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Differentiation Of Human Embryonic Stem Cells

A technology of pluripotent stem cells and cells, applied in the direction of embryonic cells, artificial cell constructs, artificially induced pluripotent cells, etc., can solve the problem of reducing the ability of insulin-expressing cells

Active Publication Date: 2012-05-30
JANSSEN BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0023] NGN3 expression in cells expressing markers characteristic of the pancreatic endoderm lineage reduces the ability of cells to further differentiate into insulin-expressing cells

Method used

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  • Differentiation Of Human Embryonic Stem Cells
  • Differentiation Of Human Embryonic Stem Cells
  • Differentiation Of Human Embryonic Stem Cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0158] Human pluripotent stem cells express markers characteristic of the pancreatic endoderm lineage (Table 1 up to PDX1, NKX6.1, but not CDX2 and NGN3) differentiation

[0159] This example demonstrates that activin A can be used in combination with noggin and retinoic acid to promote the upregulation of NKX6.1 expression. Briefly, cells of the human embryonic stem cell line H1 were incubated in MATRIGEL TM (1:30 dilution) coated plate and supplemented with 2% BSA, 100ng / ml activin A, 20ng / ml WNT-3a, 8ng / ml bFGF cultured on the RPMI medium for one day, and then supplemented with 2%BSA , 100ng / ml activin A, 8ng / ml bFGF in RPMI medium for two days (phase 1), then

[0160] a. Treated with DMEM / F12+2%BSA+50ng / ml FGF7 for three days (stage 2), then

[0161] b. Use DMEM-high glucose+1% B27+50ng / ml FGF7+0.25mM cyclopamine-KAAD+2mM retinoic acid (RA)+100ng / ml noggin+20ng / ml activin A or 50ng / ml Activin A treatment for four days (phase 3).

[0162] As a control, separate cell...

example 2

[0180] Expression of markers characteristic of the pancreatic endoderm lineage (co-expression of PDX1, NKX6.1, but Differentiation of cells that do not express CDX2 and NGN3) into pancreatic endocrine precursors

[0181] Previous studies have shown that cells expressing markers characteristic of the pancreatic endoderm lineage are more likely to give rise to glucagon-expressing cells than insulin-expressing cells when undergoing further differentiation. This may be partly due to the expression of NGN3 in pancreatic endoderm cells. The methods of the invention can generate a population of pancreatic endoderm cells that do not express NGN3 and therefore will be more likely to differentiate into insulin expressing cells. However, NGN3 expression is essential for the formation of pancreatic endocrine cells or pancreatic endocrine precursor cells (cells that can form, for example, glucagon expressing cells or insulin expressing cells). Thus, temporal regulation of NGN3 is impo...

example 3

[0193] Expression of markers characteristic of the pancreatic endoderm lineage (co-expression of PDX1, NKX6.1, but Differentiation of cells that do not express CDX2 and NGN3) into pancreatic endocrine cells

[0194] This example was designed to test the ability of cells expressing markers characteristic of the glandular endoderm lineage (coexpressing PDX1 and NKX6.1 but not CDX2 and NGN3) to further differentiate from pancreatic endocrine precursors into pancreatic endocrine cells.

[0195] Briefly, cells of the human embryonic stem cell line H1 were incubated in MATRIGEL containing RPMI medium + 2% BSA + 100ng / ml Activin A + 20ng / ml WNT-3a + 8ng / ml bFGF TM Coated plates (1:30 dilution) were cultured for one day, then treated with RPMI medium+2%BSA+100ng / ml activin A+8ng / ml bFGF for two days (stage 1), and then

[0196] a. Treated with DMEM / F12+2%BSA+50ng / ml FGF7 for three days (stage 2), then

[0197] b. Treated with DMEM-high glucose+1% B27+50ng / ml FGF7+0.25μM cyclopami...

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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 to produce cells capable of producing insulin following transplantation into an animal.

Description

[0001] Cross references to related patent applications [0002] This application claims priority to Application Serial No. 61 / 226,936, filed July 20, 2009. 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 producing cells capable of producing insulin after transplantation into an animal. 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 from pluripotent stem cells, such as embryonic stem cells. [0005] In vertebrate embryonic development, pluripotent stem cells can give rise to a cell population comprising the three germ layers (ectoderm, mesoderm and endoderm) in a process c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0735C12N5/02A61K35/39A61P3/10A61K35/12
CPCC12N2501/117C12N2501/115C12N2501/19C12N2501/415C12N2501/16A61K35/12C12N2501/41C12N2501/15C12N2501/155C12N2501/11C12N2506/02C12N2533/90C12N2501/385C12N5/0676A61P3/10A61K35/39C12N5/00C12N5/0613C12N5/0696
Inventor J·徐
Owner JANSSEN BIOTECH INC
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