for culturing pluripotent stem cells according to the present invention comprises using the culture medium for pluripotent stem cells according to the present invention to proliferate cells while maintaining the undifferentiation and pluripotency of pluripotent stem cells. It becomes. According to a preferred embodiment of the present invention, in this method, the pluripotent stem cell is derived from a mammal.

  The method for preparing a clonal population of pluripotent stem cells according to the present invention comprises culturing undifferentiated pluripotent stem cells using the culture medium for pluripotent stem cells according to the present invention, and a clonal population of pluripotent stem cells. Comprising obtaining. According to a preferred embodiment of the present invention, in this method, the pluripotent stem cell is derived from a mammal.

  According to another aspect of the present invention, differentiation obtained by differentiating cells obtained by the method for culturing pluripotent stem cells according to the present invention or the method for preparing a clonal cell population of pluripotent stem cells according to the present invention. Cells are provided.

  According to the pluripotent stem cell culture medium of the present invention, pluripotent stem cells are cultured without using any components derived from mammals, and proliferated while maintaining their undifferentiated and pluripotency. Can do. Proliferation of pluripotent stem cells is useful for various studies and regenerative medicine, but it can be performed with a well-defined medium containing a highly safe component called sericin. It can help to relieve consumers' anxiety and provide highly safe medical care. In addition, according to the present invention, since sericin, which is a safe and stable substance, is used, it is advantageous in terms of operation and handling, and further, the cost can be reduced when cell culture and clonal cell population are prepared. Can also be expected.

The figure shows the state of ES cells cultured in the medium according to the present invention (Section (3) in Example). The figure shows the results of alkaline phosphatase (ALP) staining of cultured ES cells carried out to confirm the undifferentiated nature of the cultured ES cells ((4) (a) in Example). The figure shows the measurement results of the expression level of the undifferentiated marker gene in cultured ES cells (section (4) (b) of the examples). The figure shows the results of a differentiation induction test of cultured ES cells into bone (section (5) (i) of the examples). The figure shows the results of a differentiation induction test of cultured ES cells into myocardium (section (5) (ii) of the examples).

Detailed description of the invention

Medium for pluripotent stem cell culture The medium for pluripotent stem cell culture according to the present invention comprises sericin as a serum substitute as described above.

  A stem cell refers to a cell that has the ability to self-replicate to produce the same cell as itself and pluripotency. Stem cells have a hierarchy, and the upper undifferentiated stem cells have high self-replicating ability and high pluripotency capable of differentiating into various cell lineages. It is known that it can only differentiate into cell lineages. Cells to be mainly cultured in the medium of the present invention are cells that are relatively higher in the hierarchy, that is, undifferentiated, self-replicating stem cells (pluripotent) that sufficiently retain pluripotency. Sex stem cells).

  Examples of such pluripotent stem cells include embryonic germ cells (EG cells), embryonic cancer cells (EC cells), embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and adult pluripotent cells. Competent stem cells (APS cells) and the like are included. The pluripotent stem cells may include those derived from animals, insects, etc., but are preferably derived from mammals.

  Here, “pluripotency” can be rephrased as pluripotency, but can differentiate into any differentiated cell belonging to ectoderm, mesoderm, endoderm, and ectoderm, mesoderm, endoderm. And the ability to differentiate into at least one type of differentiated cell belonging to each of the above, including the ability to differentiate into germ cells. In the future, differentiated cells can differentiate into many types of cells that constitute tissues and organs in the body.

  “Medium for pluripotent stem cell culture” refers to a culture medium or composition suitable for culturing such pluripotent stem cells, and the undifferentiated nature and pluripotency of undifferentiated pluripotent stem cells. It is possible to proliferate while maintaining the above, and it may be provided in either a powdery state or a liquid state.

  The “serum substitute” means a component that can be used in place of the serum and can exert the same effect as the serum because the serum component is an essential component in the conventional culture medium for pluripotent stem cells. .

