Method for Establishing a Placental Trophoblast Organoid Model with Correct Physiological Polarity and Its Application

Abstract

The present invention discloses a method for establishing a placental trophoblast organoid model with correct physiological polarity and its application, belonging to the technical field of organoid and stem cell model construction, aiming to solve the problem of incorrect structural polarity in current trophoblast cells. The specific steps of the present invention include: Step 1: Inducing naive embryonic stem cells to differentiate into hTSC hollow spheres; Step 2: Transferring the hTSC hollow spheres to Matrigel for culture to obtain placental trophoblast organoids; Step 3: Transferring the placental trophoblast organoids to a 2D maintenance system to maintain the growth of the placental trophoblast organoids. The present invention can simulate the internal cavity of placental villous trophoblasts, the physiological polarity structure with STB on the outside and CTB on the inside in trophoblast cells, and can construct placental trophoblast organoids simultaneously possessing three cell types, namely CTB, STB, and EVT.

Description

Technical Field

[0001] The present invention belongs to the technical fields of organoid technology and stem cell model construction technology, and particularly relates to a method for establishing a placental trophoblast organoid model with correct physiological polarity and its application. Background Art

[0002] During pregnancy, the placenta, as an important temporary organ, supports the healthy development of the fetus. The placenta is responsible for important physiological functions such as maternal-fetal material exchange, remodeling of the maternal uterine vascular system, hormone secretion, and barrier, and is very important for maintaining normal fetal development. Abnormal placental development may lead to various pregnancy diseases, such as preeclampsia, fetal growth restriction, premature birth, miscarriage, etc. Placental villi, as the main functional units of fetal functions, mainly include floating villi and anchoring villi. The floating villi immersed in the maternal blood pool include cytotrophoblasts (CTB) and syncytiotrophoblasts (STB) covering the outside of CTB, which are the main components of maternal-fetal material exchange and the placental barrier. The anchoring villi are extravillous trophoblasts (EVT) with invasive and migratory abilities differentiated from CTB, which anchor to the maternal uterus to establish maternal-fetal connection, and EVT has the function of remodeling maternal spiral arteries to ensure placental blood supply. Thus, trophoblast cells are the main cell types with the most characteristics and functions in the placenta. Understanding the development and regulatory mechanisms of placental villous trophoblast cells is very important for understanding placenta-related diseases and maintaining the healthy development of the fetus.

[0003] Placental trophoblast organoids were first reported in 2018 and have various advantages compared with traditional 2D-cultured trophoblast cell models. For example, there are various trophoblast cell types in 3D organoids, which simulate the assembly of trophoblast cells, the differentiation process of trophoblast cells, and better simulate the molecular characteristics of in vivo villous trophoblasts, etc. The method for constructing placental trophoblast organoids is applicable to trophoblast cells from various sources, including primary villous trophoblast cells isolated in the first trimester of pregnancy, human trophoblast stem cells (hTSC) isolated from human blastocysts, human trophoblast stem cells (hTSC) induced and differentiated from human embryonic stem cells (hESC), and choriocarcinoma cell lines. Currently, the culture system of placental trophoblast organoids is mainly achieved by 3D culture of trophoblast cells coated with 100% Matrigel. Placental trophoblast organoids provide a powerful tool for studying the molecular regulatory mechanisms of trophoblast cell development and differentiation, establishing pathological models, and drug screening, etc.

[0004] The main cell components in current placental trophoblast organoids include CTB, STB, and a small number of EVT cells. However, this placental trophoblast organoid has obvious limitations. The CTB and STB in the organoid have a polarity distribution opposite to that in physiological villi, resulting in the inability to mimic the structural characteristics of trophoblast cells in villi. The structural limitations restrict the use of this organoid to study maternal-fetal interface material exchange, drug transport, and pathogen transmission, etc. Therefore, to overcome the above defects, it is urgent to establish a culture system of trophoblast organoids with physiological polarity, which can mimic the development and differentiation of trophoblast cells and can also be applied to the research of placental material exchange, drug transport, and barrier function, etc. Summary of the Invention

[0005] In view of the above existing problems, the purpose of the present invention is to provide a method for establishing a model of placental trophoblast organoids with correct physiological polarity and its application. The present invention uses naïve embryonic stem cells with pluripotency as the initial cells and establishes a placental trophoblast organoid with the distribution characteristics of CTB and STB in physiological villi through a three-step construction system.

[0006] The characteristics of the placental trophoblast organoid mainly include: 1) having the gene expression characteristics and hormone secretion characteristics of primary villous trophoblasts; 2) the polarity distribution of CTB and STB in the organoid is similar to that of placental trophoblasts in vivo; 3) mimicking the syncytialization process of trophoblast cells; 4) having the ability to differentiate into EVT.

[0007] The placental trophoblast organoid is innovative in both the construction system and structural characteristics in the field. It provides a new research model for studying the development and differentiation of trophoblast cells, placental secretion function, placental barrier function, mimicking the implantation process, disease modeling, and drug screening, etc.

[0008] To achieve the above purpose, the present application provides the following technical solutions:

[0009] The first purpose of the present invention is to provide a method for establishing a model of placental trophoblast organoids with correct physiological polarity, including the following steps:

[0010] Step 1: Induce naïve embryonic stem cells to differentiate into hTSC hollow spheres;

[0011] Step 2: Transfer the hTSC hollow spheres to Matrigel for culture to obtain placental trophoblast organoids;

[0012] Step 3: Transfer the placental trophoblast organoids to a 2D maintenance system to maintain the growth of the placental trophoblast organoids.

