WO2007058401A1

WO2007058401A1 - Composition comprising okadaic acid for undifferentiated proliferation of embryonic stem cells

Info

Publication number: WO2007058401A1
Application number: PCT/KR2005/003925
Authority: WO
Inventors: Seungkwon You; Byung Sun Yoon; Ki Dong Kim; Eun Kyung Jun; Jai Hee Moon; Bona Kim; Gyuman Park; Seung Jun Yoo; Sung Sik Kwak; Issac Maeng
Original assignee: Imgen Co., Ltd.
Priority date: 2005-11-18
Filing date: 2005-11-18
Publication date: 2007-05-24
Also published as: CN101180388A; EP1948787A1; JP2009506785A; EP1948787A4

Abstract

Disclosed herein are a culture medium composition comprising okadaic acid for use in the undifferentiated proliferation of human embryonic stem cells and a method of inducing the undifferentiated proliferation of human embryonic stem cells in the culture medium.

Description

COMPOSITION COMPRISING OKADAIC ACID FOR UNDIFFERENTIATED PROLIFERATION OF EMBRYONIC STEM CELLS

Technical Field

The present invention relates to a culture medium composition comprising okadaic acid for use in the undifferentiated proliferation of human embryonic stem cells, and to a method of inducing the undifferentiated proliferation of human embryonic stem cells in the culture medium composition.

Background Art

Human embryonic stem cells (hESCs) are cells from embryos that have the potential to differentiate into derivatives constituting all organs of the human body. Since the establishment thereof by the Thomson group (Thomson JA et al., Science 1998, 282:1145-1147) and the Reubinoff group

(Reubinoff B et al., Nat Biotechnol 2000, 18:399-404), the pluripotential cells hESCs have been used as a source of cells for therapeutic treatment of diseases that had been regarded as incurable. Usually, their culture requires the support of MEF (mouse embryonic fibroblast) feeder cells. When hESCs are cocultured with the mouse cells, unfortunately, there is the risk of cross-transfer of pathogens from the xenogeneic feeders, thus limiting their medical applicability.

In order to overcome this disadvantage, extensive research aimed at finding a human cell-derived feeder culture system or a feeder-free culture system has been conducted (Richards M et al. Nat Biotechnol 2002,20:933-936; Hovatta O et al. Hum Reprod 2003, 18:1404-1409; Amit M et al. Biol

Reprod 2003, 68:2150-2156; Richards M et al. STEM CELLS 2003,

21:546-556; Xu C et al. Nat Biotechnol 2001, 19:971-974;

Rosier et al. Dev Dyn 2004,229:259-274; Carpenter et al. Dev Dyn 2004, 229:243-258; Amit et al. Biol Reprod 2004,70:837- 845; Sato N et al., Nat Med 2004, 10:55-63; Xu R-H et al. Nat Methods 2005,2:185-190; Xu C et al. STEM CELLS 2005, 23:315- 323). The Hovatta group (Hum Reprod 18:1404, 2003) reported a culture system in which human foreskin fibroblasts were used as feeder cells so as to stimulate the production of human embryonic stem cells. Amit (Biol Reprod 68:2150, 2003) and Richards (STEM CELLS 21:546, 2003) also offered human- derived feeder cell systems for the growth of undifferentiated hESCs. The Xu group (Nat Biotechnol 18:399, 2000) established a feeder-free culture system using MEF- derived CM (conditioned media) and matrices such as laminin and matrigel. Rosier (Dev Dyn 229:259, 2004) succeeded in maintaining hESCs in a feeder-free condition for one year or longer by taking advantage of the CM established by the Xu group. However, such a CM suffers from a disadvantage in that a mouse-derived component is retained in the medium. Xu et al. found in 2005 that cultures maintained in bFGF, either alone or in combination with other factors, showed characteristics similar to MEF-CM control cultures, leading to the conclusion that basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth, even without a conditioned medium (STEM CELLS 23:315, 2005). It was also reported by Sato et al. that undifferentiated mouse and human embryonic stem cells could be maintained through the activation of the Wnt pathway using the GSK-3- specific inhibitor BIO in the absence of feeder cells (Nat. Med 10:55, 2004). The Xu group also succeeded in maintaining hESCs in a feeder-free medium supplemented with bFGF and the BMP antagonist noggin (Nat Methods 2:185, 2005).