Sericin Sericin used in the present invention is a protein extracted from silk thread and has excellent affinity with water, and is used for cosmetics and textile processing.
Sericin, an amorphous protein, can be extracted from the silk thread using a hydrophilic solvent, preferably water. For example, by immersing a raw material containing a silk thread, such as silkworms and raw silk, in hot water, the sericin in the raw material can be hydrolyzed and eluted in water. At this time, if necessary, an acid, an alkali, or an enzyme may be used in combination.
Subsequently, a highly purified sericin aqueous solution can be obtained by refine | purifying an extract according to the well-known protein isolation | separation purification method. Further, it may be dried by subjecting it to a treatment such as hot air drying, reduced pressure drying, freeze drying and the like. As such sericin, for example, commercially available products such as “Pure Sericin” (available from Seiren Co., Ltd., available from Wako Pure Chemical Industries, Ltd.) can be used.

In the present invention, the average molecular weight (weight average molecular weight) of sericin is not particularly limited, but is preferably 5,000 to 100,000, and more preferably 10,000 to 50,000. If the average molecular weight is less than 5,000, the desired effect may not be obtained, and if the average molecular weight exceeds 100,000, the water solubility of itself may decrease and the handling property may decrease. The resulting effect may be reduced due to a decrease in water solubility.
Here, the average molecular weight can be measured and determined by GPC analysis using a high performance liquid chromatograph CLASS-LC10 (manufactured by Shimadzu Corporation).

  It is known that natural sericin contained in silkworms has several components having different molecular weights. For example, according to Japanese Patent Laid-Open No. 2002-128691, sericin having a molecular weight of about 400,000, about 250,000, about 200,000, and about 35,000 has been confirmed. The extract from the above-described silk thread contains these sericin in a mixed state. In some cases, sericin molecules are hydrogen-bonded to each other, increasing the apparent molecular weight. The extract obtained by hydrolyzing sericin using an acid, an alkali, or an enzyme is a mixture containing various molecular species. In the present invention, sericin having an average molecular weight in the range of 5,000 to 10,000 can be selectively prepared by controlling conditions such as the agent used, concentration, temperature, and time.

  In the present invention, the content of sericin in the culture medium for pluripotent stem cells is not particularly limited, and the type of pluripotent stem cells to be cultured (type of derived organism, type of cell, etc.), culture purpose, type of basal medium It can be appropriately changed according to the above.

  According to a preferred embodiment of the present invention, the sericin content in the medium is 0.001 to 10% by weight, more preferably 0.02 to 0.5% by weight, even more preferably, based on the total amount of the medium. Is 0.05 to 0.2% by weight. Even if the content of sericin in the medium according to the present invention is small, the present invention can exhibit a sufficient effect, but sericin is not toxic and has excellent water solubility, so usually it is added in a large amount. However, there is virtually no problem.

In the pluripotent stem cell culture medium of the present invention, sericin is used as a serum substitute. For this reason, the medium according to the present invention is a serum-free medium. Furthermore, the medium according to the present invention is not limited to sericin, and other components (for example, differentiation inhibitory factor, auxiliary component, and medium basic component) are preferably those not derived from mammals. In other words, the culture medium according to the present invention does not contain a mammal-derived component.
The medium according to the present invention enables culturing pluripotent stem cells while maintaining undifferentiation and pluripotency without using serum or a mammal-derived component. It is not excluded to use sericin as a serum substitute according to the above in order to reduce the amount of serum used in a medium containing serum. Alternatively, even when there is a possibility that a serum or a mammal-derived component is contained in a very small amount that can be said to be contaminated, the use of sericin as a serum substitute according to the present invention is the case where a serum or a mammal-derived component is used. This is beneficial in terms of risk reduction. Such a case is not excluded from the scope of the present invention.

As described above, according to a preferred embodiment of the present invention, the medium according to the present invention further comprises a differentiation inhibitor.
Here, any differentiation inhibiting factor can be used as long as it is known. A differentiation inhibitory factor is a humoral factor released by cells such as pluripotent stem cells and feeder cells, and can be obtained from a living body. In the present invention, however, the use of a mammal-derived component is excluded. Therefore, use an artificially prepared one.

