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Generation of pancreatic endoderm from pluripotent stem cells using small molecules

a technology of pluripotent stem cells and pancreatic endoderm, which is applied in the field of generating pancreatic endoderm from pluripotent stem cells, can solve the problems of inability to fully mature beta cells in vitro, restrict the use of this treatment as a clinical therapy, etc., and achieve the effect of improving the efficiency of differentiation of human ps cells, improving the fraction of nkx6.1/pdx1 double positive cells, and improving the population of pancreatic cells

Pending Publication Date: 2019-05-23
TAKARA BIO EURO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention introduces a method to increase the ratio of human PS cells that express both Nkx6.1 and PDX1, which are hallmarks of pancreatic beta cells that produce insulin. This method also creates a more even mix of pancreatic cells, which is important for further developing these cells into beta cells.

Problems solved by technology

However, the limited availability of donor beta cells constrains the use of this treatment as a clinical therapy.
Several groups have developed in vitro protocols that can differentiate PS cells into DE and PE, however they are only able to generate a modest fraction of NKX6.1 / PDX1 double positive (db+ve) cells, and importantly none of them are able to generate fully mature beta cells in vitro (Cai et al.

Method used

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  • Generation of pancreatic endoderm from pluripotent stem cells using small molecules
  • Generation of pancreatic endoderm from pluripotent stem cells using small molecules
  • Generation of pancreatic endoderm from pluripotent stem cells using small molecules

Examples

Experimental program
Comparison scheme
Effect test

example 1

Small Molecules that Induce NKX6.1 / PDX1 Co-Expression

Small Molecules

[0161]Four different libraries were included in the screen; a kinase inhibitor library (Calbiochem #539743), a bioactive lipid library (Enzo Life Sciences #BML-2800), a nuclear receptor ligand library (Enzo Life Sciences #BML-2802) and a phosphatase inhibitor library (Enzo Life Sciences #BML-2834). The compounds within the bioactive library were tested at 1 uM and 0.1 uM. Compounds from the other libraries were tested at 10 uM and 1 uM. In a second candidate based screening approach, small molecules that target the main signalling pathways involved in pancreas development were included.

NKX6.1 / PDX1 Screen

[0162]The library compounds were screened on top of a bFGF based media formulation for making PE (Ameri et al. 2010) (RPMI1640, Gibco #61870; 12% KOSR, Gibco #10828; 0.1% PEST, Gibco #15140; 64 ng / mL bFGF, Peprotech #100-18B).

[0163]The library PE screening approach was divided into an early and a late phase (FIG. 1)....

example 2

Small Molecule Hits that Induce NKX6.1 / PDX1 Co-Expression

Combining Hits from the Candidate Screening Approach with Hits from the Library Approach

[0172]DE cells were exposed to 4 days 50 nM LDN-193189, followed by 8 days AM580 (AH Diagnostics, BML GF104 0025), JNK Inhibitor II (Calbiochem, 420119), 50 nM LDN-193189 and 64 ng / ml FGF2, or AM580, JNK Inhibitor II, 50 nM LDN-193189, 64 ng / ml FGF2 and IWP2, or AM580, JNK Inhibitor II, 50 nM LDN-193189, 64 ng / ml FGF2 and Cyclopamine (FIG. 6). Media change was performed daily.

example 3

ion of Small Molecules that Induce NKX6.1 / PDX1 Co-Expression in Human Induced Pluripotent Stem Cells

[0173]Hit compounds (Tables 1 and 2) were screened on top of a bFGF based media formulation for making PE (Ameri et al. 2010) (RPMI1640, Gibco #61870; 12% KOSR, Gibco #10828; 0.1% PEST, Gibco #15140; 64 ng / mL bFGF, Peprotech #100-18B).

[0174]The screen was divided into an early and a late phase (FIG. 1). In the early phase, compounds were tested on top of the PE media for the first seven days of PE differentiation, and then continued for additional six days using PE media without compounds. In the late phase, DE cells were differentiated in the PE media for the first seven days. In the following six days compounds were tested on top of the PE media. Twelve positive control wells (PE media) and 12 negative control wells (PE media without bFGF) were included in each 96 well plate. Media change was performed daily.

[0175]Values above 200% effect were categorized as a hit (FIG. 2 and FIG. 3...

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Abstract

A method of producing pancreatic cells or pancreatic cell precursors expressing at least 5% PDX1 / NKX6.1 double positive, comprising exposing definitive endoderm cells to an effective amount of one or more small molecules, to differentiate the human definitive endoderm cells into the pancreatic cells or pancreatic cell precursors. The present invention also relates to pancreatic endoderm cells produced by said methods and uses of said pancreatic endoderm cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. Ser. No. 15 / 215,807, filed Jul. 21, 2016 (issue fee paid) which is a divisional application of U.S. Ser. No. 14 / 425,136, filed Mar. 2, 2015 (abandoned) which is a 35 U.S.C. § 371 National Stage application of International Application PCT / EP2013 / 068188 (WO 2014 / 033322), filed Sep. 3, 2013, which claims priority to European Patent Application 12182747.1, filed Sep. 3, 2012 and European Patent Application 12198820.8, filed Dec. 21, 2012; this application claims priority under 35 U.S.C. § 119 to U.S. Provisional Application 61 / 697,970, filed Sep. 7, 2012; the contents thereof which are incorporated by reference in their entirety.TECHNICAL FIELD[0002]The present invention relates to methods of generating pancreatic endoderm from pluripotent stem (PS) cells, such as human definitive endoderm.BACKGROUND[0003]Beta cell transplantation potentially provides the ultimate cure for type I diabetes. However, ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/071
CPCC12N5/0676C12N5/0678C12N2501/117C12N2506/45C12N2506/02C12N2503/02C12N2501/999C12N2501/727C12N2501/415C12N2501/41C12N2501/385C12N2501/16C12N2501/155C12N2501/115C12N2501/60C12N2501/119
Inventor EKBERG, JENNYHANSSON, MATTIASDOEHN, ULRIKHESS, KATJAFUNA, NINA
Owner TAKARA BIO EURO
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