Embryonic stem cell line and method for preparing the same

Abstract

An embryonic stem cell line derived from a nucleus-transferred oocyte prepared by transferring a nucleus of a human somatic cell into an enucleated human oocyte may differentiate into various desired cell types.

What is claimed is: 
     
         1 . An embryonic stem cell line derived from a nucleus-transferred oocyte prepared by transferring a nucleus of a human somatic cell into an enucleated human oocyte. 
     
     
         2 . The embryonic stem cell line of  claim 1 , which is a cell line deposited under the accession number of KCLRF-BP-00092. 
     
     
         3 . A method for preparing an embryonic stem 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   (5) isolating an inner cell mass from the blastocyst and culturing the inner cell mass in an undifferentiated state to establish the embryonic stem cell line.   
     
     
         4 . The method of  claim 3 , wherein the embryonic stem cell line is a cell line deposited under the accession number of KCLRF-BP-00092. 
     
     
         5 . The method of  claim 3 , wherein the reprogramming in step (4) is conducted for a time period of up to 20 hours. 
     
     
         6 . The method of  claim 3 , wherein the reprogramming in step (4) is conducted for a time period of up to 6 hours. 
     
     
         7 . The method of  claim 3 , wherein the reprogramming in step (4) is conducted for a time period of up to 3 hours. 
     
     
         8 . The method of  claim 3 , wherein the reprogramming in step (4) is conducted for a time period of about 2 hours. 
     
     
         9 . The method of  claim 3 , wherein the activation in step (4) is performed by treating the nucleus-transferred oocyte with a calcium ionophore and subsequently with 6-dimethylaminopurine. 
     
     
         10 . The method of  claim 9 , wherein the concentration of the calcium ionophore ranges from 5 μM to 15 μM. 
     
     
         11 . The method of  claim 9 , wherein the concentration of the calcium ionophore is about 10 μM. 
     
     
         12 . The method of  claim 9 , wherein the concentration of 6-dimethylaminopurine ranges from 1.5 mM to 2.5 mM. 
     
     
         13 . The method of  claim 9 , wherein the concentration of 6-dimethylaminopurine is about 2.0 mM. 
     
     
         14 . The method of  claim 3 , wherein the in vitro culturing in step (4) is performed by sequentially using at least two media, each having a different composition from the other. 
     
     
         15 . The method of  claim 14 , wherein the in vitro culturing is performed by sequentially using two media having different compositions each other. 
     
     
         16 . The method of  claim 15 , wherein the in vitro culturing is performed by sequentially using the G1.2 medium and the SNUnt-2 medium. 
     
     
         17 . The method of  claim 3 , wherein step (4) is performed by reprogramming the nucleus-transferred oocyte for a time period of up to 20 hours, treating the nucleus-transferred oocyte with a calcium ionophore at a concentration ranging from 5 μM to 15 μM and subsequently with 6-dimethylaminopurine at a concentration ranging from 1.5 mM to 2.5 mM, and sequentially culturing the nucleus-transferred oocyte in vitro in the G1.2 medium and the SNUnt-2 medium. 
     
     
         18 . The method of  claim 3 , wherein the inner cell mass is isolated from the blastocyst in step (5) by a process comprising the steps of:
 (1) removing the zona pellucida or part thereof from the blastocyst; and   (2) isolating the inner cell mass by removing the trophoblast from the resulting blastocyst.   
     
     
         19 . The method of  claim 3 , wherein the inner cell mass is cultured in step (5) on a feeder layer comprising a cell differentiated from the embryonic stem cell line of  claim 1 . 
     
     
         20 . A neuro progenitor differentiated from an embryonic stem cell line derived from a nucleus-transferred oocyte prepared by transferring a nucleus of a human somatic cell into an enucleated human oocyte. 
     
     
         21 . The neuro progenitor of  claim 20 , wherein the embryonic stem cell line is a cell line deposited under the accession number of KCLRF-BP-00092. 
     
     
         22 . A method for preparing the neuro progenitor of  claim 20 , comprising the steps of:
 (1) culturing the embryonic stem 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.   
     
     
         23 . The method of  claim 22 , wherein the embryonic stem cell line is a cell line deposited under the accession number of KCLRF-BP-00092. 
     
     
         24 . The method of  claim 22 , wherein the agent employed in step (2) is selected from the group consisting of retinoic acid; ascorbic acid; nicotinamide; N-2 supplement; B-27 supplement; and a mixture of insulin, transferrin, sodium selenite and fibronectin. 
     
     
         25 . A medium for use in carrying out the in vitro culturing in step (4) of  claim 3 , comprising:
 95 to 110 mM NaCl; 7.0 to 7.5 mM KCl; 20 to 30 mM NaHCO 3 ; 1.0 to 1.5 mM NaH 2 PO 4 ; 3 to 8 mM sodium lactate; 1.5 to 2.0 mM CaCl 2 .2H 2 O; 0.3 to 0.8 mM MgCl 2 .6H 2 O; 0.2 to 0.4 mM sodium pyruvate; 1.2 to 1.7 mM fructose; 6 to 10 mg/ml human serum albumin; 0.7 to 0.8 mg/ml kanamycin; 1.5 to 3% essential amino acids; 0.5 to 1.5% nonessential amino acids; 0.7 to 1.2 mM L-glutamine; and 0.3 to 0.7% a mixture of insulin, transferrin and sodium selenite.   
     
     
         26 . The medium of  claim 25 , which comprises:
 99.1 to 106 mM NaCl; 7.2 mM KCl; 25 mM NaHCO 3 ; 1.2 mM NaH 2 PO 4 ; 5 mM sodium lactate; 1.7 mM CaCl 2 .2H 2 O; 0.5 mM MgCl 2 .6H 2 O; 0.3 mM sodium pyruvate; 1.5 mM fructose; 8 mg/ml human serum albumin; 0.75 μg/ml kanamycin; 2% essential amino acids; 1% nonessential amino acids; 1 mM L-glutamine; and 0.5% a mixture of insulin, transferrin and sodium selenite.