Specifically, for example, differentiation inhibitory factors include leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), adenylate cyclase activity inhibitors (SQ22536, MDL-12330A, etc.), MEK1 (MAPK) / ERK kinase 1) inhibitors (PD1733074, PD184352, etc.) (where MAPK is mitogen-activated protein kinase) and ERK is an extracellular signal-regulated kinase From the group consisting of GSK3 (Glycogen Synthase Kinase 3) inhibitors (CHIR99021 etc.), FGF receptor inhibitors (SU5402 etc.), basic fibroblast growth inhibitory factor (bFGF). Is to be selected. Moreover, you may use another factor (cytokine, a low molecular weight compound, etc.) which acts in these upstream or downstream pathways as a differentiation inhibitory factor. In addition, a factor that activates or inhibits another pathway related to undifferentiation / pluripotency of pluripotent stem cells can also be used.
The differentiation inhibitory factor is appropriately selected according to the type of pluripotent stem cell (type of living organism, type of cell, etc.). For example, LIF is preferable for mouse ES cells, and bFGF is preferable for primate ES cells such as humans and monkeys.
If these factors are not derived from mammals and are cytokines, recombinants are used.

  The content of the differentiation inhibitory factor varies depending on the type of differentiation inhibitory factor selected. For example, when LIF is used for mouse ES cells, it is preferably 100 to 3000 U / ml, more preferably 500 to 2000 U / ml, and when bFGF is used for primate ES cells, 0. 1-100 ng / ml is preferable, More preferably, it is 2-20 ng / ml. When the content of the differentiation inhibiting factor in the medium according to the present invention is too small, differentiation may proceed in the cultured stem cells, and when the content of the differentiation inhibiting factor is too large, cytotoxicity may be exhibited.

  In the present specification, U (unit) indicating the unit of LIF is “300 to 400 M1-T22 cells cultured in a 1 ml soft agar culture system for 7 days, and the ratio of differentiated colonies to the total number of colonies is 50. % Is defined as “50 units” (for example, refer to Metcalf (1988) in the description of “ESGRO” (LIF trade name) (available from Millipore)).

Supplementary components The medium according to the present invention is selected from the group consisting of insulin, transferrin, ethanolamine, sodium selenite, albumin, zinc sulfide as an alternative to insulin, and ferrous sulfate as an alternative to transferrin One or more auxiliary components may further be included. In the present invention, it is desired that these auxiliary components are not directly derived from mammals, and therefore artificially prepared ones are used. In addition, ethanolamine, sodium selenite, zinc sulfide, and ferrous sulfate may be blended in the basal medium to be used.

  According to one preferred embodiment of the present invention, as described above, the medium according to the present invention further comprises one or more auxiliary components selected from the group consisting of insulin, transferrin, ethanolamine and sodium selenite. Become.

  The content of the auxiliary component varies depending on the type of auxiliary component selected. For example, in the case of insulin, 0.1 to 100 μg / ml is preferable, more preferably 1 to 20 μg / ml, and in the case of transferrin, 0.01 to 50 μg / ml is preferable, and more preferably 0. 1 to 20 μg / ml, preferably 0.1 to 10 μg / ml in the case of ethanolamine, more preferably 1 to 5 μg / ml, and 0.1 to 10 ng in the case of sodium selenite. / Ml is preferred, more preferably 0.3 to 5 ng / ml. When the content of the auxiliary component in the medium according to the present invention is within the above-described range, it is advantageous for growing the cells while maintaining the undifferentiated and pluripotent properties of the pluripotent stem cells.

Medium Basic Component According to another preferred embodiment of the present invention, as described above, the medium according to the present invention further comprises a medium basic component.

  Here, the medium basic component is usually composed of a carbon source that can be assimilated by cells, a nitrogen source that can be digested, and an inorganic salt, and specifically includes, for example, inorganic salts, amino acids, glucose, and vitamins. Moreover, you may further mix | blend trace effective substances, such as a micronutrient promoting substance and a precursor, with a culture medium basic component as needed.

