WO2022178986A1 - 一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物 - Google Patents

一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物 Download PDF

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WO2022178986A1
WO2022178986A1 PCT/CN2021/093498 CN2021093498W WO2022178986A1 WO 2022178986 A1 WO2022178986 A1 WO 2022178986A1 CN 2021093498 W CN2021093498 W CN 2021093498W WO 2022178986 A1 WO2022178986 A1 WO 2022178986A1
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differentiation
stem cells
trophoblast
bmp4
cells
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陈国凯
戈多伊·帕罗约·卡洛斯
张宇萌
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澳门大学
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Definitions

  • the present disclosure relates to the technical field of regulating the differentiation of human pluripotent stem cells into mesoderm or trophoblast, and relates to a method and medicine for inducing stem cells to differentiate into mesodermal lineage or trophoblast lineage.
  • hPSCs Human pluripotent stem cells
  • Mesoderm gives rise to cell types such as cardiomyocytes, smooth muscle, bone, and gonads, so mesoderm-derived cell types derived from hPSC differentiation provide material for regeneration and repair of a variety of tissues and for drug screening.
  • hPSCs can also differentiate into extraembryonic cell types such as primitive endoderm and trophoblasts in vitro. Extraembryonic cell types give rise to tissues such as the placenta, yolk pouch or umbilical cord, so extraembryonic cell types derived from hPSC differentiation can provide material for in vitro fertilization, early immune system and drug screening.
  • the present disclosure provides a method for inducing stem cells to differentiate into mesodermal lineage using a PKC inhibitor, comprising the following steps:
  • Stem cells were induced to differentiate into the mesodermal lineage in the presence of BMP4 using differentiation medium containing PKC inhibitors.
  • 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.
  • the PKC inhibitor includes GF109203X, and at least one of them.
  • the concentration of GF109203X is 2-5 ⁇ M.
  • the concentration is 2-5 ⁇ M.
  • the concentration is 2-5 ⁇ M.
  • the concentration of BMP4 is 5-100 ng/mL.
  • the components of the differentiation medium include DMEM/F12, magnesium L-ascorbic acid-2-phosphate, sodium selenate, transferrin and insulin.
  • no exogenous FGF2 or endogenous WNT is used in the process of inducing stem cells to differentiate into mesoderm.
  • the present disclosure provides a method of inducing stem cells to differentiate into a trophoblast lineage, comprising the steps of: inducing stem cells to differentiate into a trophoblast lineage with a differentiation medium containing a PKC activator or a DGK inhibitor.
  • the PKC activator or DGK inhibitor is added to the differentiation medium on the first day of stem cell differentiation.
  • the stem cells are cultured in differentiation medium for at least two days following the addition of the PKC activator.
  • stem cells are cultured in differentiation medium for at least six days after the addition of the DGK inhibitor.
  • the stem cells are human pluripotent stem cells.
  • human pluripotent stem cells include at least one of human embryonic stem cells and human induced pluripotent stem cells.
  • the PKC activator comprises at least one of TPA and Bryostatin 1.
  • the TPA is treated at a concentration of 25-500 nM.
  • Bryostatin 1 is treated at a concentration of 25-500 nM.
  • the DGK inhibitor includes R59949.
  • R59949 is treated at a concentration of 0.5-10 ⁇ M.
  • the components of the differentiation medium include DMEM/F12, magnesium L-ascorbic acid-2-phosphate, sodium selenate, transferrin and insulin.
  • a trophoblast inducer is further added to the differentiation medium.
  • the trophoblast inducer comprises at least one of BMP4, PD325901, Sb432542, and DAPT.
  • BMP4 is treated at a concentration of 10-100 ng/mL.
  • PD325901 is treated at a concentration of 0.5-10 ⁇ M.
  • Sb432542 is treated at a concentration of 5-20 ⁇ M.
  • DAPT is treated at a concentration of 1-10 ⁇ M.
  • the present disclosure also provides a medicament, the raw material of which contains the mesodermal lineage or trophoblast lineage obtained by the above method.
  • Figures 1 to 6 are the results of each test in Example 1, wherein in Figure 2, hESCs are human embryonic stem cells, B+GFX is BMP4 combined with PKC inhibitor GF109203X; in Figure 4, Combined PKC inhibitor for BMP4 BRACHYURY in Figures 3 and 6 is a specific protein expressed by TBXT (T-box transcription factor T, T-box transcription factor T) gene;
  • Figures 7 to 10 are graphs of each test result in Example 2.
  • Figures 14 to 16 are graphs of each test result in Example 4.
  • Figure 19 to Figure 28 are the results of each test in Comparative Example 1;
  • Figures 29 to 34 are graphs of the test results in Example 8, wherein, in Figure 34, wt is a wild-type cell line, and pkcd-/- is a cell line with PKCd knocked out;
  • cell differentiation can generally refer to the process by which cells from the same source gradually produce cell populations with different morphological, structural and functional characteristics. Status is different.
  • mammals can generally refer to the middle layer of the three main germ layers in the early embryo (the other two being the ectoderm and the endoderm), the initial precursor to cells of the skeletal system, cardiovascular system, and urinary system organs cell.
  • differentiation medium can generally refer to a medium that promotes the gradual production of cell populations with different morphological, structural and functional characteristics from cells of the same source.
  • pluripotent stem cells refer to stem cells that have pluripotency capable of differentiating into a variety of cells and also have the ability to self-proliferate.
  • a “mesodermal cell inducer” refers to a small chemical molecule or cytokine capable of inducing the differentiation of pluripotent stem cells into mesodermal cells.
  • trophoblast refers to cells with trophoblast function, derived from extraembryonic trophoblasts.
  • trophoblast-inducing agent refers to a small chemical molecule or cytokine capable of inducing pluripotent stem cells to differentiate into trophoblast cells.
  • the present disclosure provides one embodiment of a method for inducing stem cells to differentiate into mesodermal lineage using a PKC (protein kinase C) inhibitor, comprising the following steps:
  • 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. That is, only human embryonic stem cells may be differentiated, or only human induced pluripotent stem cells may be differentiated, or both human embryonic stem cells and human induced pluripotent stem cells may be differentiated.
  • the PKC inhibitor may include, for example, GF109203X, and At least one of them, optionally GF109203X.
  • the concentration of GF109203X can be 2-5 ⁇ M, 2-4 ⁇ M, 2-3 ⁇ M, 3-5 ⁇ M or 3-4 ⁇ M, including but not limited to 2 ⁇ M, 3 ⁇ M, 4 ⁇ M or 5 ⁇ M, etc.
  • the concentration can be 2-5 ⁇ M, 2-4 ⁇ M, 2-3 ⁇ M, 3-5 ⁇ M or 3-4 ⁇ M, including but not limited to 2 ⁇ M, 3 ⁇ M, 4 ⁇ M or 5 ⁇ M, etc.
  • the concentration can also be 2-5 ⁇ M, 2-4 ⁇ M, 2-3 ⁇ M, 3-5 ⁇ M or 3-4 ⁇ M, including but not limited to 2 ⁇ M, 3 ⁇ M, 4 ⁇ M or 5 ⁇ M, etc.
  • BMP4 as a mesoderm inducer, which can be used in combination with a PKC inhibitor optionally GF109203X, can replace FGF2 (fibroblast growth factor 2) as an inducer of early mesoderm cell induction in human embryonic stem cells.
  • FGF2 fibroblast growth factor 2
  • the concentration of BMP4 can be 5-100ng/mL, 10-100ng/mL, 20-100ng/mL, 30-100ng/mL, 40-1000ng/mL, 50-100ng/mL, 5-90ng/mL , 5-80ng/mL, 5-70ng/mL, 5-60ng/mL, 10-90ng/mL, 20-80ng/mL, 30-70ng/mL, including but not limited to 5ng/mL, 10ng/mL, 20ng /mL, 50ng/mL or 100ng/mL, etc.
  • the components of the differentiation medium referred to in the present disclosure 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 can be respectively: L-ascorbic acid-2-magnesium phosphate 64 mg/L, sodium selenate 13.6 ⁇ g/L, transferrin 10 mg/L and insulin 20 mg/L.
  • the above-mentioned differentiation medium can be E6 (Essential 6, basal 6) medium, which can be understood as E8 (Essential 8, basal 8) medium without FGF2 and TGF ⁇ (transforming growth factor- ⁇ ).
  • PKC inhibitors induce differentiation of stem cells into the early mesoderm lineage.
  • the process of inducing stem cells to differentiate into mesoderm does not use exogenous FGF2 or the endogenous cytokine WNT. That is, the PKC inhibitor optionally GF109203X promotes the differentiation of human embryonic stem cells H1 to mesoderm without FGF2 pathway activation. Also, the PKC inhibitor optionally GF109203X promotes the differentiation of human embryonic stem cells H1 to mesoderm without the need for the endogenous cytokine WNT.
  • the method of using a PKC inhibitor to induce stem cells to differentiate into mesodermal lineages involved in the present disclosure may, but is not limited to, refer to the following steps:
  • hPSCs were cultured to a cell density of 10-20% (the density before the initiation of differentiation), hPSCs were induced to differentiate into mesoderm using the PKC inhibitor GF109203X in the presence of BMP4. After 2 days, the formation of TBXT (T box transcription factor T) positive cells can be detected by immunostaining or FACS (flow cytometry) (ie, TBXT positive cells are measured on the third day of differentiation).
  • TBXT T box transcription factor T
  • FACS flow cytometry
  • human embryonic stem cells H1 can be cultured in E8 medium, fresh medium is replaced every day, and passage is performed when the cell density reaches 70-80% (that is, the density is the density before passage). First wash twice with DPBS-EDTA, then incubate for 5 min at room temperature, aspirate DPBS-EDTA a third time, and add E8 medium containing 5 ⁇ M ROCK (Rho-related protein kinase) inhibitor.
  • ROCK Rho-related protein kinase
  • a PKC inhibitor increases the expression levels of markers of early endoderm. Can promote the differentiation of human embryonic stem cell lines (H1 and H9) into endoderm cells.
  • E6 and E8 culture media can be obtained by referring to the components disclosed in the present disclosure, and can also be prepared by referring to the components of the relevant culture media in the prior art, which will not be repeated here.
  • Functional mesodermal cell types can be obtained by the methods described above, which can be used for regeneration and repair of tissues, such as smooth muscle cells, muscles, bones or gonads, and for drug screening.
  • the present disclosure also provides a medicine, the raw material of which contains the mesodermal lineage obtained by the above method.
  • the present disclosure provides an embodiment of the use of the above-mentioned medicament for the treatment of tissue regeneration and tissue repair.
  • the tissue is damaged by injury or disease.
  • tissue includes, but is not limited to, epithelial tissue, connective tissue, muscle tissue, neural tissue, bone, gonads.
  • muscle tissue includes skeletal muscle tissue, smooth muscle tissue, cardiac fibrous tissue.
  • the present disclosure provides one embodiment of a method of regenerating and repairing tissue in a subject in need thereof, comprising: administering to the subject in need thereof a drug as described above.
  • the subject has a bone injury or bone disease selected from the group consisting of: osteoarthritis, osteitis deformans, osteoporosis, bone fracture, fracture, bone cancer, bone cancer, myeloma Bone disease, osteopenia, osteosclerosis, renal osteodystrophy, bone cancer, osteitis cystic fibrosis, or osteoarthritis;
  • a heart injury or heart disease selected from the group consisting of heart valve disease, arrhythmia, left ventricular hypertrophy, right ventricular hypertrophy, myocardial infarction, heart failure or congenital heart defect;
  • a gonadal disorder selected from: polycystic ovary syndrome, Klinefelter syndrome, Turner syndrome, hypogonadism, androgen insensitivity syndrome (AIS)
  • muscle tissue disease selected from the group consisting of polymyositis, mitochondrial myopathy, myogenic atrophy.
  • the present disclosure provides an embodiment of a method for inducing stem cells to differentiate into the trophoblast lineage, comprising the following steps:
  • Stem cells are induced to differentiate into the trophoblast lineage with differentiation medium containing PKC activators or DGK (diacylglycerol kinase) inhibitors.
  • the stem cells are human pluripotent stem cells.
  • human pluripotent stem cells include at least one of human embryonic stem cells and human induced pluripotent stem cells. and human induced pluripotent stem cells.
  • the PKC activator is added to the differentiation medium on the first day of stem cell differentiation.
  • stem cells were cultured in differentiation medium for at least two days following the addition of the PKC activator.
  • the PKC activator may include, for example, TPA (12-O-tetradecanoylphorbol-13-acetate), a At least one of small-molecule compounds that activate PKC in vivo) and Bryostatin 1 (Bryostatin1).
  • the treatment concentrations of TPA and Bryostatin 1 can be respectively 25-500nM, 25-450nM, 25-400nM, 25-350nM, 25-300nM, 50-500nM, 100-500nM, 150-500nM or 200-500nM, including But not limited to 25nM, 50nM, 100Nm, 200nM, 300nM, 400nM or 500nM, etc.
