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PDX1 positive entoderm cell and preparation method thereof

A technology of endoderm cells and embryonic stem cells, which is applied in the field of PDX1-positive endoderm cells and their preparation, can solve the problem of low efficiency of human embryonic stem cell differentiation, and achieve the effect of strong operability, broad application prospects, simple and fast operation

Inactive Publication Date: 2010-05-26
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the efficiency of human embryonic stem cells to differentiate into pancreatic cells is still low
[0004] When using RA to induce human embryonic stem cells to produce endoderm cells, a large number of liver cells will be produced at the same time as pancreatic cells, which leads to low differentiation efficiency of human embryonic stem cells into pancreatic cells

Method used

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  • PDX1 positive entoderm cell and preparation method thereof
  • PDX1 positive entoderm cell and preparation method thereof
  • PDX1 positive entoderm cell and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1, the separation of mouse embryonic fibroblast (MEF)

[0092] 1. Matrigel's blanket

[0093] 1) After purchasing Matrigel, it must be subpackaged first to avoid repeated freezing and thawing processes each time it is used. Thaw the entire bottle of Matrigel on ice at 4 overnight;

[0094] 2) After it is completely melted, put it on ice, divide it with a cold pipette tip, and put equal volumes into sterile 1.5ml ice-bathed centrifuge tubes according to the needs of use, and then store it at -80 degrees refrigerator;

[0095] 3) Take out the centrifuge tube from the refrigerator every time you use it, and insert it on ice;

[0096] 4) Add a certain volume of cold DMEM / F12 medium, blow and suck repeatedly on ice with a pipette tip to melt Matrigel and dilute it in the medium, or let Matrigel melt on ice and then add cold DMEM / F12 for culture The base blows and sucks evenly;

[0097] 5) Add the diluted Matrigel to the culture dish or culture well, and add c...

Embodiment 2

[0126] Example 2. Embryonic stem cells were induced to differentiate into PDX1-positive endoderm cells

[0127] The embryonic stem cells used in this example are the human embryonic stem cell line H1.

[0128] 1. Preparation method

[0129] (1) Embryonic stem cells are induced to differentiate into endoderm cells, which do not express the transcription factor PDX1:

[0130] 1. Take out the differentiation medium from the refrigerator and preheat it in a 37°C water bath;

[0131] 2. On the third day after the passage of embryonic stem cells, take out the cells, suck off the medium, and wash with PBS;

[0132] 3. Add endoderm cell culture medium containing 100ng / ml Activin A to the culture dish, the volume is 3ml / 6cm culture dish. Then put back in 5% CO 2 in the cell culture incubator;

[0133] 4. After 24 hours, take out the cells from the incubator, suck off the medium, add endoderm medium containing 100ng / ml ActivinA and 0.1% ITS, the volume is 3ml / 6cm culture dish;

[...

Embodiment 3

[0171] Example 3. Embryonic stem cells were induced to differentiate into PDX1-positive endoderm cells

[0172] The embryonic stem cells used in this example are the human embryonic stem cell line H1.

[0173] 1. Preparation

[0174] The method is the same as that described in the treatment group with RA added in Example 2, except that RA is added for induction, except that in the step of "inducing endoderm cells that do not express the transcription factor PDX1 to differentiate into PDX1-positive endoderm cells" , the cell seeding density is different, as follows: The first group: 2×10 3 Cells / cm2; the second group: 5×10 3 cells / cm2; the third group: 2×10 4 Cells / cm2; the fourth group: 5×10 4 cells / cm2; the fifth group: 2×10 5 Cells / cm2; varying concentrations of all-trans retinoic acid (RA), 0.4 uM in precursor cell differentiation medium.

[0175] 2. Cell detection

[0176] The method is the same as the cell immunofluorescence staining method described in Example 2. ...

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Abstract

The invention discloses a PDX1 positive entoderm cell and a preparation method thereof. In the method, an entoderm cell which is obtained by differentiating an isolated embryonic stem cell or an isolated induction multipotential stem cell and does not express a transcription factor PDX1 is cultured to obtain the PDX1 positive entoderm cell; all trans retinoic acid is added into a system of the culture, and the cell inoculation density is 2*103 cells / square centimeter to 2*105 cells / square centimeter. The method effectively promotes the entoderm cell differentiated by the embryonic stem cell to the direction of the PDX1 positive entoderm cell, and reduces the differentiation to the direction of a liver cell. Accordingly, the method has wide application prospect in the field of induction from the human embryonic stem cell to the PDX1 positive entoderm cell.

Description

technical field [0001] The invention relates to PDX1-positive endoderm cells and a preparation method thereof. Background technique [0002] The clinical islet transplantation represented by the Edmonton scheme provides a new scheme for the treatment of type I diabetes. However, the limited source of islets limits the wide application of this method. Human embryonic stem cells have the ability to proliferate indefinitely and differentiate into various types of somatic cells, which can be a potential way to solve the source of islets. Efficient and reproducible transformation into islet cells is a prerequisite for the application of human embryonic stem cells in the treatment of diabetes. Prior to this, many protocols for step-by-step induction of pancreatic cells from human embryonic stem cells have been established. These protocols broadly mimic the key steps in embryonic pancreas development: generation of gastrulated endoderm, specification of pancreatic endoderm, sequ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/06
Inventor 蔡军于晨邓宏魁
Owner PEKING UNIV
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