NOVEL CYCLIC DEPSIPEPTIDE AND USE THEREOF TECHNICAL FIELD

[0001]

The present invention relates to a novel cyclic depsipeptide derived from the Didemnid ascidians and a method for culturing pluripotent stem cells while maintaining pluripotency by using the cyclic depsipeptide.

BACKGROUND ART

[0002]

To maintain pluripotent stem cells in an undifferentiated state, the cells are generally cultured in an undifferentiation maintenance medium containing fetal bovine serum in the presence of feeder cells or LIF (leukemia inhibitory factor for mouse)/FGF (fibroblast growth factor for human). In view of using pluripotent stem cells in medical applications, however, it is essential to develop a serum-free and feeder cell-free culture method to avoid contamination of heterogeneous animal components. At the same time, because LIF/FGF is high cost, it has been desired to develop a low molecular weight compound that can replace the functions of LIF/FGF. Ding et al. reported in 2006 that mouse embryonic stem (ES) cells are maintained in an undifferentiated state in the presence of a compound called pluripotin (ΙμΜ) (Chen S, et al., PNAS 2006, 103, 17266-17271). Smith et al. reported in 2008 that three low molecular weight inhibitors (3i: PD184352, Su5402, and CHIR99021) and two low molecular weight inhibitors (PD0325901 and CHIR99021) can replace the functions of LIF that is required for maintaining pluripotent cells in an

undifferentiated state (Ying QL, et al., Nature 2008, 453, 519-523). Later, Ding et al. showed that PD0325901, which is one of the inhibitors used by Smith, functions as a factor that enhances the efficiency of reprogramming of induced pluripotent stem (iPS) cells by 200-fold (Lin T, et al., Nature Methods 2009, 6, 805-808).

These compounds having undifferentiation maintenance ability have been found to be useful as a small molecule probe for studying the reprogramming process. Ding et al. found in 2010 that administration to human ES cells of thiazovivin (Tzv), which is a low molecular weight inhibitor of ROCK (Rho-associated kinase), improves the cell viability due to stabilization of E-cadherins (Xu Y, et al., Proc Natl Acad Sci U S A. 2010, 107, 8129-34). Furthermore, Ding et al. showed that there is a difference in properties between human ES cells and mouse ES cells. That is, human ES cells depend on FGF for the undifferentiation maintenance, while mouse ES cells depend on LIF. Furthermore, in mouse ES cells, signal transduction mediated by E-cadherin is involved in the maintenance of an undifferentiation state, while, in human ES cells, signal transduction mediated by integrin is mainly involved in the maintenance of an undifferentiated state. Then, Ding et al.

succeeded in inducing a human ES cell line of which property is changed to depending on LIF for the undifferentiation maintenance as in mouse ES cells by culturing human ES cells in a medium supplemented with LIF and two MAPK inhibitors, PD0325901 (MEK inhibitor) and SB203580 (p38 inhibitor) known to have the activity of improving the undifferentiation property of mouse ES cells. As described above, a group of low molecular weight compounds that target the maintenance of pluripotent stem cells in an undifferentiated state are highly valuable as a tool for elucidating the mechanism of maintenance of cells in a differentiated or undifferentiated state, as well as for developing a medical treatment using stem cells.

[0003]

A cyclic depsipeptide is a compound that contains at least two a-amino acids and at least one a-hydroxycarboxylic acid with a ring structure and that has at least one peptide bond and an ester bond derived from hydroxycarboxylic acid. It has been reported that this compound is extracted from natural organisms such as bacteria and has various biological activities (Taniguchi et al., Tetrahedron 2003, 59, 4533-4538).

SUMMARY OF THE INVENTION

[0004]

The present invention provides a novel cyclic depsipeptide derived from the Didemnid ascidians and a method for culturing pluripotent stem cells while maintaining pluripotency by using the cyclic depsipeptide.

[0005]

The gist of the present invention is as follows:

(1) A cyclic depsipeptide represented by the following formula:

wherein Ri and R ₂ independently represent H or Cj-C ₆ acyl, and R ₃ -Rn independently represent H or C C ₆ alkyl.

(2) The cyclic depsipeptide according to (1), wherein Ri and R ₂ are propionyl; R ₃ , R^, Rs, and R ₁₀ are methyl; and R4, R ₅ , R ₇ , R ₉ , and Rn are H.

