CN112831461B

CN112831461B - Method and medicine for inducing stem cells to differentiate into mesoderm lineage or trophoblast lineage

Info

Publication number: CN112831461B
Application number: CN202110221046.6A
Authority: CN
Inventors: 陈国凯; 卡洛斯·戈多伊·帕罗约; 张宇萌
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2021-02-26
Filing date: 2021-02-26
Publication date: 2023-08-08
Anticipated expiration: 2041-02-26
Also published as: CN112831461A; WO2022178986A1

Abstract

The invention discloses a method and a medicine for inducing stem cells to differentiate into mesoderm lineages or trophoblast lineages, belonging to the technical field of regulating differentiation of human pluripotent stem cells into mesoderm or trophoblasts. The former method comprises the following steps: differentiation of stem cells into mesodermal lineages is induced with a differentiation medium containing a PKC inhibitor in the presence of BMP4. The method can greatly improve the differentiation of the pluripotent stem cells into mesodermal lineages under the culture condition of definite chemical components, and shortens the differentiation process by half. The method improves differentiation stability, and can maintain germ layer specific differentiation without exogenous FGF2 or endogenous WNT. The latter method comprises: differentiation of stem cells into trophoblast lineages is induced with differentiation media containing PKC activators or DGK inhibitors. The method induces stem cells to differentiate into trophoblasts in an accelerated manner in a spontaneous differentiation platform using PKC activators and DGK inhibitors without the need for BMP4.

Description

Method and medicine for inducing stem cells to differentiate into mesoderm lineage or trophoblast lineage

Technical Field

The invention relates to the technical field of regulating differentiation of human pluripotent stem cells into mesodermal or trophoblastic cells, in particular to a method and a medicament for inducing stem cells to differentiate into mesodermal lineages or trophoblastic lineages.

Background

Human pluripotent stem cells (hpscs) can differentiate into all cell types in our body and are an important model system for studying human embryo development. The specific cell types generated by hPSC differentiation play an important role in drug screening and other studies. Mesoderm produces cell types such as cardiomyocytes, smooth muscle, bone and gonads, and thus mesoderm-derived cell types from hPSC differentiation provide materials for regeneration and repair of a variety of tissues and drug screens. In addition, hPSCs can also differentiate into extra-embryonic cell types, such as primitive endoderm and trophoblasts, in vitro. Extraembryonic cell types produce tissues such as placenta, yolk sac or umbilical cord, and thus extraembryonic cell types derived from hPSC differentiation can provide materials for in vitro fertilization, early immune system and drug screening.

At present, the role of PKC inhibitors in early stem cell differentiation and the role of protein kinase C in early trophoblast differentiation have not been reported. In view of this, the present invention has been made.

Disclosure of Invention

It is an object of the present invention to provide a method of inducing differentiation of stem cells into mesodermal lineages using PKC inhibitors.

It is a second object of the present invention to provide a method for inducing differentiation of stem cells into trophoblast lineages using PKC activators or DGK inhibitors.

The third object of the present invention is to provide a medicament comprising a mesodermal or feeder cell lineage obtained by the above method as a raw material.

The application can be realized as follows:

in a first aspect, the present application provides a method of inducing differentiation of stem cells into mesodermal lineages using a PKC inhibitor, comprising the steps of:

differentiation of stem cells into mesodermal lineages is induced with differentiation medium containing PKC inhibitors in the presence of BMP4.

Mesodermal lineages include mesodermal cells and endodermal precursor cells.

In an alternative embodiment, the stem cells comprise at least one of human embryonic stem cells and human induced pluripotent stem cells.

The human embryonic stem cells described herein are stem cells isolated or obtained from human embryos that have not undergone fertilization for less than 14 days of in vivo development.

In alternative embodiments, the PKC inhibitor comprises GF109203X,And->At least one of (a)。

In an alternative embodiment, the GF109203X is present in a concentration of 2-5. Mu.M.

In an alternative embodiment of the present invention,the concentration of (2) to (5) mu M.

In an alternative embodiment, the BMP4 concentration is 5-100ng/mL.

In alternative embodiments, the components of the differentiation medium include DMEM/F12, L-ascorbic acid-2-magnesium phosphate, sodium selenate, transferrin, and insulin.

In alternative embodiments, exogenous FGF2 or endogenous WNT is not used in the process of inducing stem cells to differentiate into mesoderm.

In a second aspect, the present application provides a method of inducing differentiation of stem cells into trophoblast lineages comprising the steps of: differentiation of stem cells into trophoblast lineages is induced with differentiation media containing PKC activators or DGK inhibitors.

In an alternative embodiment, the PKC activator or DGK inhibitor is added to the differentiation medium on the first day of stem cell differentiation.

In an alternative embodiment, the stem cells are cultured for at least two days in differentiation medium after addition of the PKC activator. Alternatively, the stem cells may be cultured in differentiation medium for at least six days after the addition of the DGK inhibitor.

In an alternative embodiment, the stem cells are human pluripotent stem cells.

In an alternative embodiment, the human pluripotent stem cells comprise at least one of human embryonic stem cells and human induced pluripotent stem cells.

In an alternative embodiment, the PKC activator comprises at least one of TPA and Bryostatin 1.

In alternative embodiments, the TPA is treated at a concentration of 25-500nM.

In an alternative embodiment, the concentration of Bryostatin1 is between 25 and 500nM.

