Multipotent/pluripotent cells and methods

Abstract

Described herein are multipotent stem cells, e.g., human and other mammalian pluripotent stem cells, and related methods.

1 . A human stem cell that is pluripotent, somatic, non-embryonic, and having the property of long-term self renewal. 
     
     
         2 - 132 . (canceled) 
     
     
         133 . A method of creating a clonal culture of human pluripotent stem cells, comprising the steps of:
 providing a culture of human pluripotent stem cells in culture in a culture medium in a culture vessel;   selecting a single cell from the stem cells in culture; and   initiating a new culture of the stem cells in a culture vessel containing a matrix with the single cell in a defined culture medium capable of supporting growth of pluripotent cells, the culture medium including an agent selected to increase the cloning efficiency of the stem cell culture relative to a stem cell culture that does not contain the agent, wherein the agent is a small molecule and wherein the agent is a kinase inhibitor.   
     
     
         134 . A method as claimed in  claim 133 , wherein the agent is a Rho-associated protein kinase (ROCK) inhibitor. 
     
     
         135 . A method as claimed in  claim 133 , wherein the agent is 
       
         
           
           
               
               
           
         
       
     
     
         136 . A method as claimed in  claim 133 , wherein the pluripotent stem cells are induced pluripotent stem cells. 
     
     
         137 . A method of creating a clonal culture of human pluripotent stem cells, comprising the steps of:
 providing a culture of human pluripotent stem cells in culture in a defined medium capable of supporting growth of pluripotent cells in a culture vessel;   selecting a single cell from the stem cells in culture; and   initiating a new culture of the human pluripotent stem cells in the defined medium in a culture vessel containing a matrix with the single cell, the culture medium including an agent selected to increase the cloning efficiency of the stem cell culture relative to a stem cell culture that does not contain the agent, the agent being selected from the group consisting of   
       
         
           
           
               
               
           
         
       
     
     
         138 . A method as claimed in  claim 137 , wherein the pluripotent stem cells are induced pluripotent stem cells. 
     
     
         139 . A human pluripotent stem cell culture, comprising:
 a culture vessel containing a matrix;   a defined culture medium capable of supporting growth of pluripotent cells contained in the vessel;   human pluripotent stem cells growing in the culture medium; and   the culture medium containing an agent that is a kinase inhibitor.   
     
     
         140 . A human pluripotent stem cell culture according to  claim 139 , wherein the kinase inihibitor is: 
       
         
           
           
               
               
           
         
       
     
     
         141 . A human pluripotent stem cell culture of  claim 139 , wherein the pluripotent stem cells are induced pluripotent stem cells. 
     
     
         142 . An improvement in a medium in which to culture human pluripotent stem cells, the improvement comprising including in the medium an agent selected to increase the cloning efficiency of the stem cell culture, wherein the agent is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
     
     
         143 . A method of culturing human pluripotent stem cells, comprising the steps of:
 (a) culturing human pluripotent stem cells in a culture medium in a culture vessel;   (b) selecting a single cell from the human pluripotent stem cells in culture; and   (c) initiating a new culture of the single cell in a culture vessel, wherein the culture vessel contains a matrix and a culture medium capable of supporting growth of human pluripotent cells, and wherein the culture medium includes an agent that is a kinase inhibitor.   
     
     
         144 . A method as claimed in  claim 142 , wherein the agent is a rho-associated protein kinase (ROCK) inhibitor. 
     
     
         145 . A method as claimed in  claim 142 , wherein the agent is HA-1077. 
     
     
         146 . A method as claimed in  claim 142 , wherein the pluripotent stem cells are induced pluripotent stem cells. 
     
     
         147 . A method of culturing human pluripotent stem cells, comprising the steps of:
 (a) culturing human pluripotent stem cells in a defined medium capable of supporting growth of pluripotent cells in a culture vessel;   (b) selecting a single cell from the stem cells in culture; and   (c) initiating a new culture of the single cell in a culture vessel, wherein the culture vessel contains a matrix and a culture medium capable of supporting growth of human pluripotent cells, and wherein the culture medium includes an agent that is selected from the group consisting of HA-1077 and Y-27632.   
     
     
         148 . A method as claimed in  claim 146 , wherein the human pluripotent stem cells are induced pluripotent stem cells. 
     
     
         149 . A human pluripotent stem cell culture comprising:
 (a) a culture vessel containing a matrix;   (b) a culture medium capable of supporting growth of pluripotent cells contained in the vessel;   (c) human pluripotent stem cells growing in the culture medium; and   (d) the culture medium contains a kinase inhibitor.   
     
     
         150 . A human pluripotent stem cell culture of  claim 148 , wherein the kinase inhibitor is HA-1077. 
     
     
         151 . A human pluripotent stem cell culture of  claim 148 , wherein the human pluripotent stem cell culture comprises induced pluripotent stem cells. 
     
     
         152 . An improvement in a medium in which to culture human pluripotent stem cells, the improvement comprising including in the medium an agent selected to increase the cloning efficiency of the stem cell culture, wherein the agent is selected from the group consisting of HA-1077 and Y-27632. 
     
     
         153 . A method of culturing human pluripotent stem cells, comprising the steps of:
 (a) culturing pluripotent stem cells in a culture medium in a culture vessel;   (b) selecting a single cell from the pluripotent stem cells in culture; and   (c) initiating a new culture of the single cell in a culture vessel containing a substrate, wherein the substrate is coated with fibronectin, gelatin, matrigel™ (BD Bioscience), collagen, or laminin, wherein the single cell is cultured in a culture medium that is capable of supporting growth of pluripotent cells, and wherein the culture medium includes a kinase inhibitor.   
     
     
         154 . The method of  claim 153 , wherein the culture medium is capable of supporting the growth of human pluripotent cells. 
     
     
         155 . The method of  claim 153 , wherein the kinase inhibitor is a Rho-associated kinase (ROCK) inhibitor. 
     
     
         156 . The method of  claim 153 , wherein the kinase inhibitor is selected from the group consisting of Y-27632 and HA-1077. 
     
     
         157 . The method of  claim 153 , wherein the kinase inhibitor is Y-27632. 
     
     
         158 . The method of  claim 155 , wherein the kinase inhibitor is HA-1077. 
     
     
         159 . The method of  claim 155 , wherein the pluripotent stem cells are induced pluripotent stem cells.