(3) A method for culturing pluripotent stem cells while maintaining pluripotency, which comprises culturing the pluripotent stem cells in a medium containing the cyclic depsipeptide according to (1) or (2).

(4) A method for improving the efficiency of establishment of induced pluripotent stem cells, which comprises culturing somatic cells, into which a nuclear reprogramming substance has been introduced, in a medium containing the cyclic depsipeptide according to (1) or (2).

[0006]

Pluripotent stem cells can be cultured while maintaining pluripotency by using an isolated novel cyclic depsipeptide derived from the Didemnid ascidians according to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]

FIG. 1 shows reversed phase HPLC data. The peak of the lower curve at a retention time of 4 minutes indicates sameuramide.

FIG. 2 (a) and (b) show the H NMR spectrum of sameuramide and the C NMR spectrum of sameuramide, respectively.

FIG. 3 shows analysis of the two-dimensional NMR spectra of sameuramide. FIG. 3(a) (b), and (c) show the COSY, the HMQC, and the HMBC spectra, respectively.

FIG. 4 shows the mass spectroscopic analysis of sameuramide by ESI technique.

FIG. 5 shows the infrared absorption spectrum of sameuramide.

FIG. 6 shows the structural formula of sameuramide (a), and the planar structure of sameuramide and major correlations determined by COSY and HMBC (b). β-HyLeu represents β-hydroxyleucine; N, 0-Me ₂ Thr represents N, O- dimethylthreonine; N-MeDha represents N-methyldehydroalanine; Ala represents alanine; N-MeAla represents N-methylalanine; Pla represents 3-phenyllactic acid; and Pr represents propionyl.

FIG. 7 shows the NMR analysis of sameuramide. The residues described in "Residue" correspond to the residues described in FIG. 6(a).

FIG. 8 shows a phase-contrast image showing the morphologies of ES cells cultured in the presence of sameuramide (1 nM) on individual days (photograph). FIG. 8 (a) and (e) show the phase-contrast image after 2 days of culture. FIG. 8 (b) and (f) show the phase-contrast image after 7 days of culture (3 passages). FIG. 8 (c) and (g) show the phase-contrast image after 13 days of culture (6 passages).

FIG. 8 (d) and (h) show the phase-contrast image after 19 days of culture (8 passages). FIG. 8 (a) to (d) show the images taken with a 4-fold objective, and FIG. 8 (e) to (h) show the images taken with a 10-fold objective.

FIG. 9 shows the results of the immunostaining (photograph) and the flow cytometric analysis profile of ES cells cultured in the presence of sameuramide (1 nM). FIG. 9 (a) shows the result when low serum (KSR) medium was used, and FIG. 9 (b) shows the result when the serum medium was used. The left panel shows the image of Nanog (green) and DAPI (blue) staining; the center panel shows the image of Oct3/4 (red) and DAPI (blue) staining; and the right panel shows the merged image of the left panel and the center panel. FIG. 9 (c) shows the image of SSEA1 (red) and DAPI (blue) staining. The left panel shows the result when low serum (KSR) medium was used, and the right panel shows the result when the serum medium was used. FIG. 9 (d) shows the flow cytometric analysis where the horizontal axis shows the fluorescence intensity of SSEA1 and the vertical axis shows the number of cells. The left panel shows the result of an unstained control; the center panel shows the result when the low serum (KSR) medium was used; and the right panel shows the result when the serum medium was used.

FIG. 10 shows HE staining image of teratoma generated from ES cells that were cultured using sameuramide (1 nM) (photograph).

DESCRPTION OF THE PREFERRED EMBODIMENTS

[0008]

Hereinafter, the present invention will be described in detail. [0009]

The present invention relates to a novel cyclic depsipeptide derived from the Didemnid ascidians and a method for culturing pluripotent stem cells while maintaining pluripotency by using the cyclic depsipeptide.

[0010]

As used herein, the term "depsipeptide" refers to a compound that contains at least two a-amino acids and at least one a-hydroxycarboxylic acid and that has at least one peptide bond and an ester bond derived from the hydroxycarboxylic acid. The term "cyclic depsipeptide" refers to a depsipeptide that has at least one ring structure.