  • the DGK inhibitor is added to the differentiation medium on the first day of stem cell differentiation.
  • stem cells were cultured in differentiation medium for at least six days following the addition of the DGK inhibitor.
  • the DGK inhibitor can include (is) R59949.
  • the treatment concentration of R59949 can be 0.5-10 ⁇ M, 0.5-9 ⁇ M, 0.5-8 ⁇ M, 0.5-7 ⁇ M, 0.5-6 ⁇ M, 0.5-5 ⁇ M, 1-10 ⁇ M, 2-10 ⁇ M, 3-10 ⁇ M, 4-10 ⁇ M, 5-10 ⁇ M, Including but not limited to 0.5 ⁇ M, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M or 10 ⁇ M, etc.
  • the components of the differentiation medium referred to in the present disclosure 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 can be respectively: L-ascorbic acid-2-magnesium phosphate 64 mg/L, sodium selenate 13.6 ⁇ g/L, transferrin 10 mg/L and insulin 20 mg/L.
  • the above-mentioned differentiation medium can be an E6 medium, which can be understood as an E8 medium that does not contain FGF2 and TGF ⁇ .
  • a trophoblast inducer may also be added to the differentiation medium.
  • the trophoblast inducer comprises at least one of BMP4, PD325901, Sb432542, and DAPT.
  • the treatment concentration of BMP4 can be 10-100ng/mL, 10-90ng/mL, 10-80ng/mL, 10-70ng/mL, 10-60ng/mL, 20-100ng/mL, 30-100ng/mL, 40-100ng/mL, including but not limited to 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL or 100ng/mL, etc.
  • PD325901 can be treated at concentrations of 0.5-10 ⁇ M, 1-10 ⁇ M, 3-10 ⁇ M, 5-10 ⁇ M, 0.5-9 ⁇ M, 0.5-7 ⁇ M or 0.5-5 ⁇ M, including but not limited to 0.5 ⁇ M, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M, 8 ⁇ M or 10 ⁇ M Wait.
  • the treatment concentration of Sb432542 can be 5-20 ⁇ M, 5-18 ⁇ M, 5-16 ⁇ M, 5-14 ⁇ M, 5-12 ⁇ M, 7-20 ⁇ M, 9-20 ⁇ M, 11-20 ⁇ M, including but not limited to 5 ⁇ M, 8 ⁇ M, 10 ⁇ M, 15 ⁇ M or 20 ⁇ M, etc.
  • DAPT can be treated at concentrations of 1-10 ⁇ M, 2-10 ⁇ M, 3-10 ⁇ M, 4-10 ⁇ M, 1-9 ⁇ M, 1-8 ⁇ M, 1-7 ⁇ M, 1-6 ⁇ M, including but not limited to 1 ⁇ M, 2 ⁇ M, 5 ⁇ M, 8 ⁇ M or 10 ⁇ M, etc.
  • the method for inducing stem cells to differentiate into trophoblast lineages involved in the present disclosure may, but is not limited to, refer to the following steps:
  • hPSCs were induced to differentiate into trophoblasts using PKC modulators. Differentiation using differentiation medium from day one, after 2 days of treatment (optionally on day two of differentiation), signal markers such as TROP2 (human trophoblast surface antigen) and CGB (chorionic gonadotropin) can be detected by qPCR hormone subunit ⁇ 3).
  • TROP2 human trophoblast surface antigen
  • CGB chorionic gonadotropin
  • human embryonic stem cells are cultured in E8 medium, fresh medium is replaced every day, and passage is performed when the cell density reaches 70-80% (that is, the density is the density before passage).
  • E8 medium containing 5 ⁇ M ROCK inhibitor.
  • passage at a density of 1:6-1:12 eg, 1:6, 1:8, 1:10, or 1:12, etc.
  • E6 medium E8 medium without FGF2 and TGF ⁇
  • 50 nM TPA or Bryostatin 1 50 nM TPA or Bryostatin 1 to initiate differentiation.
  • Fresh differentiation medium was added daily and trophoblast marker levels were measured on day 2 of differentiation.
  • DPBS-EDTA was aspirated and E8 medium containing 5 ⁇ M ROCK inhibitor was added.
  • E6 medium E8 medium without FGF2 and TGF[beta]
  • the DGK inhibitor R59949 was added at a concentration of 1 [mu]M to induce cell differentiation.
  • E6 and E8 culture media can be obtained by referring to the components disclosed in the present disclosure, and can also be prepared by referring to the components of the relevant culture media in the prior art, which will not be repeated here.
  • Stem cells used in the present disclosure are commercially available.
  • stem cells are commercially available from Applied StemCell.
  • the trophoblast lineage obtainable by the methods described above can provide material for early immune system and drug screening.
  • the present disclosure also provides a medicine, the raw material of which contains the trophoblast lineage obtained by the above method.
  • the present disclosure provides an embodiment to provide the use of the above-mentioned drugs for the treatment of early-stage immune system diseases and drug screening.
  • the present disclosure provides one embodiment of a method of treating an early-stage immune system disease in a subject, comprising: administering the above-mentioned drug to the subject in need thereof.
  • the immune system disease that the subject suffers from includes: autoimmune system disease and immunodeficiency system disease.
  • autoimmune system diseases include, but are not limited to, antibody-mediated autoimmune system diseases, such as toxic diffuse goiter, myasthenia gravis, granulocytopenia, systemic lupus erythematosus, etc.; T cell mediators induced autoimmune diseases, such as type 1 diabetes, neutropenia, chronic granulomas, etc.
  • diseases of the immunodeficiency system include, but are not limited to, primary immunodeficiency system diseases, such as combined immunodeficiency diseases, immune dysregulation diseases, phagocytic cell defects, innate immune defects, autoinflammatory diseases, etc.; Diseases of the immunodeficiency system, such as immunodeficiency caused by infectious agents, immunodeficiency caused by drugs, immunodeficiency caused by mycotoxicosis, nutritional deficiencies and immune responses, AIDS.
  • primary immunodeficiency system diseases such as combined immunodeficiency diseases, immune dysregulation diseases, phagocytic cell defects, innate immune defects, autoinflammatory diseases, etc.
  • Diseases of the immunodeficiency system such as immunodeficiency caused by infectious agents, immunodeficiency caused by drugs, immunodeficiency caused by mycotoxicosis, nutritional deficiencies and immune responses, AIDS.
  • the present disclosure promotes the differentiation of pluripotent stem cells into mesoderm, and promotes the expression of early mesoderm markers TBXT and MIXL1 (mixed paired homeoboxes), which can greatly improve multipotency.
  • the ability of stem cells to differentiate into mesoderm and endoderm precursor cells shortens the differentiation process in half, while also improving the stability of differentiation, providing a new application for stem cell differentiation.
  • functional mesodermal cell types can be obtained, which can be used for regeneration and repair of tissues, such as smooth muscle cells, muscles, bones or gonads, and for drug screening.
  • the present disclosure employs a method with clear components to induce hPSC differentiation into trophoblasts, wherein the reagents used are free of animal-derived components.
  • the trophoblasts obtained by this method can also express markers of mature trophoblasts, which provides a new idea for guiding the early fate decision of human pluripotent stem cells.
  • GF109203X the full name is bisindolemaleimide I, CAS No.133052-90-1, commercially available from selleckchem, item number S7208;
  • BMP4 the full name of bone morphogenetic protein 4, is commercially available from R&D Systems, item number 314-BP-500;
  • PD0325901 the full name is Mirdametinib, a MEK inhibitor, commercially available from selleckchem, Cat. No. S1036;
  • DAPT the full name is (3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester, CAS No.208255-80-5, commercially available from selleckchem, item number S2215;
  • LDN193189 full name is 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, CAS No.1062368-24-4 , commercially available from: selleckchem, item number S2618;
  • FGF2 the full name of fibroblast growth factor 2
  • peprotech item number 100-18C
  • TGF ⁇ the full name is transforming growth factor- ⁇ , commercially available from R&D Systems, item number 240-B-500;
  • Jagged-1 the full name is serrated typical Notch ligand 1, commercially available from GenScript, Item No. RP20331;
  • Human embryonic stem cells H1 purchased from WiCell, product number WA01;
  • Human embryonic stem cells H9 purchased from WiCell, item number WA09;
  • Human induced pluripotent stem cell NL4 not a human embryonic stem cell line, it is a pluripotent cell line induced by human cells in vitro.
  • human embryonic stem cells H1 are cultured in E8 medium, fresh medium is replaced every day, and passage is performed when the cell density reaches 70-80%.
  • First wash twice with DPBS-EDTA purchased from cell system, Cat. No. 4Z0-610, then incubate for 5 minutes at room temperature, aspirate DPBS-EDTA a third time, and add E8 medium containing 5 ⁇ M ROCK inhibitor. After resuspending cells, passage at a density of 1:6 was added to a 12-well plate pre-coated with Matrigel.
  • E6 medium E8 medium without FGF2 and TGF ⁇
  • BMP4 and PKC inhibitors BMP4 and PKC inhibitors
  • differentiation was started at the concentrations of 20 ng/mL and 5 ⁇ M, respectively (pre-differentiation cell density). 10-20%).
  • Fresh E6 medium was added daily.
  • Levels of early mesodermal markers of differentiated cells were measured on the first and second day of differentiation by qPCR, immunofluorescence and flow cytometry (unless otherwise stated).
  • qPCR was measured with a fluorescence quantitative PCR detector (model: Thermo Fisher QuantStudio7), total mRNA was extracted with RNAiso-plus (TAKARA, Cat. No. 108-95-2), and a high-capacity cDNA reverse transcription kit (Applied Biosystems, Cat. No. 4368813) for reverse transcription from mRNA to cDNA.
  • Real-time quantitative PCR was performed using SYBR Premix Ex Taq (TAKARA, Cat. No. RR420) and Quantstudio-7 system (Applied Biosystems). The relative amount of amplified nucleotide fragments was calculated by the 2 ⁇ (- ⁇ Ct) method. Expression levels were normalized to the housekeeping gene TBP and compared to undifferentiated hESCs.
  • Immunofluorescence was measured using an immunofluorescence analyzer (model: Thermo Fisher EVOS Cell Imaging System FL), cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, and washed with 1x PBS (5 minutes each, 3 times) , rupture the membrane with 0.5% Triton X-100 for 20 minutes, and then rinse with 1x PBS (5 minutes each, 3 times). Nuclei were stained with Hoechst 33342 (Cat. No. U0334, Abnova, 1:10,000) for 20 minutes at room temperature. Cells were then rinsed with 1x PBS (5 min each, 3 times), incubated overnight at 4°C with primary antibodies, rinsed with 1x PBS, incubated with fluorescent secondary antibodies for 2 hours, and finally rinsed with 1x PBS before imaging.
  • an immunofluorescence analyzer model: Thermo Fisher EVOS Cell Imaging System FL
  • cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, and washed with 1x PBS (5 minutes
  • FIG. 1 represents the effect of PKC inhibitor GF109203X on the expression of MIXL1 and TBXT, the marker genes of early mesoderm under BMP4 differentiation.
  • GF109203X and BMP4 were added at concentrations of 5 ⁇ M and 20 ng/mL, respectively.
  • mock control group
  • GFX represents the addition of PKC inhibitor GF109203X on the basis of mock, the same below.
  • Figure 2 represents the proportion of TBXT positive cells analyzed by immunostaining after 2 days of stem cell differentiation induced by addition of BMP4 and GF109203X.
  • gray represents positive cells for the Hoechst (purchased from Abnova, Cat. No. U0334, added at 1:10,000) and TBXT groups.
  • Figure 3 represents the proportion of TBXT positive cells analyzed by flow cytometry after adding BMP4 and GF109203X to induce stem cell differentiation for 2 days.
  • Figure 4 represents the addition of BMP4 and After induction of stem cell differentiation for 2 days, the proportion of TBXT-positive cells was analyzed by immunostaining. Added at a concentration of 5 ⁇ M In this figure, for the Hoechst group and the TBXT group, gray represents positive cells.
  • Figure 5 represents the effect of PKC inhibitor on the expression of WNT3, a marker gene of early mesoderm, one day after BMP4 differentiation.
  • Figure 6 represents the use of 5 ⁇ M PKC inhibitor And the effect of 20ng/mL BMP4 differentiation on the expression of early mesoderm marker genes WNT3, MIXL1 and TBXT.
  • GF109203X The results showed that after only 24 hours, GF109203X, and The expression levels of early mesoderm marker genes MIXL1 and TBXT can be increased.
  • TBXT is a specific marker for early mesoderm differentiation, GF109203X and The proportion of TBXT-positive cells was increased to varying degrees, indicating that PKC inhibitors could induce stem cells to differentiate into early mesoderm lineages.
