Establishment method and application of placenta trophoblast organ-like model with correct physiological polarity

Abstract

The invention discloses an establishment method and application of a placenta trophoblast organoid model with correct physiological polarity, belongs to the technical field of organoid and stem cell model establishment, and aims to solve the problem of structural polarity error in trophoblast cells at present. The establishment method comprises the following specific steps: step 1, inducing nave embryonic stem cells to differentiate hTSC hollow spheres; step 2, transferring the hTSC hollow sphere into matrigel for culture to obtain a placenta trophoblast organ; and 3, transferring the placenta trophoblast-like organs into the 2D maintenance system to maintain the growth of the placenta trophoblast-like organs. According to the invention, the internal cavity of the placenta villus trophoblast and the physiological polar structure in which STB is outside and CTB is inside in trophoblast cells can be simulated, and the placenta trophoblast organ with three cell types of CTB, STB and EVT can be constructed at the same time.

Description

Technical Field

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

[0002] During pregnancy, the placenta serves as an important temporary organ to support the healthy development of the fetus. The placenta is responsible for important physiological functions such as material exchange between mother and fetus, 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 a variety of pregnancy diseases, such as preeclampsia, fetal growth restriction, premature birth, miscarriage, etc. As the main functional unit of fetal function, the placental villi mainly include floating villi and anchored villi. The floating villi are immersed in the maternal blood pool, including the cytotrophoblast (CTB) and the syncytiotrophoblast (STB) covering the outside of the CTB, which are the main components of maternal-fetal material exchange and placental barrier, while the anchored villi are formed by the differentiation of CTB into the extravillous trophoblast (EVT) with the ability to invade and migrate, anchored in the maternal uterus, and establish maternal-fetal connection. The EVT has the function of remodeling the maternal spiral artery to ensure the blood supply of the placenta. It can be seen that trophoblast cells are the most characteristic and functional main cell type in the placenta. Understanding the development and regulatory mechanisms of placental villous trophoblast cells is very important for understanding placenta-related diseases and maintaining healthy fetal development.

[0003] Placental trophoblast organoids were first reported in 2018. Compared with the traditional 2D cultured trophoblast cell model, they have many advantages, such as multiple trophoblast cell types in 3D organoids, simulated trophoblast cell assembly, simulated trophoblast cell differentiation process, and better simulated in vivo villous trophoblast molecular characteristics. The method of constructing placental trophoblast organoids is applicable to trophoblast cells from various sources, including primary villous trophoblast cells isolated in early pregnancy, human trophoblast stem cells (hTSC) isolated from human blastocysts, human trophoblast stem cells (hTSC) induced by human embryonic stem cells (hESC), and choriocarcinoma cell lines. At present, the culture system of placental trophoblast organoids mainly relies on 100% matrix gel-coated trophoblast cells for 3D culture. 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.

[0004] The main cell components of the current placental trophoblast organoids include CTB, STB and a small amount of EVT cells. However, the placental trophoblast organoids have obvious limitations. The CTB and STB in the organoids have a polarity distribution opposite to that in the physiological villi, which makes it impossible to simulate the structural characteristics of the trophoblast cells in the villi. The structural limitations limit the use of this organoid to study material exchange at the maternal-fetal interface, drug transport, and pathogen transmission. Therefore, in order to overcome the above defects, it is urgent to establish a trophoblast organoid culture system with physiological polarity, which can simulate the development and differentiation of trophoblast cells, and can also be used in the study of placental material exchange, drug transport, and barrier function. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide a method for establishing a placental trophoblast organoid model with correct physiological polarity and its application. The present invention uses naïve embryonic stem cells with pluripotency as 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 placental trophoblast organoids mainly include: 1) having the gene expression characteristics and hormone secretion characteristics of primary villous trophoblast; 2) the polarity distribution of CTB and STB in organoids is similar to that of the placental trophoblast in vivo; 3) simulating the syncytialization process of trophoblast cells; 4) having the ability to differentiate into EVT.

[0007] Placental trophoblast organoids are innovative in their construction system and structural characteristics, providing a new research model for studying trophoblast cell development and differentiation, placental secretion function, placental barrier function, implantation simulation, disease modeling, and drug screening.

[0008] In order to achieve the above objectives, this application provides the following technical solutions: The first object of the present invention is to provide a method for establishing a placental trophoblast organoid model with correct physiological polarity, comprising the following steps: Step 1: Induce naïve embryonic stem cells to differentiate into hTSC hollow spheres; Step 2: Transfer the hTSC hollow spheres to matrix gel 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.