[0011]

A preferred cyclic depsipeptide in the present invention is represented by the following Formula (1):

wherein Rj and R ₂ independently represent H or Ci-C ₆ acyl, and R3-Rn independently represent H or C]-C ₆ alkyl.

[0012]

Examples of C]-C ₆ acyl includes formyl, acetyl, propionyl, w-butyryl, isobutyryl, valeryl, isovaleryl, and pivaloyl. Examples of C]-C ₆ alkyl includes methyl, ethyl, propyl, isopropyl, «-butyl, sec-butyl, tert-butyl, pentyl, and hexyl. A more preferred cyclic depsipeptide is a compound of Formula (2), which is named sameur amide.

[0013]

In the present invention, the cyclic depsipeptide includes stereochemical isomers and racemates of the compound represented by the formula (1).

[0014]

The cyclic depsipeptide of the present invention can be extracted from the ascidians of the Didemnidae family. For example, ascidians are soaked in methanol at room temperature, and the cyclic depsipeptide can be separated from the obtained methanol extract. The other separation processes can be used with conventional ones. For example, a solvent extraction process, an ion exchange resin process, an adsorption or partition column chromatography process, a gel filtration process, a dialysis process, a precipitation process, and the like can be used individually or in combination for extracting and purifying the cyclic depsipeptide. For example, the methanol extract can be treated with a solvent mixture of water and chloroform and the cyclic depsipeptide can be extracted from the resultant water phase. For further purification of the cyclic depsipeptide, the resultant may be, for example,

chromatographed on octadecyl silica (ODS) gel. In this case, an organic solvent such as methanol, ethanol, propanol, acetonitrile, and chloroform, or a solvent mixture of such organic solvent and water may be used. In the present invention, a process other than ODS gel chromatography, such as reversed phase HPLC, may be used for further purification. In this case, the mixture ratio of an organic solvent such as methanol, ethanol, propanol, acetonitrile, and chloroform to water can be changed to adjust the retention time of the compound of interest. The methods as described above or a combination thereof when needed can provide the cyclic depsipeptide with high purity.

[0015]

The cyclic depsipeptide obtained as described above can be used to maintain and culture pluripotent stem cells.

[0016]

As used herein, the term "pluripotent stem cell" refers to a stem cell that is capable of differentiating into cells of all three germ layers: ectoderm, endoderm, and mesoderm (pluripotent) and that are also capable of proliferating, and examples thereof includes, but not limited to, an embryonic stem (ES) cell (M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156; J.A. Thomson et al., (1999), Science 282:1145-1147; J.A. Thomson et al., (1995), Proc. Natl. Acad. Sci. USA, 92:7844- 7848; J.A. Thomson et al, (1996), Biol. Reprod., 55:254-259; and J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165), an embryonic stem cell derived from a cloned embryo by nuclear transfer (ntES cells) (Tada M et al., Curr

Biol. 11 :1553-8, 2001 ; and Cowan CA et al., Science. 2005 Aug 26;309(5739):1369- 73), germline stem cell (M. Kanatsu-Shinohara et al., (2003) Biol. Reprod., 69:612- 616; and K. Shinohara et al., (2004), Cell, 119:1001-1012), an embryonic germ cell (Y. Matsui et al., (1992), Cell, 70:841-847; and J.L. Resnick et al., (1992), Nature, 359:550-551), and an induced pluripotent stem (iPS) cell (K. Takahashi and S.

Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al., (2007) Cell, 131 : 861-872; J. Yu et al, (2007) Science, 318: 1917-1920; M. Nakagawa et al., (2008) Nat. BiotechnoL, 26: 101-106; and WO 2007/069666). A preferred pluripotent cell is an

ES cell, an ntES cell, and an iPS cell.

[0017]

In the method of the present invention, pluripotent stem cells are cultured while maintaining pluripotency. In the present invention, the maintenance of pluripotency of the pluripotent stem cells can be confirmed by using an indicator including, but not limited to, expression of alkaline phosphatase and a gene marker such as Oct3/4 or Nanog, colony formation, and differentiation into the tissues of the three germ layers after teratoma formation.

[0018]

Pluripotent stem cells can be cultured using a basal medium supplemented with the cyclic depsipeptide of the present invention. Examples of the basal medium include Doulbecco's modified Eagle's Medium (DMEM), Glasgow

Minimum Essential Medium (GMEM), and Ham's F12 medium, and a combination thereof. The medium may further contain serum, or may be serum-free.

