NZ583003A - Embryonic stem cell line and method for preparing the same - Google Patents

Abstract

Provided is a medium comprising: 95 to 110mM NaCl; 7.0 to 7.5mM KCI; 20 to 30mM NaHCO3 ; 1.0 to 1.5mM NaH2PO4; 3 to 8mM sodium lactate; 1.5 to 2.0mM CaCI2-2H2O; 0.3 to 0.8mM MgCl-6H2O; 0.2 to 0.4mM sodium pyruvate; 1.2 to 1.7mM fructose; 6 to 10mg/ml human serum albumin; 0.7 to 0.8 ug/ml kanamycin; 1.5 to 3% essential amino acids; 0.5 to 1.5% nonessential amino acids; 0.7 to 1.2mM L-glutamine; and 0.3 to 0.7% a mixture of insulin, transferrin and sodium selenite.

Description

8 3 0 0 3 PATENTS FORM NO. 5 Our ref: TIS508165NZPR Divisional of NZ 548159 Antedating requested to 30 December 2004 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION EMBRYONIC STEM CELL LINE AND METHOD FOR PREPARING THE SAME We, SEOUL NATIONAL UNIVERSITY INDUSTRY FOUNDATION, of San 4-2, Bongcheon-dong, Gwanak-gu, Seoul, 151-050, Republic of Korea hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: The next page is numbered "1/ 301332408_1.DOC:TIS:hew EMBRYONIC STEM CELL LINE AND METHOD FOR PREPARING THE SAME FIELD OF THE INVENTION The present invention relates to an embryonic stem cell line and a method for preparing the same and, more particularly, to an embryonic stem cell line prepared by transferring a nucleus of a human somatic cell into an enucleated human oocyte, culturing the resulting nucleus-transferred oocyte to form a blastocyst, and culturing an inner cell mass isolated from the blastocyst, and a method for preparing the same.

BACKGROUND OF THE INVENTION A stem cell is normally taken to mean an undifferentiated cell capable of differentiating into all types of mature fiinctional cells constituting a body. For example, a hematopoietic stem cell can differentiate into various corpuscular cells. An embryonic stem (ES) cell derived from an embryo has pluripotency to differentiate and develop into all types of organs, tissues and cells that form a body.

A mouse ES cell line constructed in 1981 has provided a technique and paradigm for developing a human ES cell. The development of the ES cell has been studied using a mouse teratocarcinoma, a tumor that occurs in a gonad of a closely bred mouse strain (Evans & Kaufman, Nature, 292:154-156 (1981)).

Bongso et al reported a method for culturing and maintaining cells isolated 1A from a human embryo derived from in vitro fertilization for a short-term period (Bongso et al, Human Reproduction, 9:2110-2117 (1994)). The cells isolated by Bongso et al had a moiphology expected in a pluripotent stem cell; however, they could not be cultured for a long-term period apparently because a proper feeder 5 layer was not used.

Primate ES cells have been prepared from a blastocyst of a rhesus monkey and a marmoset monkey. The primate ES cells are diploid, and veiy similar to a human ES cell.

The study of ES cells prepared from a monkey and a human has suggested 10 that a pluripotent stem cell might be derived from a human blastocyst, although the ES cells from the monkey and the human are somewhat different from that of a mouse in terms of phenotype (Thomson et al, Proc. Natl Acad. Sci. USA, 92:7844-7848(1995)).

Hie characteristic features of human pluripotent ES cells developed by 15 Thomson et al. in 1998 (Thomson et al., Science, 282:1145-1147 (1998)) are as follows: (1) expression of stage-specific embryonic antigen-3 (SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), and alkaline phosphatase; 20 (2) high telomerase activity; (3) differentiation into three types of blastodermal cells when injected into mice; (4) dependency on feeder cells; and (5) no response to a human leukemia inhibitory factor (hLIF). 2 Thomson et al. obtained the above ES cells from a blastocyst donated by a couple under sterility treatment. Specifically, a trophectoderm known to inhibit the establishment of an ES cell was removed immunosurgically, an inner cell mass 5 (ICM) was plated on a fibroblast feeder layer derived from a mouse embryo, and the ICM was replated on another feeder layer after a short attachment and expansion period. Thomson's method was not significantly different from the mouse ES cell protocol in terms of the medium or culture system; and yet a relatively high success rate was achieved.

The isolation of human pluripotent ES cells and breakthroughs in somatic cell nuclear transfer (SCNT) in mammals (Solter, Nat. Rev. Genet., 1:199-207 (2000)) have raised the possibility of performing human SCNT to generate virtually unlimited sources of undifferentiated cells for research, with potential applications in tissue repair and transplantation medicine. This concept, known as "therapeutic 15 cloning," employs a nuclear transfer of a somatic cell into an enucleated oocyte (Lanza et al, Nat. Med., 5:975-977 (1999)). Previous studies on such therapeutic cloning dealt with the production of bovine ES-like cells (Cibelli et al., Nat. Biotechnol., 16:642-646 (1998)) and mouse ES cells from ICMs of cloned blastocysts (Munsie et al., Cvarr. Biol., 10:989-992 (2000); Wakayama et al., 20 Science, 292:740-743 (2001)) and development of cloned human embryos until 8 to 10 cell stages (Cibelli et al., J. Regen. Med., 2:25-31 (2001)).

Although several reports have indicated that an ES cell line can be established by employing a non-human mammalian oocyte, no ES cell line developed from a human oocyte utilizing the nuclear transfer technology has been 3 reported yet.

SUMMARY OF THE INVENTION Through extensive research and development efforts, however, the present inventors have successfully established an ES cell line by culturing a nucleus-transferred human oocyte.

Accordingly, it is an object of the present invention to provide an ES cell line derived from a nucleus-transferred oocyte prepared by transferring a nucleus of 10 a human somatic cell into an enucleated human oocyte.

It is another object of the invention to provide a method for preparing an ES cell line, comprising the steps of: (1) culturing a human somatic cell to prepare a nuclear donor cell; (2) enucleating a human oocyte to prepare a recipient oocyte; (3) preparing a nucleus-transferred oocyte by transferring a nucleus of the nuclear donor cell into the recipient oocyte and fusing the nucleus of the nuclear donor cell and the recipient oocyte; (4) subjecting the nucleus-transferred oocyte to reprogramming, activation and in vitro culturing to form a blastocyst; and 20 (5) isolating an ICM from the blastocyst and culturing the ICM in an undifferentiated state to establish the ES cell line.

It is a further object of the invention to provide a medium suitable for an in vitro culturing of a nucleus-transferred oocyte prepared by transferring a nucleus of a human somatic cell into an enucleated human oocyte. 4 508165NZPR 3Q196899B It is still another object of the invention to provide a nerve cell or a neuro progenitor differentiated from an ES cell line derived from a nucleus-transferred oocyte prepared by transferring a nucleus of a human somatic cell into an enucleated human oocyte.

It is a still further object of the invention to provide a method for preparing a neuro progenitor differentiated from an ES cell line derived from a nucleus-transferred oocyte prepared by transferring a nucleus of a human somatic cell into an enucleated human oocyte, comprising the steps of: (1) culturing the ES cell line to form an embryoid body; (2) culturing the embryoid body in the presence of an agent suitable for differentiating a cell of the embryoid body into the neuro progenitor; and (3) selecting a cell expressing a marker of the neuro progenitor and culturing the selected cell to obtain the neuro progenitor.

It is a further object of the invention to provide a medium that overcomes or ameliorates at least one of the disadvantages of the prior art.

It is a farther or alternative object to at least to provide the public with a useful choice.

