Application of recombinant human Notch1 protein in preparation of neural stem cells and cortical neurons

A neural stem cell and neuron technology, applied in the field of biomedicine, can solve problems such as genotype gap, and achieve the effects of increased number, increased activity, and stable function

Active Publication Date: 2020-07-24
QINGDAO HAIER BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the literature "Single-cell study of neural stem cells derived from human iPSCs reveals distinct progenitor populations with neurogenic and gliogenic potential" (Matti Lam, Tsukasa Sanosaka, Anders Lundin, et al., Genes to Cells, 2019: 24), the human The neural stem cells induced by iPSCs and the primary cultured human embryonic stem cells were subjected to single-cell sequencing. Although both have the potential to differentiate into nerves and glia, there is still a large gap in their genotypes.

Method used

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  • Application of recombinant human Notch1 protein in preparation of neural stem cells and cortical neurons
  • Application of recombinant human Notch1 protein in preparation of neural stem cells and cortical neurons
  • Application of recombinant human Notch1 protein in preparation of neural stem cells and cortical neurons

Examples

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Embodiment 1

[0056] A kind of preparation method of neural stem cells

[0057] The U1 iPSCs used in Example 1 were derived from urinary renal epithelial cells of healthy male adults, and the cells used in the experiment were at passage 22, specifically including the following steps:

[0058] (1) Configure culture medium

[0059] Neural stem cell induction medium: Neurobasal medium+1×N2+1×B27+1×GlutaMAX-I+500ng / ml human recombinant Noggin+20μM SB431542+4ng / ml FGF-2;

[0060] Improved neural stem cell maintenance medium: Neurobasal medium+1×N2+1×B27+10ng / ml EGF+10ng / ml FGF-2+20ng / ml BDNF+100ng / ml recombinant human Notch1 protein;

[0061] (2) When the confluence of U1 iPSCs cells reaches 100% or more, start to induce differentiation, discard the iPSCs medium, wash once with PBS, add the neural stem cell induction medium in step (1), and put it in at a volume concentration of 5% CO 2 Cultured in a 37°C constant temperature cell culture incubator, half of the fresh medium was added on the 3...

Embodiment 2

[0104] A kind of preparation method of neural stem cells

[0105] The DY iPSCs used in Example 2 were derived from healthy male adult skin fibroblasts, and the cells used in the experiment were at passage 18, specifically including the following steps:

[0106] (1) Configure culture medium

[0107] Neural stem cell induction medium: Neurobasal medium+1×N2+1×B27+1×GlutaMAX-I+500ng / ml human recombinant Noggin+20μM SB431542+4ng / ml FGF-2;

[0108] Improved neural stem cell maintenance medium: Neurobasal medium+1×N2+1×B27+10ng / ml EGF+10ng / ml FGF-2+20ng / ml BDNF+100ng / ml recombinant human Notch1 protein;

[0109] (2) When the confluence of DY iPSCs cells reaches 100% or more, start to induce differentiation, discard the iPSCs medium, wash once with PBS, add the neural stem cell induction medium in step (1), and put it in at a volume concentration of 5% CO 2 Cultured in a constant temperature cell culture box at 37°C, half the amount of fresh medium was added on the 2nd day and 4th...

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Abstract

The invention relates to a preparation method of neural stem cells and application. In the invention, the high-efficiency expansion problem of the neural stem cells is solved through a method for adding a Notch signalling pathway receptor Notch1; the neural stem cells steady in function and high in activity can be obtained, and can be further differentiated into a large number of cortical functional neurons; the foundation is laid for obtaining the cortical functional neurons; and the purpose of screening multiple drugs at the same time can be satisfied.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of recombinant human Notch1 protein in preparing neural stem cells and cortical neurons. Background technique [0002] Stroke is a group of diseases that cause brain tissue damage due to cerebrovascular blockage or rupture, including ischemic stroke and hemorrhagic stroke. Its death rate ranks second in the world and it is also the leading cause of disability in Chinese adults. Among them, the incidence of ischemic stroke is relatively high, accounting for 60-70% of the total number of strokes. At present, the acute treatment of ischemic stroke is the application of thrombolytic drug recombinant human tissue plasminogen activator (rt-PA), but the application of this drug has a strict time limit, within 4.5 hours of onset, Moreover, the side effects of secondary intracranial hemorrhage caused by it also limit its application, so it is urgent to find...

Claims

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Application Information

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IPC IPC(8): C12N5/0797C12N5/0793C12Q1/02
CPCC12N5/0623C12N5/0619G01N33/5073G01N33/5058C12N2506/45C12N2501/115C12N2501/42C12N2501/11C12N2501/13G01N2500/10
Inventor 杨潘朱玮王琳娜李慧
Owner QINGDAO HAIER BIOTECH CO LTD
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