In alternative embodiments, the DGK inhibitor includes R59949.

In an alternative embodiment, the treatment concentration of R59949 is from 0.5 to 10. Mu.M.

In an alternative embodiment, a feeder cell inducer is also added to the differentiation medium during differentiation.

In alternative embodiments, the trophoblast inducer comprises at least one of BMP4, PD325901, sb432542, and DAPT.

In an alternative embodiment, the treatment concentration of BMP4 is 10-100ng/mL.

In an alternative embodiment, the treatment concentration of PD325901 is from 0.5 to 10. Mu.M.

In an alternative embodiment, the treatment concentration of Sb432542 is 5-20 μm.

In an alternative embodiment, the treatment concentration of DAPT is 1-10. Mu.M.

In a third aspect, the present application also provides a medicament, the starting material of which comprises a mesodermal or feeder cell lineage obtained by the method described above.

The beneficial effects of this application include:

according to the application, the PKC inhibitor is used for promoting the differentiation of the pluripotent stem cells into mesoderm in a BMP4 differentiation platform, promoting the expression of the premature germ layer markers TBXT and MIXL1, greatly improving the differentiation of the pluripotent stem cells into mesoderm and endoderm precursor cells, shortening the differentiation process by half, improving the differentiation stability, and providing a new application for stem cell differentiation. Functional mesodermal cell types can be obtained by this method, can be used for regeneration and repair of tissues, such as smooth muscle cells, muscle, bone or gonads, and for drug screening.

In addition, the present application employs a method with clear composition to induce differentiation of hpscs into trophoblasts, wherein the agent used is free of animal-derived components. The pioneering use of PKC activation and DGK inhibition to induce hPSC differentiation into trophoblasts, describes PKC activation as the primary mechanism of action downstream of any other trophoblast-induced platform, and finds new applications for stem cell differentiation. In addition, the trophoblasts obtained by the method can express markers of mature trophoblasts, which provides a new idea for guiding early fate decisions of human pluripotent stem cells.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIGS. 1 to 6 are graphs showing the results of the respective tests in example 1;

fig. 7 to 10 are graphs showing the results of the respective tests in example 2;

fig. 11 to 13 are graphs showing the results of the respective tests in example 3;

fig. 14 to 16 are graphs showing the results of the respective tests in example 4;

fig. 17 to 18 are graphs showing the results of the respective tests in example 7;

fig. 19 to 28 are graphs showing the respective test results in comparative example 1;

fig. 29 to 34 are graphs showing the results of the respective tests in example 8;

fig. 35 to 37 are graphs showing the results of the respective tests in example 9.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The method and drug for inducing differentiation of stem cells into mesodermal or trophoblast lineages provided herein are described in detail below.

A method of inducing differentiation of stem cells into mesodermal lineages using PKC inhibitors as set forth herein, comprising the steps of:

differentiation of stem cells into mesodermal lineages is induced with differentiation medium containing PKC inhibitors in the presence of BMP4. Mesodermal lineages include mesodermal cells and endodermal precursor cells.

Wherein the stem cells comprise at least one of human embryonic stem cells and human induced pluripotent stem cells. Namely, only human embryonic stem cells, only human induced pluripotent stem cells, and also human embryonic stem cells and human induced pluripotent stem cells can be used for differentiation.

The BMP4 as described above, in combination with PKC inhibitors, especially GF109203X, can replace FGF2 as an inducer of early mesodermal cell induction in human embryonic stem cells. By reference, the BMP4 concentration may be 5-100ng/mL, such as 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, etc.

In alternative embodiments, the PKC inhibitor may comprise GF109203X, for example,And->Preferably GF109203X.

By reference, the GF109203X concentration may be 2-5. Mu.M, such as 2. Mu.M, 3. Mu.M, 4. Mu.M, 5. Mu.M, etc.The concentration of (C) may be 2-5. Mu.M, such as 2. Mu.M, 3. Mu.M, 4. Mu.M, 5. Mu.M, etc. />The concentration of (2) may also be 2-5. Mu.M,such as 2. Mu.M, 3. Mu.M, 4. Mu.M, or 5. Mu.M, etc.

In alternative embodiments, the components of the differentiation medium referred to herein may include DMEM/F12, L-ascorbic acid-2-magnesium phosphate, sodium selenate, transferrin, and insulin. The contents of the above components in the differentiation medium may be respectively: l-ascorbic acid-2-magnesium phosphate 64mg/L, sodium selenate 13.6. Mu.g/L, transferrin 10mg/L and insulin 20mg/L. In particular, the differentiation medium may be E6 medium, which medium is understood to mean that E8 medium is free of FGF2 and TGF beta.

In a preferred embodiment, the present application provides that exogenous FGF2 or endogenous WNT is not used in the process of inducing stem cells to differentiate into mesoderm. That is, PKC inhibitors, particularly GF109203X, promote differentiation of human embryonic stem cells H1 into mesoderm without FGF2 pathway activation. Furthermore, PKC inhibitors, in particular GF109203X, promote differentiation of human embryonic stem cells H1 into mesoderm without endogenous WNT.

In alternative embodiments, methods of inducing stem cells to differentiate into mesodermal lineages involving the use of PKC inhibitors according to the present application may be, but are not limited to, reference to the following steps:

after culturing hpscs to a cell density of 10-20% (which is the density before initiation of differentiation), hpscs were induced to differentiate into mesoderm using PKC inhibitor GF109203X in the presence of BMP4. After 2 days, the formation of TBXT positive cells can be detected by immunostaining or FACS (i.e., TBXT positive cells can be measured on the third day of differentiation).

