Method for in vitro production of mammalian neurons

A mammalian and neuron technology, applied in the direction of supporting/immobilizing microorganisms, biochemical equipment and methods, botany equipment and methods, etc., can solve the problem of undetectable protein subtypes, etc.

Pending Publication Date: 2022-05-27
UNIV DE BORDEAUX +4
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the corresponding protein isoforms could not yet be detected

Method used

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  • Method for in vitro production of mammalian neurons
  • Method for in vitro production of mammalian neurons
  • Method for in vitro production of mammalian neurons

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Experimental program
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Embodiment

[0074] Materials and methods

[0075] 1. iPSC cell line and encapsulation

[0076]The BC-1 cell line (WT XY, passage 15-25, MTI-Globalstem, Gaithersburg, MD) was maintained in the absence of feeder cells. Plates were coated with Matrigel matrix (Corning, NY, 1 / 100 dilution in DMEM medium) for 2 hours at 37°C. BC-1 colonies were dissociated with ReLeSR (Stemcell Technologies, Vancouver, Canada) and then cultured in mTESR1 (STEMCELL Technologies) supplemented with 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA). The cultures were fed daily and passaged every 5 to 7 days.

[0077] The encapsulation procedure used is described in Alessandri et al., 2016.

[0078] 2. Production of Mature Cortical Neurons

[0079] Neural induction was performed in DMEM / F12 and 10 μM SB431542 (Tocris Bioscience, Bristol, UK) supplemented with B27 and N2 (Thermo Fischer Scientific Inc., Waltham, MA), 1 μM LDN-193189 (Sigma Aldrich, St. Louis, MO) and 10 μM SB431542 (Tocris Bioscience, Bristo...

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Abstract

The present invention relates to a method for the in vitro production of mammalian neurons expressing 6 subtypes (2N4R, 1N4R, 0N4R, 2N3R, 1N3R, 0N3R) of Tau protein, comprising a neuronal differentiation step in which the cell microcompartment is cultured for a period of 5 to 100 weeks, the neuronal differentiation step is carried out in a bioreactor, each cell microcompartment comprising a hollow hydrogel capsule surrounding post-mitotic neuronal cells and extracellular matrix, the cell microcompartments remaining suspended in a housing of the bioreactor containing a neuronal differentiation medium.

Description

technical field [0001] The present invention relates to a method for producing mammalian neurons in vitro. More specifically, the method according to the present invention enables the production of neurons expressing 6 isoforms of Tau protein (2N4R, 1N4R, ON4R, 2N3R, 1N3R, ON3R). The present invention has application in relation to neurodegenerative diseases, particularly in the fields of diagnostics and cell therapy, and also for the identification and development of new therapeutic methods. Background technique [0002] Tau is a multifunctional protein that was originally identified as a microtubule-associated cytoplasmic protein. Six isoforms of Tau are expressed in the adult brain, resulting from alternative splicing of the MAPT gene. Tau splicing is regulated during development such that, unlike the adult brain, which expresses the six isoforms, only the shortest isoform of Tau is expressed in the fetal brain. [0003] The accumulation of pathological intracellular d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/0793C12N5/10C12N15/12C12M3/00
CPCC12N5/0619C12N5/0622C07K14/47C12M25/16C12N2506/45C12N2513/00C12N2533/74C12N2533/90C12N2500/10C12N2500/32
Inventor 马克西姆·法耶尤科斯凯文·亚历山德里马加里·勒库托伊斯莱蒂西亚·米格尔皮埃尔·纳索伊安妮·罗夫莱特勒克鲁斯
Owner UNIV DE BORDEAUX
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