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Generation of pancreatic endoderm from pluripotent stem cells using small molecules

An endoderm, definitive endoderm technology, applied in non-embryonic pluripotent stem cells, artificially induced pluripotent cells, embryonic cells, etc.

Active Publication Date: 2015-09-09
NOVO NORDISK AS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Several in vitro protocols have been developed to differentiate PS cells into DE and PE, however only modest fractions of NKX6.1 / PDX1 double positive (db +ve) cells were produced and importantly none of them were able to generate in vitro Fully mature β cells (Cai et al (2010); D'Amour et al (2006); Kunisada et al (2012); Schulz et al (2012); Zhang et al (2009); Ameri et al (2010))

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
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Effect test

Embodiment approach

[0088] 1. A method of producing pancreatic cells or pancreatic cell precursors, wherein at least 5% of the cells co-express PDX1 and NKX6.1, comprising exposing embryonic stem cells to an effective amount of at least one compound from the group consisting of:

[0089] a. BMP inhibitors

[0090] b. Kinase inhibitors

[0091] c. Retinoic acid receptor agonists,

[0092] To differentiate human embryonic stem cells into pancreatic cells or pancreatic cell precursors.

[0093] 2. The method of embodiment 1, wherein the compound is listed in Table 1 or 2.

[0094] 3. The method of embodiment 1 or 2, wherein the kinase inhibitor is an isomer of 1,9-pyrazoloanthrone with or without N-alkylation.

[0095] 4. The method of embodiments 1-3, wherein the kinase inhibitor is JNK inhibitor II.

[0096] 5. The method of embodiments 1-2, wherein the retinoic acid receptor agonist is AM580.

[0097] 6. The method of embodiments 1-5, wherein the JNK inhibitor II is combined with AM580.

[00...

Embodiment 1

[0175] Example 1 – Screening of small molecules that induce NKX6.1 / PDX1 co-expression

[0176] Small molecule

[0177] Four different libraries were included in this screen: Kinase Inhibitor Library (Calbiochem #539743), Bioactive Lipid Library (Enzo Life Sciences #BML-2800), Nuclear Receptor Ligand Library (Enzo Life Sciences #BML-2802) and phosphatase inhibitor library (Enzo Life Sciences #BML-2834). Compounds within the biologically active library were detected at 1 μM and 0.1 μM. Compounds from other libraries were tested at 10 μM and 1 μM. In a second candidate-based screening approach, small molecules targeting major signaling pathways involved in pancreatic development were included.

[0178] NKX6.1 / PDX1 Screening

[0179] Library compounds in a bFGF-based media formulation (Ameri et al. 2010) (RPMI1640 Gibco #61870, 12% KOSR Gibco #10828, 0.1% PEST Gibco #15140, 64ng / mL bFGF Peprotech #100-18B) for preparation of PE to filter.

[0180] Library PE screening ...

Embodiment 2

[0189] Example 2 - Combination of small molecule hits that induce NKX6.1 / PDX1 co-expression

[0190] Combining hits from candidate screening methods with hits from library methods

[0191] DE cells were exposed to 50nM LDN-193189 for 4 days, followed by AM580 (AH Diagnostics, BML GF104 0025), JNK inhibitor II (Calbiochem, 420119), 50nM LDN-193189 and 64ng / ml FGF2, or AM580, JNK inhibitor II, 50nM LDN-193189, 64ng / ml FGF2 and IWP2, or AM580, JNK inhibitor II, 50nM LDN-193189, 64ng / ml FGF2 and cyclopamine for 8 days ( Image 6 ). Media changes were performed daily.

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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

technical field [0001] The present invention relates to methods for generating pancreatic endoderm from pluripotent stem (PS) cells, such as human definitive endoderm. Background of the invention [0002] Beta cell transplantation potentially offers the ultimate treatment for type 1 diabetes. However, the limited availability of donor β cells limits the use of this therapy as a clinical treatment. Pluripotent stem cells can immortalize and differentiate into a variety of cell types; thus, PS cells are a promising source of beta cells. However, before PS cells can be used to treat diabetes, they need to differentiate efficiently and reproducibly into pancreatic cells. [0003] During vertebrate embryonic development, pluripotent cells give rise to the three germ layers: ectoderm, mesoderm and endoderm. For the formation of endoderm-derived tissues, induction of definitive endoderm (DE) is the first step; generation of pancreatic endoderm (PE) from DE cells is necessary for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
CPCC12N5/0676C12N2501/999C12N2501/415C12N2501/385C12N2501/727C12N2501/155C12N2506/02C12N2503/02C12N2501/16C12N2506/45C12N2501/115C12N2501/60C12N2501/117C12N5/0678C12N2501/41C12N2501/119
Inventor J.埃克伯格M.汉斯森U.多恩K.赫斯N.富纳
Owner NOVO NORDISK AS
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