CN111321110A - Method for differentiating human pluripotent stem cells into mesoderm - Google Patents
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Abstract
The invention provides a method for differentiating human pluripotent stem cells into mesoderm, which belongs to the technical field of stem cell differentiation culture, and comprises the steps of resuspending human pluripotent stem cells in a complete culture medium, inoculating the cells in a carrier, and replacing a mesoderm differentiation first-stage culture medium after 24 hours; after induction culture for 48h, mesodermal differentiation of human pluripotent stem cells was achieved. After the cell inoculation, the liquid is changed for 2 times, only four components are contained, expensive serum and cell factors are not needed, the experimental period is short, and the result specificity is good.
Description
Technical Field
The invention belongs to the technical field of stem cell differentiation culture, and particularly relates to a method for differentiating human pluripotent stem cells into mesoderm.
Background
Pluripotent Stem Cells (PSCs), including Embryonic Stem Cells (ESCs) and induced pluripotent stem cells (ipscs), are a cell population with self-renewal and multi-directional differentiation potential, are models for studying the molecular mechanisms regulating cell fate transition and animal development, and can also be used for tissue or organ regeneration and drug screening after in vitro induced differentiation into functional cells. PSC can differentiate to form inner, middle and outer 3 germ layers under the interaction of various cytokines. The ectoderm may further develop into the epidermis and nervous system of the body. Differentiation of PSCs into ectoderm is the first step of directed differentiation into corresponding organ cell types, and is also an important indicator for verifying the multipotentiality of PSCs, which is of great significance for PSC quality detection and transformation applications.
The currently commonly used PSC germ layer Differentiation method includes monolayer cytokine induction (monolayered) the monolayer induction culture method provides a relatively simple and rapid way to induce germ layer Differentiation, and the current popular is STEM diff Trilinkage Differentiation Kit product of STEMCELL Technology company, which adopts PSC single cells planted in a matrigel-coated culture dish at high density, and changes the Differentiation culture medium every day to induce endoderm Differentiation within 5 days, however, the product is expensive and the requirement of initial planting cell density is high (2 × 10)5/cm2) And the liquid needs to be changed every day during the differentiation process, and usually only 25-hole PSC differentiation experiments can be performed in one kit (100mL), so that the time and labor cost are high.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for differentiating human pluripotent stem cells into mesoderm, which only needs 2 liquid changes after being inoculated with the human pluripotent stem cells, does not need expensive serum and cytokines, and has a short differentiation cycle.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for differentiating human pluripotent stem cells into mesoderm, which comprises the following steps:
1) after being resuspended in a complete culture medium, the human pluripotent stem cells are inoculated in a carrier, and after 24 hours, the culture medium of the first stage of mesoderm differentiation is replaced;
2) after induction culture for 48h, mesoderm differentiation of the human pluripotent stem cells is realized;
the mesoderm differentiation first-stage culture medium takes a DMEM/F12 culture medium or an RPMI 1640 culture medium as a basic culture medium and comprises 1mg/ml serum albumin, 0.66-1.32mM ascorbic acid and 5-12 mu M CHIR 99021;
the serum albumin comprises bovine serum albumin or human serum albumin.
Preferably, the step 2) further comprises, after 48 hours of induction culture: replacing a mesoderm differentiation second-stage culture medium, and performing induced culture for 24h to realize mesoderm differentiation of the human pluripotent stem cells;
the mesoderm differentiation second-stage culture medium takes DMEM/F12 culture medium or RPMI 1640 culture medium as basic culture medium and comprises 1mg/ml serum albumin and 0.66-1.32mM ascorbic acid.
Preferably, the human pluripotent stem cells are digested into single cells by TrypLE select or Accutase and then inoculated into a carrier.
Preferably, the complete culture medium is an E8 complete culture medium containing 5-10 mu M Y27632 or an mTeSR1 complete culture medium containing 5-10 mu MY 27632.
Preferably, the carrier comprises a culture plate.
Preferably, the plate is coated with laminin 521, matrigel, laminin 511, Geltrex, or vitronectin.
Preferably, the inoculation amount of the human pluripotent stem cells is 2-5 × 104Per cm2。
Preferably, the temperature of the induction culture in the step 1) is 37 ℃, and the CO of the induction culture2The concentration was 5%.