Specifically, it may be: human embryonic stem cells H1 were cultured in E8 medium, replaced with fresh medium every day, and passaged when the cell density reached 70-80% (i.e., the density was that before passaging). DPBS-EDTA was first washed twice, then incubated at room temperature for 5 minutes, DPBS-EDTA was aspirated 3 rd time, and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells are passaged at a density of 1:6 to 1:12 (e.g., 1:6, 1:8, 1:10, or 1:12, etc.), and a 12 well plate pre-coated with Matrigel is added. After 1 day of cell adhesion to the plate, the medium was changed to E6 medium (E8 medium without FGF2 and TGF-. Beta.) along with BMP4 and PKC inhibitors, and differentiation was started at 20ng/mL and 5. Mu.M, respectively. Fresh E6 medium was added daily.

It should be noted that the E6 and E8 media mentioned above may be obtained by referring to the components disclosed in the present application, or may be formulated by referring to the components of the related media in the prior art, and will not be described in detail herein.

Functional mesodermal cell types are obtained by the above methods, can be used for regeneration and repair of tissues, such as smooth muscle cells, muscle, bone or gonads, and for drug screening. Correspondingly, the application also provides a medicine, and the raw materials of the medicine contain mesoderm lineages obtained by the method.

A method of inducing differentiation of stem cells into a trophoblast lineage, comprising the steps of:

differentiation of stem cells into trophoblast lineages is induced with differentiation media containing PKC activators or DGK inhibitors.

Wherein the stem cells are human pluripotent stem cells. By reference, the human pluripotent stem cells include at least one of human embryonic stem cells and human induced pluripotent stem cells, i.e., only human embryonic stem cells, only human induced pluripotent stem cells, and also differentiation can be performed with both human embryonic stem cells and human induced pluripotent stem cells.

In an alternative embodiment, the PKC activator is added to the differentiation medium on the first day of stem cell differentiation. By reference, stem cells continue to be cultured for at least two days in differentiation medium after addition of PKC activators.

In alternative embodiments, the PKC activator can comprise at least one of TPA and Bryostatin1, for example.

Wherein, the treatment concentration of TPA and Bryostatin1 can be 25-500nM, such as 25nM, 50nM, 100nM, 200nM, 300nM, 400nM or 500nM, respectively.

In an alternative embodiment, the DGK inhibitor is added to the differentiation medium on the first day of stem cell differentiation. By reference, after the addition of the DGK inhibitor, the stem cells were cultured for at least six days in the differentiation medium.

In alternative embodiments, the DGK inhibitor may include (be) R59949. The treatment concentration of R59949 can be 0.5-10. Mu.M, such as 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M, etc.

In alternative embodiments, the components of the differentiation medium referred to herein may be DMEM/F12, magnesium L-ascorbate-2-phosphate, sodium selenate, transferrin, and insulin. The contents of the above components in the differentiation medium may be respectively: l-ascorbic acid-2-magnesium phosphate 64mg/L, sodium selenate 13.6. Mu.g/L, transferrin 10mg/L and insulin 20mg/L. In particular, the differentiation medium may be E6 medium, which medium is understood to mean that E8 medium is free of FGF2 and TGF beta.

In the differentiation process, a trophoblast inducer can be added into the differentiation medium.

The treatment concentration of BMP4 may be 10-100ng/mL, such as 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL, 100ng/mL, etc.

The treatment concentration of PD325901 can be 0.5-10. Mu.M, such as 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 8. Mu.M, 10. Mu.M, etc.

The treatment concentration of Sb432542 may be 5-20. Mu.M, such as 5. Mu.M, 8. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, etc.

The treatment concentration of DAPT may be 1-10. Mu.M, such as 1. Mu.M, 2. Mu.M, 5. Mu.M, 8. Mu.M, 10. Mu.M, etc.

In alternative embodiments, the methods of inducing differentiation of stem cells into trophoblast lineages contemplated herein may be, but are not limited to, reference to the following steps:

after culturing hpscs to a cell density of 10-20%, hpscs are induced to differentiate into trophoblasts using PKC modulators. Differentiation is performed using a differentiation medium from the first day, and after 2 days of treatment (preferably the second day of differentiation), signal markers such as TROP2 and CGB can be detected by qPCR.

Specifically, it may be: human embryonic stem cells were cultured in E8 medium, replaced with fresh medium daily, and passaged when the cell density reached 70-80% (i.e., the density was that before passaging). The DPBS-EDTA was first washed twice, then incubated at room temperature for 5 minutes, finally DPBS-EDTA was aspirated, and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells are passaged at a density of 1:6-1:12 (e.g., 1:6, 1:8, 1:10, or 1:12, etc.) to 12 well plates pre-coated with Matrigel. After one day of cell adhesion to the plates, differentiation was initiated by changing to E6 medium (E8 medium without FGF2 and TGF beta) and adding 50nM TPA or Bryostatin 1. Fresh differentiation medium was added daily during this period and trophoblast marker levels were measured on day 2 of differentiation.

