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Pancreatic and liver endoderm cells and tissue by differentiation of definitive endoderm cells obtained from human embryonic stems

A technology for human embryonic stem cells and definitive endoderm, which is used in tissue culture, biochemical equipment and methods, microorganisms, etc.

Inactive Publication Date: 2009-09-23
UNIV OF GEORGIA RES FOUND INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is problematic when using hESCs for therapeutic development as FCS and KSR contain indeterminate activity

Method used

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  • Pancreatic and liver endoderm cells and tissue by differentiation of definitive endoderm cells obtained from human embryonic stems
  • Pancreatic and liver endoderm cells and tissue by differentiation of definitive endoderm cells obtained from human embryonic stems
  • Pancreatic and liver endoderm cells and tissue by differentiation of definitive endoderm cells obtained from human embryonic stems

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Culture of human ES cells

[0126] Routine culture of human ES cells

[0127] The human embryonic stem cell line BG01 (BresaGen, Inc., Athens, GA) was used in this work. BG01 cells were grown in hES medium, and the medium consisted of DMEM / F-12 (50 / 50) supplemented with 20% knockout serum replacer (KSR; Invitrogen), 0.1 mMMEM non-essential amino acids (NEAA; Invitrogen), 2 mM L-glutamine (Invitrogen), 50 U / ml penicillin, 50 μg / ml streptomycin (Invitrogen), 4 ng / ml bFGF (Sigma) and 0.1 mM β-mercaptoethanol (Sigma). Cells were grown on mouse primary embryonic fibroblast feeder layers that were mitotically inactivated with mitomycin c. Feeder cells at 1.2x10 per 35mm dish 6 Cell seeding. BG01 cells were passaged using the collagenase / trypsin method. Briefly, medium was removed from the plate, 1 ml of 200 U / ml collagenase type IV (GibcoBRL) was added, and cells were incubated at 37°C for 1-2 minutes. Collagenase was removed and 1 ml of 0.05% trypsin / 0.53mM EDTA (GIBCO...

Embodiment 2

[0133] Treatment of HES cells with PI3-kinase inhibitors leads to differentiation of HES cells

[0134] Inhibitor / Differentiation Reagent Treatment of Stem Cells

[0135] BG01 cells were passaged from feeder using the collagenase / trypsin method and cultured at 1x10 in conditioned medium (CM; MEF conditioned medium plus 8ng / ml bFGF). 5 Density of cells per 35 mm dish was seeded on matrigel-coated dishes. After approximately 24 hours, replace the medium with fresh CM, CM with inhibitor (resuspended in EtOH), CM with EtOH, or Spontaneous Differentiation medium (hES medium minus bFGF) .

[0136] In other methods, BG01 cells were seeded at different concentrations prior to exposure to CM, CM with inhibitors and CM with EtOH, cells were seeded at the following concentrations: approximately 5x10 4 Cells / 35mm dish, about 1x10 5 Cells / 35mm dish, about 2x10 5 Cells / 35mm dish, about 4x10 5 Cells / 35mm dish and about 6x10 5 Cells / 35mm dish.

[0137] The inhibitor LY 294002 (Biomo...

Embodiment 3

[0142] Characterization of cells treated with PI3-kinase inhibitors

[0143] Inhibitor studies were performed as described in Example 2.

[0144] Flow Cytometry

[0145] For flow cytometry, BG01 cells were washed with 1xPBS and fixed with 2% paraformaldehyde / 1xPBS for 10 min at room temperature. After that, the cells were washed with 1xPBS, about 2x10 5 Cells were incubated with primary antibody diluted in 1% BSA / 1xPBS. Primary antibodies used were anti-CD9 and anti-thrombomodulin antibodies (Cymbus Biotechnology), FITC-conjugated mouse monoclonal antibodies, diluted 1:10. Cells were incubated at 4°C for 30 minutes and then washed twice with 1xPBS. When appropriate, cells were resuspended in a secondary antibody, anti-mouse Alexa-488 (Molecular Probes), diluted 1:1000 in 1% BSA / PBS, incubated at 4°C for 30 min, and then washed in 1xPBS on both sides. Second-rate. Cells were resuspended in 1% BSA / 1xPBS and analyzed for surface expression using a Beckman Coulter FC500. ...

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Abstract

The invention relates to methods that allow for the efficient differentiation to form pancreatic endoderm cells from pluripotent stem cells such as human embryonic stem cells and definitive endoderm cells. The invention is directly applicable to the ultimate generation of pancreatic beta cells that could be used as part of a therapy to treat or even cure diabetes. Additionally, the present invention may be used to generate liver endoderm cells from human embryonic stem cells and definite endoderm cells as well. This invention relates to a method for generating definitive endoderm and pancreatic endoderm cells from stem cells, preferably human embryonic stem cells using defined media in the absence of feeder cells. A simply two step procedure to provide pancreatic endoderm cells from embryonic stem cells represents further embodiments of the present invention.

Description

Field of Invention [0001] The present invention relates to methods for the efficient differentiation of pluripotent stem cells such as human embryonic stem cells and definitive endoderm cells to form pancreatic endoderm cells. The invention has direct application to the preparation of pancreatic islet beta cells which can be used as part of a therapy to cure diabetes. In addition, the present invention can be used to prepare hepatic endoderm cells from human embryonic stem cells and definitive endoderm cells. [0002] The present invention relates to methods for the preparation of definitive endoderm and pancreatic endoderm cells from stem cells, preferably human embryonic stem cells, using feeder-free defined media. [0003] related application [0004] This application claims provisional application US60 / 810,424, entitled "Pancreatic and Hepatic Endoderm Cells and Tissues Derived from Differentiation of Definitive Endoderm Cells Obtained from Human Embryonic Stem Cells," f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08
Inventor 斯蒂芬·达尔顿戴维·雷诺兹迈克尔·库利克
Owner UNIV OF GEORGIA RES FOUND INC
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