Preferably, the temperature of the induction culture in the step 2) is 37 ℃, and the CO of the induction culture2The concentration was 5%.
The invention provides a method for differentiating human pluripotent stem cells into mesoderm, which comprises the following steps: 1) after being resuspended in a complete culture medium, the human pluripotent stem cells are inoculated in a carrier, and after 24 hours, the culture medium of the first stage of mesoderm differentiation is replaced; 2) after induction culture for 48h, mesoderm differentiation of the human pluripotent stem cells is realized; the mesoderm differentiation first-stage culture medium takes a DMEM/F12 culture medium or an RPMI 1640 culture medium as a basic culture medium and comprises 1mg/ml serum albumin, 0.66-1.32mM ascorbic acid and 5-12 mu M CHIR 99021; the serum albumin comprises bovine serum albumin or human serum albumin.
The mechanism of the method for shortening the differentiation period is as follows: under the condition of maintaining the most basic growth of stem cells and serum-free condition, the activator of a specific signal path required by inducing mesoderm differentiation is added, so that the influence of complex components such as serum on differentiation specificity can be reduced, the induction efficiency is improved, and the differentiation time is shortened.
The invention has the beneficial effects that:
1. the operation is simple: after cell inoculation, only 2 liquid changes are needed;
2. the components are simple: only contains four components, and does not need expensive serum and cytokines;
3. the experimental period is short: the mesoderm differentiation detection of the human induced pluripotent stem cells can be completed only 3-4 days after inoculation;
4. the result specificity is good: only expressing mesoderm specific markers, and not expressing ectoderm and ectoendoderm genes;
5. the result is reliable: after repeated experiments, the results are consistent.
Drawings
FIG. 1 is a photograph of immunofluorescence staining of differentiated cells of example 1;
FIG. 2 shows the results of qRT-PCR in example 1;
FIG. 3 is an immunofluorescent staining pattern of differentiated cells of example 2
FIG. 4 is a photograph of immunofluorescence staining of differentiated cells of example 3;
FIG. 5 is a photograph of immunofluorescent staining of differentiated cells of example 4.
Detailed Description
The invention provides a method for differentiating human pluripotent stem cells into mesoderm, which comprises the following steps:
1) after being resuspended in a complete culture medium, the human pluripotent stem cells are inoculated in a carrier, and after 24 hours, the culture medium of the first stage of mesoderm differentiation is replaced;
2) after induction culture for 48h, mesoderm differentiation of the human pluripotent stem cells is realized;
the mesoderm differentiation first-stage culture medium takes a DMEM/F12 culture medium or an RPMI 1640 culture medium as a basic culture medium and comprises 1mg/ml serum albumin, 0.66-1.32mM ascorbic acid and 5-12 mu M CHIR 99021;
the serum albumin comprises bovine serum albumin or human serum albumin.
In the present invention, the human pluripotent Stem cells are preferably digested into single cells by TrypLE select or Accutase, the complete medium is preferably E8 complete medium containing 5 to 10 μ M Y27632 or mTeSR1 complete medium containing 5 to 10 μ MY27632, the TrypLE select is preferably purchased from Gibco usa, the Accutase is preferably purchased from Stem cell technology, canada, in the present invention, the carrier preferably comprises a culture plate, the culture plate is preferably coated with laminin 521, matrigel, laminin 511, Geltrex or vitronectin, the specification of the culture plate is not particularly limited, a culture plate for culturing human pluripotent Stem cells in a conventional manner is used, the content and coating method of the coated substance in the culture plate are not particularly limited, and a conventional manner is used in the present invention, the inoculation amount of the human pluripotent Stem cells is 2 to 5 μ 5 × 10 to 5 μ 524Per cm2The human pluripotent stem cells are seeded into a culture plate, preferably at 37 ℃ with 5% CO2Incubate overnight.
In the invention, the mesoderm differentiation first-stage culture medium takes a DMEM/F12 culture medium or an RPMI 1640 culture medium as a basal culture medium and comprises 1mg/ml of serum albumin, 0.66-1.32mM of ascorbic acid and 5-12 mu M of CHIR99021, wherein the serum albumin comprises bovine serum albumin or human serum albumin. In the present invention, the DMEM/F12 medium and the RPMI 1640 medium are preferably purchased from Gibco, the serum albumin is preferably purchased from Sigma, and the CHIR99021 is preferably purchased from MedChemexpress. In the present invention, the temperature of the induction culture is preferably 37 ℃, and the CO of the induction culture2The concentration is preferably 5%. The induction culture is to transform stem cells from a pluripotent state to a mesoderm direction.