[0013] Specifically, the specific process of Step 1 is:

[0014] Step 101: Digest the cell clones of naïve embryonic stem cells into single cells with Accutase enzyme at 37°C.

[0015] Step 102: Add anti-adhesion solution to each well of the AgreeWell 400 plate, and add the differentiation induction medium THSM1 to each well for standby.

[0016] Step 103: Add the single cells in Step 101 to the AgreeWell 5 at a density of 1×10 5 -1.2×10 400 / well and culture in the plate for one day.

[0017] Step 104: From the 2nd to the 7th day, change to the differentiation induction medium THSM2 and continue to culture, changing half of the medium every day. hTSC hollow spheres can be seen on the 7th day.

[0018] Specifically, in Step 102, the dosage of anti-adhesion is 500 μl, and the addition amount of the differentiation induction medium THSM1 to each well is 500 μl.

[0019] Specifically, from the 6th to the 7th day, the structure of the formed hTSC hollow spheres is an internal cavity, and the peripheral cells are differentiated trophoblast cells.

[0020] Specifically, the differentiation induction medium THSM1 includes: DF12: Neural basal 1:1, N2 supplement 1%, B27 supplement 2%, L-GLUTAMINE 2 mM, NEAA 1%, β-2ME 0.1 mM, P-S 1%, LIF 20 ng / mL, Activin A 20 ng / mL, PD0325901 1 μM, IM-12 1 μM, SB590885 0.5 μM, WH-4-023 1 μM, CEPT 1×, FGF2 8 ng / mL.

[0021] Specifically, the differentiation induction medium THSM2 includes: DF12: Neural basal 3:1, N2 supplement 0.25%, B27 supplement 0.5%, ITS-X supplement 0.5%, L-GLUTAMINE 1 mM, NEAA 0.5%, β-2ME 0.1 mM, LIF 10 ng / mL, PD0325901 1 μM, A83-01 1 μM, SB590885 0.5 μM, WH-4-023 1 μM, CEPT 1×.

[0022] Specifically, the specific process of step 2 is as follows:

[0023] Step 201: Collect hTSC hollow spheres;

[0024] Step 202: Select hTSC hollow spheres with good morphology and transfer them to a droplet formed by 16 - 18 μl of 100% Matrigel;

[0025] After the transfer, place the culture dish containing the 100% Matrigel droplet in a 37°C incubator and let it stand for 15 minutes. After the 100% Matrigel solidifies, add the culture medium TSM for hTSC hollow spheres or the culture medium TSM - SB431542 (SB431542 is removed from TSM), and culture it in a 37°C incubator. Change the medium every other day. After 6 days of culture, placental trophoblast organoids with STB on the outside and CTB on the inside are formed.

[0026] Specifically, TSM includes: DMEM / F12 medium added with 0.1 mM 2 - mercaptoethanol, 0.2% fetal bovine serum (FBS), 0.5% penicillin - streptomycin, 0.3% bovine serum albumin, 1% insulin - transferrin - selenium - ethanolamine (ITS - X) supplement, 0.5 μM A83 - 01, 2 μM CHIR99021, 1 μM SB431542, 5 μM Y27632, and 0.8 mM VPA.

[0027] Specifically, the specific operation of step 201 is: Aspirate and discard the upper layer of the culture medium in each well of the AgreeWell 400 plate, gently pipette with a Pasteur pipette, and collect hTSC hollow spheres.

[0028] Specifically, the structural characteristics of the placental trophoblast organoids obtained in step 2 are an internal cavity, with trophoblast cells on the periphery, and the trophoblast cells are composed of CTB on the inner side and STB on the outer side.

[0029] Specifically, the specific process of step 3 is: After treating the placental trophoblast organoids obtained in step 2 with cell recovery solution at 4°C for 25 minutes, remove the Matrigel on the periphery of the placental trophoblast organoids under a stereomicroscope, transfer the placental trophoblast organoids to a 2D culture dish, and continue to culture them in TSM / TSM - SB431542. Change the culture medium every other day to maintain the development of the placental trophoblast organoids for 6 days. During this process, the trophoblast cells will further syncytialize to form more complete placental trophoblast organoids.

[0030] The second object of the present invention is to provide a placental trophoblast organoid model with correct physiological polarity, which is obtained by using the above - mentioned establishment method.

[0031] Specifically, the placental trophoblast organoid model is a 3D spherical organoid with a cavity inside and a bilayer structure composed of CTB on the inner side and STB on the outer side, forming a bilayer structure with correct trophoblast cell polarity.

[0032] The third object of the present invention is to provide the use of the placental trophoblast organoid model with correct physiological polarity in in vitro drug screening.

[0033] Specifically, the drugs include various drugs for treating pregnancy diseases, such as preeclampsia and gestational diabetes.

[0034] The fourth object of the present invention is to provide the use of the placental trophoblast organoid model with correct physiological polarity as a pathogen infection model.

[0035] Specifically, the pathogens include Zika virus, novel coronavirus, etc.

[0036] The fifth object of the present invention is to provide the use of the placental trophoblast organoid model with correct physiological polarity as a substance transport model.

[0037] Specifically, the substance transport model includes a drug transport research model.

[0038] Specifically, the drugs include various drugs for treating pregnancy diseases, such as digoxin and paclitaxel.