[0009] In particular, the specific process of step 1 is: Step 101: Digest the naïve embryonic stem cell clones into single cells using Accutase enzyme at 37°C; Step 102: AgreeWell 400Add anti-adhesion solution to each well of the well plate, and add differentiation induction medium THSM1 to each well for later use; Step 103: The single cells in step 101 are divided into 1×10 5 ~1.2×10 5 / hole density addedAgreeWell 400 Culture in the well plate for one day; Step 104: On days 2-7, the cells were cultured in differentiation induction medium THSM2, with half the medium replaced every day. On day 7, the formation of hTSC hollow spheres could be seen.

[0010] In particular, in step 102, the amount of anti-adhesion agent used is 500 µl, and the amount of differentiation induction medium THSM1 added to each well is 500 µl.

[0011] In particular, on days 6-7, the structure of the formed hTSC hollow spheres was an internal cavity, and the peripheral cells were differentiated trophoblast cells.

[0012] Specifically, the differentiation induction medium THSM1 includes: DF12: Neural basal 1:1, N2 supplement 1%, B27 supplement 2%, L-GLUTAMINE 2mM, NEAA 1%, β-2ME 0.1mM, PS 1%, LIF 20ng / mL, Activin A 20ng / mL, PD0325901 1μM, IM-12 1μM, SB590885 0.5μM, WH-4-023 1μM, CEPT 1×, FGF2 8ng / mL.

[0013] In particular, 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 1mM, NEAA 0.5%, β-2ME 0.1mM, LIF 10ng / mL, PD0325901 1μM, A83-01 1μM, SB590885 0.5μM, WH-4-023 1μM, CEPT 1×.

[0014] In particular, the specific process of step 2 is: Step 201: Collecting hTSC hollow spheres; Step 202: Select hTSC hollow spheres with good morphology and transfer them into a drop of 16-18 μl of 100% matrix gel; Step 203: After transfer, place the culture dish containing the 100% matrix gel droplets in a 37°C incubator for 15 minutes. After the 100% matrix gel solidifies, add the hTSC hollow sphere culture medium TSM or culture medium TSM-SB431542 (SB431542 is removed from TSM), culture in a 37°C incubator, change the medium every other day, and after 6 days of culture, placental trophoblast organoids with STB outside and CTB inside are formed.

[0015] Specifically, TSM included: 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-aminoethanol (ITS-X) supplement, 0.5 μM A83-01, 2 μM CHIR99021, 1 μM SB431542, 5 μM Y27632, and 0.8 mMVPA.

[0016] In particular, the specific operation of step 201 is: absorb and discard AgreeWell 400 The upper layer of culture medium in each well of the microtiter plate was gently pipetted to collect the hTSC hollow spheres.

[0017] In particular, the placental trophoblast organoids obtained in step 2 are characterized by an internal cavity and trophoblast cells on the periphery, wherein the trophoblast cells are composed of CTB on the inside and STB on the outside.

[0018] In particular, 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, the matrix gel around the placental trophoblast organoids is removed under a stereomicroscope, and the placental trophoblast organoids are transferred to a 2D culture dish, and continue to be cultured in TSM / TSM-SB431542, and the culture medium is replaced 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 a more complete placental trophoblast organoid.

[0019] The second object of the present invention is to provide a placental trophoblast organoid model with correct physiological polarity, which is obtained using the establishment method described above.

[0020] In particular, the placental trophoblast organoid model is a 3D spherical structure organoid composed of a cavity inside and a double-layer structure consisting of inner CTB and outer STB with correct trophoblast cell polarity.

[0021] The third object of the present invention is to provide a placental trophoblast organoid model with correct physiological polarity for use in in vitro drug screening.

[0022] In particular, the drugs include various drugs for treating diseases during pregnancy, such as pre-eclampsia and gestational diabetes.

[0023] The fourth object of the present invention is to provide a placental trophoblast organoid model with correct physiological polarity as a pathogen infection model.

[0024] In particular, the pathogens include Zika virus, new coronavirus, etc.

[0025] A fifth object of the present invention is to provide a placental trophoblast organoid model with correct physiological polarity as a substance transport model.

[0026] In particular, the substance transport model includes a drug transport research model.

[0027] In particular, the drugs include various drugs for treating diseases during pregnancy, such as digoxin and paclitaxel.