Preferably, the medium is serum-free. When necessary, the medium may contain one or more serum alternatives such as, for example, albumin, transferrin, knockout serum replacement (KSR), which is an alternative to FBS (fetal bovine serum) for ES cell culture, fatty acid, insulin, collagen precursor, trace element, 2-mercaptoethanol, and 3'-thiolglycerol, and may contain one or more substances such as lipid, amino acid, nonessential amino acid, vitamin, growth factor, cytokine, antibiotic, pyruvic acid, buffering agent, and inorganic salt.

[0019]

The concentration of the cyclic depsipeptide in the medium may be 0.05 nM or more, 0.1 nM or more, 0.5 nM or more, 0.6 nM or more, 0.7 nM or more, 0.8 nM or more, 0.9 nM or more, or 1.0 nM or more, and 100 μΜ or less, 50 μΜ or less, 40 μΜ or less, 30 μΜ or less, 20 μΜ or less, or 10 μΜ or less, although it is not specially limited as long as the pluripotency is maintained. Preferably, the concentration is in the range of 1.0 nM to 10 μΜ. More preferably, the

concentration is 1.0 nM.

[0020]

Furthermore, in the present invention, the cyclic depsipeptide obtained as described above can be used to improve the efficiency of establishment of iPS cells.

[0021]

iPS cells can be established by introducing a certain nuclear reprogramming substance in the form of a DNA, RNA or a protein into somatic cells.

Supplementation of the medium with a cyclic depsipeptide together with the nuclear reprogramming substance allows for enhancement of the efficiency of establishing the iPS cells. The cyclic depsipeptide may be added simultaneously with the reprogramming substance, or may be added after the introduction of the

reprogramming substance into somatic cells. The somatic cells into which the reprogramming substance has been introduced may be cultured in a medium containing the cyclic depsipeptide, until iPS cells are established, or may be cultured in a medium containing the cyclic depsipeptide for a certain period of time and then cultured in a medium not containing the cyclic depsipeptide for establishing iPS cells. The efficiency of establishing iPS cells can be determined by calculating the number of the iPS cells or the colonies made when the same number of somatic cells is used under the same condition except for using the cyclic depsipeptide.

[0022]

A nuclear reprogramming substance may be any gene that is specifically expressed in ES cells or any gene that plays an important role in maintaining ES cells in an undifferentiated state or a product thereof, and examples thereof include, but not limited to, Oct3/4, Klf4, Klfl, Klf2, Klf5, Sox2, Soxl, Sox3, Sox 15, Soxl7, Sox 18, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb, and Esrrg. Such reprogramming substances may be used in combination when establishing iPS cells, and examples of the combination include a combination that includes two, three or four of the reprogramming substances described above and preferably, a combination that includes four of the substances.

[0023]

The nucleotide sequence of the mouse and human cDNAs for each of the nuclear reprogramming substances described above, and the amino acid sequence of the protein encoded by the cDNAs are shown as NCBI (National Center for

Biotechnology Information: www.ncbi.nlm.nih.gov/) accession numbers in WO 2007/069666. The sequence of the mouse and human cDNAs for L-Myc, Lin28, Lin28b, Esrrb, and Esrrg, and the amino acid sequence of the protein encoded by the cDNAs are shown as NCBI accession numbers in the following table. A person skilled in the art can prepare a desired nuclear reprogramming substance based on the information of the corresponding cDNA or amino acid sequences using a routine procedure.

[0024]

[0025]

These nuclear reprogramming substances may be introduced in the form of a protein into somatic cells by means such as lipofection, linkage to a cell membrane permeable peptide, and microinjection etc., or may be introduced in the form of a DNA by means such as a virus, plasmid, or artificial chromosome vector, lipofection, liposome, and microinjection, etc. Examples of the virus vector include a retrovirus vector and a lentivirus vector (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861 -872, 2007; and Science, 318, pp.1917-1920, 2007), an adenovirus vector (Science, 322, 945-949, 2008), an adeno-associated virus vector, a Sendai virus vector (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009), and the like. The artificial

chromosome vector includes, for example, a human artificial chromosome (HAC) vector, a yeast artificial chromosome (Y AC) vector, a bacterial artificial