[0039] The sixth object of the present invention is to provide the use of the placental trophoblast organoid model with correct physiological polarity as a use for constructing a placental trophoblast organoid jointly having three cell types of CTB, STB, and EVT.

[0040] The seventh object of the present invention is to provide the use of the placental trophoblast organoid model with correct physiological polarity in studying the mechanisms related to placental development.

[0041] Specifically, the mechanisms include syncytialization mechanism, EVT differentiation mechanism, etc.

[0042] The eighth object of the present invention is to provide the use of the placental trophoblast organoid model with correct physiological polarity as a biochemical and biophysical microenvironment similar to the placenta in studying reproductive hormones.

[0043] Specifically, the hormones include various reproductive-related hormones, such as human chorionic gonadotropin (hCG), progesterone, estrogen, human placental lactogen (hPL), etc.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] (1) The embryonic trophoblast organoids established in the present invention can mimic the internal cavity of placental villous trophoblasts. The periphery is composed of trophoblast cells with correct polarity, and the physiological structure of the trophoblast cells is composed of CTB on the inner side and STB on the outer side, which better mimics the placental villous structure compared with the previous 3D placental trophoblast organoid model.

[0046] (2) The initial cells of the embryonic trophoblast organoids established in the present invention are not trophoblast cells or hTSCs, but start from naïve embryonic stem cells. Naïve embryonic stem cells have totipotent characteristics similar to the inner cell mass of pre-implantation embryos and have the ability to differentiate into TE.

[0047] (3) In the embryonic trophoblast organoids established in the present invention, STB is formed by spontaneous fusion without adding small molecules to promote syncytialization, mimicking the process of CTB syncytialization, and can be used to study the regulatory mechanism of trophoblast cell syncytialization.

[0048] (4) The CTB of the present invention still has the potential for proliferation and differentiation and can differentiate into EVT cells.

[0049] (5) The embryonic trophoblast organoids established in the present invention can be used to study the relationship between trophoblast cell polarity and syncytialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 . Schematic diagram of the process for constructing placental trophoblast organoids with correct structure in the present invention.

[0051] Figure 2 . Detection of the development of placental trophoblast organoids on the 6th day of step 2 and on the 12th day of step 3 by immunofluorescence staining, including the characterization of CTB (GATA3), STB (HCG) cell types and cell nuclei (DAPI); among them, A. Detection of the development of placental trophoblast organoids on the 6th day of step 2 by immunofluorescence staining; B. Detection of the development of placental trophoblast organoids on the 12th day of step 3 by immunofluorescence staining; C. Detection of the expression characteristics of early placental trophoblasts by immunofluorescence staining; D. Statistics Figure 2 The fluorescence intensities of immunofluorescence staining of placental trophoblast organoids on the 12th day of step 3 and early placental trophoblasts in B and 2C.

[0052] Figure 3 . Expression of trophoblast-related genes in placental trophoblast organoids, human trophoblast stem cells and choriocarcinoma cells (JEG-3).

[0053] Figure 4. Detect the cell populations, cell differentiation trajectories, and gene expression characteristics of placental trophoblast organoids on day 6 of step 2 using single-cell transcriptome sequencing technology; A. Detect the cell populations and cell differentiation trajectories of placental trophoblast organoids on day 6 of step 2 using single-cell transcriptome sequencing technology; B. Detect the genes specifically expressed in each cell population of placental trophoblast organoids on day 6 of step 2 using single-cell transcriptome sequencing technology.

[0054] Figure 5 . Detect the levels of GDF15 and hCG hormones secreted by placental trophoblast organoids on day 6 of step 2 using ELISA, and detect the level of hCG hormone secreted by placental trophoblast organoids on days 2 and 6 of step 2 using a pregnancy test strip.

[0055] Figure 6 . Compare the development of placental trophoblast organoids on day 6 of step 2 and placental trophoblast organoids derived from trophoblast stem cells.

[0056] Figure 7 . Induce the differentiation of EVT cells from placental trophoblast organoids on day 6 of step 2 to form trophoblast organoids with three cell types: CTB, STB, and EVT.

[0057] Figure 8 . Detect the distribution of polarity molecules in trophoblast cells of placental trophoblast organoids by immunofluorescence staining, including polarity protein (aPKC), STB (hCG) cell type, and cell nucleus (DAPI); a. Detect the distribution of polarity molecules in trophoblast cells of placental trophoblast organoids in the present invention by immunofluorescence staining; b. Detect the distribution of polarity molecules in trophoblast cells of other placental trophoblast organoids by immunofluorescence staining.

[0058] Figure 9 . Detect the distribution of the drug transporter p-gp in the placental trophoblast model (STB-out), other placental trophoblast models (STB-in), and primary placental villi in the present invention by immunofluorescence staining. Detailed implementation

[0059] The following will describe the implementation of the present invention in detail in combination with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0060] The established placental trophoblast organoid model with correct physiological polarity in the present invention has the following advantages:

[0061] (1) Advantages of the initial cells: The initial cells of the placental trophoblast organoids constructed in the present invention are not trophoblast cells or hTSCs, but start from naïve embryonic stem cells. Naïve embryonic stem cells have totipotency characteristics similar to the inner cell mass of pre-implantation embryos and have the ability to differentiate into TE. Using naïve embryonic stem cells as the starting cells has high homogeneity, high reproducibility, and low ethical restrictions, and can simulate the process of TE specialization during the peri-implantation period. The naïve embryonic stem cells were gratuitously provided by the laboratory of Ali H. Brivanlou.