[0028] The sixth objective of the present invention is to provide a placental trophoblast organoid model with correct physiological polarity for use in constructing placental trophoblast organoids that have three cell types: CTB, STB, and EVT.

[0029] A seventh object of the present invention is to provide a placental trophoblast organoid model with correct physiological polarity for use in studying mechanisms related to placental development.

[0030] In particular, the mechanisms include syncytialization mechanism, EVT differentiation mechanism, and the like.

[0031] The eighth object of the present invention is to provide a placental trophoblast organoid model with correct physiological polarity as a placenta-like biochemical and biophysical microenvironment for studying reproductive hormones.

[0032] Particularly, the hormones include various reproduction-related hormones, such as human chorionic gonadotropin (hCG), progesterone, estrogen, human placental lactogen (hPL), and the like.

[0033] Compared with the prior art, the present invention has the following advantages: (1) The embryonic trophoblast organoids established in the present invention can simulate the internal cavity of the placental villus trophoblast, with trophoblast cells of correct polarity on the periphery. The trophoblast cells have physiological structural characteristics consisting of CTB on the inside and STB on the outside. Compared with the previous 3D placental trophoblast organoid model, it better simulates the placental villus structure.

[0034] (2) The initial cells of the embryonic trophoblast organoids established in the present invention are not trophoblast cells or hTSCs, but are initiated from naïve embryonic stem cells. Naïve embryonic stem cells have omnipotent characteristics similar to the inner cell mass of the pre-implantation embryo and have the ability to differentiate into TEs.

[0035] (3) The STBs in the embryonic trophoblast organoids established in the present invention are formed by spontaneous fusion without the addition of small molecules that promote syncytialization. This simulates the process of CTB syncytialization and can be used to study the regulatory mechanism of trophoblast cell syncytialization.

[0036] (4) The CTB of the present invention still has the potential for proliferation and differentiation and can differentiate into EVT cells.

[0037] (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

[0038] Figure 1 . Schematic diagram of the process of constructing structurally correct placental trophoblast organoids according to the present invention.

[0039] Figure 2 . Immunofluorescence staining was used to detect the development of placental trophoblast organoids on day 6 in step 2 and on day 12 in step 3, including the characterization of CTB (GATA3), STB (HCG) cell types and cell nuclei (DAPI); A. Immunofluorescence staining was used to detect the development of placental trophoblast organoids on day 6 in step 2; B. Immunofluorescence staining was used to detect the development of placental trophoblast organoids on day 12 in step 3; C. Immunofluorescence staining was used to detect the expression characteristics of early placental trophoblast; D. Statistics Figure 2 Fluorescence intensity of immunofluorescence staining of placental trophoblast organoids at day 12 and early placental trophoblasts from step 3 in B and 2C.

[0040] Figure 3 . Expression of trophoblast-related genes in placental trophoblast organoids and human trophoblast stem cells and choriocarcinoma cells (JEG-3).

[0041] Figure 4 . Single-cell transcriptome sequencing technology was used to detect the cell populations, cell differentiation trajectories, and gene expression characteristics of placental trophoblast organoids on day 6 in step 2; A. Single-cell transcriptome sequencing technology was used to detect the cell populations and cell differentiation trajectories of placental trophoblast organoids on day 6 in step 2; B. Single-cell transcriptome sequencing technology was used to detect genes specifically expressed in each cell population of placental trophoblast organoids on day 6 in step 2.

[0042] Figure 5. ELISA was used to detect the levels of GDF15 and hCG hormone secretion by placental trophoblast organoids on day 6 in step 2, and pregnancy test strips were used to detect the levels of hCG hormone secretion by placental trophoblast organoids on days 2 and 6 in step 2.

[0043] Figure 6 . Compare the development of day 6 placental trophoblast organoids from step 2 and trophoblast stem cell-derived placental trophoblast organoids.

[0044] Figure 7 . Step 2 On day 6, placental trophoblast organoids are induced to differentiate into EVT cells, forming trophoblast organoids that have three cell types: CTB, STB, and EVT.

[0045] Figure 8 . Immunofluorescence staining to detect the distribution of polarity molecules in trophoblast cells of placental trophoblast organoids, including polarity proteins (aPKC), STB (hCG) cell types, and cell nuclei (DAPI); a. Immunofluorescence staining to detect the distribution of polarity molecules in trophoblast cells of placental trophoblast organoids of the present invention; b. Immunofluorescence staining to detect the distribution of polarity molecules in trophoblast cells of other placental trophoblast organoids.