Alternatively, human embryonic stem cells H1 are cultured in E8 medium, replaced with fresh medium daily, and passaged when the cell density reaches 70-80%. The cells were first washed twice with DPBS-EDTA and then incubated for 5 minutes at room temperature. DPBS-EDTA was aspirated and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells were passaged at a density of 1:6-1:12 into 12 well plates pre-coated with Matrigel. One day after the cells were adhered to the plates, they were changed to E6 medium (E8 medium without FGF2 and TGF beta) and DGK inhibitor R59949 was added at a concentration of 1. Mu.M to promote cell differentiation. Fresh E6 medium was added daily. Trophoblast marker levels were measured on day 6.

The feeder cell lineages obtainable by the above method may provide materials for early immune system and drug screening. Correspondingly, the application also provides a medicine, and the raw materials of the medicine contain the trophoblast lineages obtained by the method.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

To investigate the role of different PKC inhibitors in early mesodermal differentiation, we selected GF109203X in the first stage of differentiation,and->These three PKC inhibitors and BMP4.

The specific implementation is as follows: human embryonic stem cells H1 were cultured in E8 medium, replaced with fresh medium daily, and passaged when cell density reached 70-80%. DPBS-EDTA was first washed 2 times, then incubated at room temperature for 5 minutes, DPBS-EDTA was aspirated 3 rd time, and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells were passaged at a density of 1:6 and added to 12 well plates pre-coated with Matrigel. After 1 day of cell attachment growth on plates, the cells were replaced with BMP4 and PKC inhibitors in E6 medium (E8 medium without FGF2 and tgfβ) and differentiation was started at 20ng/mL and 5 μm concentration, respectively (cell density 10-20% before differentiation). Fresh E6 medium was added daily. The level of early mesodermal markers of differentiated cells was measured by qPCR, immunofluorescence and flow cytometry (unless otherwise indicated) on the first and second days of differentiation.

Referring to FIGS. 1 to 6, FIG. 1 shows the effect of PKC inhibitor GF109203X on the expression of the early mesoderm marker genes MIXL1 and TBXT gene under BMP4 differentiation. GF109203X and BMP4 were added at concentrations of 5. Mu.M and 20ng/mL, respectively. In the figure, mock represents E6 medium+bmp 4, GFX represents addition of PKC inhibitor GF109203X on the basis of mock, the same applies below.

FIG. 2 represents the proportion of TBXT positive cells analyzed by immunostaining 2 days after induction of stem cell differentiation by addition of BMP4 and GF109203X. In the figure, grey represents positive cells for Hoechst and TBXT groups.

Figure 3 represents the proportion of TBXT positive cells analyzed by flow cytometry 2 days after BMP4 and GF109203X addition to induce stem cell differentiation.

FIG. 4 represents the addition of BMP4 andthe proportion of TBXT positive cells was analyzed by immunostaining 2 days after induction of stem cell differentiation. Add +.5. Mu.M concentration>In the figure, grey represents positive cells for Hoechst and TBXT groups.

FIG. 5 represents the effect of PKC inhibitors on WNT3 gene expression one day after BMP4 differentiation.

FIG. 6 represents the use of 5. Mu.M PKC inhibitorsAnd 20ng/mL BMP4 differentiation on the early mesoderm marker genes WNT3, MIXL1 and TBXT expression. In the figure, the darker the rectangle color represents the higher the expression level of the corresponding gene.

The results showed that, after only 24 hours, GF109203X,and->The expression level of the early mesoderm marker genes MIXL1 and TBXT can be improved. TBXT is a specific marker of early mesodermal differentiation, GF109203X andthe proportion of TBXT positive cells was increased to varying degrees, indicating that PKC inhibitors can induce differentiation of stem cells into the mesodermal early lineage. GF109203X may be added to BMP4 induced differentiation to promote WNT pathway and accelerate early mesodermal differentiation of human embryonic stem cells.

Example 2

GF109203X promotes differentiation of human embryonic stem cells H1 into mesoderm without FGF2 pathway activation.

Human embryonic stem cells H1 are cultured in E8 culture medium, fresh culture medium is replaced every day, and the cell density reaches 70-80%. The cells were resuspended in E8 medium containing 5. Mu.M Rock inhibitor, passaged at a density of 1:6, added to a 12-well plate pre-coated with Matrigel, added to E6 medium containing 20ng/mL BMP4, 100ng/mL FGF2 and 5. Mu.M PKC inhibitor (E8 medium contains neither FGF2 nor TGF beta.) and started to differentiate. Fresh E6 medium was added daily. The level of early mesodermal markers of differentiated cells was measured by qPCR, immunofluorescence and flow cytometry (unless otherwise indicated) on the first and second days of differentiation.

Referring to fig. 7 to 10, fig. 7 shows the effect of PKC inhibitor GF109203X on the expression of the mix l1 and TBXT genes under BMP4 or BMP4+ FGF2 differentiation, which are markers of early mesoderm. Wherein mock represents E6 medium+bmp 4, GFX represents GFX (PKC inhibitor GF 109203X) added on the basis of mock, FGF2 represents FGF2 added on the basis of mock, FGF2+gfx represents FGF2+gfx added on the basis of mock. GF109203X, BMP4 and FGF2 were added at concentrations of 5. Mu.M, 20ng/mL and 100ng/mL, respectively.

FIG. 8 shows the proportion of TBXT positive cells analyzed by flow cytometry 2 days after addition of BMP4/GF109203X (indicated as GFX in the figures) or BMP4/FGF2/GF109203X to induce stem cell differentiation.