[0062] (2) In existing placental trophoblast organoids, the polar position of the cell structure in trophoblast cells is incorrect, and this problem is solved in the present invention. The present invention constructs hTSC hollow spheres and induces the expression position of polar proteins to tend to the outside of the structure, thereby forming a physiological structure with CTB on the inside and STB on the outside, which is the same as the structural polarity of primary placental trophoblasts.

[0063] (3) Overcomes the limitations of placental trophoblast organoids. Using naïve embryonic stem cells with pluripotency as the initial cells, through a three-step construction system, a placental trophoblast organoid with the distribution characteristics of CTB and STB in physiological villi is established.

[0064] (4) Based on this type of organoid, a placental trophoblast organoid containing a cavity and having three cell types, namely CTB, STB, and EVT, can be constructed.

[0065] As Figure 1 shown, this example specifically provides a method for establishing a placental trophoblast organoid model with correct physiological polarity, including the following steps:

[0066] Step 1: Induce naïve embryonic stem cells to differentiate into hTSC hollow spheres;

[0067] Digest the cell clones of naïve embryonic stem cells into single cells with Accutase enzyme at 37 °C, and place the single cells in a 6-well plate pre-coated with 0.1% gelatin to remove feeder cells. Add 500 μl of anti-adhesion solution (AARS) to each well of the AgreeWell 400 well plate, and add 500 μl of THSM1 to each well for use. Add single-cell naïve embryonic stem cells to the pretreated AggreWell 5 at a density of 1×10 5 - 1.2×10 400 / well and perform 3D culture. Culture in THSM1 medium on the first day. From the second to the seventh day, culture in THSM2 and change half of the medium every day. On the seventh day, the formation of hTSC hollow spheres can be seen.

[0068] On the 6th - 7th day, the structure of the hTSC hollow sphere formed has an internal cavity, and the peripheral cells are differentiated trophoblast cells.

[0069] The initial cells for inducing hTSC hollow spheres can be naïve embryonic stem cells and induced pluripotent stem cells.

[0070] The culture media for inducing hTSC hollow spheres are THSM1 and THSM2 (developed in the study).

[0071] THSM1 includes: DF12: Neural basal 1:1 (23.5 mL of both DF12 and Neural basal are added in 50 mL, 47%), N2 supplement 1%, B27 supplement 2%, L - GLUTAMINE 2 mM, NEAA 1%, β - 2ME 0.1 mM, P - S 1%, LIF 20 ng / mL, Activin A 20 ng / mL, PD0325901 1 μM, IM - 12 1 μM, SB590885 0.5 μM, WH - 4 - 023 1 μM, CEPT 1×, FGF2 8 ng / mL; (where the percentages are volume ratios).

[0072] THSM2 includes: DF12: Neural basal 3:1 (37.5 mL of DF12 is added in 50 mL, 75%, 12.5 mL of Neural basal is added, 25%), N2 supplement 0.25%, B27 supplement 0.5%, ITS - X supplement 0.5%, L - GLUTAMINE 1 mM, NEAA 0.5%, β - 2ME 0.1 mM, LIF 10 ng / mL, PD0325901 1 μM, A83 - 01 1 μM, SB590885 0.5 μM, WH - 4 - 023 1 μM, CEPT 1×.

[0073] Alternatively, use culture media developed by others that can achieve the induced culture of naïve embryonic stem cells and form a hollow structure composed of hTSC, such as N2B27 + APY medium, N2B27 + APLY medium, TDM, PALLY medium, N2B27 + NaPy medium, eHDM + eTDM, mTDM, medium TSM, initial medium, and the medium replaced on the second day. These media are all used in step 1.

[0074] N2B27 + APY Medium: DF12: Neural basal 1:1, N2 1%, B27 2%, L-GLUTAMINE 2 mM, NEAA 1%, β-2ME 0.1 mM, P-S 1%, A83-01 1 μM, PD0325901 1.5 μM, Y27632 10 μM;

[0075] N2B27 + APLY Medium: DF12: Neural basal 1:1, N2 1%, B27 2%, L-GLUTAMINE 2 mM, NEAA 1%, β-2ME 0.1 mM, P-S 1%, A83-01 1 μM, PD0325901 1.5 μM, LPA 0.5 μM, Y27632 10 μM);

[0076] TDM: 1:1 (v / v) mixture of DMEM / F12 and neurobasal medium, 0.5×N2 supplement, 0.5×B27 supplement, 0.5% ITS-X, 0.5×GlutaMAX, 0.5×nonessential amino acids, 0.1 mM β-mercaptoethanol, 0.5% knockout serum replacement, 0.1% FBS, 50 mg / mL BSA, 0.5% penicillin–streptomycin, 1 μM PD0325901, 0.5 μM A83-01, 0.25 μM SB590885, 0.5 μM WH-4-023, 0.25 μM IM-12, 1 μM CHIR99021, 0.5 μM SB431542, 10 ng / mL recombinant human LIF, 25 ng / mL EGF, 0.75 μg / mL l-ascorbic acid and 0.4 mM VPA;

[0077] PALLY Medium: PD0325901 (1 μM), A 83-01 (1 μM), LPA (500 nM), hLIF (10 ng / mL) and Y-27632 (10 μM) are added to the N2B27 basal medium;