[0046] Fig. 9 Immunofluorescence staining was used to detect the distribution of drug transporter p-gp in the placental trophoblast model (STB-out) of the present invention, other placental trophoblast models (STB-in) and primary placental villi. DETAILED DESCRIPTION

[0047] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0048] The placental trophoblast organoid model with correct physiological polarity established by the present invention has the following advantages: (1) Advantages of initial cells. The initial cells of the placental trophoblast organoids constructed by the present invention are not trophoblast cells or hTSCs, but are derived from naïve embryonic stem cells. Naïve embryonic stem cells have omnipotent characteristics similar to the inner cell mass of the preimplantation embryo and have the ability to differentiate into TEs. Using naïve embryonic stem cells as starting cells has high uniformity, high reproducibility, and low ethical restrictions, and can simulate the process of peri-implantation TE specialization. The naïve embryonic stem cells were donated free of charge by the laboratory of Ali H. Brivanlou.

[0049] (2) The polarity of the cell structure in the trophoblast cells in the existing placental trophoblast organoids is incorrect, and this problem is solved in the present invention. The present invention constructs hTSC hollow spheres to induce the expression of polarity 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 has the same polarity as the primary placental trophoblast structure.

[0050] (3) We changed the limitations of placental trophoblast organoids and established a placental trophoblast organoid with the distribution characteristics of CTB and STB in physiological villi through a three-step construction system using naïve pluripotent embryonic stem cells as the initial cells.

[0051] (4) Based on this organoid, a placental trophoblast organoid with a cavity and three cell types, namely CTB, STB, and EVT, can be constructed.

[0052] like Figure 1 As shown, this embodiment specifically provides a method for establishing a placental trophoblast organoid model with correct physiological polarity, comprising the following steps: Step 1: Induce naïve embryonic stem cells to differentiate into hTSC hollow spheres; The naïve embryonic stem cell clones were digested into single cells using Accutase at 37°C and placed in a 6-well plate pre-coated with 0.1% gelatin to remove the feeder cells. 400 Add 500µl of anti-adhesion solution (AARS) to each well of the plate, and add 500µl of THSM1 to each well for later use. Single-cell naïve embryonic stem cells were cultured at a rate of 1×10 5 ~1.2×10 5 / The density of the holes is added to the pre-treated AggreWell 400 3D culture was performed in well plates. On day 1, cells were cultured in THSM1 medium. On days 2-7, cells were cultured in THSM2 medium, with half of the medium replaced every day. On day 7, the formation of hTSC hollow spheres was observed.

[0053] On day 6-7, the structure of the formed hTSC hollow spheres was an internal cavity, and the peripheral cells were differentiated trophoblast cells.

[0054] The initial cells for inducing hTSC hollow spheres can be naïve embryonic stem cells and induced pluripotent stem cells.

[0055] The culture medium for inducing hTSC hollow spheres is THSM1 and THSM2 (developed in research).

[0056] THSM1 includes: DF12: Neural basal 1:1 (23.5 mL of DF12 and Neural basal were added to 50 mL, 47%), N2 supplement 1%, B27 supplement 2%, L-GLUTAMINE 2mM, NEAA 1%, β-2ME 0.1 mM, PS 1%, LIF 20 ng / mL, Activin A 20ng / mL, PD0325901 1μM, IM-12 1μM, SB590885 0.5μM, WH-4-023 1μM, CEPT 1×, FGF2 8ng / mL; (the percentages are volume ratios).

[0057] THSM2 includes: DF12: Neural basal 3:1 (DF12 plus 37.5mL, 75% in 50mL, Neural basal plus 12.5mL, 25%), N2 supplement 0.25%, B27 supplement 0.5%, ITS-X supplement 0.5%, L-GLUTAMINE 1mM, NEAA 0.5%, β-2ME 0.1mM, LIF 10ng / mL, PD0325901 1μM, A83-01 1μM, SB590885 0.5μM, WH-4-023 1μM, CEPT 1×.

[0058] Alternatively, you can use a culture medium developed by others that can achieve naïve embryonic stem cell induction culture to form a hollow structure composed of hTSC, such as N2B27+APY culture medium, N2B27+APLY culture medium, TDM, PALLY culture medium, N2B27+NaPy culture medium, eHDM+eTDM, mTDM, culture medium TSM, initial culture medium, and culture medium replaced on the second day. These culture media are all used in step 1.