chromosome (BAC, PAC) vector, and the like. As the plasmid, a mammalian cell plasmid (Science, 322:949-953, 2008) may be used. The vectors can contain a regulatory sequence such as a promoter, an enhancer, a ribosomal binding sequence, a terminator, and a polyadenylation site to permit the vector for expressing the nuclear reprogramming substance. Examples of the promoter include an EFla promoter, a CAG promoter, an SRa promoter, an SV40 promoter, an LTR promoter, a CMV (cytomegalovirus) promoter, an RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, an HSV-TK (herpes simplex virus thymidine kinase) promoter, and the like. Among them, the EFla promoter, the CAG promoter, the MoMuLV LTR, the CMV promoter, and the SRa promoter are preferably used. Furthermore, the vector can contain a selection marker sequence such as a drug resistance gene (for example, a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, or the like), a thymidine kinase gene, and a diphteria toxin gene; and the sequence of a reporter gene such as a green fluorescent protein (GFP) gene, a β -glucuronidase (GUS) gene, and FLAG, when necessary. To excise a gene encoding the nuclear reprogramming substance or a promoter and a gene encoding the nuclear reprogramming substance after introduction of the vector into somatic cells, the vector may also have a loxP sequence preceding and following the gene or the promoter. In another preferred embodiment, transposon may be used to integrate the transgene into the chromosome, and then a plasmid or adenovirus vector may be used to allow a transferase to act on the cell to completely remove the transgene from the chromosome. A preferred transposon that may be used includes, for example, piggyBac, which is the transposon derived from a Lepidopteran insect (Kaji, K. et al., Nature, 458: 771-775 (2009); Woltjen et al., Nature, 458: 766-770 (2009); and WO 2010/012077). The vector may also contain the origin of lymphotrophic herpes virus, BK virus, and Bovine papilloma virus, and a sequence involved in the replication, so that the vector may be replicated without integration into a chromosome while remaining episomal. For example, the vector may contain EBNA-1 and oriP or Large T and SV40ori sequences (WO 2009/115295, WO 2009/157201, and WO 2009/149233). To simultaneously introduce a plurality of nuclear reprogramming substances, an expression vector that polycistronically expresses them may be used. For the polycistronic expression, the sequences encoding the genes may be separated by IRES or foot-and-mouth disease virus (FMDV) 2A coding region (Science, 322:949- 953, 2008; WO 2009/092042; and WO 2009/152529).

[0026]

Examples of the basal medium for establishing iPS cells include (1) DMEM, DMEM/F12, or DME medium that contains 10 to 15% FBS (the medium optionally containing LIF, penicillin/streptomycin, puromycin, L-glutamine, nonessential amino acids, β-mercaptoethanol, and the like); (2) medium for culturing ES cells that contains bFGF or SCF such as a medium for culturing mouse ES cells (for example, TX-WES medium available from Thrombo-X) and the basal medium for culturing primate ES cells (for example, medium for primate (human and monkey) ES cells available from ReproCELL in Kyoto, Japan); and the like.

[0027]

For example, nuclear reprogramming substances are contacted with somatic cells in DMEM or DMEM/F 12 medium containing 10% FBS at 37°C and in the presence of 5% C0 ₂ , and cultured for about 4 to 7 days. Then, the cells are replated on feeder cells (for example, mitomycin C treated STO cells, SNL cells, and the like), and after about 10 days of the contact of the somatic cells with the nuclear

reprogramming substances, the cells are cultured in a medium for culturing primate ES cells that contains bFGF. About 30 days to 45 days or more after contacting the nuclear reprogramming substances, iPS-like colonies can be obtained. The cells may also be cultured under a condition of low oxygen concentration of 5 to 10% to enhance the efficiency of establishing the iPS cells.

[0028]

As an alternative culture method, the cells may be cultured on feeder cells

(for example, mitomycin C-treated STO cells, SNL cells, and the like) in DMEM medium that contains 10% FBS (the medium may optionally contain LIF,

penicillin/streptomycin, puromycin, L-glutamine, nonessential amino acids, β- mercaptoethanol, and the like) to allow formation of ES cell-like colonies in about 25 days to about 30 days or more.

[0029]

During the culture as described above, the medium is replaced with fresh medium once a day from day 2 of the culture. The number of the somatic cells used in the nuclear reprogramming is, but not limited to, in the range from about 5x 10 ³ to about 5 x 10 ⁶ per 100 cm ² of the culture dish.