  As such a medium basic component, a basal medium known to those skilled in the art can be used. Specifically, MEM (Minimum Essential Medium), BME (Basal Medium Eagle), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's minimal essential medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glas-gow's MEM), F12 (Ham's F12 Medium), DMEM / F12, RPMI 1640, BMC-3 (Brinster's BMOC-3 Medium), CMRL- Media such as 1066, L-15 medium (Leibovitz's L-15 medium), McCoy's 5A, Media 199, MEM αMedia, MCDB105, MCDB131, MCDB153, MCDB201, Williams' medium E can be used. In the present invention, knockout DMEM medium (available from Invitrogen) and Daigo T medium (manufactured by Nippon Pharmaceutical Co., Ltd., available from Wako Pure Chemical Industries, Ltd.) are preferably used.

In addition, if necessary, the medium according to the present invention may further contain one or more components selected from the group consisting of nucleic acids, non-essential amino acids, and reducing agents. Among these, as the reducing agent (antioxidant), monothioglycerol, 2-mercaptoethanol and the like can be used, and 2-mercaptoethanol is particularly preferable.
Further, if necessary, optional components such as a pH adjuster, a buffer component, a humectant, a preservative, and a viscosity adjuster can be used.

  The present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

(1) Preparation of medium
(a) Medium according to the present invention (sericin medium)
In commercially available Daigo T medium (manufactured by Nippon Pharmaceutical Co., Ltd., obtained from Wako Pure Chemical Industries, Ltd.), 0.1% by weight sericin, 10 μg / ml insulin, 0.34 ng / ml sodium selenite, 2 μg / ml ethanolamine 0.1 mM non-essential amino acid (L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline, L-serine mixed solution, obtained from Invitrogen), 0.055 mM 2- Mercaptoethanol and 1000 U / ml leukemia inhibitory factor (LIF) (obtained from Wako Pure Chemical Industries, Ltd.) were added and dissolved (hereinafter sometimes abbreviated as “sericin medium”). Here, sericin is a pure sericin (trade name) (manufactured by Seiren Co., Ltd., obtained from Wako Pure Chemical Industries, Ltd.) dissolved in phosphate buffered saline (PBS) to a final concentration of 10% by weight. It added to the culture medium so that it might become 0.1 weight%.

(b) Control medium (serum medium)
As a control, knockout DMEM (obtained from Invitrogen) was added to 15 volume% fetal bovine serum (FBS) (manufactured by biowest), 2 mM L-glutamine, 0.1 mM non-essential amino acids (L-alanine, L-asparagine, L- Aspartic acid, L-glutamic acid, glycine, L-proline, L-serine mixed solution, obtained from Invitrogen), 0.055 mM 2-mercaptoethanol, and 1000 U / ml leukemia inhibitory factor (LIF) (Wako Pure Chemical Industries, Ltd.) What added (obtained from the corporation | Co., Ltd.) was used (henceforth abbreviated as "serum culture medium").

(2) Mouse ES cells used for cultured cell experiments were obtained from ATCC (American Type Culture Collection) (CRL-1934).

(3) Passage culture Mouse ES cells were cultured using the above-described sericin medium (the present invention) (hereinafter this cell may be abbreviated as “ES-S”).
As a control, mouse ES cells were also cultured using a serum medium (control) (hereinafter this cell may be abbreviated as “ES-F”).

Using a petri dish having a diameter of 60 mm coated with 0.1 wt% gelatin (obtained from DS Pharma Biomedical Co., Ltd.), the cells were passaged once every 2 to 3 days.
The medium was removed from the petri dish, washed once with PBS, 1 ml of a 0.05 wt% trypsin / EDTA solution (Sigma) was added, and the mixture was incubated at 37 ° C. for 3 minutes. After confirming cell detachment, the cells were pipetted with a Pasteur pipette, and the colonies were broken into single cells. The cell suspension was collected in a tube previously dispensed with a trypsin inhibitor (obtained from Invitrogen). The surface of the petri dish was washed with PBS, and after collection, centrifuged at 1200 rpm for 3 minutes.
After removing the supernatant, add 5 to 10 ml of the medium to suspend it, count the number of cells, and inoculate in a 2 day culture to 1 million cells / φ60 mm petri dish and a 3 day culture to 500,000 cells / φ60 mm petri dish. And cultured.