[0059] N2B27+APY medium: DF12: Neural basal 1:1, N2 1%, B27 2%, L-GLUTAMINE 2mM, NEAA 1%, β-2ME 0.1mM, PS 1%, A83-01 1μM, PD0325901 1.5μM, Y27632 10μM; N2B27+APLY medium: DF12: Neural basal 1:1, N2 1%, B27 2%, L-GLUTAMINE 2mM, NEAA 1%, β-2ME 0.1mM, PS 1%, A83-01 1μM, PD0325901 1.5μM, LPA 0.5μM, Y27632 10μM); TDM: 1:1 (v / v) mixture of DMEM / F12 and neurobasal medium, 0.5×N2supplement, 0.5×B27 supplement, 0.5% ITS-X, 0.5×GlutaMAX, 0.5×nonessentialamino acids, 0.1mM β-mercaptoethanol, 0.5% knockout serum replacement, 0.1% FBS, 50mg / 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, 0.75μg / mL l-ascorbic acid and 0.4mM VPA; PALLY medium: N2B27 basal medium supplemented with PD0325901 (1 μM), A 83-01 (1 μM), LPA (500 nM), hLIF (10 ng / mL), and Y-27632 (10 μM); 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; 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]]; mTDM: 1:1 (v / v) mixture of DMEM / F12 and neurobasal medium, 0.5xN2supplement, 0.5xB27 supplement, 0.5% ITS-X, 0.5x GlutaMAX, 0.5xnonessentialamino acids, 0.1 mM b-mercaptoethanol, 0.5x Na-pyruvate, 0.5% knockout serumreplacement, 0.1% FBS, 50 mg / mL BSA, 0.5% penicillin–streptomycin, 1mM PD0325901, 0.5mM A83-01, 1mM CHIR99021, 0.5mM SB431542, 2.5mM IWR-1, 2.5nM TSA, 5nM DZNep, 10ng / mL recombinant human LIF, 10ng / mL Human activin A, 25ng / mL EGF, 0.1% (v / v)Geltrex, 25.75mg / mL L-ascorbic acid and 0.4mM VPA; TSM medium: 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-aminoethanol (ITS-X) supplement, 0.5 μM A83-01, 2 μM CHIR99021, 1 μM SB431542, 5 μM Y27632, and 0.8 mM VPA.

[0060] Initial medium: Ndiff227 supplemented with 2 μM A83-01 (ALK4 / 5 / 7 inhibitor; Tocris) and 2 μM PD0325901. Medium change on the second day: Ndiff227 supplemented with 2 μM A83-01, 2 μM PD0325901 and 1 μg / mL JAK inhibitor I.

[0061] Step 2: hTSC hollow spheres are transferred to matrix gel for culture, and syncytialization is initiated under 3D conditions to obtain placental trophoblast organoids; Absorb AgreeWell 400The upper layer of culture medium in the well plate was gently pipetted with a Pasteur pipette to collect the hTSC hollow spheres. Under a stereomicroscope, hTSC hollow spheres with large cavities and good development morphology were selected and transferred to 16-18 μl of 100% matrix gel per drop using a mouth pipette. After transfer, the culture dish containing the matrix gel droplets was placed in a 37°C incubator for 15 minutes. After the matrix gel solidified, TSM (DMEM / F12 medium supplemented with 0.1mM 2-mercaptoethanol, 0.2% fetal bovine serum (FBS), 0.5% Penicillin-Streptomycin, 0.3% BSA, 1% ITS-X supplement, 0.5μM A83-01, 2μM CHIR99021, 1μM SB431542, 5μM Y27632, and 0.8mM VPA) was added and cultured in a 37°C incubator for 6 days, with the medium replaced every other day. Step 2 initiated the outward syncytialization of the trophoblast cells, forming a cavity structure with STB outside and CTB inside. The second stage was completed in step 2 after about 6 days of culture.

[0062] The culture medium for culturing hTSC hollow spheres can be TSM or TSM-SB431542, or other culture medium capable of maintaining the growth of hTSC.

[0063] The structural characteristics of the placental trophoblast organoids formed in step 2 are an internal cavity and trophoblast cells with correct polarity on the periphery. The trophoblast cells form a 3D spherical structure composed of CTB on the inside and STB on the outside.