  • GF109203X can be added to BMP4-induced differentiation to promote the WNT pathway and accelerate the early mesoderm differentiation of human embryonic stem cells.
  • GF109203X promotes the differentiation of human embryonic stem cells H1 to mesoderm without FGF2 pathway activation.
  • Human embryonic stem cells H1 were cultured in E8 medium, and the medium was replaced with fresh medium every day, and the cell density reached 70-80%. First washed twice with DPBS-EDTA, then incubated at room temperature for 5 min, removed DPBS-EDTA a third time, and resuspended cells in E8 medium containing 5 ⁇ M Rock inhibitor, passaged at a density of 1:6, added with Matrigel-coated 12-well plates were added with E6 medium (E8 medium without FGF2 and TGF ⁇ ) containing 20 ng/ml BMP4, 100 ng/mL FGF2 and 5 ⁇ M PKC inhibitor and differentiation was initiated. Fresh E6 medium was added daily.
  • Figure 7 shows the effect of PKC inhibitor GF109203X on gene expression of MIXL1 and TBXT, which are markers of early mesoderm, under BMP4 or BMP4+FGF2 differentiation.
  • mock represents E6 medium + BMP4
  • GFX represents the addition of GFX (PKC inhibitor GF109203X) on the basis of mock
  • FGF2 represents the addition of FGF2 on the basis of mock
  • FGF2+GFX represents the addition of FGF2+GFX on the basis of mock.
  • GF109203X, BMP4 and FGF2 were added at concentrations of 5 ⁇ M, 20 ng/mL and 100 ng/mL, respectively.
  • Figure 8 shows the proportion of TBXT positive cells analyzed by flow cytometry 2 days after addition of BMP4/GF109203X (indicated as GFX in the figure) or BMP4/FGF2/GF109203X to induce stem cell differentiation.
  • Figure 9 shows the effect of PKC inhibitor GF109203X on the expression of WNT3, MIXL1 and TBXT genes under differentiation of early mesoderm markers BMP4+FGF2 or BMP4+FGF2+PD0173074 (indicated as PD173 in the figure, the same below).
  • PD0173074 was added at a concentration of 100 nM.
  • Figure 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 (PD173) or both.
  • mock represents E6 medium+BMP4+FGF2
  • GFX represents the addition of GFX on the basis of mock
  • PD173 represents the addition of PD173 on the basis of mock
  • PD173+GFX represents the addition of PD173+GFX on the basis of mock.
  • gray represents positive cells.
  • GF109203X increased the expression levels of early mesoderm marker genes MIXL1 and TBXT after only 24 hours, even in the presence of the FGFR inhibitor PD0173974.
  • the FGF2 pathway inhibitor PD0173074 was used at 100 nM.
  • TBXT is a specific marker of early mesoderm differentiation
  • GF109203X increased the proportion of TBXT-positive cells even in the presence of PD0173074, suggesting that PKC inhibitors can induce stem cells to bypass FGF2 pathway activation and differentiate into early mesodermal lineages . That is, GF109203X combined with BMP4 can replace FGF2 as an inducer of early mesoderm cell induction in human embryonic stem cells.
  • GF109203X promotes the differentiation of human embryonic stem cells H1 to mesoderm without endogenous WNT.
  • Human embryonic stem cells H1 were cultured in E8 medium, and the medium was replaced with fresh medium every day, and the cell density reached 70-80%. First wash twice with DPBS-EDTA, then incubate for 5 min at room temperature, aspirate DPBS-EDTA a third time, and add E8 medium containing 5 ⁇ M Rock inhibitor. After resuspending cells, passage at a density of 1:6 was added to a 12-well plate pre-coated with Matrigel. One day after the cells adhered to the plate, the medium was changed to E6 medium (E8 medium without FGF2 and TGF ⁇ ) and 20 ng/mL BMP4 and 5 ⁇ M PKC inhibitor were added to initiate differentiation. Fresh E6 medium was added daily.
  • E6 medium E8 medium without FGF2 and TGF ⁇
  • Figure 11 shows the effect of the PKC inhibitor GF109203X (GFX) on the expression of WNT3, MIXL1 and TBXT genes under BMP4 or BMP4+IWP-2 (IWP2) differentiation, which are hallmarks of early mesoderm.
  • GF109203X, BMP4 and IWP-2 were added at concentrations of 5 ⁇ M, 20 ng/mL and 2.5 ⁇ M, respectively.
  • Figure 12 shows that GF109203X (GFX) can promote ⁇ -catenin nuclear localization under BMP4 induction after 6 hours of differentiation as analyzed by immunostaining.
  • GFX GF109203X
  • gray represents positive cells.
  • Figure 13 shows the quantification of BMP4 and BMP4+GF109203X (GFX)-induced nuclear localization of ⁇ -catenin in signal intensity after 6 hours of differentiation.
  • GF109203X can bypass the WNT pathway and promote the nuclear localization of ⁇ -catenin in human embryonic stem cells. It indicated that GF109203X could increase the expression level of early mesoderm markers.
  • GF109203X promotes the differentiation of human embryonic stem cells H1 into endoderm.
  • Human embryonic stem cells H1 were cultured in E8 medium, and the medium was replaced with fresh medium every day, and the cell density reached 70-80%. First wash twice with DPBS-EDTA, then incubate for 5 min at room temperature, aspirate DPBS-EDTA a third time, and add E8 medium containing 5 ⁇ M Rock inhibitor. After resuspending cells, passage at a density of 1:6 was added to a 12-well plate 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 ⁇ ) and started by adding 100ng/mL Activin A, 20ng/mL BMP4, 100ng/mL FGF2 and 5 ⁇ M PKC inhibitor differentiation.
  • E6 medium E8 medium without FGF2 and TGF ⁇
  • Figure 14 represents the early stage of endoderm cell differentiation, that is, from day 0 to day 1, usually Acivin A (activin A) + BMP4 + FGF2 can be used to induce embryonic stem cells to primitive streak cells (PS ) differentiation, PS cells can be induced to form specific endoderm cells (DE, DE is the precursor of human respiratory and digestive organs) under the conditions of Activin+LDN193189+IWP2 (Wnt signaling pathway inhibitor).
  • activin A activin A
  • BMP4 + FGF2 can be used to induce embryonic stem cells to primitive streak cells (PS ) differentiation
  • PS cells can be induced to form specific endoderm cells (DE, DE is the precursor of human respiratory and digestive organs) under the conditions of Activin+LDN193189+IWP2 (Wnt signaling pathway inhibitor).
  • the PKC inhibitor GF109203X combined with Acivin A+BMP4+FGF2 could significantly increase the expression of SOX17, which is a biomarker of early endoderm, from day 2 to day 3 of differentiation .
  • GF109203X, Activin A, BMP4, LDN193189 and IWP-2 were added at concentrations of 5 ⁇ M, 100 ng/mL, 20 ng/mL, 100 nM and 2.5 ⁇ M, respectively.
  • Figure 15 represents that GF109203X (GFX) can promote the differentiation of H9 and H1 cell lines to DE after 2-3 days of differentiation by quantitative PCR analysis.
  • Figure 16 shows that GF109203X (GFX) can promote the differentiation of H9 cell line to DE by flow cytometry analysis after 2-3 days of differentiation, and can obtain almost 100% of SOX17 positive DE cells.
  • GFX GF109203X
  • GF109203X could promote the differentiation of human embryonic stem cell lines (H1 and H9) into endoderm cells.
  • This example provides a method for inducing human induced pluripotent stem cells to differentiate into mesodermal lineages using a PKC inhibitor:
  • iPSCs Human induced pluripotent stem cells (iPSCs, such as NL-1 or NL-4) were cultured in E8 medium, with fresh medium replaced daily, and passaged when the cell density reached 70-80%. First wash 2 times with DPBS-EDTA, then incubate for 5 min at room temperature, 3rd aspirate DPBS-EDTA, and add E8 medium containing 5 ⁇ M ROCK inhibitor. After resuspending the cells, passage at a density of 1:10 was added to a 12-well plate pre-coated with Matrigel.
  • E6 medium E8 medium did not contain FGF2 and TGF ⁇
  • BMP4 and GF109203X BMP4 and GF109203X
  • differentiation was started at the concentrations of 20 ng/mL and 2 ⁇ M, respectively.
  • Fresh E6 medium was added daily.
  • Levels of early mesodermal markers of differentiated cells were measured on the first and second day of differentiation by qPCR, immunofluorescence and flow cytometry (unless otherwise stated). The determination methods and instrument models of qPCR, immunofluorescence and flow cytometry are the same as those in Example 1.
  • This example provides a method for inducing stem cells to differentiate into the mesodermal lineage using a PKC inhibitor:
  • 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%. First wash 2 times with DPBS-EDTA, then incubate for 5 min at room temperature, 3rd aspirate DPBS-EDTA, and add E8 medium containing 5 ⁇ M ROCK inhibitor. After resuspending cells, passage at a density of 1:12 was added to a 12-well plate pre-coated with Matrigel. One day after the cells adhered to the plate, they were changed to E6 medium (E8 medium did not contain FGF2 and TGF ⁇ ) together with BMP4 and GF109203X, and differentiation was started at the concentrations of 20 ng/mL and 4 ⁇ M, respectively.
  • E6 medium E8 medium did not contain FGF2 and TGF ⁇
  • E6 medium E8 medium without FGF2 and TGF ⁇
  • TPA or Bryostatin 1 50 nM TPA or Bryostatin 1 to initiate differentiation.
  • Fresh differentiation medium was added daily during the day and levels of trophoblast markers (TROP2 and CGB) and primitive endoderm markers (SOX17 and GATA6) were measured on day 3 of differentiation, as shown in Figures 17 and 18.
  • TROP2 and CGB trophoblast markers
  • SOX17 and GATA6 primitive endoderm markers
  • trophoblast markers (TROP2 and CGB) and endoderm markers (SOX17 and GATA6) were determined by qPCR detection of signal markers.
  • 17 shows the effect of the PKC activator TPA on the gene expression of primitive endoderm markers (SOX17 and GATA6) and trophoblast markers (TROP2 and CGB) on day 3 of differentiation.
  • TPA was added at a concentration of 50 nM.
  • NANOG is a biomarker of stem cell stemness. The higher the expression level, the higher the stemness of the cell; on the contrary, the lower the expression level, the lower the stemness of the cell, that is, the cell has differentiated.
  • Figure 18 shows the effect of the PKC activator Bryostatin 1 on the gene expression of primitive endoderm markers (SOX17 and GATA6) and trophoblast markers (TROP2 and CGB) on days 2-10 of differentiation.
  • Bryostatin 1 was added at a concentration of 50 nM.
  • NANOG is a biomarker of stem cell stemness. The higher the expression level, the higher the stemness of the cell; on the contrary, the lower the expression level, the lower the stemness of the cell, that is, the cell has differentiated.
  • Mock represents E6 medium + BMP4, GFX represents the addition of GFX (PKC inhibitor GF109203X) on the basis of mock, the same below.
  • TPA and Bryostatin 1 can increase the expression levels of primitive endoderm marker genes SOX17 and GATA6 and trophoblast marker genes TROP2 and CGB after 3 days.
  • PKC inhibitor GF109203X inhibited TPA and Bryostatin 1 to promote the expression of trophoblast-specific markers TROP2 and CGB, and PKC activator could induce stem cells to differentiate into trophoblast lineage. This suggests that activation of PKC by TPA and Bryostatin 1 promotes differentiation into extraembryonic cell types, such as primitive endoderm and trophoblast of human embryonic stem cells.
  • This comparative example provides a method for inhibiting the differentiation of human pluripotent stem cells H1, H9 and NL4 into trophoblasts promoted by any other pathway using the PKC inhibitor GF109203X:
  • Human embryonic stem cells H1 were cultured in E8 medium, and the medium was replaced with fresh medium every day. When the cell density reached 70-80%, the passage was started. First wash twice with DPBS-EDTA, then incubate for 5 min at room temperature, finally aspirate DPBS-EDTA and add E8 medium containing 5 ⁇ M ROCK inhibitor. After resuspending the cells, passage at a density of 1:6-1:12 into 12-well plates precoated with Matrigel.
  • E6 medium E8 medium without FGF2 and TGF ⁇
  • BMP4 20 ng/ml BMP4, 1 ⁇ M PD0325901, 10 ⁇ M Sb431542 or 5 ⁇ M DAPT together with PKC inhibitor to initiate differentiation.
  • Add fresh differentiation medium daily.
  • Differentiated cells were detected for trophoblast marker levels by qPCR and immunofluorescence on day 6 (TPA differentiated cells were detected on day 3), as shown in Figures 19-28.
  • TPA differentiated cells were detected on day 3
  • FIG. 19 shows the effect of PKC inhibitor GF109203X on the gene expression of trophoblast cell markers TROP2 and CGB under BMP4 differentiation.
