Method for inducing embryonic stem cell into pancreatic tissue-like cells

A technology of embryonic stem cells and cells, applied in animal cells, vertebrate cells, artificial cell constructs, etc.

Inactive Publication Date: 2013-07-10
于涛 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been many studies on the induction and differentiation of embryonic stem cells into insuli

Method used

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  • Method for inducing embryonic stem cell into pancreatic tissue-like cells
  • Method for inducing embryonic stem cell into pancreatic tissue-like cells
  • Method for inducing embryonic stem cell into pancreatic tissue-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1E14

[0034] Embodiment 1E14TG2a line mouse ESC expansion and EB culture

[0035] 1. Culture of mouse E14TG2a line ESC:

[0036] (1) ESC recovery: routinely sterilize the ultra-clean workbench, add 10ml of mouse ESC medium into a sterile 15ml centrifuge tube, take out a tube of frozen mouse E14TG2a ESC from the liquid nitrogen tank, and place it immediately at 37°C Put it in a water bath box and shake it to observe the thawing of the cell suspension. After about 2 / 3 of the thawing, put the cryopreservation tube into the ultra-clean bench after spraying with 75% alcohol, and transfer the cell suspension to a container containing 10ml of culture medium in a centrifuge tube, and blow evenly, centrifuge at room temperature at 1000rpm for 5min, discard the supernatant, add 5ml of ESC medium, blow evenly and transfer to 25cm 2 culture flask at 37°C, 5% CO 2 in the cell culture incubator.

[0037] ⑵Cell medium change and subculture: decide whether to change the medium according to the con...

Embodiment 1

[0043] In Example 1, mouse ES-E14TG2a line ESCs were purchased from American Type Culture Collection (ATCC).

[0044] The formula of ESC medium is:

[0045] High-glucose DMEM was used as the basal medium, and sodium bicarbonate (NaHCO 3 ), HEPES, non-essential amino acids, fetal bovine serum, β-mercaptoethanol, penicillin / streptomycin, LIF, the final concentration of each additive is: 10% fetal bovine serum, 10mM HEPES, 0.12%NaHCO 3 , 0.1mM non-essential amino acids, 0.1mM β-mercaptoethanol, 100U / ml penicillin, 100μg / ml streptomycin, 1000U / ml LIF.

[0046] Sterilize through a 0.22 μm filter and store at 4°C.

[0047] The formula of EB medium is:

[0048] High-glucose DMEM was used as the basal medium, and sodium bicarbonate (NaHCO 3 ), HEPES, non-essential amino acids, fetal bovine serum, β-mercaptoethanol, penicillin / streptomycin, the final concentration of each additive is: 10% fetal bovine serum, 10mM HEPES, 0.12%NaHCO 3 , 0.1mM non-essential amino acids, 0.1mM β-merca...

Embodiment 2

[0050] Example 2 Differentiation of definitive endoderm during EB formation and the promotion of Activin A on its differentiation

[0051] 1. The medium used in this example:

[0052] (1) Activin A serum-free medium (SF-Activin A):

[0053] High-glucose DMEM was used as the basal medium, and sodium bicarbonate (NaHCO 3 ), HEPES, non-essential amino acids, KSR, β-mercaptoethanol, penicillin / streptomycin, recombinant Activin A, the final concentration of each additive is: 10% KSR, 10mM HEPES, 0.12% NaHCO 3 , 0.1mM non-essential amino acids, 0.1mM β-mercaptoethanol, 100U / ml penicillin, 100μg / ml streptomycin, 50ng / ml Activin A.

[0054] Sterilize through a 0.22 μm filter and store at 4°C.

[0055] (2) Serum Control Medium (SF):

[0056] High-glucose DMEM was used as the basal medium, and sodium bicarbonate (NaHCO 3 ), HEPES, non-essential amino acids, KSR, β-mercaptoethanol, penicillin / streptomycin, the final concentration of each additive is: 10% KSR, 10mM HEPES, 0.12% NaHCO...

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Abstract

The invention discloses a method for inducing an embryonic stem cell into pancreatic tissue-like cells. The method comprises the following steps of: using a hanging-drop preparation method to differentiate an embryonic stem cell into three germ layer cells and using a immunomagnetic bead cell sorting method to purify finalized inner germ layer cells; further inducing the finalized inner germ layer cells so as to differentiate the cells into pancreatic precursor cells; and further differentiating the pancreatic precursor cells outside the body so as to form pancreatic tissue-like cells with high amylase secretion capability. The technical scheme of the invention adopts a plurality of induction factors for induced differentiation in different stages, which proves that the embryonic stem cell has the potentiality of being differentiated into pancreatic exocrine cells, and an effective way for inducting the embryonic stem cell into pancreatic tissue-like cells is found out, so that the pancreatic precursor cells are possible to recover injury of pancreas, and more cell sources are provided for recovering pancreas.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for inducing embryonic stem cells to be pancreatic tissue-like cells. Background technique [0002] The pancreas is an important endocrine and exocrine organ of the human body. It is located behind the peritoneum in the epigastric region and the left quarter rib region, and is divided into two parts: the endocrine department and the exocrine department. The exocrine part is composed of ducts (the marker molecule CK19 of duct cells) and acini, which can synthesize and secrete amylase, lipase, etc. The endocrine part is scattered among the exocrine parts, mainly composed of A cells, B cells, D cells and PP cells, which secrete glucagon, insulin, somatostatin and pancreatic polypeptide respectively. At present, the treatment of acute severe pancreatitis, chronic pancreatitis and pancreatic cancer is very difficult. The study of the differentiation and development of pancreat...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 于涛陈其奎
Owner 于涛
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