FIG. 9 shows the effect of PKC inhibitor GF109203X on the expression of WNT3, MIXL1 and TBXT genes under differentiation of early mesodermal markers BMP4+FGF2 or BMP4+FGF2+PD0173074 (denoted PD173 in the figure, hereinafter the same). PD0173074 was added at a concentration of 100 nM.

Fig. 10 shows the proportion of TBXT positive cells analyzed by immunostaining 2 days after induction of stem cell differentiation by addition of BMP4+ FGF2 and GF109203X (GFX), PD0173074 (PD 173), or both. Wherein mock represents E6 medium+bmp 4+fgf2, GFX represents GFX added on the basis of mock, PD173 represents PD173 added on the basis of mock, and PD173+gfx represents PD173+gfx added on the basis of mock. In the figure, grey represents positive cells for Hoechst and TBXT groups.

The results indicate that GF109203X increases the expression levels of early mesodermal marker genes mix 1 and TBXT only after 24 hours, even in the presence of FGFR inhibitor PD 0173974. FGF2 pathway inhibitor PD0173074 was used at 100 nM. TBXT is a specific marker of early mesodermal differentiation, and GF109203X increases the proportion of TBXT positive cells even in the presence of PD0173074, suggesting that PKC inhibitors may induce stem cells to differentiate into the early mesodermal lineage bypassing FGF2 pathway activation. That is, GF109203X binds to BMP4 and can be used as an inducer for early mesodermal cell induction in human embryonic stem cells instead of FGF2.

Example 3

GF109203X promotes differentiation of human embryonic stem cells H1 into mesoderm without endogenous WNT.

Human embryonic stem cells H1 are cultured in E8 culture medium, fresh culture medium is replaced every day, and the cell density reaches 70-80%. DPBS-EDTA was first washed twice, then incubated at room temperature for 5 minutes, DPBS-EDTA was aspirated 3 rd time, and E8 medium containing 5. Mu. MRock inhibitor was added. After resuspension of the cells, the cells were passaged at a density of 1:6 and added to 12 well plates pre-coated with Matrigel. After 1 day of cell adhesion to the plate, the medium was changed to E6 medium (E8 medium without FGF2 and TGF beta) and differentiation was initiated by the addition of 20ng/mL BMP4 and 5. Mu.M PKC inhibitor. Fresh E6 medium was added daily. The level of early mesodermal markers of differentiated cells was measured by qPCR, immunofluorescence and flow cytometry (unless otherwise indicated) on day 1 and day 2 of differentiation.

Referring to fig. 11 to 13, fig. 11 shows the effect of PKC inhibitor GF109203X (GFX) on WNT3, mix 1 and TBXT gene expression under BMP4 or BMP4+ IWP-2 (IWP 2) differentiation, which are markers of early mesoderm. GF109203X, BMP4 and IWP-2 were added at concentrations of 5. Mu.M, 20ng/mL and 2.5. Mu.M, respectively.

FIG. 12 shows that GF109203X (GFX) promotes β -catenin nuclear localization under BMP4 induction after 6 hours of differentiation by immunostaining analysis. In the figure, grey represents positive cells for Hoechst and np-. Beta.cat groups.

FIG. 13 shows the nuclear localization of β -catenin under quantitative BMP4 and BMP4+GF109203X (GFX) induction at signal intensity after 6 hours of differentiation.

The results indicate that GF109203X can bypass the WNT pathway and promote nuclear localization of β -catenin in human embryonic stem cells. It was demonstrated that GF109203X increased expression levels of early mesodermal markers.

Example 4

GF109203X promotes differentiation of human embryonic stem cells H1 into endoderm.

Human embryonic stem cells H1 are cultured in E8 culture medium, fresh culture medium is replaced every day, and the cell density reaches 70-80%. DPBS-EDTA was first washed twice, then incubated at room temperature for 5 minutes, DPBS-EDTA was aspirated 3 rd time, and E8 medium containing 5. Mu. MRock inhibitor was added. After resuspension of the cells, the cells were passaged at a density of 1:6 and added to 12 well plates pre-coated with Matrigel. After 1 day of cell adhesion to the plate, the medium was changed to E6 medium (E8 medium was free of FGF2 and TGF-. Beta.) and differentiation was initiated by the addition of 100ng/mL of Activin A, 20ng/mL of BMP4, 100ng/mL of FGF2 and 5. Mu.M PKC inhibitor. Thereafter fresh E6 medium and 100ng/mL Activin A were added daily. The level of early endoderm markers of differentiated cells was measured by qPCR, immunofluorescence and flow cytometry (unless otherwise indicated) on day 4 of differentiation. The results indicate that GF109203X can increase the expression level of early endodermal markers.

Referring to fig. 14 to 16, fig. 14 represents early stages of endoderm cell differentiation, i.e., days 0 to 1, embryonic stem cells (PS) can be induced to differentiate into primitive streak cells (PS) by using Acivin a+bmp4+fgf2, and PS cells can be induced to form specific endoderm cells (DE, DE is a precursor of human respiratory tract and digestive organs) under the conditions of activin+ldn 193189+iwp2. As a result, it was found that PKC inhibitor GF109203X in combination with Acivin a+bmp4+fgf2 was able to significantly increase SOX17 expression on days 2 to 3 of differentiation, which is a biomarker of early endoderm, compared to Acivin a+bmp4+fgf 2. GF109203X, activity A, BMP4, LDN193189 and IWP-2 were added at concentrations of 5. Mu.M, 100ng/mL, 20ng/mL, 100nM and 2.5. Mu.M, respectively.