[0030]

By using a gene that contains a drug resistance gene as a marker gene and culturing in a medium containing the corresponding drug (selection medium), cells that express the marker gene can be selected. When the marker gene is a

fluorescent protein gene, observation with a fluorescence microscope allows for detection of the cells that express the marker gene. When the marker gene is a luciferase gene, addition of the iluminescent substrate allows for detection of the cells that express the marker gene. When the marker gene is a chromogenic enzyme gene, addition of the chromogenic substance allows for detection of the cells that express the marker gene.

[0031]

The somatic cells used herein may be any mammalian (for example, human, mouse, monkey, pig, and rat) cells other than the germ cells, and examples thereof include keratinizing epithelial cells such as keratinizing epidermic cells; mucosal epithelial cells such as epithelial cells of a tongue surface layer; exocrine epithelial cells such as breast cells; hormone secreting cells such as adrenomedullary cells; metabolism and storage cells such as liver cells; luminal epithelial cells constituting the interface such as type I pneumocytes; luminal epithelial cells of internal body cavities such as vascular endothelial cells; ciliated cells having a transporting capacity such as tracheal epithelial cells; extracellular matrix secretion cells such as fibroblasts; contractile cells such as smooth muscle cells; blood and immune system cells such as T lymphocytes; sensory transducer cells such as rod cells; autonomic neurons such as cholinergic neurons; sense organ and peripheral neuron supporting cells such as satellite cells; central nervous system neurons and glial cells such as astrocytes; and pigment cells such as retinal pigment epithelial cells; precursor cells (tissue precursor cells) thereof; and the like. The degree of the cell differentiation and the age of the animals from which the somatic cells are harvested are not particularly limited, and either of undifferentiated precursor cells including somatic stem cells or terminally-differentiated mature cells may be used as the origin of the somatic cells in the present invention. Examples of the undifferentiated precursor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

[0032]

In the present invention, although the mammalian subject from which the somatic cells are harvested is not particularly limited, the subject is preferably human.

EXAMPLES

[0033]

The present invention will be described more specifically with reference to the following examples, although the examples are not intended to limit the scope of the present invention.

[0034]

Method for Experiments

<Cell Culture>

Mouse ES cells (CCE) were cultured in the serum medium for maintaining

ES cells (85: 15 mixture of knockout DMEM containing 2 mM L-glutamine, 100 μΜ non essential amino acid, 100 μΜ 2-mercaptoethanol and 5000 U/mL

penicillin/streptomycin, and fetal bovine serum (FBS)) that contains 1000 U/mL LIF on a gelatin coated dish or in a 96-well microplate, while maintaining the cells in an undifferentiated state.

[0035]

As a low serum medium, a mixture of GMEM, 1% non essential amino acid, 1% pyruvic acid, 100 μΜ 2-mercaptoethanol, 50 U/mL penicillin, 50 mg/mL streptomycin, 1% FBS, and 15% Knock out serum replacement (KSR™) was used.

[0036]

<Colony Maintenance Activity Test>

50 μΜ/well of 0.1% gelatin solution was aliquoted into a 96 well plate and then left to stand for 20 minutes to allow gelatin-coating of the plate. After removal of the gelatin solution, 200 \iL of the medium for maintaining ES cells not containing LIF was added, and ES cells (400 cells/well) were seeded. Each sample was added to the wells, and after incubation for 4 days (37°C, 5% C0 ₂ ), the morphologic change was examined under a microscope to study the colony maintenance activity. [0037]

<Immunostaining and Flow Cytometry>

The expression of Oct3/4, Nanog, and SSEA1, and the expression of SSEA1 were studied with immunostaining and flow cytometry, respectively.

[0038]

In the immunostaining, the cells were fixed in 4% PFA and permeabilized with PBS containing 0.5% Triton-X, and then the cells were stained using anti- mouse Oct3/4 and anti-human Nanog antibodies as primary antibodies, and anti- mouse IgG-Alexa 546 and anti-rabbit IgG-Alexa 488 antibodies as secondary antibodies. When Phycoerythrin-conjugated anti-SSEAl antibody was used, no secondary antibody was used. In each case, the cells were co-stained with DAPI.