  • Differentiation conditions were GF109203X (5 ⁇ M), BMP4 (20ng/ml), LDN193189 (100nM) (expressed as LDN), FGF2 (100ng/ml), TGF ⁇ (2ng/ml) and Jagged-1 (1 ⁇ M) in differentiation medium ) (represented by JAG1, the same below).
  • Figure 20 shows the effect of PKC inhibitor GF109203X on the expression of trophoblast marker genes TROP2 and CGB under PD0325901 conditions.
  • Differentiation conditions were GF109203X (5 ⁇ M), LDN193189 (100 nM), FGF2 (100 ng/ml), PD0325901 (1 ⁇ M), TGF ⁇ (2 ng/ml) and Jagged-1 (1 ⁇ M) in differentiation medium.
  • Figure 21 shows the effect of the PKC inhibitor GF109203X on the gene expression of trophoblast marker genes TROP2 and CGB under the condition of Sb431542.
  • Differentiation conditions were GF109203X (5 ⁇ M), LDN193189 (100 nM), FGF2 (100 ng/ml), TGF ⁇ (2 ng/ml), Sb431542 (10 ⁇ M) and Jagged-1 (1 ⁇ M) in differentiation medium.
  • Figure 22 shows the effect of PKC inhibitor GF109203X on the expression of trophoblast marker genes TROP2 and CGB under DAPT conditions.
  • Differentiation conditions were GF109203X (5 ⁇ M), LDN193189 (100 nM), FGF2 (100 ng/ml), TGF ⁇ (2 ng/ml), DAPT (5 ⁇ M) and Jagged-1 (1 ⁇ M) in differentiation medium.
  • Figure 23 shows the effect of the PKC inhibitor GF109203X on the expression of trophoblast marker genes TROP2 and CGB under TPA conditions. Differentiation conditions were TPA (50 nM), GF109203X (5 ⁇ M), LDN193189 (100 nM), FGF2 (100 ng/ml), TGF ⁇ (2 ng/ml) and Jagged-1 (1 ⁇ M) in differentiation medium.
  • Figure 24 shows the PKC inhibitors GF109203X and Effects on BMP4-induced expression of trophoblast marker genes TROP2 and CGB.
  • the differentiation condition is adding GF109203X (5 ⁇ M) to the differentiation medium, (5 ⁇ M) and BMP4 (20 ng/ml).
  • Figure 25 shows PKC inhibitors The effect on BMP4-induced expression of trophoblast marker proteins TROP2 and CGB, where Phase is the white field without any dye or antibody, Hoechst is the nucleus dye, and the nucleus will be stained blue (in this figure, due to the picture Modified to black and white background, the nucleus is light), the same below.
  • Figure 26 shows the effects of GF109203X and LDN193189 on BMP4-induced trophoblast marker protein TROP2 and CGB protein expression.
  • Figure 27 shows the effect of PKC inhibitor GF109203X on BMP4-induced expression of trophoblast marker genes TROP2, GATA3, GATA2, KRT18, CDX2, ELF5, ITGA6, CGB, CSH1, CSH2, GCM1, HLAG and ITGA5.
  • Figure 28 shows the effect of PKC inhibitor GF109203X on BMP4-induced expression of trophoblast marker genes TROP2, GATA3, CDX2, CGB and HLAG.
  • Figures 19-28 show that GF109203X can inhibit the expression levels of trophoblast marker genes TROP2 and CGB after 6 days of promotion by any known inducer.
  • GF109203X and The ratio of TROP2/CGB-positive cells was suppressed during BMP4-induced trophoblastic differentiation, suggesting that PKC inhibitors impair stem cell differentiation into the trophoblastic lineage.
  • PKC-delta knockout impairs trophoblast differentiation of human embryonic stem cells H1:
  • Human embryonic stem cells H1 were cultured in E8 medium, and the medium was replaced with fresh medium every day. When the cell density reached 70-80%, the passage was started. First wash 2 times with DPBS-EDTA, then incubate for 5 min at room temperature, finally aspirate DPBS-EDTA and add E8 medium containing 5 ⁇ M ROCK inhibitor. After resuspending the cells, passage at a density of 1:6-1:12 into 12-well plates precoated with Matrigel. After 1 day of cell adhesion on the plate, it was changed to E6 medium (E8 medium without FGF2 and TGF ⁇ ) and BMP4 was added to start differentiation, and PRKCD (Protein Kinase C Delta), ie PKC delta, was measured.
  • E6 medium E8 medium without FGF2 and TGF ⁇
  • BMP4 was added to start differentiation
  • PRKCD Protein Kinase C Delta
  • PKC isoforms gene expression levels, fresh differentiation medium was added daily. Levels of the trophoblast marker TROP2 in differentiated cells were detected during qPCR on the first day. Wherein, the determination method of qPCR and the instrument model are the same as those in Example 1.
  • Fig. 29 shows the effect of BMP4 on PRKCD gene expression.
  • Figure 30 shows the effect of TPA on PRKCD and TROP2 gene expression.
  • Figure 31 shows the effect of knockdown of PRKCD gene on PKC-delta protein expression in H1 cells.
  • Figure 32 shows the effect of TPA on gene expression of primitive endoderm and trophoblast markers SOX17, GATA6, TROP2 and CGB in PRKCD knockout cell lines.
  • Figure 33 shows the effect of BMP4 on gene expression of primitive endoderm and trophoblast marker CGB in PRKCD knockout cell lines.
  • Figure 34 shows the effect of BMP4 on the expression of primitive endoderm and trophoblast marker CGB protein in PRKCD knockout cell lines.
  • FIGS 29-34 show the effect of BMP4 and TPA on the expression level of PRKCD gene.
  • PRKCD knockout impairs differentiation of TPA in primitive endoderm markers SOX17 and GATA6 as well as trophoblast markers TROP2 and CGB.
  • PRKCD knockout impairs differentiation of BMP4 in the trophoblast marker CGB. That is, PRKCD controls the differentiation of human embryonic stem cells into primitive endoderm cells and trophoblast cells.
  • DGK inhibitor to block DGK function to verify its availability in trophoblast differentiation.
  • the specific operations are as follows: human embryonic stem cells H1 are cultured in E8 medium, fresh medium is replaced every day, and passage is carried out when the cell density reaches 70-80%. First wash 2 times with DPBS-EDTA and then incubate for 5 min at room temperature. DPBS-EDTA was aspirated and E8 medium containing 5 ⁇ M ROCK inhibitor was added. After resuspending the cells, passage at a density of 1:6-1:12 into 12-well plates precoated with Matrigel.
  • E6 E8 medium-FGF2-TGF ⁇
  • DGK inhibitor R59949 was added at a concentration of 1 ⁇ M to induce cell differentiation.
  • Fresh E6 medium was added daily.
  • qPCR was performed on day 6 to detect trophoblast marker levels. Among them, the determination method and instrument model of qPCR are the same as
  • Fig. 35 shows the effects of BMP4 and BMP4+GF109203X on the gene expression of DGK subtypes after 1 day of differentiation.
  • Figure 36 shows the effect of BMP4 and BMP4+GF109203X on DGKA (diacylglycerol kinase alpha) gene expression after 1 day of differentiation.
  • DGKA diacylglycerol kinase alpha
  • Figure 37 shows the effect of DGK inhibitor R59949 on gene expression of trophoblast markers TROP2 and CGB after 6 days of differentiation.
  • Figures 35-37 show that the DGK inhibitor R59949 can increase the expression levels of 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.
  • BMP4+GF109203X inhibited the gene expression of DGKA and DGKZ, promoted the gene expression of DGKB, and had no significant effect on the gene expression of DGKQ.
  • the present disclosure pioneered the use of PKC inhibitors in the BMP4 differentiation platform to promote the differentiation of pluripotent stem cells into mesoderm, and promote the expression of early mesoderm markers TBXT and MIXL1, which can greatly improve the differentiation of pluripotent stem cells into mesoderm.
  • mesoderm and endoderm precursor cells and shorten the differentiation process in half, while also improving the stability of differentiation, providing new applications for stem cell differentiation.
  • functional mesodermal cell types can be obtained, which can be used for regeneration and repair of tissues, such as smooth muscle cells, muscles, bones or gonads, and for drug screening.
  • the present disclosure employs a method with clear components to induce hPSC differentiation into trophoblasts, wherein the reagents used are free of animal-derived components.
  • the trophoblasts obtained by this method can also express markers of mature trophoblasts, which provides a new idea for guiding the early fate decision of human pluripotent stem cells.
  • the present disclosure discloses a method and medicine for inducing stem cells to differentiate into mesoderm lineage or trophoblast lineage.
  • the former method improves the stability of differentiation and provides a new application for stem cell differentiation.
  • functional mesodermal cell types can be obtained, which can be used for regeneration and repair of tissues, such as smooth muscle cells, muscles, bones or gonads, and for drug screening.
  • the latter approach pioneered the use of PKC activation and DGK inhibition to induce hPSC differentiation into trophoblasts, described PKC activation as a major mechanism of action downstream of any other trophoblast-induced platform, and uncovered new applications for stem cell differentiation.
  • the trophoblasts obtained by this method can also express markers of mature trophoblasts, which provides a new idea for guiding the early fate decision of human pluripotent stem cells.