[0064] Step 3: Transfer the placental trophoblast organoids to a 2D maintenance system to maintain the growth of the placental trophoblast organoids; Treat the placental trophoblast organoids obtained in step 2 with Cell Recovery Solution at 4°C for 25 min. Carefully remove the matrix gel around the placental trophoblast organoids under a stereomicroscope, transfer the placental trophoblast organoids to a 2D culture dish, and continue to culture them in the TSM medium of step 2. Replace the medium every other day. The third stage can maintain the development of placental trophoblast organoids for 6 days. During this process, the trophoblast cells will further syncytialize to form a more complete organoid structure.

[0065] The structure of the placental trophoblast organoid obtained in step 3 is that the bottom is in contact with the bottom of the culture dish, the interior of the formed structure is still a cavity, and the periphery is a trophoblast layer, and the trophoblast layer is composed of CTB on the inside and STB on the outside.

[0066] The maintenance medium for the placental trophoblast organoids obtained in step 3 is TSM or TSM-SB431542, or other culture medium capable of maintaining the growth of hTSC.

[0067] Embodiment 1: This example characterizes the structure and function of the established placental trophoblast organoid model, and the characterization method is as follows: (1) Visually monitor the growth rate and developmental status of placental trophoblast organoids under bright field microscopy; (2) Immunofluorescence staining to identify the formation of trophoblast structure and the differentiation of different types of trophoblast cells during the development of placental trophoblast organoids; (3) Real-time fluorescence quantitative PCR method was used to detect the expression of trophoblast-related genes during the development of placental trophoblast organoids; (4) Verify the transcriptome characteristics of placental trophoblast organoids using single-cell transcriptome sequencing technology; (5) ELISA and pregnancy test strips were used to detect hormone secretion in placental trophoblast organoids.

[0068] The placental trophoblast organoids formed in step 2 were taken for 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 3 times with PBS buffer. Treat with blocking solution (3% BSA) for one hour, and then incubate with the primary antibody at 4°C overnight. The primary antibody (GATA3, hCG) was diluted at 1:200. Then wash the samples 3 times with PBS, incubate with the secondary antibody (diluted at 1:200) at room temperature for 1 hour, and wash the samples 3 times with PBS. The results are as follows Figure 2 As shown in A, B, C, and D, Scale bars: 50µm, GATA3 is a marker protein for CTB and STB, and HCG is a marker protein for STB. Placental trophoblast organoids have significant expression of GATA3 and hCG, and the cells expressing HCG are 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 trophoblast cell types: cytotrophoblast cells and syncytiotrophoblast cells, which are similar to the placental villus tissue in vivo and can simulate the early placental development process.

[0069] The placental trophoblast organoids, human trophoblast stem cells, and choriocarcinoma cells (JEG-3) formed in step 2 were subjected to 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). Figure 3As shown, the results of qPCR showed that the data of each characteristic gene were at normal transcription levels compared with the control group. The transcriptional expression of the characteristic genes (CDH, TEAD4) of CTB in placental trophoblast organoids was lower than that of human trophoblast stem cells and slightly higher than that of JEG-3; while the characteristic genes (hCG) of STB and the characteristic genes related to CTB fusion (GCM1, ERVW1, ERVFRD) were significantly higher than those of the control group as a whole. This shows that the expression of CTB and STB-related genes can be detected at the transcriptional level in the placental trophoblast organoids of the present invention, and the culture system of the present invention promotes the spontaneous syncytialization of CTB.

[0070] The placental trophoblast organoids formed in step 2 were used for single-cell transcriptome analysis. Figure 4 The placental trophoblast organoids shown in A and B include four cell types, including hTSC, CTB, transitional cells from CTB to STB (CTB fusion), and STB. These cells express corresponding characteristic genes and have cell differentiation trajectories similar to those in vivo. This shows that the placental trophoblast organoids in the present invention have a cell composition similar to that of the placental trophoblast in vivo and can simulate the syncytialization process of the placental trophoblast in vivo.

[0071] The culture supernatant of the placental trophoblast organoids formed in step 2 on day 6 was taken, and the secretion of GDF15 and HCG in the culture supernatant was detected by ELISA. At the same time, the secretion of hCG in the culture supernatant on days 2 and 6 was detected by pregnancy test strips. Figure 5 As shown, a certain concentration of GDF15 and hCG can be detected in the supernatant of placental trophoblast organoids, and the positive reaction bands on the pregnancy test paper show that the placental trophoblast organoids have good secretory function.

[0072] Embodiment 2: Comparison of the development of placental trophoblast organoids derived from naïve embryonic stem cells and trophoblast stem cells.