  ES-S was able to be subcultured for 2 months or longer without a growth stop, although a decrease in the growth rate was observed compared to ES-F.

The morphology of ES cells in the first month of culture was as shown in FIG.
The ES-S colony has a dome-like shape with a raised center, which is consistent with the characteristics of the colony of undifferentiated ES cells that the edges of the colony are clear, and the undifferentiated state is maintained. It was shown that
In ES-S, the whole colony may be detached from the substrate during the culture, and adhesion may be weaker than that in ES-F.

(4) Confirmation of undifferentiation of cultured ES cells
(a) Cell staining In order to confirm whether or not the undifferentiated state of cultured ES cells was maintained, alkaline phosphatase (ALP) staining of the cells was performed. ALP is generally used as an undifferentiated marker whose expression is confirmed in undifferentiated ES cells.

  Specifically, the ALP staining test was performed according to the following procedure using a Leukocyte Alkaline Phosphatase Kit (obtained from Sigma).

Mix 13 ml of acetone with 5 ml of citrate solution (18 mmol / l citric acid, 9 mmol / l sodium citrate, 12 mmol / l sodium chloride, pH 3.6) and 1.6 ml of 37 wt% formaldehyde. Citrate Working Solution was obtained.
0.5 ml of FRV alkaline solution (0.4 mol / l hydrochloric acid containing 5 mg / ml Fast red violet LB Base) is mixed with 0.5 ml of 0.1 mmol / l sodium nitrate and allowed to stand at room temperature for 2 minutes, followed by deionization. Dilution with 22 ml of water (Milli Q water) gave a diazonium solution. To 4.8 ml of diazonium solution, 200 μl of naphthol solution (2 mol / l AMPD (2-amino-2-methyl-1,3-propanediol) buffer containing 4 mg / ml naphthol AS-BI phosphate, pH 9.5) Was added to obtain a staining solution.
ALP staining was performed using these reagents.

The ES cells of the first month of culture were subcultured in a 24-well multiwell plate, the medium was removed from the wells, and the cells were washed once with PBS. Next, 600 μl of the citrate experimental solution was added thereto, and after fixing for 30 seconds, the citrate experimental solution was removed and washed twice with deionized water.
Next, 400 μl of the staining solution was added to the petri dish, light-shielded with an aluminum foil, and allowed to stand at room temperature for 1 hour. After 1 hour, the staining solution was removed, washed twice with deionized water, air-dried, and then observed with a microscope.

The results of staining were as shown in FIG.
When ES-S and ES-F at 1 month of culture were stained with ALP, it was found that almost all colonies were ALP positive and maintained an undifferentiated state.

(b) Expression of undifferentiated marker gene Furthermore, the expression of a gene (undifferentiated marker gene) specifically expressed in undifferentiated ES cells was confirmed by real-time PCR.

RNA extraction was performed according to the following procedure.
After adding 1 ml of TRIzol reagent (obtained from Invitrogen) per φ35 mm petri dish and pipetting several times, the entire amount was transferred to a 1.5 ml tube and allowed to stand at room temperature for 5 minutes.
Next, 200 μl of chloroform was added and the mixture was shaken and allowed to stand for a few minutes. Thereafter, the mixture was centrifuged at 16000 rpm for 15 minutes at 4 ° C.
The upper layer was transferred to a new tube, 500 μl of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, the mixture was further centrifuged at 16000 rpm for 15 minutes at 4 ° C.
The supernatant was removed and 1 ml of 75% by volume ethanol was added, followed by centrifugation at 10,000 rpm for 5 minutes at 4 ° C.
The obtained RNA precipitate was air-dried. DEPC water (diethylpyrocarbonate-treated water) was added in an amount of 50 to 100 μl, and pipetting twice or three times, followed by incubation at 55 to 60 ° C. for 10 minutes to dissolve RNA, and then stored at −80 ° C.