Leading to the present invention, intensive and thorough research leading to the proliferation of hESCs in a feeder-free condition, conducted by the present inventors with this background, resulted in the finding that okadaic acid allows undifferentiated hESCs to proliferate in the absence of feeder cells and to exhibit characteristics including normal stem cell marker and karyotypic stability.

Disclosure of the Invention

It is therefore an object of the present invention to provide a culture medium composition comprising okadaic acid for inducing the undifferentiated proliferation of embryonic stem cells.

Another object of the present invention is to provide a method for inducing the undifferentiated proliferation of embryonic stem cells in the culture medium composition.

Brief Description of the Drawings

FIG. 1 shows the effects of okadaic acid on embryonic stem cells grown in a feeder-free condition. Okadaic acid was added in an amount of 1 nM (A, B) and 0.1 nM (C), and was not added (D) .

FIG. 2 shows human embryonic stem cells grown on a feeder cell layer (A) and in the absence of feeder cells (B- D).

FIG. 3 shows human embryonic stem cells grown on a feeder cell layer in a bFGF-free culture system (A) and in a bFGF-free culture system containing 1 nM okadaic acid (B) .

FIG. 4 shows that the human embryonic stem cells grown in a feeder-free condition according to the present invention have the normal karyotype. FIG. 5 shows that the human embryonic stem cells grown in a feeder-free condition according to the present invention have undifferentiated markers expressed thereon.

Best Mode for Carrying Out the Invention It is a very important technical goal in the study of stem cells and the use thereof to maintain and proliferate embryonic stem cells in an undifferentiated state prior to the initiation of differentiation. In accordance with an embodiment thereof, the present invention is directed to a culture medium composition comprising okadaic acid capable of inducing the undifferentiated proliferation of embryonic stem cells.

As used herein, the term "embryonic stem cell" means a cell from an embryo which has the potential to proliferate while maintaining its pluripotency.

Usable in the present invention are embryonic stem cells derived from any animal including humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rats, etc., with preference for mammals, and with further preference for humans .

The term "proliferation", as used herein, means an increase in cell number, having the same meaning as "growth". By the term "undifferentiated proliferation", as used herein, it is meant that embryonic stem cells proliferate not into specific cells but into cells having the same properties as the embryonic stem cells, that is, into pluripotent cells. It will be obvious to practitioners of the art that undifferentiated cells can be readily discerned from differentiated cells. For instance, undifferentiated cells feature a high ratio of nucleus to cytoplasm and prominent nucleoli.

It was observed by the present inventors that embryonic stem cells can proliferate in an undifferentiated state in a culture medium free of feeder cells in the presence of okadaic acid.

Serving as an inhibitor of protein phosphatase 2A (PP2A) , okadaic acid is known to inhibit the degradation of and increase the stability of c-myc (Yeh E et al. Nat Cell Biol 2004, 6:308-318). However, nowhere has information on the role of okadaic acid in the proliferation of embryonic stem cells been found. In an embodiment of the present invention, embryonic stem cells were proven to remain undifferentiated even after 15 passages in a culture medium containing okadaic acid through the detection of all of the undifferentiated ES markers nanog, oct-4, rexl and Tert, and the observation of a normal karyotype. Hence, okadaic acid allows embryonic stem cells to undergo stable undifferentiated proliferation even in the absence of feeder cells.

Further, okadaic acid can function in the place of the growth factor bFGF, which is typically used for the proliferation of embryonics stem cells. Therefore, okadaic acid can be used instead of or in combination with typical differentiation suppressors, such as bFGF, for conventional embryonic stem cell cultures employing feeder cells and matrices .

As used herein, the term "culture media" means media which assure the growth and survival of stem cells in vitro, and which may include all of the pertinent media typically used in the art. The culture media and conditions depend on the kind of stem cells. Preferable is a cell culture minimum medium (CCMM) , which generally comprises a carbon source, a nitrogen source and trace elements. Examples of the CCMM include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal Essential Medium), GMEM (Glasgow's Minimal Essential Medium), and Iscove's Modified Dulbecco's Medium. In the CCMM, an antibiotic, such as penicillin, streptomycin, gentamicin, etc., may be added.