[0078] N2B27 + NaPy medium; 1:1 DMEM / F12:Neurobasal, 1xN2 100x supplement, 1xB27 50x supplement, 1xGlutaMAX, 1xMEM NEAA, 0.1 mM b-mercaptoethanol, 1 mM NaPy and BIM: 1:1 DMEM / F12:Neurobasal, 0.5xN2 100X supplement, 0.5xB27 50X supplement, 0.5xGlutaMAX, 0.5xMEM NEAA, 1 mM NaPy, 0.1 mM b-mercaptoethanol, 0.5% ITS-X, 0.5% Knock-out Serum Replacement, 0.1% FBS, 1 mM PD0325901, 1 mM A83-01, 0.5 mM WH-4-023, 0.25 mM IM-12, 25 ng / mL rhEGF, 3 mg / mL Ascorbic Acid, and 400 mg / mL Valproic acid;

[0079] eHDM: A 1:1 (v / v) mixture of DMEM / F12 and Neurobasal medium, 1× N2 supplement, 1× B27 supplement, 1× GlutaMAX, 1× non-essential amino acids, 0.1 mM β-mercaptoethanol, 0.5% penicillin–streptomycin, 20 ng mL-1 bFGF, 20 ng / mL activin A, 3 μM CHIR99021 and CEPT cocktail [50 nM Chroman1, 5 μM Emricasan, 1X polyamine supplement, and 0.7 μM TransISRIB and eTDM: A 3:1 (v / v) mixture of DMEM / F12 and neurobasal medium, 0.25× N2 supplement, 0.25× B27 supplement, 0.5× GlutaMAX, 0.5× non-essential amino acids, 0.1 mM β-mercaptoethanol, 0.5% knockout serum replacement, 0.5% penicillin–streptomycin, 1 μM PD0325901, 2 μM A83-01, 0.5 μM SB590885, 1 μM WH-4-023, 10 ng / mL-1 recombinant human LIF, 0.5 μM LPA and CEPT cocktail [50 nM Chroman1, 5 μM Emricasan, 1X polyamine supplement, and 0.7 μM TransISRIB];

[0080] mTDM: A 1:1 (v / v) mixture of DMEM / F12 and neurobasal medium, 0.5xN2 supplement, 0.5 xB27 supplement, 0.5% ITS-X, 0.5 x GlutaMAX, 0.5x nonessential amino acids, 0.1 mM b-mercaptoethanol, 0.5 x Na-pyruvate, 0.5% knockout serum replacement, 0.1% FBS, 50 mg / mL BSA, 0.5% penicillin–streptomycin, 1 mM PD0325901, 0.5 mM A83-01, 1 mM CHIR99021, 0.5 mM SB431542, 2.5 mM IWR-1, 2.5 nM TSA, 5 nM DZNep, 10 ng / mL recombinant human LIF, 10 ng / mL Human activin A, 25 ng / mL EGF, 0.1% (v / v) Geltrex, 25.75 mg / mL L-ascorbic acid and 0.4 mM VPA;

[0081] Medium TSM: DMEM / F12 medium supplemented with 0.1 mM 2-mercaptoethanol, 0.2% fetal bovine serum (FBS), 0.5% penicillin–streptomycin, 0.3% bovine serum albumin, 1% insulin-transferrin-selenium-alcoholamine (ITS-X) supplement, 0.5 μM A83-01, 2 μM CHIR99021, 1 μM SB431542, 5 μM Y27632, and 0.8 mM VPA.

[0082] Initial medium: Ndiff227 supplemented with 2 μM A83-01 (ALK4 / 5 / 7 inhibitor; Tocris) and 2 μM PD0325901. Medium replaced on the second day: Ndiff227 supplemented with 2 μM A83-01, 2 μM PD0325901 and 1 μg / mL JAK inhibitor I.

[0083] Step 2: Transfer the hTSC hollow spheres to Matrigel for culture, initiate syncytialization under 3D conditions to obtain placental trophoblast organoids;

[0084] Aspirate and discard AgreeWell 400For the upper layer of the culture medium in the orifice plate, gently pipette with a Pasteur pipette to collect hTSC hollow spheres. Under a stereomicroscope, select hTSC hollow spheres with larger cavities and good developmental morphology, and transfer them to 100% Matrigel at 16 - 18 μl per drop using a mouth pipette. After transfer, place the culture dish containing the Matrigel droplets in an incubator at 37 °C and let it stand for 15 minutes. After the Matrigel solidifies, add TSM (DMEM / F12 medium supplemented with 0.1 mM 2 - mercaptoethanol, 0.2% fetal bovine serum (FBS), 0.5% Penicillin - Streptomycin, 0.3% BSA, 1% ITS - X supplement, 0.5 μM A83 - 01, 2 μM CHIR99021, 1 μM SB431542, 5 μM Y27632, and 0.8 mM VPA), and culture in an incubator at 37 °C for 6 days, changing the culture medium every other day. Step 2 initiates the syncytialization of trophoblast cells outward to form a cavity structure with STB on the outside and CTB on the inside. The second stage is completed in about 6 days of culturing in Step 2.

[0085] The culture medium for culturing hTSC hollow spheres can be TSM or TSM - SB431542, or other culture media that can maintain the growth of hTSC.

[0086] The structural characteristics of the placental trophoblast organoids formed in Step 2 are an internal cavity, and the periphery is composed of trophoblast cells with correct polarity, which is a 3D spherical structure composed of CTB on the inner side and STB on the outer side.

