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Method for efficiently inducing human cells to be reprogrammed into neuronal cells

A technology of neuron cells and human cells, which is applied in the field of efficiently inducing the reprogramming of human cells into neuron cells, achieving the effects of clear action pathways, improved induction efficiency and short induction time.

Pending Publication Date: 2021-08-20
GUANGXI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the key regulatory sites of reprogramming mentioned in the present invention have not been reported before

Method used

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  • Method for efficiently inducing human cells to be reprogrammed into neuronal cells
  • Method for efficiently inducing human cells to be reprogrammed into neuronal cells
  • Method for efficiently inducing human cells to be reprogrammed into neuronal cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Human dermal fibroblasts (BJ) have been successfully reprogrammed into functional neurons using this induced reprogramming method.

[0056] The overall experimental induction such as figure 1 shown.

[0057] The specific operation is as follows:

[0058] Human skin fibroblasts were divided into 5×10 5 Inoculate into a 60mm culture dish at a density of 60mm, replace the neuron induction medium (N2B27+KSR+10μM Forskolin) within 24 hours of inoculation, place at 37°C, 5% CO 2 Incubator for 48 hours. The morphological changes of the cells during the induction process are as follows: figure 2 As shown, induced neurons (CiNCs) with TUJ1 positive rate of 80% were obtained after 48 hours of induction.

[0059] After 48 hours of induction, replace with neuron maturation medium to further promote the maturation of CiNCs, and after 72 hours, replace with neuron medium for long-term culture.

[0060] We performed immunofluorescence detection of neuronal marker antigens on hu...

Embodiment 2

[0063] The difference from Example 1 is that the neuron induction medium used is N2B27+KSR+10mM cAMP. The results of immunofluorescence detection showed that the induced neurons expressed neuronal marker antigens TUJ1, MAP2 and NeuN (see Figure 5 ).

Embodiment 3

[0065] The difference from Example 1 is that the neuron induction medium used is N2B27+KSR+10 μ M 8-Bromo-cAMP. The results of immunofluorescence detection showed that the induced neurons expressed neuron marker antigens TUJ1, MAP2 and NeuN (see Figure 5 ).

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PUM

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Abstract

The invention provides a method for efficiently inducing human cells to be reprogrammed into neuronal cells. The concentration of cAMP is improved or the expression of PKA and CREB is up-regulated or the expression of AMPK, ALK2, ALK3, P38 and JNK is inhibited through a single small molecule compound or gene knockout or gene overexpression. The induced small molecule compound is single and safe, short in induction time, high in induction efficiency, clear in induction action site and gene and clear in molecular regulation mechanism, can be applied to clinical treatment of human neurodegenerative diseases, and provides a safer and more efficient treatment means for treatment of the neurodegenerative diseases. As neurons do not have division and proliferation capacities, fibroblasts and astrocytes with division and proliferation capacities can be induced in vivo and in vitro by utilizing the method; and a large number of induced neurons can be continuously obtained in vivo and in vitro.

Description

technical field [0001] The invention belongs to the field of stem cells, in particular to a method for efficiently inducing the reprogramming of human cells into neuron cells. Background technique [0002] Using induction methods such as transfection of exogenous transcription factors and combination of small molecule compounds, terminally differentiated somatic cells have been reprogrammed into neurons in many species such as humans and mice. In 2015, Pei Gang's research group reported the use of a combination of seven small molecule compounds to reprogram fibroblasts from Alzheimer's patients into neurons. In 2005, Brueckner B et al used a combination of six small molecule compounds to induce human astrocytes into neurons. These small molecular compounds inhibit the expression of non-neuronal genes, promote the expression of neuron-specific genes, and finally obtain mature functional neurons through prolonged culture of induced cells. [0003] Although there are many met...

Claims

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Application Information

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IPC IPC(8): C12N5/0797
CPCC12N2501/01C12N5/0619C12N2506/09C12N2506/1307C12N2501/727C12N2501/115C12N2501/13Y02A50/30
Inventor 黄奔王国栋张丹丹刘权辉吴玉莲吕丹薇胡吉刚谢小莲
Owner GUANGXI UNIV
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