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Human iPSC-sourced pyramidal nerve cell precursor cell as well as preparation method and application thereof

A technology of nerve cells and precursor cells, which is used in the field of spinal cord injury treatment to improve motor function and facilitate transplantation and application.

Inactive Publication Date: 2021-05-28
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The currently researched cells for the treatment of spinal cord injury mainly focus on the nutritional support and protection of the nerves, mainly reflected in the fact that the transplanted cells can secrete neurotrophic factors or support the regeneration of the host nerve cells. Robust regeneration over long distances and subsequent clinical improvement remains a specific challenge

Method used

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  • Human iPSC-sourced pyramidal nerve cell precursor cell as well as preparation method and application thereof
  • Human iPSC-sourced pyramidal nerve cell precursor cell as well as preparation method and application thereof
  • Human iPSC-sourced pyramidal nerve cell precursor cell as well as preparation method and application thereof

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preparation example Construction

[0047] The present invention provides a method for preparing pyramidal nerve cell precursor cells derived from human iPSCs, comprising the following steps: 1) inoculating human iPSCs on matrigel matrigel, and pre-cultivating them in iPSC-specific medium for 2 days; 2) inoculating The pre-cultured cells are placed in the first induction differentiation medium for the first induction differentiation culture to obtain the first differentiated cells; 3) the first differentiation cells are placed in the second induction differentiation medium for the second induction differentiation culture Obtaining second differentiated cells; 4) placing the second differentiated cells in a third induction differentiation medium for third induction differentiation culture to obtain pyramidal nerve cell precursor cells.

[0048] In the present invention, human iPSCs were seeded on matrigel, and pre-cultured in iPSC-specific medium for 2 days. The present invention has no special limitation on the ...

Embodiment 1

[0061] Preparation of pyramidal neuron precursor cells derived from human iPSCs

[0062]Referring to the differentiation methods of published articles, human iPSCs were differentiated into pyramidal neurons under the action of the small molecule Dorsomorphin (Espuny-Camacho, I., Michelsen, Kimmo A., Gall, D., Linaro, D., Hasche ,A.,Bonnefont,J.,et al.(2013).Pyramidal Neurons Derived from Human PluripotentStem Cells Integrate Efficiently into Mouse Brain Circuits InVivo.Neuron 77(3),440-456.doi:10.1016 / j.neuron.2012.12 .011.). The present invention uses enhanced green fluorescent protein (GFP) expression lentivirus to infect iPSCs

[0063] The isolated single cells were inoculated in Matrigel matrigel and pre-cultured for 2 days; the surface density of inoculated cells was 8000-10000 cells / cm 2 ; Medium is the E8 medium that adds the ROCK Inhibitor of 10mmol / L;

[0064] After 2 days of pre-culture, replace the medium with the first induction differentiation medium: DDM mediu...

Embodiment 2

[0069] The first differentiation induction medium was changed to: DDM medium + Dorsomorphin at a final concentration of 0.2 μmol / L + B27 at 20 mL / L; other operations were the same as in Example 1.

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Abstract

The invention provides a human iPSC-sourced pyramidal nerve cell precursor cell as well as a preparation method and application thereof, and belongs to the technical field of spinal cord injury treatment, human iPSC is induced to be differentiated into the pyramidal nerve cell precursor cell through a Dorsomorphin small molecule compound, and the differentiation proportion is up to 90% or above. According to the pyramidal nerve cell precursor cell, after the transplanted rat strikes the model, the movement function of the rat can be obviously improved. The pyramidal nerve cell precursor cell is transplanted to an immunodeficient mouse and a rat injected with an immunosuppressive agent, and the differentiated pyramidal nerve cell precursor cell can survive after being transplanted and shows relatively high axon projection capacity.

Description

technical field [0001] The invention belongs to the technical field of spinal cord injury treatment, and in particular relates to a pyramidal nerve cell precursor cell derived from human iPSC and its preparation method and application. Background technique [0002] Spinal cord injury (Spinal Cord Injury, SCI) is a serious neurological injury disease. It is caused by external trauma that causes damage to the gray matter and white matter of the spinal cord. Inflammation occurs in the injured area, and the formed thrombus aggravates spinal cord ischemia. The scar tissue formed by the massive proliferation of plasmocytes hinders the regeneration of nerve fibers, resulting in the deformation and necrosis of neurons and nerve fibers. Spinal cord injuries can lead to varying degrees of paraplegia in patients. Spinal cord injury will not only bring serious physical and psychological harm to the patients themselves, but also cause a huge economic burden to the whole society. Due to...

Claims

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Application Information

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IPC IPC(8): C12N5/0793C12N5/071A61K35/30A61P25/00
CPCC12N5/062A61K35/30A61P25/00C12N2506/45C12N2533/90C12N2501/405C12N2501/155C12N2501/727C12N2533/32C12N2533/52
Inventor 陈志国李默
Owner XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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