A method for inducing the differentiation of human spinal cord motor neuron precursor cells into spinal cord motor neurons

A motor neuron and motor nerve technology, applied to nervous system cells, vertebrate cells, animal cells, etc., can solve the problems of long differentiation time, introduction of pathogenic microorganisms, low differentiation efficiency, etc., achieve clear chemical composition, eliminate batches The effect of quality differences

Active Publication Date: 2021-01-15
BEIJING TRANSGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional differentiation of neural precursor cells into neurons generally has the problems of long differentiation time and low differentiation efficiency
Moreover, the use of animal-derived components and extracted proteins will be involved in the differentiation process. Animal-derived components will potentially introduce the risk of pathogenic microorganisms. It is difficult to control the uniformity of extracted protein batches, which will lead to instability of the differentiation system.
In the traditional process of differentiation of spinal cord motor nerve precursor cells into spinal cord motor neurons, the above problems also exist. The differentiation time needs at least seven days, and the differentiation efficiency is less than 40%.

Method used

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  • A method for inducing the differentiation of human spinal cord motor neuron precursor cells into spinal cord motor neurons
  • A method for inducing the differentiation of human spinal cord motor neuron precursor cells into spinal cord motor neurons
  • A method for inducing the differentiation of human spinal cord motor neuron precursor cells into spinal cord motor neurons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Method for Differentiation of Human Spinal Motor Nerve Precursor Cells into Spinal Motor Neurons

[0042] 1. Preparation of culture medium

[0043] 1.1. Coating of PLO-Laminin culture dish:

[0044] Dilute poly-L-ornithine (PLO) with PBS to 15 μg / mL, add to the culture dish until the bottom of the culture dish is submerged, incubate at 37°C for 2 hours or overnight at 4°C, do not let the Dry the bottom of the culture dish; discard PLO, rinse twice with PBS, and once with DMEM / F12; See Table 1 for the amount added, and incubate at 37°C for 2 hours or overnight at 4°C.

[0045] Table 1 The amount of PLO and Laminin required to coat the culture dish

[0046] Petri dish specification Growth area (cm 2 )

Amount added (mL) 6-well plate 10cm 2 / well

1mL / well 12-well plate 4cm 2 / well

0.4mL / well 24-well plate 2cm 2 / well

0.2mL / well 35mm culture dish 10cm 2

1mL 60mm culture dish 20cm 2

...

Embodiment 2

[0069] Example 2 Method for Differentiation of Human Spinal Motor Nerve Precursor Cells into Spinal Motor Neurons

[0070] 1. Preparation of culture medium

[0071] 1.1 The coating of petri dish is the same as embodiment 1

[0072] 1.2 Spinal cord motor nerve precursor cell culture medium

[0073] Basal medium (volume ratio): 50% DMEM / F12 medium and 50% Neurobasal medium;

[0074] Nutritional additives: the components and final use concentrations of the nutritional additives are: human insulin 10mg / L, vitamin C 50mg / L, glutathione 50mg / L, linolenic acid 1mg / L, carnitine 20mg / L, N-acetylcysteine ​​5μM, ethanolamine 5mg / L, linoleic acid 1mg / L.

[0075] 1.3 Basic complete medium

[0076] Basal medium (volume ratio): 50% DMEM / F12 medium and 50% Neurobasal medium;

[0077] Nutritional additives: the components and final use concentrations of the nutritional additives are: human insulin 10mg / L, vitamin C 50mg / L, glutathione 50mg / L, linolenic acid 1mg / L, carnitine 20mg / L, N-ace...

Embodiment 3

[0090] Example 3 Method for Differentiation of Human Spinal Motor Nerve Precursor Cells into Spinal Motor Neurons

[0091] 1. Preparation of culture medium

[0092] 1.1 The coating of petri dish is the same as embodiment 1

[0093] 1.2 Spinal cord motor nerve precursor cell culture medium

[0094] Basal medium (volume ratio): 50% DMEM / F12 medium and 50% Neurobasal medium;

[0095] Nutritional additives: the components and final use concentrations of the nutritional additives are: human insulin 20mg / L, vitamin C 200mg / L, glutathione 100mg / L, linolenic acid 5mg / L, carnitine 20mg / L, N-acetylcysteine ​​500μM, ethanolamine 10mg / L, linoleic acid 5mg / L.

[0096] 1.3 Basic complete medium

[0097] Basal medium (volume ratio): 50% DMEM / F12 medium and 50% Neurobasal medium

[0098] Nutritional additives: the components and final use concentrations of the nutritional additives are: human insulin 20mg / L, vitamin C 200mg / L, glutathione 100mg / L, linolenic acid 5mg / L, carnitine 20mg / L, N...

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PUM

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Abstract

Provided is a method for inducing human spinal motor neuron progenitors to differentiate into spinal motor neurons, comprising the following steps: 1) culture of the spinal motor neuron progenitors: digesting the spinal motor neuron progenitors into single cells, inoculating the single cells into a coated petri dish for adherent culture with a spinal motor neuron progenitor culture medium, and culturing in an incubator at 37°C, 5% CO2 and saturated humidity; and 2) induction of the spinal motor neurons: culturing the spinal motor neuron progenitors obtained from step 1) in a basic complete medium, adding different space-time-specific signaling pathway modulating small molecules and / or growth factors at different time points to induce differentiation, culturing for 3-30 days in an incubator at 37°C, 5% CO2 and saturated humidity, changing the medium every other day.

Description

technical field [0001] The invention relates to the technical field of cell differentiation and culture. More specifically, it relates to a method for inducing the differentiation of human spinal cord motor neuron precursor cells into spinal cord motor neurons. Background technique [0002] In recent years, the number of spinal cord injury patients due to car accidents, falls, contusions and other reasons has been increasing year by year. Due to damage to the internal nerves of the spinal cord, patients with spinal cord injury generally suffer from paralysis of the lower limbs, making it difficult to take care of themselves. Under normal physiological conditions, damaged nerve cells in the human body cannot repair themselves, so traditional medical methods are difficult to completely cure the disease. Cell replacement therapy provides a new therapeutic approach for patients with spinal cord injury. Spinal motoneurons are one of the ideal cell types for disease treatment o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2500/32C12N2500/36C12N2500/38C12N2500/40C12N2500/46C12N2501/065C12N2501/13C12N2501/33C12N2501/385C12N2501/405C12N2501/734C12N2501/999C12N2533/32C12N2533/52
Inventor 王娟马静辛文
Owner BEIJING TRANSGEN BIOTECH CO LTD
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