Next, cDNA was synthesized from the extracted RNA.
cDNA synthesis was performed according to the following procedure.
The RNA was dissolved on ice and the concentration was measured. The PCR tube was prepared by adding DEPC water to 5 μg of RNA and a total of 11 μl to 50 ng of random hexamer. And after 70 degreeC and 10-minute incubation, it left still on ice for 1 minute or more. 5 × First Srand Buffer (obtained from TAKARA BIO INC.) 4 μl, 10 mM dNTP 1 μl, 0.1 M DTT 2 μl, and 10 U / ml RNase inhibitor 1 μl are prepared as a sample for 1.1 times the number of samples in an Eppendorf tube. 8 μl each was added to the reaction solution (total 19 μl). After incubation at 25 ° C. for 5 minutes, 1 μl of SuperScript II reverse transcriptase (SuperScript II RT) (obtained from Invitrogen) was added.
Subsequently, after incubation at 25 ° C. for 10 minutes, 42 ° C. for 50 minutes, and 70 ° C. for 15 minutes, the mixture was allowed to stand on ice. Thereto was added 1 μl of 2U / μl RNaseH, incubated at 37 ° C. for 20 minutes, and stored at −80 ° C.
Real-time PCR was performed using the synthesized cDNA as a template.

Real-time PCR was performed according to the following procedure.
PCR grade water 4 μl, Taq MAN PCR master MIX (obtained from Applied Biosystems) 10 μl, primer (obtained from Applied Biosystems) 1 μl as one reaction, required amount x 1.1 times for each target gene Reagents were prepared and placed at 15 μl / well in 96-well plates for real-time PCR.
The cDNA was diluted 20-fold with PCR grade water and added in 5 μl / well increments.
Seal the plate and centrifuge, after 2 minutes at 50 ° C, 10 minutes at 95 ° C, (95 ° C for 15 seconds, 60 ° C for 1 minute) x 45 cycles, 50 ° C for 15 seconds in real time PCR was applied.

Among the genes specifically expressed in undifferentiated mouse ES cells, the most frequently used “Oct-4” and “Nanog” were examined for expression levels in ES-S and ES-F.
ES-S and ES-F at the second month of subculture were collected on the second day after subculture and RNA was extracted. The expression levels of undifferentiated marker genes Oct-4 and Nanog were quantified and compared by real-time PCR.

The result was as shown in FIG.
In the figure, the vertical axis (relative to ALAS) means the ratio of the expression level of the target gene of each sample to ALAS (5-aminolevulinate synthase) used as an internal standard. Specifically, the value in the graph means the relative value (unit: fold) of the DNA amount before amplification, which is obtained from the difference between the Cp value of ALAS and the Cp value of the target gene. Here, the internal standard is a gene whose expression level is always constant regardless of the state of the cell. The Cp value is the number of cycles required to amplify the DNA to a level detectable by a measuring instrument when DNA is amplified by a PCR reaction (theoretically, the amount of DNA doubles in one cycle). .

  In both genes, ES-S maintained the same expression as ES-F. For comparison, expression of both genes was decreased in ES-F (RA induction) induced by differentiation for 1 week by adding 1 μM retinoic acid (obtained from Sigma) except LIF.

(5) Confirmation of pluripotency of cultured ES cells Confirmation that ES cells ("ES-S" above) cultured in the medium (sericin medium) according to the present invention maintain pluripotency. did. That is, ES-S cultured for 2 months or longer in sericin medium was induced to differentiate into bone and myocardium. Bone and myocardium are tissues and organs that develop from the mesoderm.

(i) Induction of differentiation into bone Differentiation of cultured ES cells into bone was performed according to the following procedure.
As a bone differentiation-inducing medium, 1% by weight penicillin / streptomycin, 10% by volume FBS, 100 nM dexamethasone (obtained from Wako Pure Chemical Industries, Ltd.), 10 mM β-glycerophosphate (obtained from Tokyo Chemical Industry Co., Ltd.), 50 μg / ml ascorbic acid-2-phosphate (manufactured by Sigma) was added and used.