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Abstract

提供了一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物，属于调节人多能干细胞向中胚层或滋养细胞分化的技术领域。分化成中胚层谱系方法包括：在BMP4存在的条件下，用含PKC抑制剂的分化培养基诱导干细胞分化成中胚层谱系。该方法提高了分化的稳定性，可在没有外源FGF2或内源性WNT的情况下仍保持胚层特异性分化。分化成滋养细胞谱系方法包括：用含PKC激活剂或DGK抑制剂的分化培养基诱导干细胞分化成滋养细胞谱系。该方法在自发分化平台中使用PKC激活剂和DGK抑制剂诱导干细胞以加速的方式分化为滋养细胞，而无需BMP4。

Description

一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物
相关申请的交叉引用
本公开要求于2021年02月26日提交中国专利局的申请号为“202110221046.6”名称为“一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及调节人多能干细胞向中胚层或滋养细胞分化技术领域，涉及一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物。
背景技术
人类多能干细胞(hPSC)可以分化为我们体内的所有细胞类型，并且是研究人类胚胎发育的重要模型***。hPSC分化产生的特定细胞类型在药物筛选等研究中起着重要作用。中胚层会产生诸如心肌细胞，平滑肌，骨骼和性腺等细胞类型，因此来自hPSC分化的中胚层衍生的细胞类型为再生和修复多种组织和药物筛选提供了材料。此外，hPSCs还可在体外分化为胚外细胞类型，如原始内胚层和滋养细胞。胚外细胞类型会产生胎盘，卵黄袋或脐带等组织，因此来自hPSC分化的胚外细胞类型可为体外受精，早期免疫***和药物筛选提供材料。
目前，尚未有报道研究PKC抑制剂在早期干细胞分化中的作用以及蛋白激酶C在早期滋养层分化中的作用。鉴于此，特提出本公开。
公开内容
本公开的一实施方式可以这样实现：
本公开提供一种使用PKC抑制剂诱导干细胞分化成中胚层谱系的方法，包括以下步骤：
在BMP4存在的条件下，用含有PKC抑制剂的分化培养基诱导干细胞分化成中胚层谱系。
中胚层谱系包括中胚层细胞和内胚层前体细胞。
在可选的实施方式中，干细胞包括人胚胎干细胞和人诱导的多能干细胞中的至少一种。
在可选的实施方式中，PKC抑制剂包括GF109203X、
Figure PCTCN2021093498-appb-000001
和
Figure PCTCN2021093498-appb-000002
中的至少一种。
在可选的实施方式中，GF109203X的浓度为2-5μM。
在可选的实施方式中，
Figure PCTCN2021093498-appb-000003
的浓度为2-5μM。
在可选的实施方式中，
Figure PCTCN2021093498-appb-000004
的浓度为2-5μM。
在可选的实施方式中，BMP4的浓度为5-100ng/mL。
在可选的实施方式中，分化培养基的成分包括DMEM/F12、L-抗坏血酸-2-磷酸镁、硒酸钠、转铁蛋白和胰岛素。
在可选的实施方式中，诱导干细胞分化成中胚层的过程中，不使用外源FGF2或内源性WNT。
本公开提供一种诱导干细胞分化成滋养细胞谱系的方法，包括以下步骤：用含有PKC激活剂或DGK抑制剂的分化培养基诱导干细胞分化成滋养细胞谱系。
在可选的实施方式中，PKC激活剂或DGK抑制剂于干细胞分化的第一天加入分化培养基中。
在可选的实施方式中，加入PKC激活剂后，干细胞至少在分化培养基中继续培养两天。或，加入DGK抑制剂后，干细胞至少在分化培养基中继续培养六天。
在可选的实施方式中，干细胞为人多能干细胞。在可选的实施方式中，人多能干细胞包括人胚胎干细胞和人诱导的多能干细胞中的至少一种。
在可选的实施方式中，PKC激活剂包括TPA和Bryostatin 1中的至少一种。
在可选的实施方式中，TPA的处理浓度为25-500nM。
在可选的实施方式中，Bryostatin 1的处理浓度为25-500nM。
在可选的实施方式中，DGK抑制剂包括R59949。
在可选的实施方式中，R59949的处理浓度为0.5-10μM。
在可选的实施方式中，分化培养基的成分包括DMEM/F12、L-抗坏血酸-2-磷酸镁、硒酸钠、转铁蛋白和胰岛素。
在可选的实施方式中，分化过程中，分化培养基中还添加有滋养细胞诱导剂。
在可选的实施方式中，滋养细胞诱导剂包括BMP4、PD325901、Sb432542以及DAPT中的至少一种。
在可选的实施方式中，BMP4的处理浓度为10-100ng/mL。
在可选的实施方式中，PD325901的处理浓度为0.5-10μM。
在可选的实施方式中，Sb432542的处理浓度为5-20μM。
在可选的实施方式中，DAPT的处理浓度为1-10μM。
本公开还提供一种药物，其原料含有上述方法得到的中胚层谱系或滋养细胞谱系。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1至图6为实施例1中的各测试结果图，其中图2中，hESCs为人类胚胎干细胞、B+GFX为BMP4联合PKC抑制剂GF109203X；其中图4中，
Figure PCTCN2021093498-appb-000005
为BMP4联合PKC抑制剂
Figure PCTCN2021093498-appb-000006
其中图3和6中的BRACHYURY为TBXT(T-box transcription factor T,T盒转录因子T)基因所表达的特定蛋白；
图7至图10为实施例2中的各测试结果图；
图11至图13为实施例3中的各测试结果图；
图14至图16为实施例4中的各测试结果图；
图17至图18为实施例7中的各测试结果图；
图19至图28为对比例1中的各测试结果图；
图29至图34为实施例8中的各测试结果图，其中，图34中，wt为野生型细胞系，pkcd-/-为敲除了PKCd的细胞系；
图35至图37为实施例9中的各测试结果图。
实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
术语定义
如本文所用，“细胞分化”通常可以指同一来源的细胞逐渐产生出形态结构、功能特征各不相同的细胞类群的过程，其结果是在空间上细胞产生差异，在时间上同一细胞与其从前的状态有所不同。
如本文所用，“中胚层”通常可以指在早期胚胎中三个主要胚层的中间层(其他两层为外胚层和内胚层)，是骨骼***、心血管***、泌尿***器官细胞的最初前体细胞。
如本文所使用，“分化培养基”通常可以指促进同一来源的细胞逐渐产生出形态结构、功能特征各不相同的细胞类群的培养基。
如本文所用，“多能干细胞”是指具有能够分化成多种细胞的多能性以及还具有自我增殖能力的干细胞。
如本文所用，“中胚层细胞诱导剂”是指能够诱导多能干细胞向中胚层细胞分化的化学小分子或细胞因子。
如本文所用，“滋养细胞”是指具有滋养功能的细胞，来自胚胎外的滋养层。
如本文所用，“滋养细胞诱导剂”是指能够诱导多能干细胞向滋养层细胞分化的化学小分子或细胞因子。
下面对本公开提供的诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物进行说明。
(I)诱导干细胞分化成中胚层谱系
本公开提供一实施方式，使用PKC(蛋白激酶C)抑制剂诱导干细胞分化成中胚层谱系的方法，包括以下步骤：
在BMP4(骨形态发生蛋白4)存在的条件下，用含有PKC抑制剂的分化培养基诱导干细胞分化成中胚层谱系。中胚层谱系包括中胚层细胞和内胚层前体细胞。
其中，干细胞包括人胚胎干细胞和人诱导的多能干细胞中的至少一种。也即可以仅为人胚胎干细胞进行分化，或者也可仅为人诱导的多能干细胞进行分化，或者还可以同时以人胚胎干细胞和人诱导的多能干细胞进行分化。
(A)PKC抑制剂
在可选的实施方式中，PKC抑制剂例如可包括GF109203X、
Figure PCTCN2021093498-appb-000007
和
Figure PCTCN2021093498-appb-000008
中的至少一种，可选地为GF109203X。
可参照地，GF109203X的浓度可以为2-5μM、2-4μM、2-3μM、3-5μM或3-4μM,，包括但不限于2μM、3μM、4μM或5μM等。
Figure PCTCN2021093498-appb-000009
的浓度可以为2-5μM、2-4μM、2-3μM、3-5μM或3-4μM,，包括但不限于2μM、3μM、4μM或5μM等。
Figure PCTCN2021093498-appb-000010
的浓度也可以为2-5μM、2-4μM、2-3μM、3-5μM或3-4μM,，包括但不限于2μM、3μM、4μM或5μM等。
上述BMP4作为中胚层诱导剂，其与PKC抑制剂可选地是GF109203X联用，可代替FGF2(成纤维细胞生长因子2)作为人类胚胎干细胞中早期中胚层细胞诱导的诱导剂。可参考地，BMP4的浓度可以为5-100ng/mL、10-100ng/mL、20-100ng/mL、30-100ng/mL、40-1000ng/mL、50-100ng/mL、5-90ng/mL、5-80ng/mL、5-70ng/mL、5-60ng/mL、10-90ng/mL、20-80ng/mL、30-70ng/mL，包括但不限于5ng/mL、10ng/mL、20ng/mL、50ng/mL或100ng/mL等。
(B)分化培养基的成分
在可选的实施方式中，本公开中所涉及的分化培养基的成分可包括DMEM/F12、L-抗坏血酸-2-磷酸镁、硒酸钠、转铁蛋白和胰岛素。上述各成分在分化培养基中的含量可以分别为：L-抗坏血酸-2-磷酸镁 64mg/L、硒酸钠 13.6μg/L、转铁蛋白 10mg/L以及胰岛素 20mg/L。上述分化培养基可以为E6(Essential 6，基础6)培养基，该培养基可理解成E8(Essential 8，基础8)培养基不含FGF2和TGFβ(转化生长因子-β)。
据信，不受理论的约束，PKC抑制剂可诱导干细胞分化为中胚层早期谱系。
在可选的实施方式中，本公开提供的诱导干细胞分化成中胚层的过程中，不使用外源FGF2或内源性细胞因子WNT。也即，PKC抑制剂可选地是GF109203X促进人类胚胎干细胞H1向中胚层的分化，无需FGF2途径激活。并且，PKC抑制剂可选地是GF109203X促进人类胚胎干细胞H1向中胚层的分化，无需内源性细胞因子WNT。
(C)诱导干细胞分化成中胚层谱系的方法
在可选的实施方式中，本公开所涉及的使用PKC抑制剂诱导干细胞分化成中胚层谱系的方法可以但不限于参照 以下步骤：
将hPSC培养至10-20％的细胞密度(该密度为开始分化前的密度)后，在BMP4存在下使用PKC抑制剂GF109203X诱导hPSC分化为中胚层。2天后，可通过免疫染色或FACS(流式细胞荧光分选技术)检测TBXT(T盒转录因子T)阳性细胞的形成(也即可在分化的第三天，测量TBXT阳性细胞)。
可选地，可以是：在E8培养基中培养人胚胎干细胞H1，每天更换新鲜培养基，当细胞密度达到70-80％时进行传代(也即该密度为传代前的密度)。首先用DPBS-EDTA洗涤两次，然后在室温下孵育5分钟，第3次抽吸DPBS-EDTA，并添加含有5μM ROCK(Rho相关的蛋白激酶)抑制剂的E8培养基。重悬细胞后，以1:6至1:12、1:7至1:12、1:8至1:12、1:9至1:12、1:6至1:11、1:6至1:10或1:6至1:9(包括但不限于1：6、1：8、1：10或1：12等)的密度传代，加入预先包被Matrigel(基底胶)的12孔板。细胞粘附在平板上的1天后，与BMP4和PKC抑制剂一起换成E6培养基(E8培养基不含FGF2及TGFβ)，分别以20ng/mL和5μM的浓度开始分化。每天添加新鲜E6培养基。
据信，不受理论的约束，PKC抑制剂，可选地GF109203X可提高内胚层早期标志物的表达水平。可以促进人胚胎干细胞系(H1和H9)向内胚层细胞分化。
值得说明的是，上述所提的E6和E8培养基可参照本公开所公开的成分得到，也可参照现有技术中相关培养基的成分进行配制，在此不做过多赘述。
通过上述方法可获得功能性中胚层细胞类型，可用于组织的再生和修复，如平滑肌细胞、肌肉、骨骼或性腺，并用于药物筛选。对应地，本公开还提供了一种药物，其原料含有上述方法得到的中胚层谱系。
本公开提供一实施方式，提供上述药物用于治疗组织再生、组织修复的用途。
在可选的实施方式中，组织因损伤或疾病而受损。
在可选的实施方式中，组织包括但不限于上皮组织、***、肌组织、神经组织、骨骼、性腺。
在可选实施方式中，肌组织包括骨骼肌组织、平滑肌组织、心肌纤维组织。
本公开提供一实施方式，提供对有此需要的受试者中组织再生和修复的方法，包括：向所述有此需要的受试者施用上述药物。
在可选的实施方式中，受试者患有选自以下的骨损伤或骨疾病：骨关节炎、变形性骨炎、骨质疏松症、骨断裂、骨折、骨癌症、骨癌、骨髓瘤骨病、骨质减少、骨硬化、肾性骨营养不良、骨癌、囊性纤维性骨炎或骨关节炎；
和/或患有选自以下的心脏损伤或心脏疾病：心脏瓣膜疾病、心律不齐、左心室肥大、右心室肥大、心肌梗塞、心力衰竭或先天性心脏缺陷；
和/或患有选自以下的性腺疾病：***、Klinefelter(克氏)综合征、Turner(特纳)综合征、性腺功能减退症、雄激素不敏感综合征(AIS)