[0087] Step 3: Transfer the placental trophoblast organoids to a 2D maintenance system to maintain the growth of the placental trophoblast organoids;

[0088] Treat the placental trophoblast organoids obtained in Step 2 with Cell Recovery Solution at 4 °C for 25 min. Carefully remove the Matrigel on the periphery of the placental trophoblast organoids under a stereomicroscope, transfer the placental trophoblast organoids to a 2D culture dish, and continue to culture in the TSM culture medium of Step 2, changing the culture medium every other day. The third stage can maintain the development of the placental trophoblast organoids for 6 days. During this process, the trophoblast cells will further syncytialize to form a more complete organoid structure.

[0089] The structure of the placental trophoblast organoids obtained in Step 3 is that the bottom contacts the bottom of the culture dish, and the formed structure still has an internal cavity and a trophoblast cell layer on the periphery, and the trophoblast cell layer is composed of CTB on the inner side and STB on the outer side.

[0090] The maintenance medium for the placental trophoblast organoids obtained in Step 3 is TSM or TSM-SB431542, or other media that can maintain the growth of hTSCs.

[0091] Example 1:

[0092] In this example, the structure and function of the established placental trophoblast organoid model were characterized as follows:

[0093] (1) Intuitively monitor the growth rate and developmental status of placental trophoblast organoids under bright-field microscopy conditions;

[0094] (2) Immunofluorescence staining was used to identify the formation of trophoblast structures and the differentiation of different types of trophoblast cells during the development of placental trophoblast organoids;

[0095] (3) Real-time fluorescence quantitative PCR was used to detect the expression of trophoblast-related genes during the development of placental trophoblast organoids;

[0096] (4) Single-cell transcriptome sequencing technology was used to verify the transcriptome characteristics of placental trophoblast organoids;

[0097] (5) ELISA and pregnancy test strips were used to detect the hormone secretion of placental trophoblast organoids.

[0098] The placental trophoblast organoids formed in Step 2 were subjected to immunofluorescence staining as follows: fixed with 4% paraformaldehyde (PFA) for 20 minutes, washed with PBS buffer for 5 minutes, and repeated 3 times. Permeabilized with 0.3% Triton X-100 for 30 minutes and the samples were washed 3 times with PBS buffer. Treated with blocking solution (3% BSA) for one hour, and then incubated with the primary antibody overnight at 4°C. Both primary antibodies (GATA3, hCG) were diluted at 1:200. Then the samples were washed 3 times with PBS, incubated with the secondary antibody (diluted at 1:200) for 1 hour at room temperature, and the samples were washed 3 times with PBS. The results are shown in Figure 2 A, B, C, and D in the figure. Scale bars: 50µm. Among them, GATA3 is a marker protein for CTB and STB, and HCG is a marker protein for STB. The placental trophoblast organoids showed significant expression of GATA3 and hCG, and the cells expressing HCG were arranged on the outside, forming a cavity inside, similar to the placental villus structure. This indicates that the formed placental trophoblast organoids contain the main types of trophoblast cells: cytotrophoblast cells and syncytiotrophoblast cells, which are similar to the placental villus tissue in vivo and can simulate the early placental development process.

[0099] The placental trophoblast organoids, human trophoblast stem cells, and choriocarcinoma cells (JEG-3) formed in step 2 were taken for real-time fluorescence quantitative PCR detection, including the expression of genes related to methylation (ELF5), CTB (TEAD4, CDH1), STB (hCG), and CTB syncytialization (GCM1, ERVFRD-1, ERVW-1). As Figure 3 shown, the results of qPCR showed that the data of each characteristic gene were at the normal transcription level compared with the control group. The transcriptional levels of the characteristic genes (CDH, TEAD4) of CTB in placental trophoblast organoids were lower than those in human trophoblast stem cells and slightly higher than those in JEG-3; while the characteristic genes (hCG) of STB and the characteristic genes (GCM1, ERVW1, ERVFRD) related to CTB fusion were significantly higher than those in the control group as a whole. This indicates that the placental trophoblast organoids in the present invention can detect the expression of CTB- and STB-related genes at the transcriptional level, and the culture system in the present invention promotes the spontaneous syncytialization of CTB.

[0100] The placental trophoblast organoids formed in step 2 were taken for single-cell transcriptome detection. As Figure 4 shown in A and B, the placental trophoblast organoids contain four cell types: hTSC, CTB, the transitional cells from CTB to STB (CTB fusion), and STB. These cells express corresponding characteristic genes and have a cell differentiation trajectory similar to that in vivo. This indicates that the placental trophoblast organoids in the present invention have a cell composition similar to that of the placental trophoblast in vivo and can mimic the syncytialization process of the placental trophoblast in vivo.

[0101] The culture supernatant of the placental trophoblast organoids formed in step 2 on the 6th day was taken, and the secretion of GDF15 and HCG in the culture supernatant was detected by ELISA method. At the same time, the secretion of hCG in the culture supernatant on the 2nd and 6th days was detected by a pregnancy test strip. As Figure 5 shown, a certain concentration of GDF15 and hCG can be detected in the supernatant of the placental trophoblast organoids, and the positive reaction strip of the pregnancy test strip indicates that the placental trophoblast organoids have good secretory function.

[0102] Example 2:

[0103] Compare the development of placental trophoblast organoids derived from naïve embryonic stem cells and trophoblast stem cells.