[0073] The placental trophoblast organoids initiated by naive embryonic stem cells (the present invention) and the placental trophoblast organoids initiated by trophoblast stem cells formed in step 2 were taken for 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 3 times with PBS buffer. Treat with blocking solution (3% BSA) for one hour, and then incubate with the primary antibody at 4°C overnight. The primary antibodies (GATA3, SDC1) were diluted at 1:200. Then the samples were washed with PBS 3 times, incubated with the secondary antibody (diluted at 1:200) at room temperature for 1 hour, and washed with PBS 3 times. The results are as follows. Figure 6As shown, Scale bars: 50µm, where GATA3 is a marker protein for CTB and STB, and SDC1 is a marker protein for STB. In the model of placental trophoblast organoids initiated by naive embryonic stem cells, SDC1 is mainly expressed on the outside of the trophoblast organoid structure, which is similar to the real villus structure in vivo. In the placental trophoblast organoid model initiated by trophoblast stem cells, SDC1 appears inside the structure, which is opposite to the real villus structure in vivo. This shows that compared with the previous placental trophoblast organoid model based on trophoblast stem cells, the STB-outside organoid model established based on the present invention can better simulate the structure of the physiological placental trophoblast.

[0074] Embodiment 3: Placental trophoblast organoids with correct physiological polarity were used to construct placental trophoblast organoids containing three cell types: CTB, STB and EVT.

[0075] Take the placental trophoblast organoids formed in step 2 and transfer them to the 2D culture system. Apply a thin layer of matrix gel on the bottom and add EVT differentiation medium. After four days of culture, remove the inducible small molecule NRG1. Continue to culture for 2 days and then perform 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 sample 3 times with PBS buffer. Treat with blocking solution (3% BSA) for one hour, and then incubate with the primary antibody at 4°C overnight. The primary antibody (GATA3, HCG, HLA-G) was diluted 1:200. Then wash the sample 3 times with PBS, incubate with the secondary antibody (diluted 1:200) at room temperature for 1 hour, and wash the sample 3 times with PBS. The results are as follows Figure 7 As shown, Scale bars: 50µm, where 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 6 days of culture in a 2D environment, it can be seen that GATA3, hCG, and HLA-G are all significantly expressed, indicating that the organoid has three cell types, CTB, STB, and EVT, and that the placental trophoblast organoids in the present invention can be constructed with three cell types, CTB, STB, and EVT.

[0076] Embodiment 4: Placental trophoblast organoids with correct physiological polarity were used to investigate the relationship between polarity and syncytialization.

[0077] Placental trophoblast organoids with correct physiological polarity on days 0, 2, 4, and 6 formed in step 2 and placental trophoblast organoids with incorrect polarity structure on days 2, 4, 6, and 8 were taken for 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 3 times with PBS buffer. Treat with blocking solution (3% BSA) for one hour, and then incubate with the primary antibody at 4°C overnight. The primary antibody (HCG, aPKC) was diluted 1:200. The samples were then washed 3 times with PBS, incubated with the secondary antibody (diluted 1:200) at room temperature for 1 hour, and washed 3 times with PBS. The results are as follows. Figure 8 As shown in a and b, Scale bars: 50µm, where 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 is always expressed on the outside of the trophoblast organoid structure, and STB appears on the outside. During the development of the placental trophoblast organoid model with incorrect polarity structure, aPKC is always expressed on the inside of the trophoblast organoid structure, and STB also appears on the inside. This shows that polarity is related to the location where STB appears, and the placental trophoblast organoid with correct physiological polarity in the present invention can be used to study the relationship between polarity and syncytialization.

[0078] Embodiment 5: Mimic the barrier function of the placental trophoblast using placental trophoblast organoids with correct physiological polarity.

[0079] The placental trophoblast organoids formed in step 2 and the placental trophoblast organoids on the inner side of the trophoblast stem cell-derived STB were taken for immunofluorescence staining as follows: fix with 4% paraformaldehyde (PFA) for 30 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 3 times with PBS buffer. Treat with blocking solution (3% BSA) for one hour, and then incubate with the primary antibody at 4°C overnight. The primary antibodies (GATA3, HCG, P-gp) were all diluted at 1:200. The samples were then washed with PBS 3 times, incubated with the secondary antibody (diluted at 1:200) at room temperature for 1 hour, and washed with PBS 3 times. The results are as follows. Fig. 9As 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 in the periphery of the model, which is similar to the distribution of placental villi in human body. P-gp is a drug transporter protein mainly distributed on the surface of syncytiotrophoblast in villi in the placenta. 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 human placenta. It can be used as a drug screening and virus infection model in vitro.