In a first aspect, the invention provides a medium comprising: 95 to llOmM NaCl; 7.0 to 7.5mM KC1; 20 to 30mM NaHC03>; 1.0 to l,5mM NaH2P04; 3 to 8mM sodium lactate; 1.5 to 2.0mM CaCl2'2H20; 0.3 to 0.8mM MgCl2'6H20; 0.2 to 0.4mM sodium pyruvate; 1.2 to 1.7mM fructose; 6 to 10rng'/m£ human serum albumin; 0.7 to 0.8/i.g/mi' kanamycin; 1.5 to 3% essential amino acids; 0.5 to 1.5% nonessential amino acids; 0.7 to 1.2mM L-glutamine; and 0.3 to 0.7% a mixture of insulin, transferrin and sodium selenite.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which: Fig. 1 shows photographs of an undifferentiated colony of ES cells derived from a nucleus-transferred oocyte in accordance with the present invention (A: xlOO, B: x200); Fig, 2 represents a photograph of a fluorescence-stained neuro progenitor differentiated from an undifferentiated colony obtained in accordance with the 5A present invention by adding a mixture of insulin, transferrin, sodium selenite and fibronectin (x400); Fig. 3 depicts the incision process of the zona pellucida of an oocyte (3) with a holding pipette (1) and an incision pipette (2); Fig. 4 presents a photograph showing the removal of the first polar body and the nucleus of the oocyte (3) with the holding pipette (1) and the incision pipette (2); Fig. 5 offers a photograph showing the transfer of a nuclear donor cell into an enucleated recipient oocyte (3) with the holding pipette (1) and a transfer pipette 10 (4); Figs. 6A to 6D diagrammatically summarize the results of a karyotype analysis of an ES cell line derived from a nucleus-transferred oocyte prepared in accordance with the present invention and that of a somatic cell obtained from a female, said somatic cell providing the nucleus used for establishing the ES cell 15 line; Fig. 7 illustrates three types of blastodermal cells identified within a teratoma formed by injecting an undifferentiated cell colony obtained in accordance with the present invention into a gonad of an immune deficiency mouse (A: cartilage, B: intestinal tract, C: neural tube (A,B,C: x200)); and 20 Fig. 8 provides photographs confirming the formation of an embryoid body from an ES cell line in accordance with the present invention (A,B,C: endoderm; D,E,F: mesoderm; ectoderm; A: alpha-l-fetoprotein; B: cytokeratin; C: HNF-2-alpha; D: BMP-4; E: Myo D; F: desmin; G: neurofilament; H: S-100; and I: NCAM). 6 DETAILED DESCRIPTION OF THE INVENTION The term "nuclear transfer" as used herein means a process of transferring a nucleus of a somatic cell (or referred to as "nuclear donor cell") into an enucleated oocyte (or referred to as "recipient oocyte"). The resulting cell obtained by the nuclear transfer is referred to as a "nucleus-transferred oocyte" or "nuclear transfer oocyte." The term "somatic cell" as used herein means any cell constituting a 10 body that has two sets of chromosomes (2n), excluding a germ cell that has a single set of chromosomes (n).

The term "autologous nucleus-transferred oocyte" used herein means a nucleus-transferred oocyte obtained by transferring a nucleus of a somatic cell into an enucleated oocyte where the somatic cell is isolated from a human who is 15 expected to receive a stem cell derived from the nucleus-transferred oocyte, or a specific cell or tissue differentiated from the stem cell.

Accordingly, one of the salient advantages or benefits to be derived from the present invention resides in the fact that the person who receives a specific cell or tissue derived from the autologous nucleus-transferred oocyte would not exhibit 20 immunorejection or suffer adverse reaction since such cell or tissue is to carry the genetic characteristics of the person.

The term "embryonic stem cell (ES cell)" means an undifferentiated cell derived from an embryo, which has the capability of differentiating into various types of mature cells. Here, "embryo" means a fertilized egg up to eight (8) weeks 7 after its fertilization or a nucleus-transferred oocyte in the corresponding developmental stage. An embryo is created by a repetitive division of such fertilized egg or nucleus-transferred oocyte, and comprises a blastocyst containing an ICM and an outer trophectoderm.

The term "ES cell line derived from an autologous nucleus-transferred oocyte" or "autologous nucleus-transferred ES cell line" means a stem cell line derived from an ICM isolated from an autologous nucleus-transferred oocyte.

The term "neuro progenitor" refers to cells to be differentiated into nerve cells including neurons and glia such as astrocytes, oligodendrocytes, Schwann cells, 10 satellite cells, ependymal cells and microglia.

In accordance with one aspect of the present invention, there is provided a method for preparing an ES cell line, comprising the steps of: (1) culturing a human somatic cell to prepare a nuclear donor cell; 15 (2) enucleating a human oocyte to prepare a recipient oocyte; (3) preparing a nucleus-transferred oocyte by transferring a nucleus of the nuelear donor cell into the recipient oocyte and fusing the nucleus of the nuclear donor cell and the recipient oocyte; (4) subjecting the nucleus-transferred oocyte to reprogramming, activation 20 and in vitro culturing to form a blastocyst; and (5) isolating an ICM from the blastocyst and culturing the ICM in an undifferentiated state to establish the ES cell line.

Hereinafter, the method for preparing an ES cell line in accordance with the 8 present invention will be described in detail.

Sten 1 : Preparation of nuclear donor cell A human somatic cell is cultured to function as a nuclear donor cell.

A somatic cell from a human is amenable for such nuclear donor cell, and a nucleus thereof is transferred into an enucleated human oocyte.

There is no limitation on the type or source of the somatic cell as long as it is obtained from a human, and it is also possible to use a somatic cell obtained from 10 an institute storing human cells for commercial purposes. Preferred exemplary somatic cells include a dermal cell, a nerve cell, a cumulus cell, an oviduct epithelial cell, and the like.

In case of preparing an autologous nucleus-transferred oocyte in accordance with the present inventicm, the nuclear donor cell is taken from an individual who is 15 expected to receive a stem cell derived from the nucleus-transferred oocyte, or a specific cell or tissue differentiated from the stem cell.

The somatic cell can be cultured to establish a cell line by using the Mather and Barnes method (Animal Cell Culture Methods: vol.57 of Methods in Cell Biology (Mather & Barnes eds., Academic Press, 1998)). 20 In accordance with a preferred embodiment of the present invention, a uterus fluid and a phosphate buffered saline (PBS) containing P/S antibiotic (penicillin 10,000iu, streptomycin 10mg) are added to a somatic cell. Such somatic cell is centrifuged and washed, and cultured in a DMEM medium containing human serum,'nonessential amino acids (NEAAs) and the P/S antibiotic 9 at, e.g., 39 °C in 5% C02 atmosphere.

Especially, in case of using a cumulus cell as a nuclear donor cell, Hie cumulus cell can be prepared by treating a cumulus-oocyte complex with hyaluronidase to isolate a cumulus cell layer surrounding an oocyte, adding a 5 trypsin-EDTA solution to the cumulus cell layer and placing the resulting solution at, e.g., 39 °C in 5% C02 atmosphere under saturated humidity. After centtifuging and washing, the collected cumulus cells can be cultured under the same condition described above.

Step 2 : Preparation of recipient oocyte A recipient oocyte as used in the present invention means an oocyte that lacks its own nucleus and receives a foreign nucleus from a human somatic cell.

A mature oocyte may be prepared by collecting a superovulated oocyte from 15 a human ovary or obtaining an oocyte from an institute storing human oocytes for commercial purposes and culturing the oocyte using a method known in the art (Yuzpe et al., J. Reprod. Med., 34:937-942 (1989)). For example, an oocyte may be matured by culturing the oocyte in the G1.2 medium, marketed by Vitro Life of Goteborg, Sweden, supplemented with 5% human serum albumin (HSA) under the 20 condition of, e.g., 5% CO2 for 4 hours.

Next, an enucleated recipient oocyte is prepared by removing the surrounding cumulus cells from the oocyte, and eUminating part of the zona pellucida and the cytoplasm containing the first polar body.

