Method for differentiation of embryonic stem cells into nerve cells through in vitro induction

A technology for embryonic stem cells and cell differentiation, which is applied in the neural cell differentiation of Olig2+-GFP+-mES induced by purine derivatives, neural cell induction, and in vitro induction of embryonic stem cells into the field of neural cells, to promote recovery, improve recovery, and promote myeloid Effects of sheath regeneration and recovery of nerve function

Inactive Publication Date: 2013-01-30
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no information about the use of Purmorphamine instead of SHH to induce embryonic stem cell differentiation to obtain high-purity, large number, and functional spinal motor neurons and oligodendrocytes, and whether Purmorphamine can induce SHH signaling pathways, dorsal- Changes in related genes in

Method used

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  • Method for differentiation of embryonic stem cells into nerve cells through in vitro induction
  • Method for differentiation of embryonic stem cells into nerve cells through in vitro induction
  • Method for differentiation of embryonic stem cells into nerve cells through in vitro induction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Olig2 + -GFP + -Cultivation and Characterization of mES

[0075] 1. Cells used in the experiment: mouse (CF-1) embryonic fibroblasts (MEF), primary culture;

[0076] ,2,Olig2 + -GFP + -The mES cell line was provided by Professor Zhang Suchun, Changjiang Scholars Distinguished Professor of Fudan University.

[0077] , 3, the main reagents:

[0078] Gelatin, DAPI, DMSO: purchased from Sigma (USA);

[0079] DME / F12 glutamine, β-mercaptoethanol, non-essential amino acids, sodium pyruvate, trypsin-EDTA, fetal bovine serum (FBS): purchased from Gibco BRL (USA);

[0080] 6-well and 24-well cell culture plates, centrifuge tubes, and Pasteur pipettes: purchased from Greiner (Germany);

[0081] Fluorescent mounting medium: purchased from Vector company (U.S.);

[0082] Mouse anti-Oct-3 / 4 antibody (Santa Cruz, SC-5279), goat anti-Sox2 antibody (R&D, AF2018), goat anti-Sox1 antibody (R&D, AF3369);

[0083] Donkey Anti-Goat Fluorescent Alexa 594-labeled IgG second...

Embodiment 2

[0134] Example 2 Purmorphamine induces Olig2 + -GFP + - Directed differentiation of mESCs into spinal motor neurons

[0135] 1) cells

[0136] Same as Example 1.

[0137] 2) Main reagents and supplies

[0138] RA: purchased from Sigma (USA);

[0139] N2, B27, Neurobasal medium: purchased from Gibco BRL company (USA);

[0140] Purmorphamine: purchased from Calbiochem (USA);

[0141] bFGF, NT3, GDNF, BDNF, PDGF-AA: purchased from R&D Company (USA);

[0142] RNase inhibitors, Oligo (dT), dNTP, DNA polymerase: purchased from Takara Company (Dalian, China);

[0143] Primers: Synthesized by Shanghai Sangong Company;

[0144] M-MLV reverse transcription kit, Trizol for Total RNA extraction reagent: purchased from Invitrogen (USA);

[0145] Rabbit anti-Tuj1 antibody (Convance, PRB-435P), mouse anti-Tuj1 antibody (Sigma, T8660), goat anti-Hb9 antibody (Santa cruz, SC-22542), mouse anti-MNR2 antibody (DHSB, 81.5C10), rabbit anti- Isl1 antibody (Chemicon, AB5754), mouse anti-Ho...

Embodiment 3

[0196] Example 3 Purmorphamine induces directed differentiation of Olig2+-GFP+-mES into oligodendrocytes

[0197] Experimental cells are the same as in Example 1. Main experimental apparatus is identical with embodiment 2.

[0198] Main reagents and supplies:

[0199] Insulin, Transferrin, BSA, Progeserone, Na Solenite, Putrescine, T3, Biotin, NAC: purchased from Sigma (USA);

[0200] Rabbit anti-NG2 antibody (Chemicon, MAB5320), mouse anti-O4 antibody (Chemicon, MAB345), mouse anti-MBP antibody (Chemicon, MAB389), rabbit anti-PDGFR-a antibody (Santacruz, SC-338), mouse anti-Nkx2 .2 Antibody (DHSB, 74.5A5), rabbit anti-GFAP antibody (Invitrogen, 709135A);

[0201] Donkey Anti-Rabbit Fluorescent Alexa 594-labeled IgG secondary antibody, donkey anti-mouse fluorescent Alexa 594-labeled IgG secondary antibody: purchased from Invitrogen (USA);

[0202] Preparation of main reagents:

[0203] Table 10100×SATO medium

[0204]

[0205]

[0206] Table 11 Improved SATO me...

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Abstract

The invention belongs to the field of biomedicine, and relates to a method for differentiation of embryonic stem cells into nerve cells through in vitro induction, especially to a method for induction of Olig2<+>-GFP<+>-mES nerve cell differentiation through a purine derivative Purmorphamine, and uses thereof. According to the present invention, an embryoid body mediated nerve induction method is adopted, Olig2-GFP<+>-mES is adopted as a cell model, a purine derivative Purmorphamine and all-trans retinoic acid are combined to carry out directed induction on the Olig2-GFP<+>-mES cells to obtain spinal motor neurons and oligodendrocyte progenitor cells through differentiation; experiment results show that the Purmorphamine can be used as a substitute of SHH, can effectively induce Olig2<+>-GFP<+>-mES differentiation to obtain high purity and function spinal motor neurons and oligodendroglial cells, and can cause expression changes of related genes; and transplant experiment results show that the induced nerve cells can promote partial function and morphology restoration after rat spinal cord injury, and have effects of spinal cord injury regeneration promotion and function reconstruction.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a method for inducing nerve cells, in particular to a method for inducing differentiation of embryonic stem cells into nerve cells in vitro, and in particular to purine derivatives (Purmorphamine) inducing Olig2 + -GFP + - A method for neural cell differentiation of mES and its use. Background technique [0002] Spinal cord injury (SCI) is one of the common clinical traumatic diseases. The occurrence of SCI is mostly caused by traffic injuries, falling injuries or sports injuries, etc., and it has been on the rise in recent years. Once SCI is formed, it often leads to severe behavioral dysfunction and brings great burden to patients, families and society. In recent years, regenerative medicine has brought new hope to the treatment of spinal cord injury. Embryonic stem cells (ESCs), with their strong ability to proliferate and differentiate, can be induced to differentiate into neurons...

Claims

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Application Information

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IPC IPC(8): C12N5/0735C12N5/10A61K35/12A61P25/00A61K35/30
Inventor 孙燕彭裕文张素春李瑞锡
Owner FUDAN UNIV
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