[0080] The information of reagents and antibodies in the above examples is as follows: 1. Reagents: ; 2. Antibodies: ; It can be understood that the above specific description of the present invention is only used to illustrate the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents to achieve the same technical effects; as long as the use requirements are met, they are 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: The following steps are involved: Step 1: Induce naïve embryonic stem cells to differentiate into hTSC hollow spheres; Step 2: Transfer the hTSC hollow spheres to matrix gel 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 specific process of step 1 is: Step 101: Digest the naïve embryonic stem cell clones into single cells using Accutase enzyme at 37°C; Step 102: AgreeWell 400 Add anti-adhesion solution to each well of the well plate, and add differentiation induction medium THSM1 to each well for later use; Step 103: The single cells in step 101 are divided into 1×10 5 ~1.2×10 5 / hole density addedAgreeWell 400 Culture in the well plate for one day; Step 104: On days 2-7, the cells were cultured in differentiation induction medium THSM2, with half the medium replaced every day. On day 7, the formation of hTSC hollow spheres could be seen.

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

4. The method for establishing a placental trophoblast organoid model with correct physiological polarity according to claim 2, characterized in that: Differentiation induction medium THSM1 includes: DF12: Neural basal 1:1, N2 supplement 1%, B27supplement 2%, L-GLUTAMINE 2mM, NEAA 1%, β-2ME 0.1mM, PS 1%, LIF 20ng / mL, ActivinA 20 ng / mL, PD0325901 1μM, IM-12 1μM, SB590885 0.5μM, WH-4-023 1μM, CEPT 1×, FGF28ng / mL.

5. The method for establishing a placental trophoblast organoid model with correct physiological polarity according to claim 2, characterized in that: Differentiation induction medium THSM2 includes: DF12: Neural basal 3:1, N2 supplement 0.25%, B27supplement 0.5%, ITS-X supplement 0.5%, L-GLUTAMINE 1mM, NEAA 0.5%, β- 2ME 0.1mM, LIF 10ng / mL, PD0325901 1μM, A83-01 1μM, SB590885 0.5μM, WH-4-023 1μM, CEPT 1×.

6. 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 2 is: Step 201: Collecting hTSC hollow spheres; Step 202: hTSC hollow spheres with good morphology are selected and transferred into a drop formed by 16-18 μl of 100% Matrigel; Step 203: After the transfer, the culture dish containing the 100% matrix gel droplets was placed in a 37°C incubator for 15 minutes. After the 100% matrix gel solidified, the hTSC hollow sphere culture medium TSM or culture medium TSM-SB431542 was added and cultured in a 37°C incubator. The medium was changed every other day. After 6 days of culture, an internal cavity was formed, with the placental trophoblast organoids with STB outside and CTB inside on the periphery.

7. The method for establishing a placental trophoblast organoid model with correct physiological polarity according to claim 6, characterized in that: 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 min, the matrix gel around the placental trophoblast organoids was removed under a stereomicroscope, and the placental trophoblast organoids were transferred to a 2D culture dish and continued to be cultured in culture medium TSM or culture medium TSM-SB431542. The culture medium was replaced every other day to maintain the development of the placental trophoblast organoids for 6 days.

8. A placental trophoblast organoid model with correct physiological polarity, characterized in that: Obtained by the establishment method described in any one of claims 1-7.

9. The placental trophoblast organoid model with correct physiological polarity according to claim 8, characterized in that: The placental trophoblast organoid model is a 3D spherical structure organoid with a cavity inside and a double-layer structure composed of inner CTB and outer STB with correct trophoblast cell polarity.

10. Use of the placental trophoblast organoid model with correct physiological polarity according to claim 8 in in vitro drug screening.

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

12. Use of the placental trophoblast organoid model with correct physiological polarity according to claim 8 as a substance transport model.

13. The use according to claim 12, characterized in that The substance transport model includes a drug transport research model.

14. Use of the placental trophoblast organoid model with correct physiological polarity according to claim 8 as a method for constructing placental trophoblast organoids having three cell types: CTB, STB, and EVT.

15. Use of the placental trophoblast organoid model with correct physiological polarity according to claim 8 in studying mechanisms related to placental development.

16. Use of the placental trophoblast organoid model with correct physiological polarity according to claim 8 as a placenta-like biochemical and biophysical microenvironment in studying reproductive hormones.

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