Alternative Compositions and Methods for the Culture of Stem Cells

Abstract

Methods and cell culture medium for the generation and maintenance of human pluripotent embryonic stem cells are disclosed. Human embryonic stem cells are cultured with human feeder cell conditioned medium, and the embryonic stem cells maintain their pluripotent phenotype. The human pluripotent embryonic stem cells can be cultured without feeder cells, and in the presence of supplemental growth factors.

1 . A human pluripotent stem cell culture, comprising a human embryonic stem cell and a human feeder cell conditioned medium, wherein the human feeder cell conditioned medium maintains the human embryonic stem cell in an undifferentiated state for at least two passages. 
     
     
         2 . The human pluripotent stem cell culture of  claim 1 , wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 
     
     
         3 . The human pluripotent stem cell culture of  claim 2 , wherein the fibroblast cell is a fetal skin fibroblast cell. 
     
     
         4 . The human pluripotent stem cell culture of  claim 2 , wherein the fibroblast cell is a skin keloid fibroblast. 
     
     
         5 . The human pluripotent stem cell culture of  claim 4 , wherein the skin keloid fibroblast cell is ATCC Deposit Number CRL-1762. 
     
     
         6 . The human pluripotent stem cell culture of  claim 2 , wherein the human feeder cell is a bone marrow stromal cell. 
     
     
         7 . The human pluripotent embryonic stem cell culture of claim  22 , wherein the bone marrow stromal cell is ATCC Deposit Number CRL-11882. 
     
     
         8 . The human pluripotent stem cell culture of  claim 1 , wherein the human pluripotent embryonic stem cell culture further comprises one or more supplemental growth factors. 
     
     
         9 . The human pluripotent stem cell culture of  claim 8 , wherein one or more supplemental growth factors are selected from the group consisting of SCF, OSM, CNTF, IL-6, IL-6R, FGF, BMP, TNF, and GM-CSF. 
     
     
         10 . A tissue generated from the cell culture of  claim 1 . 
     
     
         11 . A method of maintaining a human pluripotent embryonic stem cell culture in an undifferentiated state for at least two passages, comprising culturing a human embryonic stem cell in a human feeder cell conditioned medium. 
     
     
         12 . The method of  claim 11 , wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 
     
     
         13 . The method of  claim 12 , wherein the wherein the human feeder cell is a fetal skin fibroblast cell. 
     
     
         13 . The method of  claim 11 , comprising the steps of:
 a) isolating cells from the inner cell mass of a blastocyst;   b) plating the inner cell mass cells, wherein inner cell mass-derived cell masses are formed;   c) re-plating the human embryonic stem cell colony in the absence of a feeder cell; and   d) adding a human feeder cell conditioned medium;   
       thereby maintaining a human pluripotent embryonic stem cell in an undifferentiated state for at least two passages. 
     
     
         14 . The method of  claim 13 , wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 
     
     
         15 . The method of  claim 13 , wherein the wherein the human feeder cell is a fetal skin fibroblast cell. 
     
     
         16 . The method of  claim 13 , wherein the inner cell mass-derived cells are dissociated into clusters, and re-plated on a human feeder cell, and a colony is selected with the characteristics of a human embryonic stem cell prior to re-plating the selected human embryonic stem cell colony and adding a human feeder cell conditioned medium. 
     
     
         17 . A method of isolating and maintaining a human pluripotent embryonic stem cell culture, comprising the steps of:
 a) isolating cells from the inner cell mass of a blastocyst;   b) plating the inner cell mass cells on a human feeder cell, wherein inner cell mass-derived cell masses are formed;   c) re-plating the colony in the absence of a feeder cell; and   d) adding a human feeder cell conditioned medium to thereby isolate and maintain a human pluripotent embryonic stem cell in an undifferentiated state for at least two passages.   
     
     
         18 . The method of  claim 17 , wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell. 
     
     
         19 . The method of  claim 17 , wherein the human feeder cell is a fetal skin fibroblast cell. 
     
     
         20 . The method of  claim 17 , wherein the inner cell mass-derived cells are dissociated into clusters, and re-plated on a human feeder cell, and a colony is selected with the characteristics of a human embryonic stem cell prior to re-plating the selected human embryonic stem cell colony and adding a human feeder cell conditioned medium.