Method for quickly, directly and directionally inducing differentiation from mouse embryonic stem cells to neuroepithelial cells

Abstract

The invention discloses a method for quickly, directly and directionally inducing differentiation from mouse embryonic stem cells to neuroepithelial cells. The method comprises the steps of adding an induction medium containing dorsomorphin, noggin, SB431542 and CHIR99021 to a bulk clone of the mouse embryonic stem cells subjected to adherent culture, to induce the mouse embryonic stem cells to form rosette neurospheres; then adding a second stage of induction medium containing bFGF and performing suspension culture to form suspended neurospheres; and finally, digesting the neurospheres into single cells, and then performing adherent culture with a third stage of induction medium to form the neuroepithelial cells with long-term self proliferation and update. By adopting the method, the traditional EB method is abandoned, the differentiation time is shortened, and the differentiation efficiency reaches over 95%. The method can be used for quickly inducing NE cells to substitute NSC cells to treat central system injury, and has very high clinical applicability.

Description

technical field

[0001] The invention belongs to the field of biomedicine, in particular to a composition for rapidly and directly inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells, especially a method for rapidly and directly inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells using the composition, and A method for further directing neuroepithelial cells to differentiate into neuron cells or glial cells. Background technique

[0002] Neural stem cells (NSCs) are a kind of mother cells with division potential and self-renewal ability, which exist in adult brain tissue as a kind of stem cells. Mainly distributed in the ependyma, subventricular zone, striatum, hippocampal dentate gyrus and other regions, it is a kind of pluripotent cell that can self-renew and differentiate into a variety of neural cells in the adult nervous system. neurons, astrocytes, and oligodendrocytes. The discovery of neural stem ce...

Examples

Embodiment 1

[0079] Example 1 Rapid and direct induction of mouse embryonic stem cells to differentiate into neuroepithelial cells

[0080] The purpose of the experiment: mouse embryonic stem cells differentiate into neuroepithelial cells with high efficiency and the generated neuroepithelial cells can carry out self-replication and long-term proliferation. Mouse embryonic stem cells (mESCs) were purchased from Shanghai Stance Biotechnology Co., Ltd., strain M1.

[0081] First, 24-well plates (Corning) were coated with Matrigel (BD). After cell counting, mouse embryonic stem cells (mESCs) were plated at 1x10 3 The number per well was seeded on a cell culture plate. Add 400 μl of culture medium to each well, and change the medium every other day. After 3-5 days, the cells grow into clump-like clones, and then start induction, which is divided into 3 stages.

[0082] 1) The first stage: the medium of the adherent cultured mouse embryonic stem cell cluster clones was replaced with the fir...

Embodiment 2

[0089] Example 2 Immunological identification of mouse neuroepithelial cells

[0090] The first to third generation of neuroepithelial cells obtained in Example 1 were subjected to immunofluorescence detection, and the specific steps included fixation, membrane rupture, and blocking solution for blocking for 1 hour, and then adding primary antibodies at the ratio of Nestin (Beyotime) 1:10 , Sox2 (CST) 1:200, Sox1 (R&D) 1:10, Pax6 (Abcam) 1:20, CD133 (Bioss) 1:100 or Sox9 (Millipore) 1:5000 overnight at 4°C, after washing with PBS, add a Anti-appropriate fluorescent secondary antibody FITC (ImmunoResearch), CY3 (ImmunoResearch) was incubated at room temperature for 2 hours. Finally, after washing with PBS, the cells were stained with Hoechst or DAPI and mounted with a mounting agent. Detection was performed under a fluorescence microscope (Zeiss) and a scanning laser confocal imaging system (Leica). Immunofluorescent staining of NE revealed expression of the neural progenitor...