和/或患有选自以下的肌组织疾病：多发性肌炎、线粒体肌病、肌源性萎缩。
(II)诱导干细胞分化成滋养细胞谱系
本公开提供一实施方式，诱导干细胞分化成滋养细胞谱系的方法，包括以下步骤：
用含有PKC激活剂或DGK(二酰基甘油激酶)抑制剂的分化培养基诱导干细胞分化成滋养细胞谱系。其中，干细胞为人多能干细胞。可参考地，人多能干细胞包括人胚胎干细胞和人诱导的多能干细胞中的至少一种，也即可以仅为人胚胎干细胞，也可仅为人诱导的多能干细胞，还可以同时以人胚胎干细胞和人诱导的多能干细胞进行分化。
(A)激活剂
在可选的实施方式中，PKC激活剂于干细胞分化的第一天加入分化培养基中。可参考地，加入PKC激活剂后，干细胞至少在分化培养基中继续培养两天。
在可选的实施方式中，PKC激活剂例如可包括TPA(12-氧-十四烷酰佛波醇-13-乙酸酯(12-O-tetradecanoylphorbol-13-acetate)，一种能够在细胞内激活PKC的小分子化合物)和苔藓虫素1(Bryostatin1)中的至少一种。
其中，TPA及Bryostatin 1的处理浓度均可分别为25-500nM、25-450nM、25-400nM、25-350nM、25-300nM、50-500nM、100-500nM、150-500nM或200-500nM，包括但不限于25nM、50nM、100Nm、200nM、300nM、400nM或500nM等。
(B)DGK抑制剂
在可选的实施方式中，DGK抑制剂于干细胞分化的第一天加入分化培养基中。可参考地，加入DGK抑制剂后，干细胞至少在分化培养基中继续培养六天。
在可选的实施方式中，DGK抑制剂可包括(为)R59949。R59949的处理浓度可以为0.5-10μM、0.5-9μM、0.5-8μM、0.5-7μM、0.5-6μM、0.5-5μM、1-10μM、2-10μM、3-10μM、4-10μM、5-10μM，包括但不限于0.5μM、1μM、2μM、5μM或10μM等。
(C)分化培养基的成分
在可选的实施方式中，本公开中所涉及的分化培养基的成分可包括DMEM/F12、L-抗坏血酸-2-磷酸镁、硒酸钠、转铁蛋白和胰岛素。上述各成分在分化培养基中的含量可以分别为：L-抗坏血酸-2-磷酸镁 64mg/L、硒酸钠 13.6μg/L、转铁蛋白 10mg/L以及胰岛素 20mg/L。上述分化培养基可以为E6培养基，该培养基可理解成E8培养基不含FGF2和TGFβ。
(D)滋养细胞诱导剂
在可选的实施方式中，本公开上述分化过程中，分化培养基中还可添加有滋养细胞诱导剂。在可选的实施方式中，滋养细胞诱导剂包括BMP4、PD325901、Sb432542以及DAPT中的至少一种。
其中，BMP4的处理浓度可以为10-100ng/mL、10-90ng/mL、10-80ng/mL、10-70ng/mL、10-60ng/mL、20-100ng/mL、30-100ng/mL、40-100ng/mL，包括但不限于10ng/mL、20ng/mL、50ng/mL、80ng/mL或100ng/mL等。
PD325901的处理浓度可以为0.5-10μM、1-10μM、3-10μM、5-10μM、0.5-9μM、0.5-7μM或0.5-5μM，包括但不限于0.5μM、1μM、2μM、5μM、8μM或10μM等。
Sb432542的处理浓度可以为5-20μM、5-18μM、5-16μM、5-14μM、5-12μM、7-20μM、9-20μM、11-20μM，包括但不限于5μM、8μM、10μM、15μM或20μM等。
DAPT的处理浓度可以为1-10μM、2-10μM、3-10μM、4-10μM、1-9μM、1-8μM、1-7μM、1-6μM，包括但不限于1μM、2μM、5μM、8μM或10μM等。
(E)诱导干细胞分化成滋养细胞谱系的方法
在可选的实施方式中，本公开所涉及的诱导干细胞分化成滋养细胞谱系的方法可以但不限于参照以下步骤：
将hPSC培养至10-20％的细胞密度后，使用PKC调节剂诱导hPSC分化为滋养细胞。从第一天开始就使用分化培养基进行分化，处理2天后(可选在分化的第二天)，可以通过qPCR检测信号标记，例如TROP2(人滋养细胞表面抗原)和CGB(绒毛膜***亚基β3)。
可选地可以是：在E8培养基中培养人胚胎干细胞，每天更换新鲜培养基，当细胞密度达到70-80％时进行传代(也即该密度为传代前的密度)。首先用DPBS-EDTA洗涤两次，然后在室温下孵育5分钟，最后抽吸DPBS-EDTA，并添加含有5μM ROCK抑制剂的E8培养基。重悬细胞后，以1:6-1:12(如1：6、1：8、1：10或1：12等)的密度传代至预先涂有Matrigel的12孔板。细胞粘附在平板上一天后，换成E6培养基(E8培养基不含FGF2与TGFβ)并加入50nM的TPA或Bryostatin 1开始分化。期间每天添加新鲜分化培养基，并在分化的第2天测量滋养细胞标志物水平。
或者，在E8培养基中培养人胚胎干细胞H1，每天更换新鲜培养基，当细胞密度达到70-80％时进行传代。首先用DPBS-EDTA洗涤两次，然后在室温下孵育5分钟。抽吸DPBS-EDTA，并添加含有5μM ROCK抑制剂的E8培养基。重悬细胞后，以1:6-1:12的密度传代到预先涂有Matrigel的12孔板。细胞粘附在平板上一天后，换成E6培养基(E8培养基不含FGF2与TGFβ)，并以1μM的浓度添加DGK抑制剂R59949以促使细胞分化。每天添加新鲜E6培养基。在第6天检测滋养细胞标志物水平。
值得说明的是，上述所提的E6和E8培养基可参照本公开所公开的成分得到，也可参照现有技术中相关培养基的成分进行配制，在此不做过多赘述。
本公开所使用的干细胞可以商购。例如，干细胞可以商购自Applied StemCell。
(III)用途及药物
通过上述方法可获得的滋养细胞谱系可为早期免疫***和药物筛选提供材料。对应地，本公开还提供了一种药物，其原料含有上述方法得到的滋养细胞谱系。
本公开提供一实施方式，提供上述药物用于治疗早期免疫***疾病和药物筛选的用途。
本公开提供一实施方式，提供治疗受试者中早期免疫***疾病的方法，包括：向所述有此需要的受试者施用上述药物。
在可选的实施方式中，受试者患有的免疫***疾病包括：自身免疫***疾病和免疫缺陷***疾病。在可选的实施方式中，自身免疫***疾病包括但不限于抗体介导的自身免疫***疾病，诸如毒性弥漫性甲状腺肿、重症肌无力、粒细胞减少症、***性红斑狼疮等；T细胞介导的自身免疫***疾病，诸如1型糖尿病、粒细胞减少症、慢性肉芽肿等。
在可选的实施方式中，免疫缺陷***疾病包括但不限于原发性免疫缺陷***疾病，诸如联合免疫缺陷病、免疫失调性疾病、吞噬细胞缺陷、天然免疫缺陷、自身炎症性疾病等；继发性免疫缺陷***疾病，诸如传染性因子引起的免疫缺陷、由药物导致的免疫缺陷、由霉菌中毒引起的免疫缺陷、营养缺陷与免疫应答、AIDS。
本公开的有益效果包括：
本公开通过开拓性地在BMP4分化平台中使用PKC抑制剂促进多能干细胞分化成中胚层，促进早期中胚层标记物TBXT和MIXL1(混合成对的同源盒)的表达，能大幅度提高多能干细胞分化为中胚层和内胚层前体细胞，并使分化过程缩短一半时间，同时还提高了分化的稳定性，为干细胞分化提供了新应用。通过该方法可以获得功能性中胚层细胞类型，可用于组织的再生和修复，如平滑肌细胞、肌肉、骨骼或性腺，并用于药物筛选。
此外，本公开采用具有清晰成分的方法来诱导hPSC分化为滋养细胞，其中所使用的试剂不含动物来源的成分。开拓性地使用PKC激活和DGK抑制来诱导hPSC分化为滋养细胞，描述了PKC活化是任何其他滋养细胞诱导的平台下游的主要作用机制，并发现了干细胞分化的新应用。另外，通过该方法获得的滋养细胞还可表达成熟滋养细胞的标志物，这为指导人类多能干细胞的早期命运决定提供了新思路。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例
材料及试剂
GF109203X，全称为双吲哚马来酰亚胺I，CAS No.133052-90-1，商购自selleckchem，货号S7208；
Figure PCTCN2021093498-appb-000011
全称为12-(2-氰乙基)-6,7,12,13-四氢-13-甲基-5-氧代-5H-吲哚并[2,3-a]吡咯并[3,4-c]咔唑，CAS No.136194-77-9，商购自Cayman，货号13310；
Figure PCTCN2021093498-appb-000012
全称为3-[1-[3-(二甲基氨基)丙基]-5-甲氧基-1H-吲哚-3-基]-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮，CAS No.133053-19-7，商购自Cayman，货号13311；
BMP4，全称为骨形态发生蛋白4，商购自R&D Systems，货号314-BP-500；
PD0325901，全称为Mirdametinib，一种MEK抑制剂，商购自selleckchem，货号S1036；
Sb432542，为一种ALK5抑制剂，商购自selleckchem，货号S1067；
DAPT，全称为(3,5-二氟苯乙酰基)-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯，CAS No.208255-80-5，商购自selleckchem，货号S2215；
LDN193189，全称为4-(6-(4-(哌嗪-1-基)苯基)吡唑并[1,5-a]嘧啶-3-基)喹啉，CAS No.1062368-24-4，商购自：selleckchem，货号S2618；
FGF2，全称成纤维细胞生长因子2，商购自peprotech，货号100-18C；
TGFβ，全称为转化生长因子-β，商购自R&D Systems，货号240-B-500；
Jagged-1，全称为锯齿状典型Notch配体1，商购自GenScript，货号RP20331；
人胚胎干细胞H1：购自WiCell，货号WA01；
人胚胎干细胞H9：购自WiCell，货号WA09；
人类诱导多能干细胞NL4：并非人胚胎干细胞系，其是一种经人体细胞在体外诱导产生的多能细胞系。
实施例1
为了研究不同的PKC抑制剂在中胚层早期分化中的作用，在分化的第一阶段，我们选择了GF109203X，
Figure PCTCN2021093498-appb-000013
和
Figure PCTCN2021093498-appb-000014
这三种PKC抑制剂以及BMP4。
具体实现如下：在E8培养基中培养人胚胎干细胞H1，每天更换新鲜培养基，当细胞密度达到70-80％时进行传代。首先用DPBS-EDTA(购自cell system，货号4Z0-610)洗涤2次，然后在室温下孵育5分钟，第3次抽吸DPBS-EDTA，并添加含有5μM ROCK抑制剂的E8培养基。重悬细胞后，以1:6的密度传代，加入预先包被Matrigel的12孔板。细胞贴壁生长在平板上的1天后，与BMP4和PKC抑制剂一起换成E6培养基(E8培养基不含FGF2及TGFβ)，分别以20ng/mL和5μM的浓度开始分化(分化前细胞密度为10-20％)。每天添加新鲜E6培养基。在分化的第一天和第二天通过qPCR，免疫荧光和流式细胞术(除非另有说明)测量分化细胞的早期中胚层标志物的水平。
qPCR采用荧光定量PCR检测仪(型号为：赛默飞QuantStudio7)进行测量，用RNAiso-plus(TAKARA，货号108-95-2)提取总mRNA，并使用高容量cDNA逆转录试剂盒(Applied Biosystems，货号4368813)进行从mRNA到cDNA的逆转录。使用SYBR Premix Ex Taq(TAKARA，货号RR420)和Quantstudio-7***(Applied Biosystems)进行实时定量PCR。通过2^(-ΔCt)方法计算扩增的核苷酸片段的相对量。将表达水平相对于管家基因TBP进行标准化，并与未分化的hESC进行比较。
免疫荧光采用免疫荧光分析仪(型号为：赛默飞EVOS Cell Imaging System FL)进行测量，细胞在室温下用4％多聚甲醛固定20分钟，用1x PBS冲洗(每次5分钟，3次)，用0.5％Triton X-100破膜20分钟，再用1x PBS冲洗(每次5分钟，3次)。细胞核在室温下用Hoechst 33342(货号U0334，Abnova，1：10,000)染色20分钟。然后将细胞用1x PBS漂洗(每次5分钟，3次)，在4℃下与一抗孵育过夜，用1xPBS漂洗，与荧光二抗孵育2小时，最后用1xPBS漂洗后进行成像。
流式细胞术采用流式细胞仪(型号为：贝克曼库尔特CytoFLEX)进行测量，通过TrypLE(37℃，5min)收获细胞，并用DMEM/5％FBS中和。用具有1％BSA的DPBS洗涤后，将细胞在DPBS中的0.2％Triton X-100和5％FBS中破膜1小时。将一抗和二抗孵育，洗涤后，将细胞重悬于DPBS中，通过CytoFLEX进行分析。。请参照图1至图6，图1代表PKC抑制剂GF109203X对BMP4分化下早期中胚层的标志基因MIXL1和TBXT基因表达的影响。分别以5μM和20ng/mL的浓度添加GF109203X和BMP4。图中，mock(对照组)代表E6培养基+BMP4，GFX代表在mock的基础上添加PKC抑制剂GF109203X，下同。
