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Method for amplifying human neural precursor cells through regulation of Wnt signals and/or Notch signals

A technology of precursor cells and human nerves, applied in the biological field, can solve the problems of limited effect on disease progression, high disability rate, and expensive monitoring costs

Pending Publication Date: 2020-09-22
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] As the most common severe neurological diseases in clinical practice, Alzheimer's disease and epilepsy have high disability rates and expensive monitoring costs
Although there are currently some conventional treatments for this type of disease, including anticholinesterase inhibitors or antiepileptic drugs, surgical treatment, and nursing rehabilitation, these methods can only have a limited therapeutic effect in the early stage of the disease. It cannot fundamentally solve the problem, and has limited effect on the development of the disease

Method used

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  • Method for amplifying human neural precursor cells through regulation of Wnt signals and/or Notch signals
  • Method for amplifying human neural precursor cells through regulation of Wnt signals and/or Notch signals
  • Method for amplifying human neural precursor cells through regulation of Wnt signals and/or Notch signals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Recovery and culture of human embryonic stem cells:

[0049] (1) Preheat DMEM / F12 medium and human embryonic stem cell (hES) medium in a water bath at 37°C.

[0050] (2) Take the 6-well plate covered with MEF, suck away the MEF medium, add 2ml DMEM / F12 medium to each well, shake gently and suck off the DMEM / F12 medium, add hES medium to each well 2ml and put back into the 37°C incubator.

[0051] (3) Take out the frozen hES cells from the liquid nitrogen tank, and shake them gently in a water bath at 37°C to quickly thaw them.

[0052] (4) Add the thawed hES cells into a 15ml centrifuge tube containing 6ml hES medium, mix gently, and centrifuge at 1100rpm for 1min.

[0053] (5) Aspirate the supernatant, take out the MEF plate added with hES medium, resuspend the cells with 0.5ml hES medium, add the cells into the 6-well plate, mix well, and culture in a 37°C incubator.

[0054] (6) After 12-15 hours, suck off the culture medium, then add 2.5ml of fresh hES medium ...

Embodiment 2

[0265] Human embryonic stem cells can be directed to differentiate into human forebrain cortex precursor cells and MGE precursor cells. For the specific schematic diagram, see figure 1 a. The resource of human ES cells comes from the international commonly used human embryonic stem cell line H9. Human ES cells were cultured on mouse embryonic fibroblasts (MEF) treated with radiation (the formula of the culture medium: 392.5ml DMEM / F12, 100ml Knockout serum replacer, 5ml MEM nonessential aminoacids solution, 2.5ml 200mM L- glutamine solution, 3.5ml 14.3M β-Mercaptoethanol). The cell clones were passaged every 5 days through a combination of mechanical and chemical methods. The method of neural differentiation of human embryonic stem cells refers to the third part of Example 1. The culture medium contains bFGF factor. When it differentiates towards neuroectodermal cells, ES cells are gathered into embryoid bodies (Embroynic Body, EBs) were suspended cultured in a medium witho...

Embodiment 3

[0269] In order to clarify the role of Wnt signaling, we knocked out β-catenin, an important effector in the Wnt signaling pathway, in human embryonic stem cells (KO cells, this cell line has been published; Liu, Z., Hui, Y., Shi, L ., Chen, Z., Xu, X., Chi, L., Fan, B., Fang, Y., Liu, Y., Ma, L., et al. (2016). Efficient CRISPR / Cas9-Mediated Versatile , Predictable, and Donor-Free Gene Knockout in Human Pluripotent StemCells.Stem Cell Reports 7,496-507.), and Western blot experiments verified the absence of β-catenin protein ( figure 2 A), for the experimental method, refer to Part 7 of Example 1 for details. We performed transcriptome sequencing (RNA-seq) on the MGE precursor cells differentiated from normal (WT, i.e. human embryonic stem cell line H9 in Example 2) and gene knockout (KO) embryonic stem cells. The specific method See Parts 8 to 11 of Example 1. We found that compared with WT cells, KO-MGE precursor cells had a total of 960 differential genes, including 400...

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Abstract

The invention relates to the technical field of biology, in particular to a method for amplifying human nerve precursor cells through regulation of Wnt signals and / or Notch signals. The culture methodof the human neural precursor cells comprises the step of culturing the human neural precursor cells, of which Wnt signals and / or Notch signals are exogenously regulated, under proper conditions. Ina culture system of directionally differentiated human neural cells, exogenous activation, inhibition or over-expression is performed on the Wnt signals and / or Notch signals, so that the development process of the neural precursor cells is interfered exogenously, the proliferation ability of the neural precursor cells is enhanced, and large-scale and long-time proliferation of the neural precursorcells is promoted; and meanwhile, the normal differentiation potential of the neural precursor cells into multiple specific types of neurons is maintained, and therefore, the method is provided for in-vitro and large-scale culture of the neural precursor cells which have continuous proliferation ability and normal neural differentiation potential.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for amplifying human neural precursor cells by regulating Wnt signal and / or Notch signal. Background technique [0002] As the most common severe neurological diseases in clinical practice, Alzheimer's disease and epilepsy have a high disability rate and expensive monitoring costs. Although there are currently some conventional treatments for this type of disease, including anticholinesterase inhibitors or antiepileptic drugs, surgical treatment, and nursing rehabilitation, these methods can only have a limited therapeutic effect in the early stage of the disease. It cannot fundamentally solve the problem, and has a limited effect on the development of the disease. From the perspective of disease pathology, the cause of Alzheimer's disease is that β-amyloid first damages basal forebrain cholinergic neurons, while epilepsy is mainly due to the dysfunction of GABAergic intern...

Claims

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Application Information

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IPC IPC(8): C12N5/079A61K35/30A61P9/10A61P25/00A61P25/08A61P25/28A61P25/16A61P25/14A61P21/00
CPCC12N5/0618A61K35/30A61P9/10A61P25/00A61P25/08A61P25/28A61P25/16A61P25/14A61P21/00C12N2501/415C12N2501/42
Inventor 章小清刘玲马琳
Owner TONGJI UNIV
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