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.

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 a blastocyst derived from a human somatic cell and a human oocyte, comprising: 
 (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; and    (4) subjecting the nucleus-transferred oocyte to reprogramming, activation and in vitro culturing to form a blastocyst.    
   
   
       4 . The method of  claim 45 , wherein the embryonic stem cell line is a cell line deposited under the accession number of KCLRF-BP-00092.  
   
   
       5 . The method of  claim 45 , wherein the reprogramming in step (4) is conducted for a time period of up to 20 hours.  
   
   
       6 . The method of  claim 45 , wherein the reprogramming in step (4) is conducted for a time period of up to 6 hours.  
   
   
       7 . The method of  claim 45 , wherein the reprogramming in step (4) is conducted for a time period of up to 3 hours.  
   
   
       8 . The method of  claim 45 , wherein the reprogramming in step (4) is conducted for a time period of about 2 hours.  
   
   
       9 . The method of  claim 45 , 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 45 , 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 45 , 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 45 , 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 45 , 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: 
 (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 μg/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  comprising: 
 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 acnonessential amino aciL-glutamine; and mixture of insulin, transferrin and sodium selenite.    
   
   
       27 . The method according to  claim 3 , wherein the step (2) comprises incising a part of zona pellucida of the oocyte and removing the cytoplasm containing first polar body by pressing the oocyte.  
   
   
       28 . The method according to  claim 27 , wherein the step (2) further comprises removing surrounding cumulus cells from the oocyte before the incising and the removing.  
   
   
       29 . The method according to  claim 3 , wherein the step (2) comprises holding said human oocyte with a holding pipette; incising a part of zona pellucida of the oocyte with an incision pipette; removing first polar body and nucleus from the oocyte supported by the holding pipette through a hole made by the incision process by pressing the oocyte with the incision pipette.  
   
   
       30 . The method according to  claim 3 , wherein the step (2) comprises removing part of cytoplasm containing first polar body corresponding to 10 to 15 percent of total cytoplasm.  
   
   
       31 . The method according to  claim 3 , wherein the reprogramming in step (4) is conducted for a time period of up to 20 hours.  
   
   
       32 . The method according to  claim 3 , wherein the reprogramming in step (4) is conducted for a time period of up to 6 hours.  
   
   
       33 . The method according to  claim 3 , wherein the reprogramming in step (4) is conducted for a time period of up to 3 hours.  
   
   
       34 . The method according to  claim 3 , wherein the reprogramming in step (4) is conducted for a time period of up to 2 hours.  
   
   
       35 . The method according to  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.  
   
   
       36 . The method of  claim 35 , wherein the concentration of the calcium ionophore ranges from 5 μM to 15 μM.  
   
   
       37 . The method of  claim 36 , wherein the concentration of the calcium ionophore is about 10 μM.  
   
   
       38 . The method of  claim 35 , wherein the concentration of 6-dimethylaminopurine ranges from 1.5 mM to 2.5 mM.  
   
   
       39 . The method of  claim 38 , wherein the concentration of 6-dimethylaminopurine is about 2.0 mM.  
   
   
       40 . 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.  
   
   
       41 . The method of  claim 40 , wherein the in vitro culturing is performed by sequentially using two media having different compositions from each other.  
   
   
       42 . The method of  claim 41 , wherein the in vitro culturing is performed by sequentially using G1.2 medium and SNUnt-2 medium.  
   
   
       43 . 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 G1.2 medium and SNUnt-2 medium.  
   
   
       44 . A blastocyst prepared by the method according to  claim 3 .  
   
   
       45 . A method for preparing an embryonic stem cell comprising: 
 (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.    
   
   
       46 . The method according to  claim 45 , wherein the step (2) comprises incising a part of zona pellucida of the oocyte and removing the cytoplasm containing first polar body by pressing the oocyte.  
   
   
       47 . The method according to  claim 44 , wherein the step (2) further comprises removing surrounding cumulus cells from the oocyte before the incising and the removing.  
   
   
       48 . The method according to  claim 45 , wherein the step (2) comprises holding said human oocyte with a holding pipette; incising a part of zona pellucida of the oocyte with an incision pipette; removing first polar body and nucleus from the oocyte supported by the holding pipette through a hole made by the incision process by pressing the oocyte with the incision pipette.  
   
   
       49 . The method according to  claim 45 , wherein the step (2) comprises removing part of cytoplasm containing first polar body corresponding to 10 to 15% of total cytoplasm.  
   
   
       50 . A medium for use in carrying out the in vitro culturing in step (4) of  claim 45 , 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 μg/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.    
   
   
       51 . A stem cell line prepared by the method according to  claim 3.