图2代表在添加BMP4和GF109203X诱导干细胞分化2天后，通过免疫染色分析了TBXT阳性细胞的比例。该图中，对于Hoechst(购自Abnova，货号U0334，按1：10,000加入)组和TBXT组，灰色代表阳性细胞。
图3代表在添加BMP4和GF109203X以诱导干细胞分化2天后，通过流式细胞术分析了TBXT阳性细胞的比例。
图4代表添加BMP4和
Figure PCTCN2021093498-appb-000015
诱导干细胞分化2天后，通过免疫染色分析了TBXT阳性细胞的比例。以5μM的浓度添加
Figure PCTCN2021093498-appb-000016
该图中，对于Hoechst组和TBXT组，灰色代表阳性细胞。
图5代表PKC抑制剂对BMP4分化一天后早期中胚层的标志基因WNT3基因表达的影响。
图6代表使用5μM PKC抑制剂
Figure PCTCN2021093498-appb-000017
及20ng/mL BMP4分化对早期中胚层的标志基因WNT3、MIXL1和TBXT表达的影响。该图中，矩形颜色越深代表对应基因的表达量越高。
结果显示，仅在24小时后，GF109203X，
Figure PCTCN2021093498-appb-000018
和
Figure PCTCN2021093498-appb-000019
即可提高早期中胚层标记基因MIXL1和TBXT的表达水平。TBXT是中胚层早期分化的特异性标志物，GF109203X和
Figure PCTCN2021093498-appb-000020
不同程度地增加TBXT阳性细胞的比例，表明PKC抑制剂可诱导干细胞分化为中胚层早期谱系。GF109203X可以添加到BMP4诱导分化中，以促进WNT途径并加速人胚干细胞的中胚层早期分化。
实施例2
GF109203X促进人类胚胎干细胞H1向中胚层的分化，而无需FGF2途径激活。
将人类胚胎干细胞H1培养在E8培养基中，每天更换新鲜培养基，细胞密度达到70-80％。首先用DPBS-EDTA洗涤两次，然后在室温下孵育5分钟，第三次除去DPBS-EDTA，并用含有5μM Rock抑制剂的E8培养基重悬细胞后，以1:6的密度传代，加入预先包被Matrigel的12孔板，加入含有20ng/ml BMP4，100ng/mL FGF2和5μM PKC抑制剂的E6培养基(E8培养基不含FGF2和TGFβ)并开始分化。每天添加新鲜E6培养基。在分化的第一天和第二天通过qPCR，免疫荧光和流式细胞术(除非另有说明)测量分化细胞的早期中胚层标志物的水平。qPCR、免疫 荧光和流式细胞术的测定方法以及仪器型号同实施例1。
请参照图7至图10，图7表示PKC抑制剂GF109203X对BMP4或BMP4+FGF2分化下的MIXL1和TBXT基因表达的影响，MIXL1和TBXT是早期中胚层的标志物。其中，mock代表E6培养基+BMP4，GFX代表在mock的基础上添加GFX(PKC抑制剂GF109203X)，FGF2代表在mock的基础上添加FGF2,FGF2+GFX代表在mock的基础上添加FGF2+GFX。分别以5μM、20ng/mL和100ng/mL的浓度添加GF109203X、BMP4和FGF2。
图8表示在添加BMP4/GF109203X(图中以GFX表示)或BMP4/FGF2/GF109203X以诱导干细胞分化后2天，通过流式细胞术分析TBXT阳性细胞的比例。
图9表示PKC抑制剂GF109203X对早期中胚层标志物BMP4+FGF2或BMP4+FGF2+PD0173074(图中以PD173表示，下同)分化下WNT3、MIXL1和TBXT基因表达的影响。以100nM的浓度添加PD0173074。
图10表示在添加BMP4+FGF2和GF109203X(GFX)、PD0173074(PD173)或两者诱导干细胞分化后2天，通过免疫染色分析了TBXT阳性细胞的比例。其中，mock代表E6培养基+BMP4+FGF2，GFX代表在mock的基础上添加GFX，PD173代表在mock的基础上添加PD173,PD173+GFX代表在mock的基础上添加PD173+GFX。该图中，对于Hoechst组和TBXT组，灰色代表阳性细胞。
结果表明，即使在存在FGFR抑制剂PD0173974的情况下，仅在24小时后，GF109203X即可提高早期中胚层标记基因MIXL1和TBXT的表达水平。FGF2途径抑制剂PD0173074以100nM使用。TBXT是中胚层早期分化的特异性标志物，即使在存在PD0173074的情况下，GF109203X也会增加TBXT阳性细胞的比例，这表明PKC抑制剂可以诱导干细胞绕过FGF2途径活化而分化为早期中胚层谱系。也即GF109203X与BMP4结合，可以代替FGF2作为人类胚胎干细胞中早期中胚层细胞诱导的诱导剂。
实施例3
GF109203X促进人类胚胎干细胞H1向中胚层的分化，而无需内源性WNT。
将人类胚胎干细胞H1培养在E8培养基中，每天更换新鲜培养基，细胞密度达到70-80％。首先用DPBS-EDTA洗涤两次，然后在室温下孵育5分钟，第3次抽吸DPBS-EDTA，并添加含有5μM Rock抑制剂的E8培养基。重悬细胞后，以1:6的密度传代，加入预先包被Matrigel的12孔板。细胞粘附在平板上的1天后，培养基换成E6培养基(E8培养基不含FGF2及TGFβ)并加入20ng/mL BMP4和5μM PKC抑制剂开始分化。每天添加新鲜E6培养基。在分化的第1天和第2天通过qPCR，免疫荧光和流式细胞术(除非另有说明)测量分化细胞的早期中胚层标志物的水平。qPCR、免疫荧光和流式细胞术的测定方法以及仪器型号同实施例1。
请参照图11至图13，图11表示PKC抑制剂GF109203X(GFX)对BMP4或BMP4+IWP-2(IWP2)分化下的WNT3、MIXL1和TBXT基因表达的影响，这是早期中胚层的标志。分别以5μM、20ng/mL和2.5μM的浓度添加GF109203X、BMP4和IWP-2。
图12表示通过免疫染色分析，经过6个小时的分化后，GF109203X(GFX)可以在BMP4诱导下促进β-联蛋白(β-catenin)核定位。该图中，对于Hoechst组和np-βcat(即非磷酸化β-联蛋白(Non-phosphoβ-Catenin)，代表一种激活型β-Catenin)组，灰色代表阳性细胞。
图13表示分化6小时后，在信号强度下定量BMP4和BMP4+GF109203X(GFX)诱导下β-catenin的核定位。
结果表明GF109203X可以绕过WNT途径，并促进人胚胎干细胞中β-catenin的核定位。说明GF109203X可提高中胚层早期标志物的表达水平。
实施例4
GF109203X促进人类胚胎干细胞H1向内胚层的分化。
将人类胚胎干细胞H1培养在E8培养基中，每天更换新鲜培养基，细胞密度达到70-80％。首先用DPBS-EDTA洗涤两次，然后在室温下孵育5分钟，第3次抽吸DPBS-EDTA，并添加含有5μM Rock抑制剂的E8培养基。重悬细胞后，以1:6的密度传代，加入预先包被Matrigel的12孔板。细胞粘附在平板上的1天后，培养基换成E6培养基(E8培养基不含FGF2及TGFβ)并加入100ng/mL Activin A、20ng/mL BMP4、100ng/mL FGF2和5μM PKC抑制剂开始分化。此后每天添加新鲜E6培养基和100ng/mL Activin A。在分化的第4天通过qPCR，免疫荧光和流式细胞术(除非另有说明)测量分化细胞的早期内胚层标志物的水平。结果表明GF109203X可提高内胚层早期标志物的表达水平。qPCR、免疫荧光和流式细胞术的测定方法以及仪器型号同实施例1。
请参照图14至图16，图14代表在内胚层细胞分化的早期，即第0天至第1天，通常可以用Acivin A(激活素A)+BMP4+FGF2诱导胚胎干细胞向原条细胞(PS)分化，PS细胞可在Activin+LDN193189+IWP2(Wnt信号通路抑制剂)的条件下诱导形成特异性内胚层细胞(DE，DE是人体呼吸道和消化器官的前体)。结果发现，相比于Acivin A+BMP4+FGF2，PKC抑制剂GF109203X联合Acivin A+BMP4+FGF2能够在分化的第2天至第3天显著提高SOX17的表达，这是早期内胚层的生物标志物。分别以5μM、100ng/mL、20ng/mL、100nM和2.5μM的浓度添加GF109203X、Activin A、BMP4、LDN193189和IWP-2。
图15代表通过定量PCR分析，经过2-3天的分化后，GF109203X(GFX)可以促进H9和H1细胞系向DE分化。
图16代表通过流式细胞术分析，经过2-3天的分化后，GF109203X(GFX)可以促进H9细胞系向DE分化，且能够得到几乎100％的SOX17阳性DE细胞。
结果表明GF109203X可以促进人胚胎干细胞系(H1和H9)向内胚层细胞分化。
实施例5
本实施例提供了一种使用PKC抑制剂诱导人诱导多能干细胞分化成中胚层谱系的方法：
在E8培养基中培养人诱导多能干细胞(iPSC，如NL-1或NL-4)，每天更换新鲜培养基，当细胞密度达到70-80％时进行传代。首先用DPBS-EDTA洗涤2次，然后在室温下孵育5分钟，第3次抽吸DPBS-EDTA，并添加含有5μM ROCK抑制剂的E8培养基。重悬细胞后，以1:10的密度传代，加入预先包被Matrigel的12孔板。细胞粘附在平板上的1天后，与BMP4和GF109203X一起换成E6培养基(E8培养基不含FGF2及TGFβ)，分别以20ng/mL和2μM的浓度开始分化。每天添加新鲜E6培养基。在分化的第一天和第二天通过qPCR，免疫荧光和流式细胞术(除非另有说明)测量分化细胞的早期中胚层标志物的水平。qPCR、免疫荧光和流式细胞术的测定方法以及仪器型号同实施例1。
实施例6
本实施例提供了一种使用PKC抑制剂诱导干细胞分化成中胚层谱系的方法：
在E8培养基中培养人胚胎干细胞H1，每天更换新鲜培养基，当细胞密度达到70-80％时进行传代。首先用DPBS-EDTA洗涤2次，然后在室温下孵育5分钟，第3次抽吸DPBS-EDTA，并添加含有5μM ROCK抑制剂的E8培养基。重悬细胞后，以1:12的密度传代，加入预先包被Matrigel的12孔板。细胞粘附在平板上的1天后，与BMP4和GF109203X一起换成E6培养基(E8培养基不含FGF2及TGFβ)，分别以20ng/mL和4μM的浓度开始分化。每天添加新鲜E6培养基。在分化的第一天和第二天通过qPCR，免疫荧光和流式细胞术(除非另有说明)测量分化细胞的早期中胚层标志物的水平。qPCR、免疫荧光和流式细胞术的测定方法以及仪器型号同实施例1。
实施例7
本实施例提供了一种使用PKC激活剂促进人类胚胎干细胞向滋养细胞的分化的方法：
为了研究不同PKC激活在滋养细胞分化中的作用，我们选择了TPA，Bryostatin 1这两种PKC激活剂对hPSCs的分化。具体如下：在E8培养基中培养人胚胎干细胞，每天更换新鲜培养基，当细胞密度达到70-80％时进行传代。首先用DPBS-EDTA洗涤两次，然后在室温下孵育5分钟，最后抽吸DPBS-EDTA，并添加含有5μM ROCK抑制剂的E8培养基。重悬细胞后，以1:6-1:12的密度传代至预先涂有Matrigel的12孔板。细胞粘附在平板上1天后，换成E6培养基(E8培养基不含FGF2与TGFβ)并加入50nM的TPA或Bryostatin 1开始分化。期间每天添加新鲜分化培养基，并在分化的第3天测量滋养细胞标志物(TROP2和CGB)水平以及原始内胚层标志物(SOX17和GATA6)水平，如图17和图18所示。
其中，滋养细胞标志物(TROP2和CGB)水平以及内胚层标志物(SOX17和GATA6)水平通过qPCR检测信号标记测定，其方法以及仪器型号同实施例1。其中，图17表示PKC激活剂TPA在分化第3天对原始内胚层标志物(SOX17和GATA6)和滋养细胞标志物(TROP2和CGB)的基因表达的影响。以50nM的浓度添加TPA。NANOG为干细胞干性的生物标志物，其表达水平越高，则表示细胞的干性越高；反之，其表达水平越低，则表示细胞的干性越低，即细胞发生了分化。
图18表示PKC激活剂Bryostatin 1在分化第2-10天对原始内胚层标志物(SOX17和GATA6)和滋养细胞标志物(TROP2和CGB)的基因表达的影响。以50nM的浓度添加Bryostatin 1。NANOG为干细胞干性的生物标志物，其表达水平越高，则表示细胞的干性越高；反之，其表达水平越低，则表示细胞的干性越低，即细胞发生了分化。mock代表E6培养基+BMP4，GFX代表在mock的基础上添加GFX(PKC抑制剂GF109203X)，下同。
图17和图18的结果显示：TPA和Bryostatin 1可以提高3天后原始内胚层标记基因SOX17和GATA6以及滋养层标记基因TROP2和CGB的表达水平。PKC抑制剂GF109203X抑制TPA和Bryostatin 1促进滋养细胞特异性标志物TROP2和CGB的表达，PKC激活剂可以诱导干细胞分化为滋养细胞谱系。说明TPA和Bryostatin 1对PKC的激活促进了分化为胚外细胞类型的分化，例如人类胚胎干细胞的原始内胚层和滋养细胞。
对比例1
本对比例提供了一种使用PKC抑制剂GF109203X抑制人类多能干细胞H1、H9和NL4向任何其他途径促进的滋养细胞的分化的方法：