In accordance with a preferred embodiment of the present invention, the enucleation process is performed as follows.

A mature oocyte is placed in a washing solution containing hyaluronidase, and the cumulus cell is physically removed. Next, the mature oocyte is washed with the G1.2 medium. Subsequently, the zona pellucida of the oocyte is 5 penetrated to form a small hole therein. The oocyte is enucleated by removing part of the cytoplasm containing the first polar body corresponding to 10 to 15% of the total cytoplasm through the small hole. After this removal, the enucleated oocyte is washed with the G1.2 medium and placed in the G1.2 medium for culturing.

The enucleation can be confirmed by investigating cytoplasm stained with 10 Hoechst 33342 (Sigma Co., St Louis, MO, U.S.A.) using a UV detector.

Step 3 : Preparation of nucleus-transferred oocyte and electrofiision The nuclear donor cell prepared by step 1 is transferred into the enucleated 15 recipient oocyte obtained in step 2, and the nucleus-transferred oocyte is treated with electrofiision.

The nuclear transfer of a somatic cell into a recipient oocyte may be realized by transferring either the nucleus of the somatic cell or the whole somatic cell into the recipient oocyte.

In accordance with a preferred embodiment of the present invention, the nuclear transfer and electrofiision are performed as follows.

First, the enucleated oocyte is washed with the G1.2 medium. The nuclear donor cell is injected into the ©nucleated oocyte in a phytohemagglutin-P (PHA-P) solution via a small hole formed in the zona pellucida using a transfer pipette to 11 produce a nucleus-transferred oocyte. Next, the resulting nucleus-transferred oocyte is washed with the Gl .2 medium and placed in Ihe same medium.

Subsequently, the nucleus-transferred oocyte is treated with electrofiision with the aid of a cell manipulator. A mannitol solution is added to the G1.2 5 medium containing the nucleus-transferred oocyte. The resulting mannitol solution containing the nucleus-transferred oocyte is placed between two electrodes of the cell manipulator and is positioned such that the nuclear donor cell faces the (+) electrode. The nucleus-transferred oocyte is electrofiised by treating it with a direct current ranging from 0.75 to 2.00kV/cm for 10 to 15|xs, 1 to 5 times at an 10 interval of, e.g., 1 second.

Hie fused nucleus-transferred oocyte is washed with a mannitol solution and the G1.2 medium. The mannitol solution used in this step is prepared by dissolving bovine serum albumin (BSA) and mannitol in a 4-(2-hydroxyethyl)-1 -perazine ethanesulfonic acid (HEPES) buffer at apH ranging from 7.2 to 7.4 Step 4: Repropramming. activation and in vitro culturing of nucleus-transferred oocyte In order to allow the nucleus-transferred oocyte prepared in step 3 to 20 undergo a same developmental procedure as a normal fertilized oocyte formed as a result of fusion between a sperm and an oocyte, several critical factors, such as reprogramming time, activation method and in vitro culturing conditions, should be judiciously chosen.

The present invention provides unique fertilization and development 12 WO 2005/063972 PCT/KR2004/003528 procedures conducive for activating and culturing the nucleus-transferred oocyte. Specifically, the nucleus-transferred oocyte prepared by electrofiision in step 3 is subjected to reprogramming, activation, and in vitro culturing to form a blastocyst.

The reprogramming time means the time lapsed between the electrofiision 5 and the activation, and the length of the reprogramming time may affect the developmental capacity (in particular, the blastocyst formation rate) of the nucleus-transferred oocyte. This reprogramming time is required to allow the gene expression pattern of the somatic cell to turn into one that is appropriate and necessary for the development of the nucleus-transferred oocyte. Such 10 reprogramming time plays a critical role in chromatin remodeling, and it is known to determine the developmental competence in vivo and in vitro of the nucleus-transferred oocyte.

The reprogramming time of the present invention may be 20 hours or below, preferably, 6 hours or below, more preferably 3 hours or below, and, most 15 preferably, about 2 hours.

After the reprogramming, the nucleus-transferred oocyte may be activated by various chemical, physical and mechanical stimuli, such as calcium ionophore, ionomycin, ethanol, Tyrode's solution (Sigma-AIdrich, St. Louis, MO, U.S.A.) puromycin, and the like. In the present invention, it is preferable to treat the 20 nucleus-transferred oocyte with calcium ionophore for its activation. It is more preferable to treat the nucleus-transferred oocyte with calcium ionophore and then with 6-dimethylaminopurine (6-DMAP). Specifically, the calcium ionophore may be used at a concentration ranging from 5 to 15jjM, and, preferably, about IOjliM. In addition, said 6-DMAP may be employed at a concentration ranging from 1.5 to 13 2.5mM, and, preferably, about 2.0mM. If the concentrations of the calcium ionophore and the the 6-DMAP are within the above respective ranges, the nucleus-transferred oocyte may be activated effectively. Both calcium ionophore and 6-DMAP are preferably dissolved in an in vitro culture medium.

A representative example of the in vitro culture medium is the G1.2 medium (Vitro Life, Goteborg, Sweden) comprising NaCl, KC1, NaHC03, NaH2PC>4, CaCl2, sodium lactate, glucose, phenol red, BSA, kanamycin, essential amino acids (EAAs), NEAAs, and glutamine.

Further, for an efficient in vitro culturing of the nucleus-transferred oocyte, 10 it is preferable to supplement the culture medium with various energy substrates known in the art or employ a sequential culturing system using at least two media having different compositions suitable for each stage of the embryonic development. The sequential culturing system useful in the present invention may be any one of commercially available culturing systems. Preferably, said in vitro 15 culturing is performed by sequentially using two media having different compositions each other, such as the G1.2 and the G2.2 media (Vitro Life, Goteborg, Sweden).

Such in vitro culture medium preferably contains a human modified synthetic oviductal fluid with amino acids (hmSOFaa), which has been designated 20 as "SNUnt-2 medium." The hmSOFaa is prepared by supplementing a modified synthetic oviductal fluid with amino acids (mSOFaa) (Ghoi et al., Theriogenology, 58:1187-1197 (2002)) with HSA and fructose instead of BSA and glucose, respectively. The mSOFaa medium has been widely used for culturing bovine embryos.

In particular, the SNUnt-2 medium comprises 95 to 1 lOmM NaCl; 7.0 to 7.5mM KC1; 20 to 30mM NaHC03; 1.0 to 1.5mM NaH2P04; 3 to 8mM sodium lactate; 1.5 to 2,0mM CaCl2- 2H20; 0.3 to 0.8mM MgCl2- 6H20; 0.2 to 0.4mM sodium pyruvate; 1.2 to 1.7mM fructose; 6 to lOmg/mt, HSA; 0,7 to 0.8#g/mfe kanamycin; 1.5 to 3% EAAs; 0.5 to 1.5%NEAAs; 0.7 to 1.2 mML-glutamine; and 0.3 to 0.7% a mixture of insulin, transferrin and sodium selenite. Preferably, the SNUnt-2 medium comprises the ingredients as listed in Table 1.

Table 1 Ingredient Concentration NaCl 99.1~106mM KC1 7.2mM NaHC03 25mM NaH2P04 1.2mM sodium lactate 5mM CaCl2- 2H20 1.7mM MgCl2- 6H20 0.5mM sodium pyruvate 0.3mM fructose 1.5mM HSA 8mgM kanamycin 0.75^g/m?.

EAAs 2% NEAAs 1% L-glutamine ImM ITS* 0.5% * ITS: a mixture of l.Og/L insulin, 0.55g/L transferrin and 0.67mg/L sodium selenite The sequential culturing system of the present invention may employ any combination of the different media. For example, in the two-step culturing system, the first culturing may be conducted in the G1.2 medium, and the second culturing, 5 in the SNUnt-2 medium.

