US2020190477A1PendingUtilityA1

Efficient induction of definitive endoderm from pluripotent stem cells

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Assignee: NOVO NORDISK ASPriority: Jun 21, 2011Filed: Nov 25, 2019Published: Jun 18, 2020
Est. expiryJun 21, 2031(~4.9 yrs left)· nominal 20-yr term from priority
Inventors:Nina FunaKatja HessJenny EkbergHenrik Semb
C12N 5/0606C12N 5/0678C12N 5/0696C12N 2506/02A61P 3/08C12N 5/0676C12N 2501/16C12N 2501/115C12N 2501/40
59
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Claims

Abstract

The present invention relates to a method to differentiate pluripotent stem cells to a primitive streak cell population, in a stepwise manner for further maturation to definitive endoderm.

Claims

exact text as granted — not AI-modified
1 . A method for differentiation of stem cells into definitive endoderm comprising the steps of:
 incubating stem cells in a medium comprising at least 2 μM CHIR, wherein activin A is not present; and   subsequently incubating stem cells in a medium comprising activin A.   
     
     
         2 . The method according to  claim 1 , wherein said medium is RPMI-1640. 
     
     
         3 . The method according to  claim 1 , wherein said stem cells are embryonic stem cells or induced pluripotent stem cells. 
     
     
         4 . The method according to  claim 1 , wherein the concentration of CHIR is at least about 2.5 μM. 
     
     
         5 . The method according to  claim 1 , wherein the concentration of CHIR is at least about 3.1 μM. 
     
     
         6 . The method according to  claim 1 , wherein the concentration of CHIR is in a range selected from the group consisting of about 2.5-15 μM, about 3.1-15 μM, about 3.1-7 μM, 3.5-7 μM, about 3.5-6 μM, and about 3.5-5 μM. 
     
     
         7 . The method according to  claim 1 , wherein the concentration of CHIR is at least about 3.5 μM. 
     
     
         8 . The method according to  claim 1 , wherein said incubation with CHIR is at least 24 hours. 
     
     
         9 . The method according to  claim 1 , wherein said incubation with activin A is at least 24 hours. 
     
     
         10 . The method according to  claim 1 , wherein said incubation with activin A is 48 to 72 hours. 
     
     
         11 . The method according to  claim 1 , wherein endodermal cells are obtained from said definitive endoderm cells. 
     
     
         12 . The method according to  claim 11 , wherein said endoderm cells are pancreatic endoderm cells. 
     
     
         13 . Pancreatic endoderm cells obtainable by the method of  claim 12 . 
     
     
         14 . The method according to  claim 1 , wherein the concentration of CHIR in the culture medium is at least 3 μM. 
     
     
         15 . The method according to  claim 1 , wherein the concentration of CHIR in the culture medium is in a range selected from the group consisting of about 2-15 μM, about 3-15 μM, about 3.1-15 μM, about 3.1-7 μM, about 3.5-15 μM, and about 3.5-7 μM. 
     
     
         16 . A method for inducing primitive streak cells from embryonic stem cells by using CHIR in a concentration of at least 2 μM in the culture medium. 
     
     
         17 . A method for inducing definitive endoderm cells from embryonic stem cells by using CHIR in a concentration in the range of 3.5-7 μM.

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