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A method for differentiating human pluripotent stem cells to ectoderm

A technology for human pluripotent stem cells and ectoderm, which is applied in the field of ectoderm differentiation of human pluripotent stem cells, can solve the problems of uncertain differentiation effect, high culture cost, complicated process, etc., and achieves short experimental period, simple components and reliable results. Effect

Active Publication Date: 2021-11-16
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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Problems solved by technology

This method has a long period (2 weeks), complicated process (suspension transfer to the wall), high culture cost (a differentiation experiment needs to change the medium no less than 7 times, and uses a high-concentration serum medium), and the differentiation effect is uncertain (the existence of finding The risk of less than specific germ layer cells) and other disadvantages are gradually abandoned by people

Method used

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  • A method for differentiating human pluripotent stem cells to ectoderm
  • A method for differentiating human pluripotent stem cells to ectoderm
  • A method for differentiating human pluripotent stem cells to ectoderm

Examples

Experimental program
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Effect test

Embodiment 1

[0044]Method for differentiating human pluripotent stem cells to ectoderm:

[0045] Day-1: Human iPS cells were digested into single cells with TrypLE select, resuspended in E8 complete medium containing 5 μM Y27632 and counted according to 2×10 4 piece / cm 2 Inoculate onto laminin 521-coated culture plates at 37°C in 5% CO 2 Culture overnight;

[0046] Day 0: After 24 hours, replace with the medium of the first stage of ectoderm differentiation; the medium of the first stage of ectoderm differentiation is based on DMEM / F12 medium, containing 1 mg / ml bovine serum albumin and 2 μM AM580;

[0047] Day 2: After 48 hours of induction culture, replace with the medium of the second stage of ectoderm differentiation; the medium of the second stage of ectoderm differentiation is based on DMEM / F12 medium, including 1mg / ml bovine serum albumin;

[0048] Day 3: After re-induction culture for 24 hours, immunofluorescent staining was performed on the cells using the antibodies of ectoder...

Embodiment 2

[0051] Method for differentiating human pluripotent stem cells to ectoderm:

[0052] Day-1: Human iPS cells were digested into single cells with TrypLE select, resuspended in mTeSR1 complete medium containing 5 μM Y27632, and counted according to 2×10 4 piece / cm 2 Inoculate onto laminin 521-coated culture plates at 37°C in 5% CO 2 Culture overnight;

[0053] Day 0: After 24 hours, replace with the medium of the first stage of ectoderm differentiation; the medium of the first stage of ectoderm differentiation is based on DMEM / F12 medium, containing 1 mg / ml human serum albumin and 2 μM AM580;

[0054] Day 2: After 48 hours of induction culture, replace with the medium of the second stage of ectoderm differentiation; the medium of the second stage of ectoderm differentiation is based on DMEM / F12 medium, including 1mg / ml human serum albumin;

[0055] Day 3: After re-induction culture for 24 hours, immunofluorescent staining was performed on the cells using the antibodies of ect...

Embodiment 3

[0057] Method for differentiating human pluripotent stem cells to ectoderm:

[0058] Day-1: Human iPS cells were digested into single cells with TrypLE select, resuspended in E8 complete medium containing 5 μM Y27632 and counted according to 2×10 4 piece / cm 2 Inoculate onto laminin 521-coated culture plates at 37°C in 5% CO 2 Culture overnight;

[0059] Day 0: After 24 hours, replace with the medium of the first stage of ectoderm differentiation; the medium of the first stage of ectoderm differentiation is based on DMEM / F12 medium, containing 1 mg / ml bovine serum albumin and 2 μM AM580;

[0060] Day 2: After induction and culture for 48 hours, immunofluorescent staining was performed on the cells using antibodies to ectoderm-specific markers PAX6 and NESTIN, and the results are shown in Figure 4 , Figure 4 Shown in ectoderm-directed differentiation of cells, ectoderm markers PAX6 and NESTIN staining positive (10X).

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Abstract

The invention provides a method for human pluripotent stem cells to differentiate into ectoderm, which belongs to the technical field of stem cell differentiation and culture. Phase medium; after 48 hours of induction culture, the ectoderm differentiation of human pluripotent stem cells was achieved. After the cells are inoculated, the invention only needs to change the medium twice, contains only three components, does not need expensive serum and cytokines, has a short experimental period, and has good specificity of results.

Description

technical field [0001] The invention belongs to the technical field of stem cell differentiation and cultivation, and in particular relates to a method for human pluripotent stem cells to differentiate into ectoderm. Background technique [0002] Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are a type of cell population with the potential of self-renewal and multidirectional differentiation. Models that regulate cell fate transition and molecular mechanisms of animal development can also be used for tissue or organ regeneration and drug screening after induction into functional cells in vitro. Under the interaction of various cytokines, PSC can differentiate into three germ layers: inner, middle and outer. The ectoderm can further develop into the epidermis and nervous system of the body. The differentiation of PSCs to the ectoderm is the first step in directed differentiation into corresponding organ cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10
CPCC12N5/0696C12N2501/998C12N2510/00
Inventor 刘中民贾文文鲁济真汤红明
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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