Step 5: Removal of zona pellucida or part thereof In order to obtain an ES cell derived from the blastocyst obtained in step 4, the zona pellucida or part thereof has to be removed from the blastocyst. This removal may be carried out by using one of the methods known in the art, e.g., pronase treatment, incubation in acidic Tyrode's solution, or a physical method such as laser dissetiion. It is preferable to use pronase dissolved in a suitable medium such as PBS, G2 medium (Vitro Life, Goteborg, Sweden) or S2 medium (Scandinavian IVF Sciences, Goteborg, Sweden). In a preferred embodiment, pronase is dissolved in a mixture of PBS and the S2 medium at equal volumes. The blastocyst is treated with 0.1% pronase for about 1 to 2 minutes, preferably 1 to 1.5 minutes, to remove the zona pellucida therefrom.

Step 6: Removal of trophoblast and isolation of ICM Once the zone pellucida is removed from the blastocyst as described above, the trophoblast is exposed. It is preferable to completely separate the trophoblast 16 from the ICM. The trophoblast may be separated from the ICM using one of the methods known in the art, such as an immunosurgical method employing an antibody or a mechanical method using a pipette.

In a preferred embodiment, the trophoblast is removed by an 5 immunosurgical method that treats the trophoblast with an antibody responsive to an epitope located on a surface of the trophoblast It is more preferable to carry out the immunosurgical method together with a complement treatment. In this case, an antibody and a complement may be used independently or simultaneously. A preferred combination between the antibody and the complement may include anti-10 placental alkaline phosphatase antibody (anti-AP) and baby rabbit complement, or anti-human serum antibody and guinea pig complement The antibody and the complement may be diluted with a suitable medium such as SNUnt-2, G2.2 or S2 medium. Preferably, the anti-AP may be diluted with the S2 medium at the ratio of 1:20; and other antibodies and complements, at the 15 ratio of 1:1.

It is preferable to treat the zona pellucida-removed blastocyst with an antibody and then with a complement. Preferably, the blastocyst may be treated with the antibody for about 30 minutes, washed with a suitable medium, e.g., SNUnt-2, G2.2 or S2 medium, and then, treated with the complement for about 30 20 minutes.

Moreover, the trophoblast or part thereof may be removed from the blastocyst by washing the blastocyst with a suitable medium such as SNUnt-2, G2.2 or S2. In such case, the trophoblast may be removed by a mechanical method known in the art, e.g., pipetting a solution containing the blastocyst using a pipette 17 having a small bore.

Through these steps, the trophoblast is removed from the blastocyst; and the ICMs, i.e., the remaining part thereof, are obtained.

Step 7: Culturing of ICMs on fibroblast feeder layer ICMs isolated in step 6 are cultured on a fibroblast feeder layer since ICMs maintain their undifferentiated state when cultured on the fibroblast feeder layer. Sometimes, hLIF has been suggested for maintaining the undifferentiated 10 morphology of ICMs instead of the feeder layer. However, it is practically impossible for a human cell to remain in its undifferentiated state without using a fibroblast feeder layer. Accordingly, the condition that does not induce extraembryonic differentiation and apoptosis in the ES cells generally requires culturing on a fibroblast feeder layer.

It is preferable to employ a mouse- and/or a human-derived fibroblast for preparing the fibroblast feeder layer. They may be used alone or in a mixture. It is more preferable to use cells differentiated from the ES cells derived from an autologous nucleus-transferred oocyte of a human as a feeder layer (this feeder layer has been designated as "auto feeder layer"). It is most preferable to use the 20 fibroblasts differentiated from the ES cells derived from an autologous nucleus-transferred oocyte of an individual. The use of such feeder layer can prevent other foreign cells from contaminating the ES cells.

Such human-derived fibroblasts are capable of inducing an optimum growth and differentiation inhibition of Ihe ES cells when appropriately mixed with mouse- 18 derived fibroblasts.

The cell density in the fibroblast feeder layer may affect its stability and capability. In case of using a mixture of mouse and human fibroblasts, it is preferable to maintain the human fibroblasts at a density of, e.g., 2.5 X104 cells/carf 5 and the mouse fibroblasts at a density of, e.g., 7.0 X104 cells/af. In case of using the mouse fibroblasts alone, it is preferable to use the same at a density ranging from 7.5 X104 to 1.0 X105 cells/cnf. It is preferable to establish such feeder layer 6 to 48 hours before the addition of ES cells thereon.

Further, it is preferable to use mouse or human fibroblasts having a low 10 passage number. Quality of the fibroblasts may affect the capability of supporting the ES cells. It is preferable to use the fibroblasts isolated from an embryo. The mouse fibroblasts are preferably obtained from 13.5-day old fetus, and the human fibroblasts, from an embryo or a fetal tissue. These fibroblasts can be cultured by using a cell culturing method known in the art.

In handling the mouse embryonic fibroblasts, it is important to minimize the use of trypsin and inhibit overcrowding. Otherwise, the mouse embryonic fibroblasts cannot support the growth of undifferentiated ES cells. Each batch of the mouse embryonic fibroblasts so prepared has to be tested first to confirm whether it is suitable for supporting and maintaining the ES cells. 20 Between fresh primary embryonic fibroblasts and fibroblasts having undergone a freezing-thawing treatment, the former is normally considered more suitable for supporting renewal of the ES cells. However, certain batches may show their capability of supporting the ES cells even after repeated freezing-thawing. 19 Certain mouse strains can produce embryonic fibroblasts more suitable for supporting the ES cells than other strains. For example, it has been demonstrated that the fibroblasts derived from the mice produced by inbreeding of 129/Sv or CBA strain or by crossbreeding of 129/Sv and C57/B16 strains are more suitable 5 for supporting the ES cells.

In addition, it is preferable to inhibit the growth of feeder cells by using any one of the methods known in the art, including irradiation and chemical treatment In a preferred embodiment, such cells are treated with mitomycin C.

The fibroblast feeder layer thus prepared is cultured on a petri dish coated 10 with gelatin, preferably 0.1% gelatin.

The fibroblast feeder layer may be maintained in an ES medium. A suitable ES medium is the DMEMZF12 medium comprising 20% serum replacement, O.lmM p-mercaptoethanol, 1% NEAAs, 2mM glutamine, lOOunits/mi penicillin, and 100/ig/mC streptomycin, and 4ng/m£ human recombinant fibroblast 15 growth factor (FGF).

Further, such ES medium may be supplemented with a soluble growth factor capable of stimulating growth or survival of the stem cells or inhibiting differentiation thereof. Representative examples of the growth factor are human pluripotent stem cell factor, ES cell renewal factor, and the like. 20 The isolated ICMs may be cultured for 6 days or longer, and cell colonies are generated therefrom. The colonies typically comprise undifferentiated stem cells. The undifferentiated stem cells may be isolated by using one of the methods known in the art. It is preferable to use a micropipette for isolating the undifferentiated stem cells. Such mechanical isolation may be supplemented with a treatment of a Ca2+/Mg2+-free PBS medium or an enzyme helpful for cell dissociation such as dispase.

Step 8: Subculturine of ES cells The ES cells cultured in step 7 are detached from the feeder layer and transferred to a fresh feeder layer. Then, the ES cells may be further cultured to propagate in a morphologically undifferentiated state.

In this case, it is preferable to culture the ES cells for 5 to 7 10 days. Undifferentiated stem cell colonies start to be observed by about the second day of culturing. The stem cells are morphologically identified by a high ratio in nucleus to cytoplasm, clear nucleoli, condensed colony formation and distinctive cell boundary.

Propagation of the undifferentiated stem cells is initiated by isolating an 15 undifferentiated stem cell clump from the stem cell colony. Such isolation may be carried out by using one of the methods known in the art, such as a chemical or mechanical method. Preferably, the stem cells are isolated from the colony by washing with a Ca2+/Mg2+-free PBS medium, a mechanical method, or a combination thereof. It is more preferable to mechanically isolate the stem cells 20 from the colony.

