Method for inducing differentiation of human pluripotent stem cells into spinal cord motor nerve precursor cells

A pluripotent stem cell and motor nerve technology, applied in the field of inducing human pluripotent stem cells to differentiate into spinal cord motor nerve precursor cells, can solve the problems of only about 60% differentiation efficiency, cumbersome and complicated operation process and low efficiency, etc. Eliminate system instability, clear chemical composition, and good repeatability

Active Publication Date: 2018-03-30
BEIJING TRANSGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional method of differentiating pluripotent stem cells into spinal motor nerve precursor cells has the following problems: (1) The operation process is cumbersome and complicated, involving the differentiation of human pluripotent stem cells into embryoid bodies in suspension culture, and the embryoid bodies are carefully The rosette-like neural tube structure was obtained by wall culture, the rosette-like structure was screened and suspended into neurospheres, and the neurospheres were adhered to the wall and differentiated to obtain spinal cord motor nerve precursor cells; (2) long time, low efficiency, Human pluripotent stem cells were induced to differentiate to obtain spinal cord motor nerve precursor cells for as long as 28 days, and the differentiation efficiency was only about 60%; (3) The culture medium used in the differentiation process added animal-derived components

Method used

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  • Method for inducing differentiation of human pluripotent stem cells into spinal cord motor nerve precursor cells
  • Method for inducing differentiation of human pluripotent stem cells into spinal cord motor nerve precursor cells
  • Method for inducing differentiation of human pluripotent stem cells into spinal cord motor nerve precursor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Method for Differentiation of Human Pluripotent Stem Cells into Spinal Motor Nerve Precursor Cells

[0036] 1. Preparation of culture medium

[0037] 1.1 Coating of Petri dishes

[0038] 1.1.1 Coating of PLO-Laminin Petri dishes

[0039] Dilute poly-L-ornithine (PLO) with PBS to 15 μg / mL, add it to the culture dish until it covers the bottom of the culture dish, see Table 1 for the amount added, and incubate at 37°C for 2 hours or 4 hours. overnight at ℃, do not let the bottom of the culture dish dry during incubation; discard PLO, rinse twice with PBS, and once with DMEM / F12; dilute laminin to 5 μg / mL with DMEM / F12, Add to the PLO-coated Petri dish to cover the bottom of the Petri dish. See Table 1 for the amount added, and incubate at 37°C for 2 hours or overnight at 4°C.

[0040] 1.1.2 Coating of Vitronectin Petri dishes

[0041] Dilute Vitronectin (Life technologies) to 5 μg / mL with DMEM / F12 and add it to the culture dish to be coated. Please refer to ...

Embodiment 2

[0085] Example 2 The method for differentiating human pluripotent stem cells into spinal cord motor nerve precursor cells

[0086] 1. Preparation of culture medium

[0087] 1.1 The coating of petri dish is the same as embodiment 1

[0088] 1.2 Basic complete medium includes

[0089] Basal medium (volume ratio): 50% DMEM / F12 medium and 50% Neurobasal medium;

[0090] Nutritional additives: the composition and final use concentration of the nutritional additives are: human insulin 0.1mg / L, vitamin C 10mg / L, glutathione 10mg / L, linolenic acid 0.05mg / L, carnitine 0.2mg / L, N-acetylcysteine ​​5 μM, ethanolamine 0.01 mg / L, linoleic acid 0.05 mg / L.

[0091] 1.3 Pluripotent stem cell culture medium

[0092] mTeSR medium

[0093] 2. Inducing human pluripotent stem cells to differentiate into spinal cord motor nerve precursor cells

[0094] 1) Culture of human pluripotent stem cells

[0095] Human pluripotent stem cells in good growth condition were digested into single cells with ...

Embodiment 3

[0103] Example 3 Method for Differentiation of Human Pluripotent Stem Cells into Spinal Motor Nerve Precursor Cells

[0104] 1. Preparation of culture medium

[0105] 1.1 The coating of petri dish is the same as embodiment 1

[0106] 1.2 Basic complete medium includes

[0107] Basal medium (volume ratio): 50% DMEM / F12 medium and 50% Neurobasal medium

[0108] 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.

[0109] 1.3 Pluripotent stem cell culture medium

[0110] E8 medium

[0111] 2. Method for Inducing Human Pluripotent Stem Cells to Differentiate into Spinal Motor Nerve Precursor Cells

[0112] 1) Culture of human pluripotent stem cells

[0113] Human pluripotent stem cells in good growth condition were digested into single cells with Accutase, ac...

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Abstract

The discloses a method for inducing differentiation of human pluripotent stem cells into spinal cord motor nerve precursor cells. The method comprises the following steps: 1) culturing the human pluripotent stem cells: digesting the human pluripotent stem cells into single cells and inoculating into a coated petri dish to perform adherent culture, wherein a culture solution is a pluripotent stem cell culture solution, and culture is performed in a saturated humidity incubator at the temperature of 37 DEG C with the CO2 concentration being 5% for 20 to 28 h; 2) inducing the differentiation of the spinal motor neural precursor cells: culturing the human pluripotent stem cells obtained in the step 1) in a basic complete medium, adding different spatiotemporal specific signal pathways at different times to regulate small molecules and / or growth factors so as to induce the differentiation, changing the solution once every two days, culturing in the saturated humidity incubator at the temperature of 37 DEG C with the CO2 concentration being 5% for 8 to 20 days, then inducing the differentiation of the human pluripotent stem cells into the spinal cord motor nerve precursor cells. The method disclosed by the invention is high-efficient, rapid, stable, safe, and easy and simple to operate, and can obtain more than 90% of the spinal cord motor nerve precursor cells after the differentiation is performed on the eighth day.

Description

technical field [0001] The invention relates to the technical field of stem cell differentiation and culture. More specifically, it relates to a method for inducing differentiation of human pluripotent stem cells into spinal cord motor nerve precursor cells. Background technique [0002] Human pluripotent stem cells have unlimited proliferation ability and the potential to differentiate into various types of somatic cells in the human body. They are important seed resource cells for the study of developmental regulation mechanisms, disease pathogenesis and regenerative medicine. However, at present, the differentiation of human pluripotent stem cells into specific functional cells generally has problems such as long differentiation time and low differentiation efficiency, which seriously affects its application in the field of biomedicine. [0003] In recent years, the number of spinal cord injury patients due to car accidents, falls, contusions and other reasons has been i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
CPCC12N5/0618C12N2501/15C12N2501/385C12N2501/415C12N2501/727
Inventor 王娟马静辛文
Owner BEIJING TRANSGEN BIOTECH CO LTD
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