FIG. 15 shows that GF109203X (GFX) promotes differentiation of H9 and H1 cell lines to DE after 2-3 days of differentiation by quantitative PCR analysis.

FIG. 16 shows that GF109203X (GFX) promotes differentiation of H9 cell lines to DE after 2-3 days of differentiation by flow cytometry and gives nearly 100% SOX17 positive DE cells.

The results indicate that GF109203X can promote differentiation of human embryonic stem cell lines (H1 and H9) into endodermal cells.

Example 5

This example provides a method of inducing differentiation of human induced pluripotent stem cells into mesodermal lineage using PKC inhibitors:

human induced pluripotent stem cells (iPSCs, such as NL-1 or NL-4) were cultured in E8 medium, fresh medium was changed daily, and passaged when cell density reached 70-80%. DPBS-EDTA was first washed 2 times, then incubated at room temperature for 5 minutes, DPBS-EDTA was aspirated 3 rd time, and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells were passaged at a density of 1:10 and added to 12 well plates pre-coated with Matrigel. After 1 day of cell adhesion to the plates, the medium was changed to E6 medium (E8 medium without FGF2 and TGF-. Beta.) along with BMP4 and GF109203X, and differentiation was started at 20ng/mL and 2. Mu.M, respectively. Fresh E6 medium was added daily. The level of early mesodermal markers of differentiated cells was measured by qPCR, immunofluorescence and flow cytometry (unless otherwise indicated) on the first and second days of differentiation.

Example 6

This example provides a method of inducing stem cells to differentiate into mesodermal lineages using PKC inhibitors:

human embryonic stem cells H1 were cultured in E8 medium, replaced with fresh medium daily, and passaged when cell density reached 70-80%. DPBS-EDTA was first washed 2 times, then incubated at room temperature for 5 minutes, DPBS-EDTA was aspirated 3 rd time, and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells were passaged at a density of 1:12 and added to 12 well plates pre-coated with Matrigel. After 1 day of cell adhesion to the plates, the medium was changed to E6 medium (E8 medium without FGF2 and TGF-. Beta.) along with BMP4 and GF109203X, and differentiation was started at 20ng/mL and 4. Mu.M, respectively. Fresh E6 medium was added daily. The level of early mesodermal markers of differentiated cells was measured by qPCR, immunofluorescence and flow cytometry (unless otherwise indicated) on the first and second days of differentiation.

Example 7

The present example provides a method for promoting differentiation of human embryonic stem cells into trophoblasts using PKC activators:

to investigate the role of different PKC activation in trophoblast differentiation, we selected the differentiation of hPSCs by two PKC activators, TPA, bryostatin 1. The method comprises the following steps: human embryonic stem cells were cultured in E8 medium, replaced with fresh medium daily, and passaged when cell density reached 70-80%. The DPBS-EDTA was first washed twice, then incubated at room temperature for 5 minutes, finally DPBS-EDTA was aspirated, and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells were passaged at a density of 1:6-1:12 to 12 well plates pre-coated with Matrigel. After 1 day of cell adhesion to the plate, differentiation was initiated by changing to E6 medium (E8 medium without FGF2 and TGF beta) and adding 50nM TPA or Bryostatin 1. Fresh differentiation medium was added daily during this period, and trophoblast marker (TROP 2 and CGB) levels and primitive endoderm marker (SOX 17 and GATA 6) levels were measured on day 3 of differentiation, as shown in fig. 17 and 18.

Among them, FIG. 17 shows the effect of PKC activator TPA on gene expression of primitive endoderm markers (SOX 17 and GATA 6) and trophoblast markers (TROP 2 and CGB) on day 3 of differentiation. TPA was added at a concentration of 50 nM. NANOG is a biomarker of stem cell stem properties, the higher its expression level, the higher the stem properties of the cells; conversely, a lower expression level indicates a lower stem property of the cell, i.e., differentiation of the cell.

FIG. 18 shows the effect of PKC activator Bryostatin1 on gene expression of primitive endoderm markers (SOX 17 and GATA 6) and trophoblast markers (TROP 2 and CGB) on days 2-10 of differentiation. Bryostatin1 was added at a concentration of 50 nM. NANOG is a biomarker of stem cell stem properties, the higher its expression level, the higher the stem properties of the cells; conversely, a lower expression level indicates a lower stem property of the cell, i.e., differentiation of the cell. mock represents E6 medium+bmp 4, GFX represents addition of GFX (PKC inhibitor GF 109203X) on the basis of mock, the same applies below.

The results of fig. 17 and 18 show that: TPA and Bryostatin1 can increase the expression level of primitive endoderm marker genes SOX17 and GATA6 and trophoblast marker genes TROP2 and CGB after 3 days. PKC inhibitor GF109203X inhibits TPA and Bryostatin1 to promote expression of trophoblast specific markers TROP2 and CGB, and PKC activators can induce stem cells to differentiate into trophoblast lineages. Demonstrating that activation of PKC by TPA and Bryostatin1 promotes differentiation into extraembryonic cell types, such as primitive endoderm and trophoblast cells of human embryonic stem cells.

