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Method for inducing astrocytes to transdifferentiate into 5-hydroxytryptaminergicneuron and application

A kind of astrocyte, serotonin technology, applied in biological field

Pending Publication Date: 2020-04-28
CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, those skilled in the art have not yet succeeded in inducing functional serotonergic neurons using astrocytes as starting cells

Method used

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  • Method for inducing astrocytes to transdifferentiate into 5-hydroxytryptaminergicneuron and application
  • Method for inducing astrocytes to transdifferentiate into 5-hydroxytryptaminergicneuron and application
  • Method for inducing astrocytes to transdifferentiate into 5-hydroxytryptaminergicneuron and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1. Screening for transcription factors to induce serotonergic neurons

[0122] 1. Isolation and purification of astrocytes

[0123] In order to induce the reprogramming of astrocytes to produce serotonergic neurons, the inventors combined a series of transcription factors (Ascl1, Nkx2.2, Gata2, Gata3, Foxa2, Lmx1b, Pet1, Insm1, Sim1) were cloned into the lentiviral vector as candidate genes. The transcription factors were all expressed through the Ubiquitin promoter, a ubiquitous promoter on the FUGW lentiviral vector. The FUGW vector expressing the reporter gene EGFP alone was used as the control group. Astrocytes from the dorsal midbrain of postnatal day 5 (P5) mice were collected, isolated and purified, and used as the initial cells for transcription factor-induced 5-HTergic neurons. The purity of the starting cells was detected by immunofluorescent staining of astrocyte-specific marker molecules GFAP and S100β ( figure 1 ). Approximately 85% and 97% of ...

Embodiment 2

[0144] Example 2. Comparison of electrophysiological characteristics between induced serotonergic neurons and in vivo serotonergic neurons

[0145] The inventors prepared acutely dissociated brain slices of the raphe nucleus of TPH2-iCreERT2-tdTomato knock-in mice at postnatal day 28-32, and recorded the electrophysiological properties of tdTomato-positive serotonergic neurons ( Figure 9 A). It was found that the five serotonergic neurons (iSNs) generated after 41 days of induction by five transcription factors (M5) could be compared with the serotonergic neurons (Native ) generate similar action potentials ( Figure 9 A, B). Statistical action potential complexity found that there was no significant difference in the proportion of cells capable of generating multiple action potentials between the two ( Figure 9 C). The frequency adaptation of the action potential (spikefrequency accommodation, SFA). There was no significant difference in the SFA values ​​of serotonerg...

Embodiment 3

[0148] Embodiment 3, optimizing culture condition improves induction efficiency and maturity

[0149] 1. Add small molecules to optimize culture conditions

[0150] Obtaining enough serotonergic neurons in an in vitro culture dish is important for studying their functions, and has the potential to improve diseases caused by dysfunction of serotonergic neurons. The present inventors tried to add small molecules (Small molecules, SM) to the induction medium, and tested whether the addition of small molecules could improve the efficiency of inducing serotonergic neurons. In the induction system for transdifferentiation to obtain serotonergic neurons, the inventors added the neurotrophic factor BDNF (20ng / ml ). The small molecule medium is based on the induction medium, and Y27632 (10 μM), PD0332991 (1 μM), neurotrophic factors GDNF (20ng / ml) and BDNF (20ng / ml) are added throughout the induction process. On day 9, the small molecules Dorsomorphin (0.5 μM) and SB431542 (5 μM) we...

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Abstract

The invention relates to a method for inducing astrocytes to transdifferentiate into 5-hydroxytryptaminergicneuron and application thereof. The invention discloses a method for generating functional 5-hydroxytryptaminergicneuron by using different transcription factor combinations to induce astrocytes to be directly reprogrammed in vitro, and the obtained neurons can survive after being transplanted into the brain. Chemical small molecules are added on the basis of application of transcription factors, so that the efficiency and maturation degree of generating 5-hydroxytryptaminergicneuron canbe further improved. The 5-hydroxytryptaminergicneuron generated by the method can be used for nerve regeneration, simulation of various nervous system diseases and drug screening.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a method and application for inducing transdifferentiation of astrocytes into serotonergic neurons. Background technique [0002] As a kind of modulation neurons, serotonergic neurons act on many different brain regions by releasing the neurotransmitter 5-Hydroxytryptamine (5-HT), and play an important regulatory function in the central nervous system. A large number of studies have shown that the function of serotonin (5-HT) neurons is related to a variety of behaviors. Pain perception, sexual orientation, sleep and cycle rhythm, food intake and various emotional behaviors are related to the function of the serotonergic nervous system. . Moreover, a series of psychiatric diseases are closely related to abnormal function of serotonergic neurons, including depression, anxiety, obsessive-compulsive disorder, schizophrenia, autism and drug addiction. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12Q1/02A61K35/30A61P25/00A61P25/28A61P25/24A61P25/18A61P25/16
CPCC12N5/0619G01N33/5058A61K35/30A61P25/00A61P25/28A61P25/24A61P25/18A61P25/16C12N2501/60C12N2506/08C12N2501/11C12N2501/115C12N2501/13G01N2500/10
Inventor 程乐平韩素娥王毅刘月光袁嘉成
Owner CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI
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