In order to achieve the object according to the present invention, which is to induce the undifferentiated proliferation of embryonic stem cells, okadaic acid is added to an embryonic stem cell culture medium without restriction to as to medium kind or culture type. In this regard, okadaic acid may be used alone or in combination with one or more differentiation suppressors.

The culture medium must comprise an effective concentration of okadaic acid. The term "effective concentration" means an amount sufficient for okadaic acid to induce the undifferentiated proliferation of okadaic acid. Because it shows no desirable effects at a concentration lower than the effective concentration and cytotoxicity at a concentration higher than the effective concentration, okadaic acid should be used within the effective concentration range. Factors determining the effective concentration of okadaic acid include the origin of the embryonic stem cells, the kind of culture media, the components of the culture media, differentiation suppressors used together therewith, etc. For instance, when okadaic acid is used as a sole differentiation suppressor in the culture of human embryonic stem cells, its effective concentration may be on the order of 0.5 to 1.5 nM.

In accordance with another embodiment, the present invention is directed to a method for inducing the undifferentiated proliferation of embryonic stem cells, comprising culturing them in a culture medium containing okadaic acid.

In a culture medium comprising okadaic acid in accordance with the present invention, embryonic stem cells can proliferate without spontaneous differentiation and remain undifferentiated until a desired time point.

Embryonic stem cells may be obtained using conventional methods. For instance, the preparation of human embryonic stem cells may be achieved according to the method of Thomson (U. S. Pat No.5843780; Science 282; 1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995). The culture medium containing okadaic acid in accordance with the present invention assures that embryonic stem cells undergo undifferentiated proliferation without being contaminated by pathogens from the xenogeneic feeders, such as those from viruses and animals.

A better understanding of the present invention may be given with the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.

EXAMPLE 1: Culture of hESCs

The hESC line HSF-β (University of California, San Francisco) was cultured while a 13.5 day-old CFl mouse fetus (ORIENT) was used as a feeder cell. The feeder cell was incubated in a DMEM (high glucose, Invitrogen) supplemented with 10% FBS (HyClone) , 0.1 mM β -mercaptoethanol, and 0.1 mM non-essential amino acids and then treated with 10 μg/ml of mitomycin C (sigma) for 1.5 hrs to terminate the mitosis thereof. Afterwards, the feeder cell treated with mitomycin C was seeded in a 0.1% gelatin-coated four-well culture dish at a density of β.lxlO⁴ cells per well. On the day after the seeding of the feeder cell, the hESC was seeded, and subcultured at time intervals of 5-7 days in a mechanical manner .

The hESC was cultured in a Dulbecco's modified Eagle's medium/F12 (DMEM/F12, without pyruvate) supplemented with 20% knockout serum replacement (SR) (Gibco/BRL, Invitrogen, Carlsbad, CA), 0.1 mM β -mercaptoenthanol, 1% non-essential amino acids (Gibco/BRL) , 100 U/ml penicillin G, 100 g/ml streptomycin, and 4 ng/ml hr-bFGF (human recombinant basic fibroblast growth factor; Invitrogen) . In FIG. 2A, the hESC on a CFl mouse feeder cell layer is shown.

EXAPLE 2: Effect of Okadaic Acid on Human Embryonic Stem Cell

The effects of okadaic acid on hESCs grown on feeder cell layers were evaluated with various concentrations (0.01,

0.1, 1, 10, 100 nM) of okadaic acid in human embryonic stem cell culture media. On the next day (24 hrs) after the addition of 10 or 100 nM of okadaic acid, both the embryonic stem cells and the feeder cells underwent lysis in the culture media as a result of the cytotoxicity of okadaic acid.

In contrast, in culture media containing 1 nM okadaic acid or less, the embryonic stem cells proliferated, showing morphology similar to that of the control cultured in okadaic acid-lacking culture media, without observation of the toxicity of okadaic acid.

EXAMPLE 3: Effect of Okadaic Acid on Human Embryonic Stem Cells in Feeder-Free Condition The effect of okadaic acid on hESCs was evaluated in the absence of feeder cells. 0.01 nM, 0.1 nM, and 1 nM of okadaic acid were added to culture systems using no CM. Without using matrices such as laminin and matrigel, in contrast to a previous culture system using such matrices, a four-well culture dish was coated at room temperature for 30 min with 0.1% gelatin, after which the embryonic stem cells were mechanically split. Thereafter, the embryonic stem cells were seeded in culture media containing 0.01 nM, 0.1 nM, and InM okadaic acid.