[0104] Immunofluorescence staining was performed on the naive embryonic stem cell-derived placental trophoblast organoids (the present invention) and trophoblast stem cell-derived placental trophoblast organoids formed in step 2 as follows: Fix with 4% paraformaldehyde (PFA) for 20 minutes, wash with PBS buffer for 5 minutes, and repeat 3 times. Permeabilize with 0.3% Triton X-100 for 30 minutes, and wash the samples with PBS buffer 3 times. Treat with blocking solution (3% BSA) for one hour, and then incubate with the primary antibody overnight at 4°C. Both primary antibodies (GATA3, SDC1) were diluted at 1:200. Then wash the samples with PBS 3 times, incubate with the secondary antibody (diluted at 1:200) for 1 hour at room temperature, and wash the samples with PBS 3 times. The results are as Figure 6 shown. Scale bars: 50µm. Among them, GATA3 is a marker protein for CTB and STB, and SDC1 is a marker protein for STB. In the model of naive embryonic stem cell-derived placental trophoblast organoids, SDC1 is mainly expressed on the outer side of the trophoblast organoid structure, which is similar to the true villous structure in vivo. In the model of trophoblast stem cell-derived placental trophoblast organoids, SDC1 appears inside the structure, which is contrary to the true villous structure in vivo. This indicates that compared with the previous model of trophoblast stem cell-derived placental trophoblast organoids, the organoid model with STB on the outside established based on the present invention can better simulate the structure of physiological placental trophoblast.

[0105] Example 3:

[0106] Construct placental trophoblast organoids with three cell types, namely CTB, STB, and EVT, using placental trophoblast organoids with correct physiological polarity.

[0107] Transfer the placental trophoblast organoids formed in step 2 to a 2D culture system, thinly coat a layer of Matrigel on the bottom, add EVT differentiation medium, remove the inductive small molecule NRG1 after culturing for four days, and continue culturing for 2 days before performing immunofluorescence staining as follows: Fix with 4% paraformaldehyde (PFA) for 20 minutes, wash with PBS buffer for 5 minutes, and repeat 3 times. Permeabilize with 0.3% Triton X-100 for 30 minutes, and wash the samples with PBS buffer 3 times. Treat with blocking solution (3% BSA) for one hour, and then incubate with the primary antibody overnight at 4°C. All primary antibodies (GATA3, HCG, HLA-G) were diluted at 1:200. Then wash the samples with PBS 3 times, incubate with the secondary antibody (diluted at 1:200) for 1 hour at room temperature, and wash the samples with PBS 3 times. The results are as Figure 7As shown, Scale bars: 50µm. Among them, GATA3 is a marker protein for CTB, STB, and EVT, hCG is a marker protein for STB, and HLA-G is a marker protein for EVT. After culturing in a 2D environment for 6 days, obvious expressions of GATA3, hCG, and HLA-G can be seen, indicating that at this time, this type of organoid has three cell types: CTB, STB, and EVT. At the same time, it shows that the placental trophoblast organoids based on the present invention can construct placental trophoblast organoids with three cell types: CTB, STB, and EVT.

[0108] Example 4:

[0109] Use placental trophoblast organoids with correct physiological polarity to explore the relationship between polarity and syncytialization.

[0110] Take the placental trophoblast organoids with correct physiological polarity on days 0, 2, 4, and 6 formed in step 2 and the placental trophoblast organoids with incorrect polarity structures on days 2, 4, 6, and 8 for immunofluorescence staining. The method is as follows: Fix with 4% paraformaldehyde (PFA) for 20 minutes, wash with PBS buffer for 5 minutes, and repeat 3 times. Then permeabilize with 0.3% Triton X-100 for 30 minutes and wash the samples with PBS buffer 3 times. Treat with blocking solution (3% BSA) for one hour, and then incubate with the primary antibody overnight at 4°C. Both primary antibodies (HCG, aPKC) are diluted at 1:200. Then wash the samples with PBS 3 times, incubate with the secondary antibody (diluted at 1:200) at room temperature for 1 hour, and wash the samples with PBS 3 times. The results are as Figure 8 shown in a and b, Scale bars: 50µm. Among them, aPKC is a marker protein for polarity, and hCG is a marker protein for STB. During the development of the placental trophoblast organoid model with correct physiological polarity, aPKC has been expressed outside the structure of the trophoblast organoid, and at the same time, STB appears on the outside. While during the development of the placental trophoblast organoid model with incorrect polarity structure, aPKC has been expressed inside the structure of the trophoblast organoid, and STB also appears inside. This shows that the polarity is related to the position where STB appears. The placental trophoblast organoids with correct physiological polarity in the present invention can be used to study the relationship between polarity and syncytialization.

[0111] Example 5:

[0112] Use placental trophoblast organoids with correct physiological polarity to simulate the barrier function of placental trophoblasts.

