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Method used for inducing differentiation of stem cells into neurone, neurone, and applications

A stem cell differentiation and neuron technology, applied in artificially induced pluripotent cells, biochemical equipment and methods, and other methods of inserting foreign genetic materials, etc., can solve the problems of slow differentiation, poor stability and hidden dangers of iPSCs, and achieve application promising effect

Active Publication Date: 2019-02-26
GUIDON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compound-based iPSC differentiation is relatively slow and has poor stability; the method of using lentivirus to generate gene integration still has safety risks in clinical transformation, and the practical operability is not ideal

Method used

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  • Method used for inducing differentiation of stem cells into neurone, neurone, and applications
  • Method used for inducing differentiation of stem cells into neurone, neurone, and applications
  • Method used for inducing differentiation of stem cells into neurone, neurone, and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Synthesis of mRNA and Culture of iPSCs

[0043] 1) Culture of stem cells

[0044] In this example, stem cells were selected from iPSCs (which can be obtained by reprogramming technology from a variety of human cells), using mTeSR TM 1 kit, culture iPSCs according to the method provided in the technical manual of the kit (https: / / cdn.stemcell.com / media / files / manual / MA28315-Maintenance_of_Human_Pluripotent_Stem_Cells_mTeSR1_CN.pdf), the morphological characteristics of the obtained iPSCs include dense clones , compact center and smooth edges, large nuclei, prominent nucleoli, and high nucleoplasmic ratio.

[0045] 2) Acquisition of target gene mRNA

[0046] 2.1 Cloning of the target gene and linearization of the vector plasmid

[0047] 2.1.1 Cloning of NEUROD1: In this example, the target gene is NEUROD1, and the cloning process is as follows: Figure 5 shown.

[0048] The coding sequence of human NEUROD1 (full name: neuronal differentiation 1, accession n...

Embodiment 2

[0071] Example 2. Transfection of mRNA and differentiation of iPSCs

[0072] The differentiation of iPSCs and the transfection of mRNA were carried out at the same time, that is, the mRNA transfection of iPSCs was carried out three times in stages during the whole differentiation process, and the interval between adjacent transfections was 24h. Taking the transfection of NEUROD1 mRNA as an example, the specific process is as follows:

[0073] Day 1: The iPSCs obtained in 1) the cultivation of iPSCs in Example 1 were prepared according to mTeSR TM 1 The technical manual of the kit was digested into single cells, and then spread it on GFR Matrigel (purchased from Corning Corporation (Corning Corporation) Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, Cat. No. 356230) coated 12-well culture plate, 6 × 10 5 cells / well, add DAPT(N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, N-[N-(3,5-difluorophenylacetyl) base)-L-alanyl-S-phenylglycin...

Embodiment 3

[0087] Embodiment 3, cryopreservation and recovery of miN

[0088] The mRNA-induced neuronal miN obtained in Example 2 can be cryopreserved in a cryopreservation solution containing 40wt% neuron medium B27, 50wt% fetal calf serum and 10% DMSO. Thaw and recover when in use, that is, thaw quickly in a 37°C water bath, shake the cryovial gently until there are still ice cubes in the tube, transfer the liquid in the tube to a centrifuge tube, and add 3-5 times the volume of the liquid for neuron culture Base B27, mix gently, centrifuge at 300g for 5 minutes, discard the supernatant, collect the precipitate and resuspend in the cell neuron culture medium (containing neurotrophic factors BDNF (10ng / ml), GDNF (10ng / ml) , TGFβ-3 (1ng / ml), cAMP (0.1mM), ascorbic acid (0.2mM) and DAPT (10μM) in neuron medium B27), and then plated on Poly-D-Lysine / Laminin-coated glass containing In the culture plate of the piece, the number of cells is 2×10 5 Gently shake the culture plate to ensure th...

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Abstract

The invention discloses a method used for inducing differentiation of stem cells into neurone, neurone, and applications. According to the method, target gene mRNA is adopted for transfection of stemcells, and induction of stem cell differentiation so as to obtain neurone which is called as mRNA induced neurone. According to the method, utilization of the biological characteristics of human induced multifunctional stem cell iPSCs is adopted, liposome transfected mRNA is adopted in expression of transcription factors capable of promoting neurone differentiation, so that orientation differentiation of stem cells into neuron in vitro is realized. The method is capable of providing guarantee for supplement of neurone damage deflection, and providing foundation for applications of replacing ofclinical treatment of neurodegenerative diseases such as Parkinson disease, Alzheimer's disease, and cerebral ischemic injury with stem cell transplantation. The method is of great effect in the field of medical science, and the application prospect is promising.

Description

technical field [0001] The present invention relates to the technical field of stem cell differentiation, in particular to a method for inducing stem cells to differentiate into neurons, in particular, a method for inducing stem cells to differentiate into dopaminergic neurons (dopaminergic neurons), cholinergic neurons (cholinergic neurons) ), gamma-aminobutyric acid neuron (GABAergic neuron), glutamatergic neuron (glutaminergic neuron) and other types of neuron fast and efficient method, and neurons obtained by this method, target gene mRNA in Application of an inducer for inducing differentiation of stem cells into neurons. Background technique [0002] Pluripotent stem cells (PSCs) have unique self-renewal ability and multilineage differentiation potential, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Human PSCs, especially iPSCs derived from patients with diseases, have a variety of applications in tissue regenerative medicine, incl...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/88
CPCC12N5/0619C12N15/88C12N2500/38C12N2501/01C12N2501/13C12N2501/15C12N2501/734C12N2506/45C12N2510/00
Inventor 张建民王靓
Owner GUIDON PHARM INC
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