[0039]

In the flow cytometry, the cells were harvested with 0.5 mM EDTA from the culture dish, and stained with Phycoerythrin-conjugated anti-SSEAl antibody to examine the expression using FACS Aria II.

[0040]

Example 1 : Isolation of sameuramide

Colonial ascidians (27g) of the Didemnidae family obtained in Samenoura bay, Ishinomaki City, Miyagi, Japan were soaked in methanol (100 mLx2) at room temperature for more than 3 hours for extraction. After concentration, the resultant was treated with water (100 mL) and chloroform (50 mL><2) to be separated into two phases. The resultant water phase was subjected to ODS column chromatography (φ 1.2x 15 cm; 50% MeOH, 70% MeOH, 70% MeCN, 85% MeCN, 100% MeOH, CHCl ₃ /MeOH/Water (ratio: 6:4:1)), and the resultant fractions were evaluated by the colony maintenance activity test for ES cells. 70% MeCN and 85% MeCN fractions, which exhibited strong colony maintenance activity, were combined and purified by reversed HPLC (Cosmosil 5C ₁₈ AR-II column, φ 1.0x25 cm, 2.0 mL/min; 50-100% MeCN) (FIG. 1), and thereby 2.3 mg of a novel compound named sameuramide was isolated.

[0041]

Example 2: Structure Determination of sameuramide

Sameuramide is white amorphous powders and exhibits an optical rotation of

[a] _D -75.9 (c 0.02, MeOH). According to several NMR data (FIG. 2 and 3) and HRESI-MS (FIG. 4), the molecular formula of sameuramide was determined to be C ₅₀ H ₇₇ N ₇ Oi ₅ ([M+Na] ⁺ , m/z 1038.5454 (C ₅₀ H ₇₇ N ₇ NaO ₁₅ , calc 1038.5375)). The infrared absorption spectrometry (FIG. 5) showed the presence of -OH and -NH (3349 cm ^"1 ), ester (1749 cm ^"1 ), and amide (1642 cm ^'1 ).

[0042]

In the Ή NMR spectrum (FIG. 2a), there are 5 exchangeable signals at 5 _H 8.5-6.5, and in the C NMR spectrum (FIG. 2b), there are 10 carbonyl carbons that are appeared to be carbons on the ester or amide at 5c 176-164. These results indicate that sameuramide is a peptide. On the other hand, there are pluralities of signals that are appeared to be a-protons of amino acids at 6 _H 5.5-3.5. For the signals at 5 _H 5.25, 5.09, and 3.57, the signals for the carbons attached to the protons (5c 78.1, 78.6, and 79.0) are shifted to lower field, and thus these have been found to be oxymethine. According to the COSY (FIG. 3a), HMQC (FIG. 3b), and HMBC (FIG. 3c) spectral analysis, these were assigned to the β-methine signal of β- hydroxyleucine (P-HyLeu)-l , -2, and -3. Although no definite COSY correlation was observed for the a- (5 _H 5.14) and β -proton (δπ 5.09) of β-HyLeu-l due to their close chemical shifts, and no HMBC correlation that confirmed the bond

therebetween was also observed, each of the 1H signals is coupled at J= 1.9 Hz, and thus they have been found to be attached to each other. According to the Ή chemical shift values for the β-position of β-HyLeu-l, -2, and-3, it has been expected that the β-oxygen atoms form an ester bond (5 _H 5.09 and 5.25) in β-HyLeu-l and -2, and that the β-oxygen atom forms a hydroxy group (6 _H 3.57) in P-HyLeu-3. These have also been observed in the COSY spectrum, because in -HyLeu-3, a correlation was observed between a hydroxy proton (δπ 6.91) and a β-methine proton (5 _H 3.57). The N- and O-methyl groups ofN O-dimethylthreonine (N 0-Me2Thr) were confirmed, because the HMBC correlation from the N-methyl to the a-carbon and the HMBC correlation from the O-methyl to the β-carbon were observed. The NMR spectrum of sameuramide has two alanine-like spin systems. It has been confirmed that one of them corresponds to alanine (Ala), and the other corresponds to N-methylalanine (N-MeAla), because the HMBC correlation from the N-methyl proton (6 _H 2.82) to the a-carbon of the alanine like spin system (CH-CH ₃ ) was observed. Because the protons at 6 _H 5.26 and 5.17 with a small coupling constant (J= 2.2 Hz) are attached to the carbon at 5c 107.5, they were found to be from exomethylene protons. The HMBC correlation from these protons to the carbon signals at 5c 146.7 and 164.7 and the HMBC correlation from the N-methyl protons (5 _H 3.20, 5c 37.0) to the signal at 6c 146.7 were observed, and thus the residue has been found to be N-methyldehydroalanine (N-MeDha).