As a result, the human embryonic stem cells in 0.01 nM and 0.1 nM okadaic acid (FIG. 1C) proliferated in a similar manner to that of the control in the absence of okadaic acid

(FIG. ID) , and after three passages, no undifferentiated stem cells were observed, because all of the cells underwent differentiation. In contrast, the human embryonic stem cells in the 1 mM okadaic acid culture media remained undifferentiated even after three passages (FIG. 2B-D) , without any sign of differentiation. It was also observed that the embryonic stem cells on the feeder cell layers had high ratios of nucleus to cytoplasm (FIG. 2D) .

EXAMPLE 4: Effect of Okadaic Acid on Embryonic Stem Cells Grown on Feeder Cell Layers in the Absence of bFGF

bFGF is usually used at a concentration of 4 ng/ml for the proliferation of undifferentiated embryonic stem cells. Embryonic stem cells were analyzed for proliferation and differentiation in culture media containing 1 nM okadaic acid in the absence of bFGF. In culture media containing neither okadaic acid nor bFGF, all of the embryonic stem cells grown on feeder cell layers were observed to differentiate after two or more passages (FIG. 3A) . After two passages, human embryonic stem cells were observed to remain undifferentiated in culture media which contained 1 nM okadaic acid, but no bFGF (FIG. 3B) . Therefore, it was proven that okadaic acid can work in place of bFGF.

EXAMPLE 5: Expression of ES Marker on hESCs and Karyotype of hESCs in Feeder-Free Condition

To examine whether hESCs grown in the absence of feeder cells expressed normal ES markers, RT-PCR was conducted. Total RNA was isolated with a Trizol reagent

(Invitrogen) . Reverse-transcription (RT) was carried out using 500 ng of the total RNA, oligo(dT) 12-18 mer primers

(Invitrogen) , and an AMV reverse transcriptase (Roche Molecular Biochemicals) to yield cDNA. For RT-PCR, 1 μl of cDNA, 10 pmol of each primer, and a PCR premix (IU Tag DNA polymerase, 250 μM dNTPs, 10 mM Tris-HCl, 40 mM KCl and 1.5 mM MgC12 Bioneer, Korea) were used.

Oligonucleotides specific for undifferentiated ES markers nanog (SEQ ID NOS.: 1 and 2), oct-4 (SEQ ID NOS.: 3 and 4) and rexl (SEQ ID NOS.: 5 and 6) and for hTert (SEQ ID NOS.: 7 and 8), expressed in ES, were used as primers in the RT-PCR. Primers for β -actin (SEQ ID NOS.: 9 and 10) were used as a negative control. As for cDNA, it was obtained from hESCs grown in the absence of feeder cells for 5, 10 and 15 passages, while hESCs grown on feeder cell layers were used as a positive control. The RT-PCR data of FIG. 5 show that all of the ES markers nanog, oct-4 and rex 1 and the ES expression protein hTert were expressed in the hESCs grown in the absence of feeder cells, as in the positive control. Karyotype analysis, entrusted to Samkwang Medical Laboratories, demonstrated that the cells obtained after 15 passages in the absence of feeder cells exhibited a normal karyotype. Therefore, okadaic acid allows the hESCs grown in a feeder-free culture system in the presence of okadaic acid to have the same gene expression behavior and karyotype as in those grown on feeder cell layers (FIG. 4) .

Industrial Applicability As described hitherto, the culture medium composition comprising okadaic acid in accordance with the present invention allows the undifferentiated proliferation of hESCs without the risk of cross-transfer of pathogens from xenogeneic feeders, such as those from viruses and other animals.

Claims

1. A culture medium composition for inducing the undifferentiated proliferation of embryonic stem cells, comprising okadaic acid.

2. The culture medium composition according to claim 1, wherein the embryonic stem cells are originated from human.

3. The culture medium composition according to claim 2, wherein the composition contains 0.5 to 1.5 nM of okadaic acid.

4. A method for inducing the undifferentiated proliferation of embryonic stem cells, comprising culturing the embryonic stem cells in the culture medium composition of claim 1.

5. The method according to claim 4, wherein the embryonic stem cells are originated from human.

6. The method according to claim 5, wherein the culture medium composition contains 0.5 to 1.5 nM of okadaic acid.