In the present invention, the induction culture further comprises, after 48 h: replacing a mesoderm differentiation second-stage culture medium, and performing induced culture for 24h to realize mesoderm differentiation of the human pluripotent stem cells; the mesoderm differentiation second-stage culture medium takes DMEM/F12 culture medium or RPMI 1640 culture medium as basic culture medium and comprises 1mg/ml serum albumin and 0.66-1.32mM ascorbic acid.
In the invention, the mesoderm differentiation second-stage culture medium takes a DMEM/F12 culture medium or an RPMI 1640 culture medium as a basic culture medium, and comprises 1mg/ml of serum albumin and 0.66-1.32mM of ascorbic acid, wherein the serum albumin comprises bovine serum albumin or human serum albumin. In the present invention, the DMEM/F12 medium is preferably purchased from Gibco, and the serum albumin is preferably purchased from Sigma. In the present invention, the temperature of the induction culture is preferably 37 ℃, and the CO of the induction culture2The concentration is preferably 5%.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Method for differentiation of human pluripotent stem cells into mesoderm:
day-1 human iPS cells were TrypLE select digested into single cells, resuspended in E8 complete medium containing 5. mu. M Y27632, and counted at 2 × 104Per cm2Inoculating to laminin 521-coated plates, 5% CO at 37 ℃2Culturing overnight;
day 0: after 24 hours, the medium was replaced with the mesoderm differentiation first stage medium; the mesoderm differentiation first-stage culture medium takes DMEM/F12 culture medium as a basic culture medium and contains 1mg/ml bovine serum albumin, 1.32mM ascorbic acid and 6 mu MCHIR 99021;
day 2: after the induction culture is carried out for 48 hours, the medium is replaced by a medium for the second stage of mesoderm differentiation; the mesoderm differentiation second-stage culture medium takes DMEM/F12 culture medium as a basic culture medium and comprises 1mg/ml bovine serum albumin and 1.32mM ascorbic acid;
day3 cells were immunofluorescent stained with antibodies to the mesoderm specific markers BRACHYURY and α -SMA after 24h of re-induction culture, and the results are shown in FIG. 1, and in FIG. 1, the mesoderm-directed differentiated cells were stained positive for the mesoderm markers BRACHYURY and α -SMA (10X).
Collecting cells, extracting total RNA, and carrying out qRT-PCR detection. And (3) detecting genes: human pluripotent stem cell marker genes Oct4 and Nanog, mesodermal marker genes T, Eomes, Tbx6, Mesp 2. The results show that compared with human iPS, the expression of the pluripotent stem cell marker gene of the cells subjected to endoderm directed differentiation is obviously reduced, while the expression of the mesoderm marker gene is obviously increased, wherein the T expression is increased by about 800 times, the Eomes expression is increased by about 1550 times, the Tbx6 expression is increased by 12 times, and the Mesp2 expression is increased by 5 times.
Example 2
Method for differentiation of human pluripotent stem cells into mesoderm:
day-1 human iPS cells were TrypLE select digested into single cells, resuspended in mTeSR1 complete medium containing 5. mu. M Y27632, and plated out as 2 × 104Per cm2Inoculating to laminin 521-coated plates, 5% CO at 37 ℃2Culturing overnight;
day 0: after 24 hours, the medium was replaced with the mesoderm differentiation first stage medium; the mesoderm differentiation first-stage culture medium takes DMEM/F12 culture medium as a basic culture medium and contains 1mg/ml human serum albumin, 1.32mM ascorbic acid and 6 mu MCHIR 99021;
day 2: after induction culture for 48h, replacing the medium with a mesoderm differentiation second-stage medium; the mesoderm differentiation second-stage culture medium takes a DMEM/F12 culture medium as a basic culture medium and comprises 1mg/ml human serum albumin 1.32mM ascorbic acid;
day3 cells were immunofluorescent stained with antibodies to the mesoderm specific markers BRACHYURY and α -SMA after 24h of re-induction culture, and the results are shown in FIG. 3, which shows that the mesoderm-directed differentiated cells were stained positively for the mesoderm markers BRACHYURY and α -SMA (10X).