[0113] Take the placental trophoblast organoids formed in step 2 and the placental trophoblast organoids with trophoblast stem cell-derived STB on the inner side for immunofluorescence staining as follows: Fix with 4% paraformaldehyde (PFA) for 30 minutes, wash with PBS buffer for 5 minutes, and repeat 3 times. Then permeabilize with 0.3% Triton X-100 for 30 minutes and wash the samples with PBS buffer 3 times. Treat with blocking solution (3% BSA) for one hour, and then incubate with the primary antibody overnight at 4°C. The primary antibodies (GATA3, HCG, P-gp) are all diluted at 1:200. Then wash the samples with PBS 3 times, incubate with the secondary antibody (diluted at 1:200) for 1 hour at room temperature, and wash the samples with PBS 3 times. The results are as Figure 9 shown, Scale bars: 50µm. Compared with the organoid model of STB-in, in the organoid model of STB-out constructed by the present invention, P-gp is mainly distributed on the periphery of the model, which is similar to the distribution of human physiological placental villi. P-gp is a drug transporter mainly distributed on the surface of syncytiotrophoblasts in placental villi, and it can transport a variety of small drug molecules, such as digoxin and paclitaxel, etc. The organoid model of STB-out constructed by the present invention can better simulate the barrier function of the human placenta. It can be used as a drug screening and virus infection model in vitro.

[0114] The information of the reagents and antibodies in the above embodiments is as follows:

[0115] 1. Reagents:

[0116] ;

[0117] 2. Antibodies:

[0118] ;

[0119] It can be understood that the above specific description of the present invention is only for explaining the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced to achieve the same technical effects; as long as the use requirements are met, they are all within the protection scope of the present invention.

Claims

1. A method for establishing a placental trophoblast organoid model with correct physiological polarity, characterized in that, It includes the following steps: Step 1: Induce naïve embryonic stem cells to differentiate into hTSC hollow spheres; Digest the cell clones of naïve embryonic stem cells into single cells with Accutase enzyme at 37 °C, and place the single cells in a 6-well plate pre-coated with 0.1% gelatin to remove feeder cells; To AgreeWell 400 Add 500 μl of anti-adhesion solution to each well of the orifice plate, and add 500 μl of THSM1 medium to each well for standby; The naïve embryonic stem cells in the single-cell state were added to the pre-treated AggreWell 5 at a density of 1×10 5 to 400 1.2×10 / well for 3D culture in the well plate. On the first day, they were cultured in THSM1 medium; From the 2nd to the 7th day, culture in THSM2 medium and change half of the medium every day. On the 7th day, the formation of hTSC hollow spheres can be seen; THSM1 medium: DF12: Neural basal 1:1, N2 supplement 1%, B27 supplement 2%, L-GLUTAMINE 2 mM, NEAA 1%, β-2ME 0.1 mM, P-S 1%, LIF 20 ng / mL, Activin A 20 ng / mL, PD0325901 1 μM, IM-12 1 μM, SB590885 0.5 μM, WH-4-023 1 μM, CEPT 1×, FGF2 8 ng / mL; THSM2 medium: DF12: Neural basal 3:1, N2 supplement 0.25%, B27 supplement 0.5%, ITS-X supplement 0.5%, L-GLUTAMINE 1 mM, NEAA 0.5%, β-2ME 0.1 mM, LIF 10 ng / mL, PD0325901 1 μM, A83-01 1 μM, SB590885 0.5 μM, WH-4-023 1 μM, CEPT 1×; Step 2: Transfer the hTSC hollow spheres to Matrigel for culture to obtain placental trophoblast organoids; Step 3: Transfer the placental trophoblast organoids to a 2D maintenance system to maintain the growth of the placental trophoblast organoids.

2. The method for establishing a placental trophoblast organoid model with correct physiological polarity according to claim 1, characterized in that, The inside of the hTSC hollow sphere is a cavity, and the outside is differentiated trophoblast cells.

3. The method for establishing a placental trophoblast organoid model with correct physiological polarity according to claim 2, characterized in that, The specific process of Step 2 is as follows: Step 201: Collect hTSC hollow spheres; Step 202: Select hTSC hollow spheres with good morphology and transfer them to a droplet formed by 16 - 18 μl of 100% Matrigel; Step 203: After transfer, place the culture dish with the 100% Matrigel droplet in a 37 °C incubator and let it stand for 15 minutes. After the 100% Matrigel solidifies, add the medium TSM or the medium TSM-SB431542 for hTSC hollow spheres, and culture in a 37 °C incubator. Change the medium every other day. After 6 days of culture, a placental trophoblast organoid with an internal cavity and STB on the outside and CTB on the inside is formed on its periphery.

4. The method for establishing a placental trophoblast organoid model with correct physiological polarity according to claim 3, characterized in that, The specific process of Step 3 is as follows: After treating the placental trophoblast organoids obtained in step 2 with the cell recovery solution at 4°C for 25 minutes, remove the Matrigel around the placental trophoblast organoids under a stereomicroscope, transfer the placental trophoblast organoids to a 2D culture dish, and continue culturing in medium TSM or medium TSM-SB431542. Replace the medium every other day and maintain the development of the placental trophoblast organoids for 6 days.

5. A placental trophoblast organoid model with correct physiological polarity, characterized in that, Obtained by using the establishment method described in any one of claims 1-4.

6. The placental trophoblast organoid model with correct physiological polarity according to claim 5, characterized in that, The placental trophoblast organoid model is a 3D spherical structure organoid with a cavity inside and a bilayer structure composed of CTB on the inner side and STB on the outer side forming a correct trophoblast cell polarity on the periphery.

7. Use of the placental trophoblast organoid model with correct physiological polarity according to claim 5 in in vitro drug screening.

8. Use of the placental trophoblast organoid model with correct physiological polarity according to claim 5 as a pathogen infection model.

9. Use of the placental trophoblast organoid model with correct physiological polarity according to claim 5 as a substance transport model.

10. The use according to claim 9, characterized in that, The substance transport model includes a drug transport research model.

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