[0043]

A phenylalanine like spin system was observed in addition to these amino acid residues, and it has been found to be phenyllactic acid (Pla), which is a hydroxy acid, according to the chemical shift of the a-carbon (5c 72.23). Furthermore, two proprionic acid units have been found to be attached to the amino group of β-HyLeu- 1 and -3, respectively.

[0044]

According to the two dimensional NMR spectral analysis including COSY,

HMQC, and HMBC analyses, sameuramide has been found to be a cyclic depsipeptide that consists of 7 amino acids: β-hydroxyleucine-l (β-HyLeu-l), β- hydroxyleucine-2 (β-HyLeu-2), P-hydroxyleucine-3 (β-HyLeu-3), N, O- dimethylthreonine (N,0-Me ₂ Thr), N-methyldehydroalanine (N-MeDha), alanine (Ala), and N-methylalanine (N-MeAla); a hydroxy acid (3-phenyllactic acid: Pla); and 2 propionyl groups (FIG. 6a and FIG. 7).

[0045]

Detailed analysis on HMBC correlations revealed the arrangement of these residues. The HMBC correlations from the protons of NH or NMe to the carbon on the adjacent carbonyl group are correlations from N-MeAla to Ala, from Ala to N- MeDha, from N-MeDha to Pla, from p-HyLeu-3 to Pr-2, from N, O-Me ₂ Thr to β- HyLeu-2, from β-^Ι^-2 to N-MeAla, and from β-HyLeu-l to Pr-1. The ester bond of and -3 has been found to link from P-HyLeu-3 to NO-Me ₂ Thr and from β-^Ι υ-2 to β-HyLeu-l, according to the HMBC correlation from the β- proton to the carbon on the carbonyl group of the adjacent site. Although the correlation between Pla and β-^Ι^ could not be confirmed, they are considered to form an ester bond, in view of only one NMR spectrum of a hydroxy group and the molecular weight. As a result, sameuramide has been found to have the planar structure shown in FIG. 6b.

[0046]

Example 3: Colony Maintenance Activity by sameuramide and Expression of

Undifferentiated Markers

When ES cells were cultured in a medium supplemented with sameuramide (1 nM) in place of LIF and passaged every 2 days, the number of the colonies decreased on day 7, and only very small or no colonies were observed on day 13. However, when the culture and the passage were continued in the presence of sameuramide, the colonies of almost same size appeared again around day 19 and since then, the colonies could be maintained in an undifferentiation maintenance medium for ES cells supplemented with sameuramide (1 nM) without LIF (FIG. 8).

[0047]

Furthermore, when the expression of undifferentiated markers was examined, it was observed that Nanog, Oct3/4, and SSEA1 were expressed in both serum and low serum medium (FIG. 9). The result has shown that addition of sameuramide in placed of LIF allows for culture of ES cells while maintaining the cells in an undifferentiated state.

[0048]

Example 4: Examination on Teratoma Formation and Differentiation into Three Germ Layers

Mouse ES cells (CCE) were cultured in the presence of sameuramide (1 nM) and maintained for about 3 months. The resultant cells were implanted

subcutaneously in nude mice. After 4 weeks, teratoma was recovered from the mice, and the paraffin sections were made and stained with HE. As a result, differentiated cells were observed while undifferentiated cells were few. The histological differentiation into ectoderm (neuroepithelium, keratinizing epithelium), mesoderm (skeletal muscle, cardiac muscle), and endoderm (mucus-producing epithelium) was observed (FIG. 10), and thus it has been confirmed that the mouse ES cells cultured in the presence of sameuramide have pluripotency.

INDUSTRIAL APPLICABILITY

[0049]

According to the present invention, pluripotent stem cells can be cultured while maintaining pluripotency by using a novel depsipeptide. The pluripotent stem cells cultured as above can be used in the tissue engineering field etc.