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Differentiation of pluripotent stem cells

A technology of pluripotent stem cells and precursor cells, applied in the field of differentiation of pluripotent stem cells, can solve problems such as not completely simulating the developmental program of higher mammals

Active Publication Date: 2013-06-12
JANSSEN BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] However, mouse models of embryonic stem cell development may not fully mimic the developmental program in higher mammals such as humans

Method used

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  • Differentiation of pluripotent stem cells
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  • Differentiation of pluripotent stem cells

Examples

Experimental program
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example 1

[0121] Cells of the human embryonic stem cell line H1 were inactivated in cells lacking FBS and containing CYP26A inhibitors pancreatic endocrine progenitor cells

[0122] Cells of the human embryonic stem cell line H1(p40-p50) were colonized as Cultured in MEF-CM (Mouse Embryo Fibroblast Conditioned Medium) on coated dishes (1:30 dilution) (BD Biosciences; Cat# 356231 ) and differentiated into pancreatic endocrine precursor cells as follows:

[0123] a. Stage I (definitive endoderm): Human embryonic stem cell line cells were supplemented with 2% fatty acid-free BSA (Catalog No. 68700, Proliant, Iowa (Proliant, IA)) and 100 ng / ml Activin A (R&D Systems, MN) plus 20ng / ml WNT-3a (catalogue number 1324-WN-002, (Andy Biotech, Minnesota)) plus 8ng / ml bFGF ( Cat. No. 100-18B, PeproTech, NJ) in RPMI medium for one day and then re-treated with RPMI medium supplemented with 2% BSA and 100 ng / ml Activin A plus 8 ng / ml bFGF two days, then

[0124] b. Stage II (gastric tube): cell...

example 2

[0129] Cells of the human embryonic stem cell line H1 were inactivated in cells lacking FBS and containing CYP26A inhibitors Alternative methods for differentiation into pancreatic endocrine precursor cells in culture

[0130] Human embryonic stem cell line H1 (p40-p52) cells were used as single cells at 100,000 cells / cm 2 planted at a density of Coated dishes (1:30 dilution) (BD Biosciences; Cat. No. 356231 ) were supplemented with 16 ng / ml FGF2 (Cat. No. 100-18B, PeproTech, NJ) and 10 μM Y-27632 (Rock Inhibitor, Cat. No. Y0503, Sigma, MO) in MEF-CM (Mouse Embryo Fibroblast Conditioned Medium). 72 hours after seeding, cultures were differentiated into definitive endoderm (DE) as follows:

[0131] a. Stage I (definitive endoderm): human embryonic stem cell line cells were supplemented with 2% fatty acid-free BSA (Cat. No. 68700, Proliant, Iowa), 0.0025 g / ml sodium bicarbonate (Cat. , Sigma Corporation of Missouri), 1X GlutaMax TM (Cat. No. 35050-079, Invitrogen, Calif...

example 3

[0138] Cells of the human embryonic stem cell line H1 were inactivated in cells lacking FBS and containing CYP26A inhibitors Alternative methods for differentiation into pancreatic endocrine cells in culture

[0139] Human embryonic stem cell line H1 (p40-p52) cells were used as single cells at 100,000 cells / cm 2 planted at a density of Coated dishes (1:30 dilution) (BD Biosciences; Cat. No. 356231 ) were supplemented with 16 ng / ml FGF2 (Cat. No. 100-18B, PeproTech, NJ) and 10 μM Y-27632 (Rock Inhibitor, Cat. No. Y0503, Sigma, MO) in MEF-CM (Mouse Embryo Fibroblast Conditioned Medium). 72 hours after seeding, cultures were differentiated into definitive endoderm (DE) as follows:

[0140] a. Stage I (definitive endoderm): Human embryonic stem cell line cells cultured as single cells on Matrigel-coated dishes were treated with supplemented with 2% fatty acid-free BSA (Catalog No. 68700, Proliant, Iowa) , 0.0025 g / ml Sodium Bicarbonate (Cat. No. S3187, Sigma, Missouri), 1...

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Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method utilizing an agent that degrades retinoic acid to produce a population of pancreatic endocrine precursor cells.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Patent Application Serial No. 61 / 378,480, filed August 31, 2010, which is hereby incorporated by reference in its entirety for all purposes. technical field [0003] The present invention provides methods for promoting the differentiation of pluripotent stem cells into insulin-producing cells. In particular, the present invention provides methods for generating pancreatic endocrine precursor cells using agents that degrade retinoic acid. Background technique [0004] Advances in cell replacement therapy for type 1 diabetes and the scarcity of transplantable islets have focused attention on developing a source of insulin-producing cells or beta cells suitable for engraftment. One approach is to generate functional beta cells from pluripotent stem cells, such as embryonic stem cells. [0005] In vertebrate embryonic development, pluripotent stem cells can give rise ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0735C12N5/02
CPCC12N2501/115C12N2501/16C12N2501/385C12N2501/727C12N2506/02C12N5/0678C12N5/00C12N5/0606C12N5/0607C12N5/0613C12N5/0018C12N2501/998C12N2501/999
Inventor A.雷扎尼亚
Owner JANSSEN BIOTECH INC
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