Neural cell system obtained by enabling human induced pluripotent stem cells (hiPSC) to differentiate by means of directional induction, and induction method and application of neural cell system

A nerve cell, directional induction technology, applied in the field of neurobiology, can solve the problems of mixed naive cells, insufficient to provide nerve cell lineages, and inability to meet various cell types, etc., to achieve the effect of treating nervous system diseases.

Active Publication Date: 2017-11-07
杨涛
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are insufficient to provide mature neural cell lineages, resulting in the intermingling of naive cells, leading to the formation of teratomas after intrace...

Method used

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  • Neural cell system obtained by enabling human induced pluripotent stem cells (hiPSC) to differentiate by means of directional induction, and induction method and application of neural cell system
  • Neural cell system obtained by enabling human induced pluripotent stem cells (hiPSC) to differentiate by means of directional induction, and induction method and application of neural cell system
  • Neural cell system obtained by enabling human induced pluripotent stem cells (hiPSC) to differentiate by means of directional induction, and induction method and application of neural cell system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Establishment of co-culture system of bone marrow stromal cell line HS5 and hiPSC cells

[0078] Human bone marrow stromal cell line HS5 (human bone marrow stromal cell line, CRL-11882 TM, American Type Culture Collection (ATCC), Manassas, VA, USA) to induce neural differentiation of hiPSC cells, the composition of the induction medium is: 20% KSR, 1% NEAA, 1 mM glutamine, 0.1 mM β-mercaptoethanol, 10 ng / ml bFGF, 10ng / ml EGF, 10ng / ml BDNF, 10ng / ml NT-3, 2% B27, 0.5mM bisbutyryl cyclic adenosine monophosphate, 1ng / ml transforming growth factor β3 (the above components were purchased from Invitrogen) and 500ng / ml Noggin (R&D Systems, Minneapolis, MN, USA). Among them, the addition of NEAA, bFGF, β-mercaptoethanol and bisbutyryl cyclic adenosine monophosphate is to effectively promote the proliferation activity of cell differentiation products synchronously during the differentiation of stem cells into neural cell lineages; B27 is a serum-free additive ( It m...

Embodiment 2

[0082] Example 2 Three-stage culture of neural differentiation of hiPSC cells

[0083] The first stage: direct co-cultivation of hiPSC cells and HS5 cells for 2 weeks. The composition of the induction medium is the same as that in Example 1. In a six-well plate, hiPSC cells 5 Each / well was directly inoculated on 2 × 10 pre-plated 1 day before 5 cells / well on the HS5 cell layer; for the parallel control group, hiPSC cells were not co-cultured with HS5 cells, but were directly inoculated into Matrigel-coated 6-well plates, and then cultured in the second and third stages by the same method. Change the solution every other day.

[0084] The second stage: continuous culture with 1:1 diluted HS5-CM medium for 2 weeks; preparation of HS5-CM medium: 1×10 7 HS5 cells were irradiated and inoculated into a culture dish containing 10ml hiPSC induction medium, and the waste liquid was collected every day for 4 consecutive days to obtain HS5-CM medium. Before use, the collected HS5-CM w...

Embodiment 3

[0086] Example 3 Direct contact co-culture activates the Notch signaling pathway in hiPSC cells

[0087] In view of the fact that the Notch ligands Delta1, delta3, Jagged1 and Jagged2 can be easily detected in HS5 cells by using the "reverse transcription PCR" method in the experimental method ( figure 2 a), while the Notch corresponding receptors Notch1, Notch2 and Notch3 can be detected in hiPSC cells and their derivatives ( figure 2 b), It is speculated that the Notch signaling pathway mediates the interaction between HS5 and hiPSC cells. The NICD protein dissociates from the Notch receptor after being acted on by γ-secretase, and targets the downstream molecules Hes1 and Hes5. After induction of differentiation for 8 days, the expression level of NICD protein in hiPSC-derived cells in each group was detected by using the "Western blot analysis" method in the experimental method. The results showed that the direct contact co-culture group was significantly higher than t...

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Abstract

The invention discloses a neural cell system obtained by enabling human induced pluripotent stem cells (hiPSC) to differentiate by means of directional induction, and an induction method and application of the neural cell system. The method comprises the step of culturing the hiPSC by stages so as to induce the neuronal differentiation of the hiPSC, wherein the stages comprises a. carrying out co-culture on the hiPSC and bone marrow stromal cells (HS5) in an induced medium; b. continuously culturing the hiPSC by using an HS5 conditioned medium; c. using a basic medium for culturing neuronal cells to continuously culture the hiPSC. The method provided by the invention can induce the hiPSC to directionally differentiate into nervous system cells and inhibit the generation of non-nervous system cells at the same time, thus obtaining a mature and broad-spectrum neural cell group. The neural cell group is not only proved to be mature neurons having electrical impulse discharge by means of in vitro validation, but is also confirmed to have a function of effectively treating nervous system diseases (such as brain stroke and brain injury) in experiments of mice in vivo.

Description

technical field [0001] The invention belongs to the field of neurobiology, and specifically relates to a neural cell system, induction method and application after directional induction of hiPSC differentiation. Background technique [0002] Nerve cells derived from human induced pluripotent stem cells (hiPSC) have a significant effect on cell transplantation in the treatment of ischemic stroke (characterized by the severe loss of a variety of different strains of nerve cells). However, the existing procedures for inducing hiPSC cells to differentiate into neural cells are inefficient and poor in stability. [0003] Recent advances in stem cell biology provide a basis for regenerative medicine, in which directed differentiation of hiPSC cells may provide a basis for cell transplantation in patients with ischemic stroke characterized by severe deficits of various neurons and glial cells. Various human neurons. Efficient induction, purification, and implantation of human neu...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/077
CPCC12N2501/11C12N2506/45C12N2510/00C12N5/0618C12N15/01C12N13/00C12N5/0669C12N2500/32C12N2501/13C12N2501/15C12N2501/998C12N2500/84C12N2500/38C12N2500/40C12N2500/44C12N2501/115C12N2501/999C12N2502/1394
Inventor 杨涛隋昳
Owner 杨涛
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