Comparative example 1

This comparative example provides a method of inhibiting differentiation of human pluripotent stem cells H1, H9 and NL4 to trophoblasts promoted by any other pathway using the PKC inhibitor GF 109203X:

human embryonic stem cells H1 were cultured in E8 medium, fresh medium was changed daily, and passage was started until cell density reached 70-80%. The DPBS-EDTA was first washed twice, then incubated at room temperature for 5 minutes, finally DPBS-EDTA was aspirated, and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells were passaged at a density of 1:6-1:12 to 12 well plates pre-coated with Matrigel. 1 day after cell adhesion to the plates, the E6 medium was changed (E8 medium was free of FGF2 and TGF-. Beta.) and differentiation was initiated by the addition of 20ng/ml BMP4, 1. Mu.M PD0325901, 10. Mu.M SB431542 or 5. Mu.M DAPT together with PKC inhibitor. Fresh differentiation medium was added daily. Trophoblast marker levels of differentiated cells were detected by qPCR and immunofluorescence on day 6 (TPA differentiated cells were detected on day 3) as shown in FIGS. 19-28.

Among them, FIG. 19 shows the effect of PKC inhibitor GF109203X on the expression of the trophoblast cell markers TROP2 and CGB genes under BMP4 differentiation. Differentiation conditions were that GF109203X (5. Mu.M), BMP4 (20 ng/ml), LDN193189 (100 nM) (expressed as LDN), FGF2 (100 ng/ml), TGF beta (2 ng/ml) and Jagged-1 (1. Mu.M) (expressed as JAG1, the same applies below) were added to the differentiation medium.

FIG. 20 shows the effect of PKC inhibitor GF109203X on the expression of the marker genes TROP2 and CGB in trophoblasts under PD0325901 conditions. Differentiation conditions were GF109203X (5. Mu.M), LDN193189 (100 nM), FGF2 (100 ng/ml), PD0325901 (1. Mu.M), TGF beta (2 ng/ml) and Jagged-1 (1. Mu.M) were added to the differentiation medium.

FIG. 21 shows the effect of PKC inhibitor GF109203X on gene expression of the marker genes TROP2 and CGB that nourish cells under SB431542 conditions. Differentiation conditions were that GF109203X (5. Mu.M), LDN193189 (100 nM), FGF2 (100 ng/ml), TGFbeta (2 ng/ml), SB431542 (10. Mu.M) and Jagged-1 (1. Mu.M) were added to the differentiation medium.

FIG. 22 shows the effect of PKC inhibitor GF109203X on the expression of the marker genes TROP2 and CGB in trophoblasts under DAPT conditions. Differentiation conditions were GF109203X (5. Mu.M), LDN193189 (100 nM), FGF2 (100 ng/ml), TGF beta (2 ng/ml), DAPT (5. Mu.M) and Jagged-1 (1. Mu.M) were added to the differentiation medium.

FIG. 23 shows the effect of PKC inhibitor GF109203X on the expression of the marker genes TROP2 and CGB that nourish cells under TPA conditions. Differentiation conditions were TPA (50 nM), GF109203X (5. Mu.M), LDN193189 (100 nM), FGF2 (100 ng/ml), TGF beta (2 ng/ml) and Jagged-1 (1. Mu.M) were added to the differentiation medium.

FIG. 24 shows PKC inhibitors GF109203X andeffect on BMP 4-induced expression of trophoblast marker genes TROP2 and CGB. The differentiation conditions were that GF109203X (5. Mu.M) and->(5. Mu.M) and BMP4 (20 ng/ml).

FIG. 25 shows PKC inhibitorsEffect on BMP 4-induced expression of trophoblast marker proteins TROP2 and CGB, where Phase is the white field without any dye or antibody, hoechst is the nuclear dye, the nucleus will be stained blue (in this figure, the nucleus is light due to the picture being modified to black and white background), the same is true below.

FIG. 26 shows the effect of GF109203X and LDN193189 on BMP 4-induced expression of the trophoblast marker proteins TROP2 and CGB proteins.

FIG. 27 shows the effect of PKC inhibitor GF109203X on the expression of BMP 4-induced trophoblast marker genes TROP2, GATA3, GATA2, KRT18, CDX2, ELF5, ITGA6, CGB, CSH1, CSH2, GCM1, HLAG and ITGA 5.

FIG. 28 shows the effect of PKC inhibitor GF109203X on BMP 4-induced expression of the trophoblast marker genes TROP2, GATA3, CDX2, CGB and HLAG.

The results in FIGS. 19-28 show: GF109203X inhibited the expression levels of the trophoblast marker genes TROP2 and CGB after 6 days of promotion by any known inducer. GF109203X andthe proportion of TROP2/CGB positive cells was inhibited during BMP 4-induced trophoblast differentiation, indicating that PKC inhibitors can impair stem cell differentiation into trophoblast lineages.

Example 8

PKC-delta knockout impairs trophoblast differentiation of human embryonic stem cell H1:

human embryonic stem cells H1 were cultured in E8 medium, fresh medium was changed daily, and passage was started until cell density reached 70-80%. The cells were first washed 2 times with DPBS-EDTA, then incubated at room temperature for 5 minutes, finally DPBS-EDTA was aspirated, and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells were passaged at a density of 1:6-1:12 to 12 well plates pre-coated with Matrigel. After 1 day of cell adhesion to the plate, the differentiation was started by changing to E6 medium (E8 medium contains no FGF2 and TGF beta) and adding BMP4, and the expression level of PRKCD gene was measured, and fresh differentiation medium was added every day. On the first day qPCR was performed, the level of trophoblast marker TROP2 of differentiated cells was detected. The results are shown in fig. 29 to 34.