In a preferred embodiment, the Ca2+/Mg2+-free PBS medium may be used for reducing intercellular adhesive power. After incubation in the above medium for about 15 to 20 minutes, the cells begin to detach themselves gradually from the feeder layer, and, finally, are isolated as a clump having a desired size. In case 21 WO 2005/063972 PCT/KR2004/003528 such isolation of the cells proves to be insufficient, a mechanical method using a sharp edge of a micropipette may be more effectively employed for isolating and cutting the clump.

A chemical method employing an enzyme may be also used. The enzyme, 5 preferably, dispase, may be used alone or in combination with a mechanical method. hi another preferred embodiment, it is possible to isolate clumps from the colony by treating with dispase after mechanical cutting of the colony. Cutting of the colony is carried out in a Ca2+/Mg2+-containing PBS medium. The colony can be mechanically cut into clumps, each clump containing about 100 cells, with the 10 aid of a sharp edge of a micropipette. As soon as a clump is isolated, it is picked up with a micropipette having a wider bore, washed with the Ca2+/Mg2+-containing FBS medium, and transferred to a fresh fibroblast feeder layer.

It is necessary to confirm whether the stem cells maintain their undifferentiated state during these culturing processes. Undifferentiated stem cells 15 can be identified by examining their typical morphological characteristic features as described above. Such stem cells can be also identified by detecting a cell marker or measuring the gene expression specific for a pluripotent cell.

Representative examples of genes specific for a pluripotent cell or a typical lineage include, but are not limited to, alkaline phosphatase, Octamer-4 (Oct-4), 20 SSEA-3 and SSEA-4 which may be used as stem cell markers. Other exemplary genes specific for stem cells may include genesis, GDF-3 and cripto. The expression profile of these genes can be analyzed by using one of the methods known in the art, including reverse transcriptian-polymerase chain reaction (RT-PCR), a differentiation gene expression method, a microarray assay, and the like. 22 Preferably, the stem cells can be identified by an immunological reaction with a human pluripotent stem cell marker such as SSEA-4, germ cell tumor marker-2 (GCTM-2) antigen, TRA-1-60, or the like. In particular, the stem cells may express Oct-4 as a transcription factor and maintain a normal diploid karyotype.

Growth progress of the stem cells and maintenance status of their differentiated or undifferentiated state can be monitored by quantitatively measuring the proteins specific for the stem cells excreting into the medium or analyzing fixed cell preparations with enzyme-linked immunosorbent assay. Representative examples of the proteins specific for the stem cells are a soluble type of CD antigen 10 and GCTM-2 antigen, and these proteins can be monitored by detecting a cell marker or measuring the gene expression.

In accordance with another aspect of the present invention, a nerve cell or a neuro progenitor is differentiated from an ES cell line derived from a nucleus-15 transferred oocyte prepared by transferring a nucleus of a human somatic cell into an enucleated human oocyte.

In accordance with a further aspect of the present invention, there is provided a method for preparing a neuro progenitor differentiated from an ES cell line derived from a nucleus-transferred oocyte prepared by transferring a nucleus of 20 a human somatic cell into an enucleated human oocyte, which comprises the steps of: (1) culturing the ES cell line to form an embryoid body; (2) culturing the embryoid body in the presence of an agent suitable for differentiating a cell of the embryoid body into the neuro progenitor; and 23 (3) selecting a cell expressing a marker of the neuro progenitor and culturing the selected cell to obtain the neuro progenitor.

Hereinafter, the inventive method for preparing the neuro progenitor from 5 the ES cell line is described in detail.

Step A: Preparation of an embrvoid body The first step for differentiating the ES cells derived from the nucleus-10 transferred oocytes (referred to as "nuclear transfer embryonic stem cells" or "ntES cells") into neuro progenitors is to generate an embryoid body by culturing the ES cells. The embryoid body can be prepared from the ES cells by using one of the methods known in Hie art (Zhang et al.s Nat. Biotechnol, 19:1129-1133 (2001)).

In a preferred embodiment, the embryoid body is obtained by transferring 15 cultured ntES cell colonies into a non-adhesive culture dish containing the DMEMZF12 medium supplemented with a 20% serum replacement and culturing them for 3 to 5 days. Typically, floating embryoid bodies start to appear about one day after the beginning of the culturing (about 40 to 60 embryoid bodies/dish). At this point, it is preferable to trans&r such embryoid bodies to a new dish while 20 removing any remaining feeder cells. Then, the embryoid bodies are plated on an adhesive dish coated with polyornithine/laminin.

Step B: Inducement of differentiation into neuro progenitors bv an agent 24 PCT7KR2004/003528 Representative agents which may be employed in the present invention for inducing differentiation of the embryoid bodies obtained in step A into neuro progenitors include, but are not limited to, retinoic acid; ascorbic acid; nicotinamide; N-2 supplement (100X, 17502-048; Gibco, Grand Island, NY, 5 U.S.A.); B-27 supplement (50X, 17504-044, Gibco, Grand Island, NY, U.S.A.); and a mixture of insulin, transferrin, sodium selenite and fibronectin (ITSF). Neuro progenitors differentiated from the ntES cells can be obtained by culturing the embryoid bodies in a medium supplemented with such agent and inducing their expansion and differentiation.

In a preferred embodiment, the embryoid bodies prepared in step A are further cultured for 1 day followed by culturing in the DMEM/F12 medium supplemented with ITSF, i.e., insulin (about 25#g/m£), transferrin (about 100#g/m£), sodium selenite (about 30nM) and fibronectin (about 5fig/vd) for 5 to 10 days, thereby inducing differentiation of the ntES cells into the neuro progenitors.

Step C: Selection and culturing of cells expressing a neuro progenitor marker The neuro progenitors differentiated from the ntES cells may be obtained by selecting cells expressing a neuro progenitor marker such as nestin among the differentiated cells obtained in step B and culturing them.

Further, the obtained neuro progenitors may be differentiated into desired specific type of nerve cells. The differentiation into the nerve cells can be carried out through conventional methods such as induction with chemicals, etc.

In a preferred embodiment, the cells exhibiting a positive signal for a neuro progenitor marker are selected; their expansion is induced by culturing the selected cells in the DMEMZF12 medium supplemented with the N-2 supplement, laminin and basic fibroblast growth factor (bFGF) for 5 to 7 days; and, then, they are further 5 cultured in the DMEM/F12 medium supplemented with only the N-2 supplement and laminin for 8 to 14 days.

It is well known that ES cells are capable of differentiating into almost any type of cells. Accordingly, the ES cell line of the present invention may be a good 10 source providing various types of cells. For instance, the ES cells may be induced to differentiate into hematopoietic cells, nerve cells, beta cells, muscle cells, liver cells, cartilage cells, epithelial cells, etc., by culturing them in a medium under conditions suitable for cell differentiation. Such medium and conditions are well known in the art.

Accordingly, the ES cell line of the present invention may have numerous therapeutic and diagnostic applications. Especially, such ES cell line may be used in cell transplantation therapies for the treatment of numerous diseases, e.g., diabetes, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), cerebral palsy and cancer. Further, the ES cell line derived from the 20 autologous nucleus-transferred oocyte can be advantageously used in the cell transplantation therapies since no adverse immunorejection reaction may occur during and after the treatment procedure.

The following Examples are intended to further illustrate the present invention 26 without limiting its scope.

Hie G1.2 medium or G1 ver.3 medium (Vitro Life, Goteborg, Sweden) used in these Examples are supplemented with 5% HSA unless indicated otherwise.

Example 1: Preparation of oocyte and nuclear donor cell Voluntary oocyte donors were screened carefully through physical and mental examinations, and administered with follicle stimulation hormone (FSH) to 10 induce superovulation.

