Method of embryonic stem cell differentiation into neurons by in vitro induction

An embryonic stem cell and neuron technology, applied in the field of in vitro induction of embryonic stem cells to differentiate into neurons and induced differentiated cells, can solve the problems of small amount of neuronal cells, difficulty, lack of clear data on the amount of use and induction time, etc. Induction rate, low cost effect

Inactive Publication Date: 2013-08-28
CYAGEN BIOSCI GUANGZHOU +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many researches and developments on ES induced neurons, most of which are to form embryoid bodies (EBs), and then induced by inducing factors such as DMSO, retinoic acid, etc., but there are still corresponding methods for the formation, culture and later maintenance of EBs. Difficulty, the induction rate of induced neurons is also relatively low, so the difficulty of research and development is also certain
[0005] The induction factors generally used for the directional induction of mouse embryonic stem cells into neurons include DMSO, RA, etc., but there is no clear data on the specific usage amount and induction time; most of the process is to form embryoid bodies (EBs), and then through the induction factors Such as retinoic acid, etc. for induction, but there are still corresponding difficulties in the formation, culture and later maintenance of EBs. The number of cells induced to form neurons is small, and the low induction rate is also a difficulty.

Method used

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  • Method of embryonic stem cell differentiation into neurons by in vitro induction
  • Method of embryonic stem cell differentiation into neurons by in vitro induction
  • Method of embryonic stem cell differentiation into neurons by in vitro induction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Separation of ES and MEF: use the differential adherence method, inoculate in a cell culture dish coated with 0.1% gelatin, incubate at 37°C for 45 minutes, collect the supernatant and centrifuge, repeat 1-3 times, and perform ES Separated from MEFs.

[0030] EB formation: add EB formation solution to resuspend cells, inoculate 2-3×10 6 Cells in 10cm bacterial culture dish, 37°C, 5% CO 2 Cultured for 4 days, the medium was changed every 2 days.

[0031] Induction of RA neurons: preparation of neural induction solution: preparation of RA for induction: 1-5×10 -7 M was induced for 4 days, and the medium was changed every 2 days.

[0032] Adhesive culture of nerve-induced EBs: use PLL-coated culture plates, inoculate EBs on the coated culture plates, add appropriate amount of EB-forming solution, 37°C, 5% CO 2 Cultured for 2 days.

[0033] Late neuron induction: 2 days later, use EB formation medium and neuron culture medium for culture, and change half of the medium ...

Embodiment 2

[0035] Separation of ES and MEF: use the differential attachment method, inoculate in a cell culture dish coated with 0.1% gelatin, incubate at 37°C for 30 minutes, and repeat twice to separate ES and MEF.

[0036] EB formation: add EB formation solution to resuspend cells, inoculate 2-3×10 6 Cells in 10cm bacterial culture dish, 37°C, 5% CO 2 Cultured for 4 days, the medium was changed every 2 days.

[0037] Induction of RA neurons: preparation of neural induction solution: preparation of RA for induction: 4×10 -7 M was induced for 4 days, and the medium was changed every 2 days. All the other steps are the same as in Example 1.

Embodiment 3

[0039] Separation of ES and MEF: using the differential adherence method, inoculate in a cell culture dish coated with 0.1% gelatin, incubate at 37°C for 45 minutes, collect the supernatant and centrifuge, and repeat once to separate ES and MEF .

[0040] EB formation: add EB formation solution to resuspend cells, inoculate 2-3×10 6 Cells in 10cm bacterial culture dish, 37°C, 5% CO 2 Cultured for 3 days, the medium was changed every 2 days.

[0041] Induction of RA neurons: preparation of neural induction solution: preparation of RA for induction: 5×10 -7 M was induced for 4 days, and the medium was changed every 2 days.

[0042] Adhesive culture of nerve-induced EBs: use PLL-coated culture plates, inoculate EBs on the coated culture plates, add appropriate amount of EB-forming solution, 37°C, 5% CO 2 Cultured for 2 days.

[0043] Late neuron induction: After 2 days, use EB formation medium and neuron culture medium for culture, and change half of the medium every day for...

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Abstract

A method of embryonic stem cell differentiation into neurons by in vitro induction comprises the following steps: separating MEF and ES by a differential adhesion method; adding EB formation liquid resuspended cells to prepare an EB suspension, wherein per 10 cm petri dish is inoculated with a concentration of 2-3*10<6>, culturing lasts for 2-4 d, and the liquid is changed for every 1-2d; inducing neurons with a RA concentration of 1-5*10<-7>M for 2-4d, wherein the liquid is changed every 2d; adherent culturing EB, including using a PLL-coated culture plate, inoculating EB on the coated culture plate, adding an appropriate amount of EB formation liquid, and culturing for 1-2d; and inducing anaphase neurons by a neuronal culture medium.

Description

technical field [0001] The invention belongs to the field of biological technology and relates to a method for inducing differentiated cells. It specifically relates to a method for inducing embryonic stem cells to differentiate into neurons in vitro. Background technique [0002] Since Thomson et al reported the establishment of human embryonic stem cell (ESC) in 1998, the study of ESC has attracted the attention of researchers in this field. Since ESC is a pluripotent cell line with self-replication ability and developmental pluripotency isolated from early mammalian embryos or primordial germ cells, it can be directional induced to differentiate into almost all types of cells under certain conditions. [0003] There are three main methods to induce cell differentiation: adding cytokines, co-cultivation and transgenic. Activate certain tissue-specific silenced genes in stem cells with exogenous factors to change the intracellular signal transduction network, or strengthe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793
Inventor 陈丽娟陈银娜
Owner CYAGEN BIOSCI GUANGZHOU
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