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Method for differentiating human pluripotent stem cells into mesoderm

A human pluripotent stem cell and germ layer technology, which is applied in non-embryonic pluripotent stem cells, artificially induced pluripotent cells, biochemical equipment and methods, etc., can solve the problems of high time and labor costs, high density requirements, and high costs. , to achieve the effect of reliable results, good specificity of results and simple ingredients

Pending Publication Date: 2020-06-23
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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  • Application Information

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Problems solved by technology

However, the cost of this product is expensive, and the initial planting cell density requirement is high (2×10 5 / cm 2 ), and the medium needs to be changed every day during the differentiation process. Usually, a kit (100mL) can only do 25 wells of PSC differentiation experiments, and the time and labor costs are also high

Method used

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  • Method for differentiating human pluripotent stem cells into mesoderm
  • Method for differentiating human pluripotent stem cells into mesoderm
  • Method for differentiating human pluripotent stem cells into mesoderm

Examples

Experimental program
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Embodiment 1

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

[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 for the first stage of mesoderm differentiation; the medium for the first stage of mesoderm differentiation is based on DMEM / F12 medium, containing 1mg / ml bovine serum albumin, 1.32mM ascorbic acid and 6 μM CHIR99021;

[0047] Day 2: After 48 hours of induction culture, replace with the medium for the second stage of mesoderm differentiation; the medium for the second stage of mesoderm differentiation is based on DMEM / F12 medium, including 1mg / ml bovine serum albumin and 1.32mM ascorbic acid ;

[0048] Day 3: After re-induction culture for 24 hours, the cells were immunofluore...

Embodiment 2

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

[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] Day0: After 24 hours, replace with the medium for the first stage of mesoderm differentiation; the medium for the first stage of mesoderm differentiation is based on DMEM / F12 medium, containing 1mg / ml human serum albumin, 1.32mM ascorbic acid and 6μM CHIR99021 ;

[0054] Day2: After 48 hours of induction culture, replace with the medium for the second stage of mesoderm differentiation; the medium for the second stage of mesoderm differentiation is based on DMEM / F12 medium, including 1mg / ml human serum albumin and 1.32mM ascorbic acid;

[0055] Day3: After re-induction culture for 24 hours, the cells were immunofluoresc...

Embodiment 3

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

[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] Day0: After 24 hours, replace with the medium for the first stage of mesoderm differentiation; the medium for the first stage of mesoderm differentiation is based on DMEM / F12 medium, containing 1mg / ml bovine serum albumin, 1.32mM ascorbic acid and 6μM CHIR99021 ;

[0060] Day2: After induction and culture for 48 hours, the cells were immunofluorescently stained with the antibody of the mesoderm-specific marker BRACHYURY, and the results are shown in Figure 4 , Figure 4 In the figure, the mesoderm marker BRACHYURY was positively stained (10X) in cells undergoing mesoderm-directed differentiation.

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Abstract

The present invention provides a method for differentiating human pluripotent stem cells into mesoderm and belongs to the technical field of stem cell differentiation and culture. The method comprisesthe following steps: inoculating human pluripotent stem cells in a carrier after resuspending the human pluripotent stem cells in a complete culture medium and replacing a first-stage mesodermal differentiation culture medium after 24 h; and after 48 h of induction culture, realizing mesoderm differentiation of the human pluripotent stem cells. After the cell inoculation, only 2 fluid exchanges are required, only four components are contained, no expensive serum and cytokines are needed, experimental period is short, and results have good specificity.

Description

technical field [0001] The invention belongs to the technical field of stem cell differentiation and culture, and in particular relates to a method for human pluripotent stem cells to differentiate into mesoderm. 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 typ...

Claims

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Application Information

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IPC IPC(8): C12N5/074
CPCC12N5/0696C12N2500/38C12N2501/998
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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