About 36 hours after the administration of human chorionic gonadotropin (hCG) to the donors, cumuhis-oocyte complexes (COCs) were recovered and cultured for 40 minutes in the G1.2 medium using an incubator maintained at 37 °C, 5% CO2 and saturated humidity. Such COCs were treated with 0.1% (w/v) 15 hyaluronidase (Sigma Co., St. Louis, MO, U.S.A.) for 1 hour to disperse cumulus cells.

The oocytes were obtained by separating such cumulus cells from the COCs. The separated cumulus cells were isolated through a mouth pipette and washed with the G1.2 medium. Those cumulus cells having a modal diameter ranging from 10 20 to 12 mm were selected as nuclear donor cells.

Example 2: Enucleation of oocyte and cell fusion One of the oocytes obtained in Example 1 was cultured in the G1.2 medium 27 for 1 to 2 hours in order to induce the maturation of its nucleus. Thereafter, enucleation, nuclear transfer and electrofiision thereof were performed as follows. (2-11 Enucleation of oocyte and nuclear transfer from somatic cell The oocyte was washed once with the G1.2 medium. Such oocyte was transferred to a hyaluronidase solution prepared by mixing lm& of the G1.2 medium with 111 fd of a solution, wherein 0.05g of hyaluronidase was dissolved in 5m£ of the G1.2 medium, and adjusted to a 0.1% (w/v) hyaluronidase concentration. The 10 oocyte was stripped of any remaining cumulus cells, washed three times with the G1.2 medium and placed in the same medium. Then, the oocyte was transferred to a cytochalasin B solution prepared by mixing lmfc of the G1.2 medium supplemented with 10% fetal bovine serum (FBS) with ltd of a solution wherein cytochalasin B was dissolved in dimethyl sulfoxide to a concentration of 7.5/zg/mL 15 The zona pellucida of the oocyte was incised by a micromanipulator to form a small hole, and the oocyte was enucleated by removing part of the cytoplasm containing the first polar body thereof and corresponding to 10 to 15% of the total cytoplasm through the small hole.

Fig. 3 shows the incision process of the zona pellucida of the oocyte (3) by 20 employing a holding pipette (1) and an incision pipette (2). Fig. 4 shows the enucleation process removing the first polar body and the nucleus from the oocyte where the oocyte (3) having the small hole vertically positioned was supported by the holding pipette (1) positioned beneath the oocyte and then lightly pressed by the incision pipette (2) to enucleate the same. Such enucleated oocyte was washed 28 three times with the G1.2 medium and placed in the same medium.

Subsequently, a nuclear donor cell in a 4jid drop of PBS supplemented with 1% BSA was transferred, using a holding pipette and a transfer pipette, into the enucleated oocyte in a 4fd drop of a solution prepared by mixing 400^0 of the 5 G1.2 medium with 100fd of a PHA-P solution wherein 5mg of PHA-P was dissolved in 10m£ of the G1.2 medium. Hie drops containing the nuclear donor cell and the enucleated oocyte were coated with a mineral oil to prevent the evaporation of the drops.

Fig. 5 describes the process used to transfer the nuclear donor cell into the enucleated oocyte. As can be seen from Fig. 5, the enucleated oocyte (3) was fixed . to a holding pipette (1), a transfer pipette (4) was injected through the small hole into the enucleated oocyte (3) and, then, the nuclear donor cell was injected into the oocyte (3) to obtain a nucleus-transferred oocyte. Such nucleus-transferred oocyte was washed three times with the G1.2 medium and placed in the same medium. (2-2) Preparation of nucleus-transferred oocyte bv electrofiision The nucleus-transferred oocyte was subjected to electrofiision through a BTX-electro cell manipulator (BTX Inc., San Diego, CA, U.S.A.).

A 20fd drop of a mannitol solution prepared by dissolving 0. ImM MgSC>4, 0.05% BSA and 0.28mM mannitol in a 0.5mM HEPES buffer (pH 7.2), a 20fd drop of a mixing solution containing 10 fd of the G1.2 medium and 10 fd of the mannitol solution, and a 20/id drop of the G1.2 medium were prepared.

First, the nucleus-transferred oocyte obtained in Example (2-1) was 29 incubated in the 20fd drop of the mixing solution for 1 minute. Next, the nucleus-transferred oocyte was transferred to the 20 fd drop of the mannitol solution via a mouth pipette and incubated therein for 1 minute. Subsequently, the nucleus-transferred oocyte was transferred to a mannitol solution having the above 5 composition and placed between two electrodes connected to the BTX-electro cell manipulator and was positioned such that the nuclear donor cell faced the (+) electrode. The nucleus-transferred oocyte was electrofused by applying a direct current of lkV/cm for 15|xs twice, at an interval of 1 second.

The fused nucleus-transferred oocyte was incubated in the 20 fd drop of the 10 mixing solution for 1 minute, transferred to the 20 fd drop of the G1.2 medium and then washed with the G1.2 medium three times.

Example 3: Reprogramming, activation and in vitro culturing of nucleus-transferred oocyte Since a sperm-mediated activation, which is one of the major factors for a normal embryonic development, was absent in case of the nucleus-transferred oocyte obtained in Example 2, an artificial stimulus was needed instead. In order to determine the optimum conditions for artificial embryogenesis, therefore, 20 nucleus-transferred. oocytes were reprogrammed, activated and in vitro cultured under various conditions as shown in Tables 2 to 4.

First, to examine the effect of the reprogramming time on the rate of blastocyst formation, the reprogramming times were set at about 2, 4, 6 and 20 hours, respectively, while applying the same conditions for activation and in vitro culturing as can be seen from Table 2. As a result, the highest rate of blastocyst formation was obtained when the reprogramming time was about 2 hours.

Table 2 Reprogramming time (hour) Activation condition In vitro con< culture ition No. of oocytes No. of ooc1 nucleus-transferred /tes developed to medium 2Bd medium 2-cell stage morula blastocyst 2 10pM ionophore* 2.0mM 6-DMAP GU SNUnt-2 16 16 4 4 4 10pM ionophore* 2.0mM 6-DMAP G1.2 SNUnt-2 16 1 0 6 lOyM ionophore* 2.0mM 6-DMAP G1.2 SNUnt-2 16 1 1 ;oM ionophore* 2.0mM 6-DMAP G1.2 SNUnt-2 16 9 1 0 * calcium ionophore A23187 Next, to find the optimal activation condition for blastocyst formation, nucleus-transferred oocytes subjected to about 2 hour-reprogramming time were treated for 5 minutes with calcium ionophore A23187 (5 or lOpM; Sigma Co., St Louis, MO, U.S.A.) or ionomycin (5 or IOjjM; Sigma Co., St. Louis, MO, U.S.A.) * • in the G1.2 medium at 37°C as can be seen from Table 3. Such nucleus-transferred oocytes were washed several times with the G1.2 medium, transferred to the G1.2 medium containing 2.0mM 6-DMAP (Sigma Co., St. Louis, MO, U.S.A.) and, then, cultured at 37*0, 5% CO2, 5% 02 and 90% N2 for 4 hours. After these 15 activation steps, the nucleus-transferred oocytes were in vitro cultured under the same condition. As can be seen from Table 3, the highest rate of blastocyst formation was observed when the oocyte was sequentially treated with 10yM calcium ionophore and 2.0mM 6-DMAP. 31 Table 3 Reprogramming time (hour) Activation condition In vitro culture condition No. of oocytes No. of nucleus-transferred oocytes developed to 1st medium 2ad medium 2-cell stage morula blastocyst 2 5fiM ionophore* 2.0mM 6-DMAP G1.2 SNUnt-2 16 11 0 0 2 lOyM ionophore* 2.0mM 6-DMAP G1.2 SNUnt-2 16 16 3 2 5fiM ionomycin 2.0mM 6-DMAP GU SNUnt-2 16 9 0 0 2 MM ionomycin 2.0mM 6-DMAP G1.2 SNUnt-2 16 12 0 0 * calcium ionophore A23187 Finally, the optimal in Vitro culture condition was determined as follows: The nucleus-transferred oocytes subjected to the above optimal reprogramming and activation conditions were washed vigorously with the G1.2 medium and cultured for 48 hours in a 10 fd drop of the G1.2 medium or SNUnt-2 medium at 37 °C in 5% C02, 5% 02 and 90% N2 atmosphere. After such culturing, the nucleus-transferred oocytes were transferred to 10 a fresh SNUnt-2 medium or G2.2 medium and cultured further for 6 days. A representative example of the in vitro culture medium is the G2.2 medium (Vitro Life, Goteborg, Sweden) comprising Alanine, Alanyl-glutamine, Arginine, Asparagine, Aspartic acid, Calcium chloride, Calcium pantothenate, Choline chloride, Cystine, Folic acid, Glucose, Glutamic acid, Glycine, Histidine, Human serum albumine, Inositol, Isoleucine, 15 Leucine, Lysine, Magnesium sulphate, Methionine, Nicotinamide, Penicillin G, ' Phenylalanine, Potassium chloride, Proline, Pyridoxal HCL, Riboflavin, Serine, Sodium bicarbonate, Sodium chloride, Sodium dihydrogen phosphate, Sodium lactate, Sodium pyruvate, Thiamine, Threonine, Tryptophan, Tyrosine, Valine and water. 32 As indicated in Table 4, the highest rate of blastocyst formation was detected when the oocyte was first cultured in the G1.2 medium and subsequently in the SNUnt-2 medium.

