Process for the multiplication of stem cells

Abstract

The use of a cellular development inhibitor in a controlled manner in order to maintain an undifferentiated stem cell state, especially one of human stem cells, whereby cell division is permitted.

1 . A method for maintaining human embryonic stem cells in a non-differentiated state while allowing cell division of the stem cells, the method comprising
 repeatedly administering to the stem cells an effective amount of an inhibitor of cell development in sequential combination with an anti-inhibitor of cell proliferation in a controlled manner to maintain the stem cells in a non-differentiated state while allowing cell division of the stem cells, until amplification of the stem cells is sufficient to obtain a desired number of cells,   wherein the inhibitor of cell development is TGFP in an amount of 0.01 pg/ml to 1 mg/ml and the anti-inhibitor of cell proliferation is anti-TGFβ in an amount of 0.1 μg/ml to 10 mg/ml.   
   
   
       2 . The method according to  claim 1 , wherein the human stem cells are in a cell concentration in a range from about 1 to about 10 10  cells per ml. 
   
   
       3 . The method according to  claim 1 , further comprising the following steps:
 a) initiating a first cycle of cell division of the stem cells, by seeding the stem cells in a resting state in the presence of one or more cytokines, and neutralizing the effect of an inhibitor of cell development present in the culture medium so that the stem cells leave their resting state by initiation of a first cell division,   b) returning the stem cells to a resting state by treating the cells with an inhibitor of cell development,   c) repeating the cycles of division and resting described in steps a) and b) until the amplification of the stem cells is sufficient to obtain the desired number of stem cells, and then   d) stopping the multiplication of the non-differentiated stem cells.   
   
   
       4 . The method according to  claim 3 , further comprising washing the stem cells obtained in step b) to remove catabolites and the inhibitor of cell development, prior to step c). 
   
   
       5 . The method according to  claim 3 , further comprising diluting the stem cells obtained in step b) to maintain a stem cell concentration in a range from about 100 to 10 10  stem cells per ml, prior to step c). 
   
   
       6 . The method according to  claim 3 , further comprising e) storing the stem cells, using the stem cells, and/or causing the stem cells to differentiate in vitro. 
   
   
       7 . The method according to  claim 3 , wherein the inhibitor of cell development in step b) is synthesized by the stem cells and/or is added to the culture medium. 
   
   
       8 . The method according to  claim 3 , wherein neutralizing the effect of the inhibitor of cell development present in the culture medium is effected by
 adding a suitable amount of an anti-inhibitor of cell proliferation to the cell culture medium, and   removing the inhibitor of cell development from the culture medium.   
   
   
       9 . The method according to  claim 3 , wherein the duration of a single resting state is in a range from about 1 hour to 3 years. 
   
   
       10 . The method according to  claim 3 , wherein the duration of a single division cycle is in a range from about 6 hours to 3 years. 
   
   
       11 . The method according to  claim 3 , wherein the number of cell divisions is in a range from 1 to about 100. 
   
   
       12 . The method according to  claim 3 , wherein the desired number of stem cells is in a range from about 2 times to about 10 12  times the number of initially seeded stem cells. 
   
   
       13 . The method according to  claim 1 , wherein the human embryonic stem cells are at the origin of somatic stem cells.