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Differentiation of human embryonic stem cells into pancreatic endoderm

A technology of human embryonic stem cells and embryonic stem cells, applied to embryonic cells, cell culture supports/coatings, liver cells, etc.

Inactive Publication Date: 2015-01-14
JANSSEN BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Although significant progress has been made in improving protocols for generating pancreatic cells from human pluripotent stem cells, there is a large degree of variability in the results reported by different groups regarding the efficiency of pancreatic cell generation from ES cells

Method used

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  • Differentiation of human embryonic stem cells into pancreatic endoderm
  • Differentiation of human embryonic stem cells into pancreatic endoderm
  • Differentiation of human embryonic stem cells into pancreatic endoderm

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0085] Seeding density of embryonic stem cells does not significantly affect the expression of definitive endoderm markers

[0086] This example was performed to see if the initial seeding density of ES cells could significantly affect the generation of cells of the definitive endoderm lineage.

[0087] Cells of the human embryonic stem cell line H1 (hESC H1) were harvested at different passages (from passage 40 to passage 52) at the following density: 0.3×10 5 cells / cm 2 , 0.5×10 5 cells / cm 2 , 0.75×10 5 cells / cm 2 , 0.9×10 5 cells / cm 2 , 1×10 5 cells / cm 2 , 1.25×10 5 cells / cm 2 , 1.5×10 5 cells / cm 2 , 1.8×10 5 cells / cm 2 and 2×10 5 cells / cm 2 Seeded on Matrigel as single cells TM (1:30 dilution; BD Biosciences, Franklin Lakes, NJ) supplemented with 10 μM Y27632 (Rock inhibitor, catalog number Y0503, SigmaAldrich, St.Louis, MO) on the coated culture dish 1 medium (StemCell Technologies, Vancouver, Canada) or MEF-CM (conditioned medium). Forty-eight hours...

example 2

[0098] The seeding density of embryonic stem cells significantly affects the expression of pancreatic endoderm and pancreatic endocrine markers reach

[0099] This example was performed to see if the initial seeding density of ES would significantly affect the generation of pancreatic endoderm / endocrine cultures.

[0100] Cells of the human embryonic stem cell line H1 (hESC H1) were harvested at different passages (from passage 40 to passage 52) at the following density: 0.5×10 5 cells / cm2, 0.75×10 5 cells / cm 2 , 1×10 5 cells / cm 2 , 1.5×10 5 cells / cm 2 , 1.8×10 5 cells / cm 2 and 2×10 5 cells / cm 2 Seeding on MATRIGEL as single cells TM (1:30 dilution; BD Biosciences, NJ) in MEF-CM (conditioned medium) supplemented with 10 μM Y27632 on coated dishes. Forty-eight hours after inoculation, cultures were washed and incubated in incomplete PBS (phosphate buffered saline without Mg or Ca) for approximately 30 seconds.

[0101] Cultures were differentiated into the pancr...

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Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides methods to produce a population of pancreatic endoderm cells, wherein the initial seeding density of undifferentiated epluripotent cells is defined.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Patent Application Serial No. 61 / 643,684, filed May 7, 2012, which is hereby incorporated by reference in its entirety for any purpose. technical field [0003] The present invention is in the field of cell differentiation. More specifically, the present invention provides various ranges of seeding densities of human pluripotent cells and / or cells expressing markers indicative of definitive endoderm, which can be used to subsequently efficiently generate cells expressing markers indicative of pancreatic endoderm and Cells expressing markers indicative of pancreatic endocrine. Background technique [0004] Advances in cell replacement therapy for type I 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 f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/02
CPCC12N5/0678C12N2506/02C12N2501/19C12N2533/90C12N5/0676C12N2501/999C07K14/505C12N5/005C12N5/06C12N2500/30C12N2501/065C12N2510/02C12N2511/00C12N5/0018C12N5/0672
Inventor A.雷扎尼亚
Owner JANSSEN BIOTECH INC