The cells were cultured until they became confluent, and then replaced with a bone differentiation-inducing medium.
Once every 2-3 days, the culture was continued for 3 weeks while changing the medium, and bone differentiation was confirmed by alizarin staining.

Alizarin staining was performed as follows.
Alizarin Red S (obtained from Wako Pure Chemical Industries, Ltd.) was dissolved in ultrapure water to 1% by weight and adjusted to pH 6.0 to 6.2 to obtain Alizarin Red staining solution.
The medium was removed, 95% by volume ethanol was added, and the mixture was allowed to stand for 10 minutes. The ethanol was then removed, and the plate was washed with ultrapure water. Here, alizarin red staining solution was added and allowed to stand for 30 minutes. Next, the operation of washing with the addition of ultrapure water was repeated about 3-6 times after removing the staining solution, and then air-dried.

  As a control, a culture prepared by exchanging with a sericin medium or a serum medium instead of the differentiation induction medium was prepared.

The result was as shown in FIG.
It was confirmed that the ES cells cultured in sericin medium (the present invention) maintained the ability to differentiate into bone.

(ii) Induction of differentiation into myocardium Differentiation of cultured ES cells into myocardium was performed according to the following procedure.
As a myocardial differentiation inducing medium, GMEM medium was supplemented with 5% by volume KSR (Knockout Serum Replacement), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids (L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, Mixed solution containing glycine, L-proline and L-serine, obtained from Invitrogen), 0.1 mM 2-mercaptoethanol, 1 μM bone morphogenetic protein 4 (BMP4, available from R & D Systems, Wako Pure Chemical Industries, Ltd.) Were added and used.

ES-S was seeded in a non-adhesive 96-well multiwell plate at 500 cells / well. After culturing for 2 days, the formed embryoid body (EB) was collected and suspended in a non-adherent petri dish using a myocardial differentiation induction medium.
Therefore, after culturing for 3 days, a 24-well multiwell plate surface-coated with 0.1% by weight gelatin (obtained from DS Pharma Biomedical Co., Ltd.) was seeded so that one EB was placed per well. After culturing for 2 days, the medium was replaced with a myocardial differentiation induction medium excluding BMP4.
On the 8th day from the start of the adhesion culture, the number of EBs with pulsations was measured by microscopic observation, and the pulsation rate was calculated.

The result was as shown in FIG.
It was confirmed that the ES cells cultured in sericin medium (the present invention) maintained the ability to differentiate into myocardium.

Claims (11)

  A culture medium for pluripotent stem cell culture comprising sericin as a serum substitute.   2. The medium according to claim 1, further comprising a differentiation inhibitor.   The culture medium of Claim 1 or 2 which does not contain a mammal origin component.   The culture medium according to any one of claims 1 to 3, further comprising one or more auxiliary components selected from the group consisting of insulin, transferrin, ethanolamine and sodium selenite.   The medium according to any one of claims 1 to 4, further comprising a medium basic component.   Differentiation inhibitors include leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), adenylate cyclase activity inhibitor, MEK1 (MAPK / ERK kinase 1) inhibitor, GSK3 (glycogen synthase kinase 3) inhibitor, The culture medium according to any one of claims 2 to 5, which is selected from the group consisting of an FGF receptor inhibitor and a basic fibroblast growth inhibitory factor (bFGF).   Using the medium for pluripotent stem cell culture according to any one of claims 1 to 6, comprising cell proliferation while maintaining undifferentiation and pluripotency of pluripotent stem cells. A method for culturing pluripotent stem cells.   The method according to claim 7, wherein the pluripotent stem cell is derived from a mammal.   Culturing undifferentiated pluripotent stem cells using the culture medium for pluripotent stem cells according to any one of claims 1 to 6 to obtain a clonal population of pluripotent stem cells. A method for preparing a clonal cell population of pluripotent stem cells.   The method according to claim 9, wherein the pluripotent stem cell is derived from a mammal.   A differentiated cell obtained by differentiation of a cell obtained by the method according to any one of claims 7 to 10.