Among them, FIG. 29 shows the effect of BMP4 on PRKCD gene expression.

FIG. 30 shows the effect of TPA on PRKCD and TROP2 gene expression.

FIG. 31 shows the effect of knockout of the PRKCD gene on PKC-delta protein expression in H1 cells.

FIG. 32 shows the effect of TPA on gene expression of the primitive endoderm and trophoblast markers SOX17, GATA6, TROP2 and CGB in PRKCD knockout cell lines.

FIG. 33 shows the effect of BMP4 on the expression of the primitive endoderm and feeder cell markers CGB genes in PRKCD knockout cell lines.

FIG. 34 shows the effect of BMP4 on the expression of primitive endoderm and feeder cell markers CGB protein in PRKCD knockout cell lines.

The results of fig. 29-34 show: the expression level of PRKCD is increased. PRKCD gene knockout attenuated TPA differentiation in primitive endodermal markers SOX17 and GATA6 and trophoblast markers TROP2 and CGB. PRKCD gene knockout impairs BMP4 differentiation in the trophoblast marker CGB. That is, PRKCD controls the differentiation of human embryonic stem cells into primitive endodermal cells and trophoblast cells.

Example 9

Inhibition of DGK promotes differentiation of human embryonic stem cells H1 into trophoblasts:

to investigate the role of different signaling pathways in trophoblast differentiation, we used DGK inhibitors to block DGK function to verify its availability in trophoblast cell differentiation. The specific operation is as follows: human embryonic stem cells H1 were cultured in E8 medium, replaced with fresh medium daily, and passaged when cell density reached 70-80%. The cells were first washed 2 times with DPBS-EDTA and then incubated for 5 minutes at room temperature. DPBS-EDTA was aspirated and E8 medium containing 5. Mu.M ROCK inhibitor was added. After resuspension of the cells, the cells were resuspended at 1:6-1: the density of 12 was passaged to 12 well plates pre-coated with Matrigel. After 1 day of cell adhesion to the plate, the E6 medium (E8 medium-FGF 2-TGF beta) was changed and DGK inhibitor R59949 was added at a concentration of 1. Mu.M to promote cell differentiation. Fresh E6 medium was added daily. Trophoblast marker levels were measured on day 6. The results are shown in FIGS. 35-37.

Among them, FIG. 35 shows the effect of BMP4 and BMP4+GF109203X on gene expression of DGK subtype after 1 day of differentiation.

FIG. 36 shows the effect of BMP4 and BMP4+GF109203X on DGKA gene expression after 1 day of differentiation.

FIG. 37 shows the effect of DGK inhibitor R59949 on gene expression of markers TROP2 and CGB of trophoblasts after 6 days of differentiation.

The results in FIGS. 35-37 show that: the DGK inhibitor R59949 increases the expression level of the trophoblast marker genes TROP2 and CGB after 6 days. TROP2 and CGB are specific markers of trophoblasts, and DGK inhibitors can induce stem cells to differentiate into trophoblasts.

In summary, the application promotes the differentiation of pluripotent stem cells into mesoderm by using PKC inhibitors in a BMP4 differentiation platform, promotes the expression of premature germ layer markers TBXT and MIXL1, can greatly improve the differentiation of pluripotent stem cells into mesoderm and endoderm precursor cells, shortens the differentiation process by half time, improves the differentiation stability, and provides a new application for stem cell differentiation. Functional mesodermal cell types can be obtained by this method, can be used for regeneration and repair of tissues, such as smooth muscle cells, muscle, bone or gonads, and for drug screening. In addition, the present application employs a method with clear composition to induce differentiation of hpscs into trophoblasts, wherein the agent used is free of animal-derived components. The pioneering use of PKC activation and DGK inhibition to induce hPSC differentiation into trophoblasts, describes PKC activation as the primary mechanism of action downstream of any other trophoblast-induced platform, and finds new applications for stem cell differentiation. In addition, the trophoblasts obtained by the method can express markers of mature trophoblasts, which provides a new idea for guiding early fate decisions of human pluripotent stem cells.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method of inducing differentiation of stem cells into mesodermal lineages using a PKC inhibitor, comprising the steps of:

inducing differentiation of stem cells into mesodermal lineages with a differentiation medium comprising a PKC inhibitor in the presence of BMP 4;

the mesodermal lineage includes mesodermal cells and endodermal precursor cells,

the stem cells include at least one of human embryonic stem cells and human induced pluripotent stem cells,

wherein the human embryonic stem cells are stem cells isolated or obtained from human embryos that have not undergone fertilization for less than 14 days of in vivo development,

the PKC inhibitor is selected from GF109203X,And->At least one of the above-mentioned materials,

exogenous FGF2 and endogenous WNT were not used in the process of inducing stem cells to differentiate into mesoderm.

2. The method of claim 1, wherein the GF109203X is at a concentration of 2-5 μΜ.

3. The method of claim 1, wherein theThe concentration of (2) to (5) mu M.

4. The method of claim 1, wherein theThe concentration of (2) to (5) mu M.

5. The method of claim 1, wherein the BMP4 is at a concentration of 5-100ng/mL.

6. The method of claim 1, wherein the components of the differentiation medium comprise DMEM/F12, magnesium L-ascorbate-2-phosphate, sodium selenate, transferrin, and insulin.