US2019218512A1PendingUtilityA1

Methods for producing mammalian pluripotent stem cell-derived endodermal cells

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Assignee: TAKARA BIO EUROPE ABPriority: Nov 29, 2012Filed: Jan 25, 2019Published: Jul 18, 2019
Est. expiryNov 29, 2032(~6.3 yrs left)· nominal 20-yr term from priority
Inventors:Barbara Küppers-MuntherJosefina Edsbagge
C12N 2506/03C12N 2506/45C12N 2501/70C12N 2501/999C12N 2501/06C12N 5/067C12N 5/0606C12N 5/0607C12N 2501/727C12N 2506/02C12N 2501/16
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Claims

Abstract

The present invention relates to the directed differentiation of mammalian pluripotent stem cells, especially human pluripotent stem (hPS) cells, into endodermal cells. In particular, the present invention relates to the treatment of mammalian pluripotent stem cells, especially hPS cells, with a DNA demethylating agent while undergoing differentiation into endodermal. The inventors have, as disclosed herein, found that exposing differentiating mammalian pluripotent stem cells, especially hPS cells, to a DNA demethylating agent leads to an improved morphology and improved yield of endodermal cells. The treatment with a DNA demethylating agent also leads to a significant down-regulation of expression of the stem cell marker Oct4 and to an improved expression of endoderm specific markers, notably sox17, cxcr4 and hhex.

Claims

exact text as granted — not AI-modified
1 . A composition comprising at least one DNA demethylating agent and activin. 
     
     
         2 . The composition according to  claim 1 , wherein the DNA demethylating agent is a cytidine analogue. 
     
     
         3 . The composition according to  claim 1 , wherein the DNA demethylating agent is selected from the group consisting of 5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine), zebularine, Pseudoisocytidine, 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine, 2′,2′-Difluoro-deoxycytidine (gemcitabine), or Cytosine-beta-D-arabinofurasonide. 
     
     
         4 . The composition according to  claim 1 , wherein the DNA demethylating agent is selected from the group consisting of 5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine), 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine and 2′,2′-Difluoro-deoxycytidine (gemcitabine). 
     
     
         5 . The composition according to  claim 1 , wherein the DNA demethylating agent is 5-aza-2-deoxycytidine (decitabine). 
     
     
         6 . The composition according to  claim 1 , wherein the DNA demethylating agent is 5-azacytidine (azacitidine). 
     
     
         7 . The composition according to  claim 1 , wherein the activin is activin A or activin B. 
     
     
         8 . The composition according to  claim 1 , further comprising one or more growth factors and/or serum. 
     
     
         9 . The composition according to  claim 8 , wherein the one or more growth factors is selected from the group consisting of FGF1, FGF2 and FGF4. 
     
     
         10 . A kit comprising at least one DNA demethylating agent and activin. 
     
     
         11 . The kit according to  claim 10 , wherein the DNA demethylating agent is a cytidine analogue. 
     
     
         12 . The kit according to  claim 10 , wherein the DNA demethylating agent is selected from the group consisting of 5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine), zebularine, Pseudoisocytidine, 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine, 2′,2′-Difluoro-deoxycytidine (gemcitabine), or Cytosine-beta-D-arabinofurasonide. 
     
     
         13 . The kit according to  claim 10 , wherein the DNA demethylating agent is selected from the group consisting of 5-aza-2-deoxycytidine (decitabine), 5-azacytidine (azacitidine), 5-fluoro-2-deoxycytidine, 5,6-dihydro-5-azacytidine, 2′-deoxy-5,6-dihydro-5-azacytidine, 6-azacytidine and 2′,2′-Difluoro-deoxycytidine (gemcitabine). 
     
     
         14 . The kit according to  claim 10 , wherein the DNA demethylating agent is 5-aza-2-deoxycytidine (decitabine). 
     
     
         15 . The kit according to  claim 10 , wherein the DNA demethylating agent is 5-azacytidine (azacitidine). 
     
     
         16 . The kit according to  claim 10 , wherein the activin is activin A or B. 
     
     
         17 . The kit according to  claim 10 , further comprising one or more growth factors and/or serum. 
     
     
         18 . The kit according to  claim 17 , wherein the one or more growth factors is selected from the group consisting of FGF1, FGF2 and FGF4. 
     
     
         19 . The kit according to  claim 10 , further comprising mammalian pluripotent stem cells. 
     
     
         20 . The kit according to  claim 19 , wherein the mammalian pluripotent stem cells are human pluripotent stem cells.

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