" Table4 Reprogramming time (hour) Activation condition In vitro corn culture ition No. of oocytes No. of nucleus-transferred oocytes developed to 1st medium 2nd medium 2-cell stage morula blastocyst 2 10pM ionophore* 2.0xnM 6-DMAP G1.2 SNUnt-2 16 16 4 3 2 10nM ionophore* 2.0mM 6-DMAP G1.2 G2.2 16 16 0 0 2 lOpM ionophore* 2.0mM 6-DMAP SNUnt-2 SNUnt-2 16 16 0 0 * calcium ionophore A23187 Based on the above results, an optimal embryogenesis of a nucleus-transferred oocyte was achieved by subjecting the oocyte to 2-hour reprogramming, 10 activation through a serial treatment with IOjjM calcium ionophore and 2.0mM 6-DMAP, and a sequential culturing in the G1.2 medium and the SNUnt-2 medium.

Under the above optimal conditions, additional 66 nucleus-transferred oocytes were reprogrammed, activated and in vitro cultured to thereby yield 19 blastocysts (equal to 29%). This percentage of the nucleus-transferred oocytes 15 developed to blastocysts in accordance with the present invention is comparable to those observed in established SCNT methods in cattle (about 25%) (Kwun et al., Mol. Reprod. Dev., 65:167-174 (2003)) and pigs (about 26%) (Hyun et al., Biol. Reprod., 69:1060-1068 (2003); Kuhholzer et al., Biol Reprod, 64:1635-1698 (2004)). 33 Example 4: Removal of zona pellucida and trophoblast, and isolation of ICMs The blastocyst obtained in Example 3 was treated with 0.1% pronase (Sigma Co., St Louis, MO, U.S.A.) for 1 minute to remove its zona pellucida. Then, it was treated with 100% anti-human serum antibody (Sigma Co., St. Louis, MO, U.S.A.) for 20 minutes, and was exposed to 10jtt6 of guinea pig complement (Life Technologies, Rockville, MD, U.S.A.) at 37°C, 5% C02for 30 minutes to 10 remove its trophoblast and isolate ICMs therefrom.

Example 5: Culturing of ICMs The ICMs isolated in Example 4 were cultured in a tissue culture dish 15 coated with 0.1% gelatin, which contained a feeder layer (7.5 x 104 cells/cm2) of mitomycin C-inactivated primary mouse (C57BL breed) embryonic / fibroblasts. DMEM/F12 medium (Life Technologies, Rockville, MD, U.S.A.) comprising 20% serum replacement, O.lmM p-mercaptoethanol, 1% NEAAs, 2mM glutamine, lOOunits/mft penicillin, and 100#g/m& streptomycin, and 4 ng/mfl bFGF 20 (Life Technologies, Rockville, MD, U.S.A.) was used as the culture medium.

At an early stage of culturing the ES cells in the ICMs, the medium was supplemented with a hLIF (100units/m4; Chemicon, Temecula, CA, U.S.A.). The culturing was conducted for more than 6 days until the colonies of undifferentiated ntES cells appeared. Hie ntES cells were mechanically isolated from the colonies 34 by using a micropipette every five or seven days after such colony formation.

The ntES cell line thus obtained from the nucleus-transferred oocyte prepared by transferring a nucleus of a female somatic cell into an enucleated human oocyte was designated "hntES" and deposited with the Korean Cell Line 5 Research Foundation (KCLRF; Address:. Cancer Research Institute, College of Medicine, Seoul National University, 28, Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea) on December 29, 2003 under the accession number of KCLRF-BP-00092, in accordance with the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of 10 Patent Procedure.

Test Example 1: Identification of human ntES cells obtained in Example 5 by karyotype analysis The colonies of the undifferentiated ntES cells obtained in Example 5 were washed with PBS containing O.lmM Ca2+ and O.lmM Mg2+, fixed with citrate-acetone-formaldehyde (the mixing ratio in volume was 25:65:8) at 4°C for 1 hour, and washed again with PBS containing O.lmM Ca2+ and O.lmM Mg2+. The alkaline phosphatase activity of the ntES cells was determined by AP kit (Sigma Co., 20 St. Louis, MO, U.S.A.). Further, an immunohistochemica! assay was performed in order to identify specific surface antigens on the ntES cells, by employing monoclonal antibodies Oct-4 (SC-5279) purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.SA..); SSEA-1 (MC480), SSEA-3 (MC63I) and SSEA-4 (MC-813-70) purchased from Developmental Studies Hybridoma Bank (Iowa City, IA, U.S.A.); and TRA-1-60 and TRA-1-80 purchased from Chemicon (Temecula, CA, U.S A.) as primary antibodies. Such primary antibodies were detected by using a Vectastatin ABC kit (Vector laboratory, Burlingame, CA, U.S.A.) containing a biotinylated secondary antibody and an avidin-horseradish peroxidase conjugate.

DNA fingerprinting analysis was performed with regard to the genomic DNA and human short tandem repeat (STR) marker using a STR AMP FLSTR PROFILER kit (Applied Biosystems, Foster City, CA, U.S A.) with an automated ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, U.S.A.). The results are shown in Figs. 6A to 6D.

As shown in Figs. 6A to 6D, it was observed that the karyotype of the ntES cells derived from the nucleus-transferred oocyte prepared in accordance with Examples 1 to 5 above was identical to that of the nuclear donor cell. This result demonstrates that the ntES cells of the present invention have been indeed derived from the nucleus-transferred oocyte prepared by transferring a nucleus of a female somatic cell into an enucleated human oocyte, not from a parthenogenetically activated oocyte.

Test Example 2: Identification of human ntES cells by teratoma anaylsis 100 colonies of the undifferentiated ntES cells obtained in Example 5 were isolated from their culture dish, injected into a testis of a SCID mouse (Korea Research Institute of Bioscience and Biotechnology, Korea) using a syringe and cultured for 8 weeks. Teratomas thus formed were paraffin-fixed and 36 examined by an immunohistochemical assay to check whether three dermal cells were formed. The result is shown in Fig. 7.