将人类胚胎干细胞H1培养在E8培养基中，每天更换新鲜培养基，细胞密度达到70-80％开始传代。首先用DPBS-EDTA洗涤两次，然后在室温下孵育5分钟，最后抽吸DPBS-EDTA，并添加含有5μM ROCK抑制剂的E8培养基。重悬细胞后，以1:6-1:12的密度传代至预先涂有Matrigel的12孔板。细胞粘附在平板上后1天，换成E6培养基(E8培养基不含FGF2及TGFβ)并加入20ng/ml BMP4，1μM PD0325901，10μM Sb431542或5μM DAPT和PKC抑制剂一起开始分化。每天添加新鲜的分化培养基。在第6天通过qPCR和免疫荧光检测分化细胞的滋养层标志物水平(TPA分化的细胞在第3天检测)，如图19-28所示。其中，qPCR、免疫荧光检测的测定方法以及仪器型号同实施例1。
其中，图19表示PKC抑制剂GF109203X对在BMP4分化下的滋养层细胞标志物TROP2和CGB基因表达的影响。分化条件为在分化培养基中加入GF109203X(5μM)，BMP4(20ng/ml)，LDN193189(100nM)(以LDN表示)，FGF2(100ng/ml)，TGFβ(2ng/ml)和Jagged-1(1μM)(以JAG1表示，下同)。
图20表示PKC抑制剂GF109203X对在PD0325901条件下滋养细胞的标志基因TROP2和CGB表达的影响。分化条件为在分化培养基中加入GF109203X(5μM)，LDN193189(100nM)，FGF2(100ng/ml)，PD0325901(1μM)，TGFβ(2ng/ml)和Jagged-1(1μM)。
图21表示PKC抑制剂GF109203X对在Sb431542条件下滋养细胞的标志基因TROP2和CGB的基因表达的影响。分化条件为在分化培养基中加入GF109203X(5μM)，LDN193189(100nM)，FGF2(100ng/ml)，TGFβ(2ng/ml)，Sb431542(10μM)和Jagged-1(1μM)。
图22表示PKC抑制剂GF109203X对在DAPT条件下滋养细胞的标志基因TROP2和CGB表达的影响。分化条 件为在分化培养基中加入GF109203X(5μM)，LDN193189(100nM)，FGF2(100ng/ml)，TGFβ(2ng/ml)，DAPT(5μM)和Jagged-1(1μM)。
图23表示PKC抑制剂GF109203X对在TPA条件下滋养细胞的标志基因TROP2和CGB表达的影响。分化条件为在分化培养基中加入TPA(50nM)，GF109203X(5μM)，LDN193189(100nM)，FGF2(100ng/ml)，TGFβ(2ng/ml)和Jagged-1(1μM)。
图24表示PKC抑制剂GF109203X和
Figure PCTCN2021093498-appb-000021
对BMP4诱导的滋养细胞标志基因TROP2和CGB表达的影响。分化条件为在分化培养基中加入GF109203X(5μM)，
Figure PCTCN2021093498-appb-000022
(5μM)和BMP4(20ng/ml)。
图25表示PKC抑制剂
Figure PCTCN2021093498-appb-000023
对BMP4诱导的滋养细胞标志蛋白TROP2和CGB表达的影响，其中Phase为没有任何染料或者抗体的情况下的白场，Hoechst为细胞核染料，细胞核会被染成蓝色(在本图中，由于图片修改为黑白背景，则细胞核为浅色)，下同。
图26表示GF109203X和LDN193189对BMP4诱导的滋养细胞标志蛋白TROP2和CGB蛋白表达的影响。
图27表示PKC抑制剂GF109203X对在BMP4诱导的滋养细胞标志基因TROP2，GATA3，GATA2，KRT18，CDX2，ELF5，ITGA6，CGB，CSH1，CSH2，GCM1，HLAG和ITGA5表达的影响。
图28表示PKC抑制剂GF109203X对BMP4诱导的滋养细胞标志基因TROP2，GATA3，CDX2，CGB和HLAG表达的影响。
图19-28结果显示：GF109203X在任何已知诱导剂促进的6天后均能抑制滋养细胞标记基因TROP2和CGB的表达水平。GF109203X和
Figure PCTCN2021093498-appb-000024
在BMP4诱导的滋养细胞分化过程中抑制了TROP2/CGB阳性细胞的比例，表明PKC抑制剂可损害干细胞分化为滋养细胞谱系。
实施例8
PKC-δ敲除损害人胚胎干细胞H1的滋养细胞分化：
将人类胚胎干细胞H1培养在E8培养基中，每天更换新鲜培养基，细胞密度达到70-80％开始传代。首先用DPBS-EDTA洗涤2次，然后在室温下孵育5分钟，最后抽吸DPBS-EDTA，并添加含有5μM ROCK抑制剂的E8培养基。重悬细胞后，以1:6-1:12的密度传代至预先涂有Matrigel的12孔板。细胞粘附在平板上的1天后，换成E6培养基(E8培养基不含FGF2和TGFβ)并加入BMP4开始分化，并测量PRKCD(蛋白激酶Cδ(Protein Kinase C Delta)，即PKC delta，一种PKC的异构体)基因的表达水平，每天添加新鲜分化培养基。在第一天进行qPCR时，检测分化细胞的滋养层标记物TROP2的水平。其中，qPCR的测定方法以及仪器型号同实施例1。
其结果如图29至34所示。
其中，图29表示BMP4对PRKCD基因表达的影响。
图30表示TPA对PRKCD和TROP2基因表达的影响。
图31表示敲除PRKCD基因对H1细胞中PKC-δ蛋白表达的影响。
图32表示TPA对PRKCD敲除细胞系中原始内胚层和滋养细胞标志物SOX17，GATA6，TROP2和CGB的基因表达的影响。
图33表示BMP4对PRKCD敲除细胞系中原始内胚层和滋养细胞标志物CGB基因表达的影响。
图34表示BMP4对PRKCD敲除细胞系中原始内胚层和滋养细胞标志物CGB蛋白表达的影响。
图29-34结果显示：BMP4和TPA对PRKCD基因的表达水平的影响。PRKCD基因敲除削弱了原始内胚层标记SOX17和GATA6以及滋养层标记TROP2和CGB中TPA的分化。PRKCD基因敲除损害了滋养细胞标志物CGB中BMP4的分化。也即PRKCD控制着人胚胎干细胞向原始内胚层细胞和滋养层细胞的分化。
实施例9
抑制DGK可促进人胚胎干细胞H1向滋养细胞的分化：
为了研究不同信号通路在滋养细胞分化中的作用，我们使用DGK抑制剂阻断DGK功能来验证其在滋养层细胞分化中的可用性。具体操作如下：在E8培养基中培养人胚胎干细胞H1，每天更换新鲜培养基，当细胞密度达到70-80％时进行传代。首先用DPBS-EDTA洗涤2次，然后在室温下孵育5分钟。抽吸DPBS-EDTA，并添加含有5μMROCK抑制剂的E8培养基。重悬细胞后，以1：6-1：12的密度传代到预先涂有Matrigel的12孔板。细胞粘附在平板上1天后，换成E6培养基(E8培养基-FGF2-TGFβ)，并以1μM的浓度添加DGK抑制剂R59949以促使细胞分化。每天添加新鲜E6培养基。在第6天进行qPCR，以检测滋养细胞标志物水平。其中，qPCR的测定方法以及仪器型号同
实施例1。其结果如图35-37所示。
其中，图35表示分化1天后，BMP4和BMP4+GF109203X对DGK亚型的基因表达的影响。
图36表示分化1天后，BMP4和BMP4+GF109203X对DGKA(二酰基甘油激酶α)基因表达的影响。
图37表示分化6天后，DGK抑制剂R59949对滋养细胞的标志物TROP2和CGB的基因表达的影响。
图35-37结果表明：DGK抑制剂R59949在6天后能增加滋养细胞标记基因TROP2和CGB的表达水平。TROP2和CGB是滋养细胞的特异性标志物，DGK抑制剂可以诱导干细胞分化为滋养细胞。其中，分化1天后，相比于BMP4，BMP4+GF109203X抑制DGKA和DGKZ的基因表达，促进DGKB的基因表达，对DGKQ的基因表达无明显影响。
综上所述，本公开通过开拓性地在BMP4分化平台中使用PKC抑制剂促进多能干细胞分化成中胚层，促进早期中胚层标记物TBXT和MIXL1的表达，能大幅度提高多能干细胞分化为中胚层和内胚层前体细胞，并使分化过程缩短一半时间，同时还提高了分化的稳定性，为干细胞分化提供了新应用。通过该方法可以获得功能性中胚层细胞类型，可用于组织的再生和修复，如平滑肌细胞、肌肉、骨骼或性腺，并用于药物筛选。此外，本公开采用具有清晰成分的方法来诱导hPSC分化为滋养细胞，其中所使用的试剂不含动物来源的成分。开拓性地使用PKC激活和DGK 抑制来诱导hPSC分化为滋养细胞，描述了PKC活化是任何其他滋养细胞诱导的平台下游的主要作用机制，并发现了干细胞分化的新应用。另外，通过该方法获得的滋养细胞还可表达成熟滋养细胞的标志物，这为指导人类多能干细胞的早期命运决定提供了新思路。
以上所述仅为本公开的可选的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开公开了一种诱导干细胞分化成中胚层谱系或滋养细胞谱系的方法及药物。前者方法提高了分化的稳定性，为干细胞分化提供了新应用。通过该方法可以获得功能性中胚层细胞类型，可用于组织的再生和修复，如平滑肌细胞、肌肉、骨骼或性腺，并用于药物筛选。后者方法开拓性地使用PKC激活和DGK抑制来诱导hPSC分化为滋养细胞，描述了PKC活化是任何其他滋养细胞诱导的平台下游的主要作用机制，并发现了干细胞分化的新应用。另外，通过该方法获得的滋养细胞还可表达成熟滋养细胞的标志物，这为指导人类多能干细胞的早期命运决定提供了新思路。

Claims (20)

  1. 一种使用PKC抑制剂诱导干细胞分化成中胚层谱系的方法，其特征在于，包括以下步骤：
    在BMP4存在的条件下，用含有PKC抑制剂的分化培养基诱导干细胞分化成中胚层谱系；
    所述中胚层谱系包括中胚层细胞和内胚层前体细胞。
  2. 根据权利要求1所述的方法，其特征在于，所述干细胞包括人胚胎干细胞和人诱导的多能干细胞中的至少一种。
  3. 根据权利要求1所述的方法，其特征在于，所述PKC抑制剂包括GF109203X、
    Figure PCTCN2021093498-appb-100001
    和
    Figure PCTCN2021093498-appb-100002
    中的至少一种。
  4. 根据权利要求3所述的方法，其特征在于，所述GF109203X的浓度为2-5μM。
  5. 根据权利要求3所述的方法，其特征在于，所述
    Figure PCTCN2021093498-appb-100003
    的浓度为2-5μM。
  6. 根据权利要求3所述的方法，其特征在于，所述
    Figure PCTCN2021093498-appb-100004
    的浓度为2-5μM。
  7. 根据权利要求1所述的方法，其特征在于，所述BMP4的浓度为5-100ng/mL。
  8. 根据权利要求1所述的方法，其特征在于，所述分化培养基的成分包括DMEM/F12、L-抗坏血酸-2-磷酸镁、硒酸钠、转铁蛋白和胰岛素。
  9. 根据权利要求1所述的方法，其特征在于，诱导干细胞分化成中胚层的过程中，不使用外源FGF2或内源性WNT。
  10. 一种诱导干细胞分化成滋养细胞谱系的方法，其特征在于，包括以下步骤：用含有PKC激活剂或DGK抑制剂的分化培养基诱导干细胞分化成滋养细胞谱系。
  11. 根据权利要求10所述的方法，其特征在于，所述PKC激活剂或所述DGK抑制剂于所述干细胞分化的第一天加入所述分化培养基中。
  12. 根据权利要求11所述的方法，其特征在于，加入所述PKC激活剂后，所述干细胞至少在所述分化培养基中继续培养两天；
    或，加入所述DGK抑制剂后，所述干细胞至少在所述分化培养基中继续培养六天。
  13. 根据权利要求10-12任一项所述的方法，其特征在于，所述干细胞为人多能干细胞；
    优选地，所述人多能干细胞包括人胚胎干细胞和人诱导的多能干细胞中的至少一种。
  14. 根据权利要求10-12任一项所述的方法，其特征在于，所述PKC激活剂包括TPA和Bryostatin 1中的至少一种。
  15. 根据权利要求14所述的方法，其特征在于，所述TPA的处理浓度为25-500nM。
  16. 根据权利要求14所述的方法，其特征在于，所述Bryostatin 1的处理浓度为25-500nM。
  17. 根据权利要求10-12任一项所述的方法，其特征在于，所述DGK抑制剂包括R59949。
  18. 根据权利要求17所述的方法，其特征在于，所述R59949的处理浓度为0.5-10μM。
  19. 根据权利要求10所述的方法，其特征在于，所述分化培养基的成分包括DMEM/F12、L-抗坏血酸-2-磷酸镁、硒酸钠、转铁蛋白和胰岛素；
    优选地，分化过程中，所述分化培养基中还添加有滋养细胞诱导剂；
    优选地，所述滋养细胞诱导剂包括BMP4、PD325901、Sb432542以及DAPT中的至少一种；
    优选地，所述BMP4的处理浓度为10-100ng/mL；
    优选地，所述PD325901的处理浓度为0.5-10μM；
    优选地，所述Sb432542的处理浓度为5-20μM；
    优选地，所述DAPT的处理浓度为1-10μM。
  20. 一种药物，其特征在于，所述药物的原料含有如权利要求1-9任一项所述的方法得到的中胚层谱系或如权利要求10-19任一项所述的方法得到的滋养细胞谱系。
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