Mammalian embryonic stem cell isolated from a homogeneous pluripotent outgrowth of a mammalian pre-implantation embryo

Abstract

The present invention relates to a method of isolating a pluripotent cell from a pre-implantation embryo including one or more pluripotent cells. The method includes propagating the one or more pluripotent cells from the embryo under conditions that allow undifferentiated growth of the one or more pluripotent cells and do not allow growth of non-pluripotent cells from the embryo.

1 - 110 . (canceled) 
     
     
         111 . A mammalian pluripotent embryonic stem cell, wherein the stem cell is isolated from a mammalian pre-implantation embryo which includes one or more pluripotent embryonic stem cells, and wherein the stem cell is isolated from the mammalian pre-implantation embryo by a method comprising the steps of:
 (i) selecting the pre-implantation embryo in a blastocyst stage of development, wherein the inner cell mass of the pre-implantation embryo has yet to differentiate into the epiblast and hypoblast;   (ii) depressing, embedding or flattening all of the pre-implantation embryo into a feeder cell layer causing all cells of the embryo to make contact with the feeder cell layer;   (iii) culturing the depressed, embedded or flattened pre-implantation embryo in a medium that is free of serum to (a) inhibit differentiation of the one or more pluripotent embryonic stem cells and allow undifferentiated growth of the one or more pluripotent embryonic stem cells resulting in formation of a homogeneous pluripotent embryonal outgrowth and (b) not allow growth of non-pluripotent cells from the pre-implantation embryo; and   (iv) isolating a mammalian pluripotent embryonic stem cell from the one or more pluripotent embryonic stem cells.   
     
     
         112 . The mammalian pluripotent embryonic stem cell of  claim 111 , wherein the medium includes a serum replacement and/or albumin. 
     
     
         113 . The mammalian pluripotent embryonic stem cell of  claim 112 , wherein the medium includes 5 to 25% serum replacement. 
     
     
         114 . The mammalian pluripotent embryonic stem cell of  claim 111 , wherein the medium includes L-glutamine, ribonucleosides, deoxyribonucleosides, sodium pyravate, bFGF, hrEGF, 1× ITS, β-mercaptoethanol, non-essential amino acids and hrLIF. 
     
     
         115 . The mammalian pluripotent embryonic stem cell of  claim 111 , wherein the method does not include isolation of the pluripotent embryonic stem cell away from other cells. 
     
     
         116 . The mammalian pluripotent embryonic stem cell of  claim 111 , wherein the method further comprises the step of establishing a homogeneous primary culture of cells from the pre-implantation embryo, the primary culture including the one or more pluripotent embryonic stem cells. 
     
     
         117 . The mammalian pluripotent embryonic stem cell of  claim 111 , wherein the feeder cell layer is treated to inhibit proliferation of the feeder cells. 
     
     
         118 . The mammalian pluripotent embryonic stem cell of  claim 111 , wherein the zona pellucida of the pre-implantation embryo is removed prior to depressing, embedding or flattening all of the pre-implantation embryo into the feeder cell layer. 
     
     
         119 . The mammalian pluripotent embryonic stem cell of  claim 111 , wherein the one or more pluripotent embryonic stem cells are cultured until the one or more pluripotent embryonic stem cells form a colony of cells. 
     
     
         120 . The mammalian pluripotent embryonic stem cell of  claim 119 , wherein the colony is substantially free of trophectoderm cells, differentiated cells and/or cells committed to differentiate. 
     
     
         121 . The mammalian pluripotent embryonic stem cell of  claim 111 , wherein the blastocyst is a blastocyst produced in vitro or is a blastocyst derived in vivo. 
     
     
         122 . The mammalian pluripotent embryonic stem cell of  claim 111 , wherein the pluripotent embryonic stem cell is free of non-apoptotic non-pluripotent cells derived from the embryo. 
     
     
         123 . A multipotent or differentiated cell derived from the pluripotent embryonic stem cell of  claim 111 . 
     
     
         124 . A mammalian pluripotent embryonic stem cell line, wherein the stem cell line is established from a mammalian pre-implantation embryo, and wherein the stem cell line is established by a method comprising the steps of:
 (i) isolating one or more pluripotent embryonic stem cells from the mammalian pre-implantation embryo which comprises one or more pluripotent embryonic stem cells, comprising the substeps of:
 (A) selecting the mammalian pre-implantation embryo in a blastocyst stage of development, wherein the inner cell mass of the preimplantation embryo has yet to differentiate into the epiblast and hypoblast; 
 (B) depressing, embedding or flattening all of the pre-implantation embryo into a feeder cell layer causing all cells of the embryo to make contact with the feeder cell layer; 
 (C) culturing the depressed, embedded or flattened pre-implantation embryo in a medium that is free of serum to (a) inhibit differentiation of the one or more pluripotent embryonic stem cells and allow undifferentiated growth of the one or more pluripotent embryonic stem cells resulting in formation of a homogeneous pluripotent embryonal outgrowth and (b) not allow growth of non-pluripotent cells from the pre-implantation embryo; and 
 (D) isolating one or more pluripotent embryonic stem cells from the one or more pluripotent embryonic stem cells; and 
   (ii) culturing the one or more isolated pluripotent embryonic stem cells in a medium that is free of serum to establish the pluripotent embryonic stem cell line.