As indicated in Fig. 7, it was found that the ntES cells obtained in Example 5 formed three dermal cells (cartilage (A): endoderm; intestinal tract (B): 5 mesoderm; neural tube (C): ectoderm) in the testis. This result demonstrates that such ntES cells are pluripotent ES cells having the ability to differentiate into various tissues.

Test Example 3: Examination of embryoid body formation through 10 immunohistochemical assay Colonies of the human ntES cells obtained in Example 5 were treated with 0.1% tiypsin/lmM EDTA to isolate the ntES cells, which were then transferred to a plastic petri dish. Hie human ntES cells were cultured for 14 days in the 15 DMEM/DMEM F12 medium devoid of hLIF and bFGF. For paraffin fixation, such ntES cells were transferred to 1% low-melting temperature agarose dissolved in PBS and cooled to 42 "G. The resulting solidified agarose containing the ntES cells was fixed by 4% paraformaldehyde dissolved in PBS and embedded in paraffin. Each 6-mm section of the paraffin-embedded cells was placed on a slide 20 and subjected to an immunohistochemical analysis. As primary antibodies, alpha-1-fetoprotein (18-0003), cytokeratin (18-0234), desmin (18-0016), neurofilament (18-0171) and S-100 (18-0046) purchased from Zemed (South San Francisco, CA, U.S.A.) and HNF-2-alpha (SC-6556), BMP-4 (SC-6896), Myo D (SC-760) and NCAM (SC-7326) purchased from Santa Cruz Biotechnology (Santa Cruz, CA, 37 U.S A.) were employed. A biotinylated anti-rabbit, anti-mouse or anti-goat antibody was used as a secondary antibody, and Hie reaction was detected by streptavidin-conjugated horseradish peroxidase and diaminobenzidine chromagen. The result is shown in Fig. 8.

As shown in Fig. 8, it was confirmed that such ntES cells could form embryoid bodies based on the fact that the marker proteins of endoderm (i.e., alpha-1-fetoprotein (A), cytokeratin (B), and HNF-2-alpha (C)), the marker proteins of mesoderm (i.e., BMP-4 (D), Myo D (E), and desmin (F)) and the marker proteins of ectoderm (i.e., neurofilament (G), S-100 (H), and NCAM 00) were expressed in the 10 ntES cells obtained in Example 5. This result demonstrates that the cells obtained in the present invention fall within the scope of an ES cell.

Example 6: Differentiation into neuro progenitors 15 (6-1') Expansion of undifferentiated ES cells The human undifferentiated ntES cells obtained in Example 5 ware cultured at 37 °C in 5% C02 atmosphere on a mouse embiyonic fibroblast feeder layer with inactivated cell division, contained in a culture plate coated with 2% gelatin. The 20 culture medium was composed of DMEM/F12 (1:1), 20% knock-out serum replacement, O.lmM NEAAs, O.lmM p-mercaptoethanol, ImML-glutamine, 100U/ penicillin G, 100#g/m& streptomycin, and 4ng/m& bFGF; and was changed everyday. 38 (6-21 Formation of embryoid body Colonies of the ntES cells cultured as above were collected and cultured on anon-adhesive culture dish at 37°C in 5% C02 atmosphere. The culture medium 5 was identical to that of Example (6-1) except that 4ng/m& bFGF was omitted therefrom. After one day, such colonies began to grow as floating embryoid bodies (about 50 embryoid bodies/dish). At that point, the embryoid bodies were transferred to a new dish, while removing any remaining feeder cells completely. After further culturing for 4 days, embryoid bodies thus formed were plated on an 10 adhesive dish coated withpolyornithine/laminin. (6-31 Selection of nestin-positive cells After 1-day culturing on the adhesive dish, embryoid bodies in the process 15 of differentiation were transferred to the DMEM/F12 medium supplemented with insulin (25#g/mU), transferrin (lOOygM), sodium selenite (30nM) and fibronectin (5 jug/nd) and cultured at 37 "C for 6 days. The resulting cells were cultured at 37 V for 40 minutes in a solution wherein anti-nestin antibody (Chemicon, Temecula, CA, U.S.A.) was diluted 1000 folds with a solution containing 0.01M PBS, 1% BSA and 20 5mM EDTA. Such cells were washed with the DMEM/F12 medium, treated with phycoerythrine (PE)-conjugated secondary antibody (Chemicon, Temecula, CA, U.S.A.) for 30 minutes and then washed three times with the DMEMZF12 medium, thereby selecting the nestin-positive cells. 39 508165NZPR 301968990 (6-4) Expansion of nestin-positive cells The nestin-positive cells selected in Example (6-3) were cultured at 37 °C in the DMEM/F12 medium supplemented with the N-2 supplement, laminin (lng/ ml) and bFGF (lOngM) for 6 days to expand those cells. (6-5) Differentiation into neuro progenitors The nestin-positive cells expanded in Example (6-4) were cultured for 10 days at 37 °C in the DMEMZF12 medium supplemented with the N-2 supplement and laminin (lng/ra&) but devoid of bFGF to induce their differentiation into neuro progenitors.

Fig. 2 shows the neuro progenitors differentiated from the nucleus-transferred oocyte prepared by transferring a nucleus of a female somatic cell into an enucleated human oocyte.

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to". 40 The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in New Zealand.

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL 40A BUDAPEST TESATT ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS TOR THE FDBPOSB OF PATENT PROCEDDRK INTERNATIONAL FORM RECEPTION IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Hale 7.1 To: Hwang Woo Suk College of Veterinary Medicine, Seoul National Unlveraity San 56-1, ShUlim-dong, Gwanak-gti, Seoul 151-742, KOREA I. IDENTIFICATION OF THE MICROORGANISM Identification reference given "by the DEPOSITOR t hnffiS Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: KCLRF-BP-00Q92 IL SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microoiganlsm identified under I above was accompanied by : [x] A scientific description [x] A proposed texonomic designation (Matfc with a cross where applicable) HL RECEIPT AND ACCEPTANCE This International Deposltasy Authority accepts the microorganism identified nnder I above, which was received by It on December 29, 2003 IV. INTERNATIONAL DEPOSITARY AUTHORITY Name : Director tv Korean Cell line Foundation Signature^) : G-vA-V Address :Cancer Research Institute I Date : 2004. 1. 31.

Seoul National University College of Medicine 28 Y ongon-dong, Chongno-Gu Seoul, 110-744, Korea.

Rob BJ/4 (KCLKP V«u M) . Page sail 41 5081S5N2PR 301968998

Claims (3)

What is claimed is:

1. A medium comprising: 95 to HOmM NaCl; 7.0 to 7.5mM KC1; 20 to 30mM NaHC03; 1.0 to 1.5mM NaH2P04; 3 to 8mM sodium lactate; 1.5 to 2.0mM CaCl2-2H20; 0.3 to 0.8mM MgCl2'6H20; 0.2 to 0.4mM sodium pyruvate; 1.2 to 1.7mM fructose; 6 to 1 Oing/mi1, human serum albumin; 0.7 to 0.8//g/M kanamycin; 1.5 to 3% essential amino acids; 0.5 to 1.5% nonessential amino acids; 0.7 to 1.2mM L-glutamine; and 0.3 to 0.7% a mixture of insulin, transferrin and sodium selenite.

2. The medium of claim 1, which comprises: 99.1 to 106mM NaCl; 7.2mM KC1; 25mM NaHC03; 1.2mM NaH2P04; 5mM sodium lactate; 1.7mM CaCl2-2H20; 0.5mM MgCl2 6H20; 0.3mM sodium pyruvate; 1.5mM fructose; 8mg/m£ human serum albumin; 0.75/ig/M kanamycin; 2% essential amino acids; 1% nonessential amino acids; ImM L-glutamine; and 0.5% a mixture of insulin, transferrin and sodium selenite.

3. A medium as claimed in claim 1, substantially as hereinbefore described with a particular reference to any one or more of the Examples and/or Figures. 42