Embodiment 3

[0091] Example 3 Identification of polar structure of neuroepithelial cells and neural rosette-shaped specific markers The neuroepithelial cells obtained in Example 1 were subjected to cell immunofluorescence staining, the operation method was the same as in Example 2, and the primary antibodies used were respectively PLZF (Santa Cruz) 1:50, ZO-1 (Santa Cruz) 1:50, SOX2 (CST) 1:200, PAX6 (Abcam) 1:200, the results show that the neuroepithelial cells obtained in Example 1 show nuclear expression transcription Factors PLZF and DACH1, and the expression of neuroepithelial polarity marker ZO-1 was also found (such as Figure 6 As shown, A. NE-specific transcription factor DACH1, also expresses neural precursor marker Nestin. B. NE can also be stained by the neural precursor transcription factor Sox2. C.NE can express the polarity marker ZO-1, indicating the polarity characteristics of neuroepithelial cells, and can also express the neural precursor marker Pax6. D.NE expresses it...

technical field

[0001] The invention belongs to the field of biomedicine, in particular to a composition for rapidly and directly inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells, especially a method for rapidly and directly inducing the differentiation of mouse embryonic stem cells into neuroepithelial cells using the composition, and A method for further directing neuroepithelial cells to differentiate into neuron cells or glial cells. Background technique

[0002] Neural stem cells (NSCs) are a kind of mother cells with division potential and self-renewal ability, which exist in adult brain tissue as a kind of stem cells. Mainly distributed in the ependyma, subventricular zone, striatum, hippocampal dentate gyrus and other regions, it is a kind of pluripotent cell that can self-renew and differentiate into a variety of neural cells in the adult nervous system. neurons, astrocytes, and oligodendrocytes. The discovery of neural stem ce...

Examples

Embodiment 1

[0079] Example 1 Rapid and direct induction of mouse embryonic stem cells to differentiate into neuroepithelial cells

[0080] The purpose of the experiment: mouse embryonic stem cells differentiate into neuroepithelial cells with high efficiency and the generated neuroepithelial cells can carry out self-replication and long-term proliferation. Mouse embryonic stem cells (mESCs) were purchased from Shanghai Stance Biotechnology Co., Ltd., strain M1.

[0081] First, 24-well plates (Corning) were coated with Matrigel (BD). After cell counting, mouse embryonic stem cells (mESCs) were plated at 1x10 3 The number per well was seeded on a cell culture plate. Add 400 μl of culture medium to each well, and change the medium every other day. After 3-5 days, the cells grow into clump-like clones, and then start induction, which is divided into 3 stages.

[0082] 1) The first stage: the medium of the adherent cultured mouse embryonic stem cell cluster clones was replaced with the fir...

Embodiment 2

[0089] Example 2 Immunological identification of mouse neuroepithelial cells

[0090] The first to third generation of neuroepithelial cells obtained in Example 1 were subjected to immunofluorescence detection, and the specific steps included fixation, membrane rupture, and blocking solution for blocking for 1 hour, and then adding primary antibodies at the ratio of Nestin (Beyotime) 1:10 , Sox2 (CST) 1:200, Sox1 (R&D) 1:10, Pax6 (Abcam) 1:20, CD133 (Bioss) 1:100 or Sox9 (Millipore) 1:5000 overnight at 4°C, after washing with PBS, add a Anti-appropriate fluorescent secondary antibody FITC (ImmunoResearch), CY3 (ImmunoResearch) was incubated at room temperature for 2 hours. Finally, after washing with PBS, the cells were stained with Hoechst or DAPI and mounted with a mounting agent. Detection was performed under a fluorescence microscope (Zeiss) and a scanning laser confocal imaging system (Leica). Immunofluorescent staining of NE revealed expression of the neural progenitor...

Embodiment 3

[0091] Example 3 Identification of polar structure of neuroepithelial cells and neural rosette-shaped specific markers The neuroepithelial cells obtained in Example 1 were subjected to cell immunofluorescence staining, the operation method was the same as in Example 2, and the primary antibodies used were respectively PLZF (Santa Cruz) 1:50, ZO-1 (Santa Cruz) 1:50, SOX2 (CST) 1:200, PAX6 (Abcam) 1:200, the results show that the neuroepithelial cells obtained in Example 1 show nuclear expression transcription Factors PLZF and DACH1, and the expression of neuroepithelial polarity marker ZO-1 was also found (such as Figure 6 As shown, A. NE-specific transcription factor DACH1, also expresses neural precursor marker Nestin. B. NE can also be stained by the neural precursor transcription factor Sox2. C.NE can express the polarity marker ZO-1, indicating the polarity characteristics of neuroepithelial cells, and can also express the neural precursor marker Pax6. D.NE expresses it...

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