Example 3
Method for differentiation of human pluripotent stem cells into mesoderm:
day-1 human iPS cells were TrypLE select digested into single cells, resuspended in E8 complete medium containing 5. mu. M Y27632, and counted at 2 × 104Per cm2Inoculating to laminin 521-coated plates, 5% CO at 37 ℃2Culturing overnight;
day 0: after 24 hours, the medium was replaced with the mesoderm differentiation first stage medium; the mesoderm differentiation first-stage culture medium takes DMEM/F12 culture medium as a basic culture medium and contains 1mg/ml bovine serum albumin, 1.32mM ascorbic acid and 6 mu MCHIR 99021;
day 2: after 48h of induction culture, the cells were subjected to immunofluorescence staining using an antibody against the mesoderm specific marker BRACHYURY, and the results are shown in FIG. 4, and in FIG. 4, the mesoderm marker BRACHYURY stained positively for the cells that were differentiated in a mesoderm-oriented manner (10X).
Example 4
Method for differentiation of human pluripotent stem cells into mesoderm:
day-1 human iPS cells were TrypLE select digested into single cells, resuspended in mTeSR1 complete medium containing 5. mu. M Y27632, and plated out as 2 × 104Per cm2Inoculating to laminin 521-coated plates, 5% CO at 37 ℃2Culturing overnight;
day 0: after 24 hours, the medium was replaced with the mesoderm differentiation first stage medium; the mesoderm differentiation first-stage culture medium takes DMEM/F12 culture medium as a basic culture medium and contains 1mg/ml human serum albumin, 1.32mM ascorbic acid and 6 mu MCHIR 99021;
day 2: after 48h of induction culture, the cells were subjected to immunofluorescence staining using an antibody against the mesoderm specific marker BRACHYURY, and the results are shown in FIG. 5, and in FIG. 5, the mesoderm marker BRACHYURY stained positively for the cells that underwent mesoderm-directed differentiation (10X).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method of differentiating human pluripotent stem cells into mesoderm, comprising the steps of:
1) after being resuspended in a complete culture medium, the human pluripotent stem cells are inoculated in a carrier, and after 24 hours, the culture medium of the first stage of mesoderm differentiation is replaced;
2) after induction culture for 48h, mesoderm differentiation of the human pluripotent stem cells is realized;
the mesoderm differentiation first-stage culture medium takes a DMEM/F12 culture medium or an RPMI 1640 culture medium as a basic culture medium and comprises 1mg/ml serum albumin, 0.66-1.32mM ascorbic acid and 5-12 mu M CHIR 99021;
the serum albumin comprises bovine serum albumin or human serum albumin.
2. The method as claimed in claim 1, wherein the step 2) further comprises, after 48 hours of induction culture: replacing a mesoderm differentiation second-stage culture medium, and performing induced culture for 24h to realize mesoderm differentiation of the human pluripotent stem cells;
the mesoderm differentiation second-stage culture medium takes DMEM/F12 culture medium or RPMI 1640 culture medium as basic culture medium and comprises 1mg/ml serum albumin and 0.66-1.32mM ascorbic acid.
3. The method of claim 1, wherein the human pluripotent stem cells are digested into single cells by TrypLE select or Accutase and then seeded into a carrier.
4. The method of claim 1, wherein the complete medium is E8 complete medium containing 5-10 μ MY27632 or mTeSR1 complete medium containing 5-10 μ MY 27632.
5. The method of claim 1, wherein the carrier comprises a culture plate.
6. The method of claim 5, wherein the culture plate is coated with laminin 521, matrigel, laminin 511, Geltrex, or vitronectin.
7. The method according to claim 1, wherein the amount of the inoculated human pluripotent stem cells is 2 to 5 × 104Per cm2。
8. The method of claim 1, wherein the temperature of the step 1) induction culture is 37 ℃, and the CO of the induction culture2The concentration was 5%.
9. The method of claim 1, wherein the temperature of the step 2) induction culture is 37 ℃, and the CO of the induction culture2The concentration was 5%.
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