A method for the production of a human embryonic stem cell line + Methods for controlled construction and advanced differentiation of human endometrial organoids from human endometrial epithelial stem cells

Abstract

The application discloses a kind of SSEA-1 + The present application discloses a controllable construction and high-level differentiation method of human endometrial organoids originated from human endometrial epithelial stem cells. + The present application discloses a controllable construction and high-level differentiation method of human endometrial organoids originated from human endometrial epithelial stem cells. The present application discloses a controllable construction and high-level differentiation method of human endometrial organoids originated from human endometrial epithelial stem cells. The present application discloses a controllable construction and high-level differentiation method of human endometrial organoids originated from human endometrial epithelial stem cells. The present application discloses a controllable construction and high-level differentiation method of human endometrial organoids originated from human endometrial epithelial stem cells.

Description

Technical Field

[0001] This invention relates to the fields of regenerative medicine and tissue engineering, specifically to a method using SSEA-1 + Methods for the controlled construction and advanced differentiation of human endometrial organoids derived from human endometrial epithelial stem cells and their applications. Background Technology

[0002] Endometrial organoids are three-dimensional models that can reconstruct the structure and function of the endometrial epithelium in vitro while preserving its genetic and phenotypic characteristics. They have significant application value in the study of endometrial-related disease mechanisms and drug screening. Currently available human endometrial organoids are mostly cultured in three dimensions starting from primary tissue fragments. While this approach can obtain hormone-responsive epithelial structures, it generally suffers from problems such as high heterogeneity of originating cells and difficulty in controlling batch-to-batch variability. Furthermore, it has limitations in terms of standardization, editability, and high-throughput compatibility. Although some literature has reported on the long-term culture of endometrial organoids and their differentiation response to steroid hormones, a methodological system that uses definitively derived human endometrial epithelial stem cells (heESCs) as the sole starting cell, achieves on-demand induction to obtain advanced differentiation phenotypes under low / optimized WNT axis conditions, and establishes quantitative thresholds for structural and functional assessment still requires further refinement and standardization.

[0003] heESCs are considered key source cells for endometrial epithelial regeneration and homeostasis, possessing the potential to form endometrial organoids. However, heESCs often face technical bottlenecks in in vitro culture, such as purification difficulties, unstable stemness maintenance, and insufficient long-term expansion consistency. These factors limit the promotion of organoid systems constructed using heESCs as a starting point in standardized modeling, gene editing, and large-scale applications. In related research, various small molecule regulation and reprogramming approaches have been proposed for the stable in vitro culture and expansion of adult-derived epithelial stem cells (e.g., using small molecule combinations to achieve long-term proliferation and directed differentiation in other organ systems). Building on this, and combining technical pathways for screening small molecule combinations that affect cell fate and optimizing culture media, studies have further explored the long-term maintenance and differentiation potential of heESCs, validating their functions in terms of clonogenesis, epithelial lineage differentiation, and three-dimensional cyst / cilia structure reconstruction, providing a feasible basis for constructing organoids using heESCs as the sole origin.

[0004] It should be noted that since 2017, studies have been conducted to construct hormone-responsive organoid models from human endometrial tissue that can be passaged long-term and can simulate phenotypes related to different menstrual cycles under the regulation of steroid hormones. However, the above methods are based on one-step three-dimensional culture after direct digestion of primary tissue, which lacks clear definition of the source cells for organoid formation and presents certain obstacles in gene editing, personalized modification, and high-throughput applications. In view of this, it is necessary to establish a "source-definable (SSEA-1)" model. + The organoid construction method, which uses heESCs as the sole origin, features a controllable process (two-stage culture, adjustable dosage / timing) and verifiable thresholds (joint determination of multiple indicators of structure, molecule, and function). This method is of great significance for improving model consistency, editability, and translational application value. Summary of the Invention

[0005] This invention addresses the shortcomings of existing organoid construction technologies, such as unclear origin, high heterogeneity, uncontrolled differentiation, and lack of judgment criteria. It provides a method for the controllable construction and advanced differentiation of human endometrial organoids with SSEA-1⁺ heESCs as the sole origin. The aim is to establish a standardized, verifiable, and scalable in vitro organoid platform for regenerative medicine research, drug screening, and precision medicine.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A type of SSEA-1 + A method for the controlled construction and advanced differentiation of human endometrial organoids derived from human endometrial epithelial stem cells includes the following steps:

[0008] (1) Obtaining and purifying human endometrial epithelial stem cells: Human endometrial tissue was minced, enzyme-digested, sieved, and cleaved, and then seeded into EEM medium for culture; P1 generation cells were purified by differential two-stage digestion and identified by flow cytometry or immunofluorescence to obtain SSEA-1 cells. + Human endometrial epithelial stem cells;

[0009] (2) Three-dimensional construction of human endometrial organoids: The SSEA-1 obtained in step (1) was used to construct the organoids. + Human endometrial epithelial stem cells were resuspended and solidified in Matrigel, then cultured in EOM-A medium with regular medium changes until they formed human endometrial organoids.

[0010] (3) Advanced differentiation of human endometrial organoids: The organoids obtained from three-dimensional culture were transferred to EOM-B medium for directional induction, and continuously cultured in a conventional culture environment with regular medium changes until advanced human endometrial organoids with ciliated and glandular structures were obtained.

[0011] Preferably, the EEM culture medium (500 mL) in step (1) has the following formulation:

[0012] Basic culture medium: Advanced DMEM / F-12 500 mL;

[0013] Component A (essential for cell survival and expansion, final concentration may vary): Y-27632 5-10 μmol / L; EGF 15-25 ng / mL; HGF 15-25 ng / mL; A83-01 0.7-1.3 μmol / L; CHIR99021 2-4 μmol / L; Nicotinamide 5-10 mmol / L; N-acetylcysteine ​​1-1.5 mmol / L; Ascorbic acid 10-15 μg / mL; Sodium pyruvate 1 mmol / L; Penicillin and streptomycin 5 mg / mL;

[0014] Component B (low-intensity WNT pathway regulatory component, final concentration fluctuation range): WNT3A 50-150 ng / mL; RSPO12-10 nmol / L; Noggin 0.8-3.0 nmol / L.

[0015] Culture conditions: 37℃, 5% CO2, fresh culture medium every 2-3 days; the above components can be finely adjusted within the given concentration range according to the differences in the donor.

[0016] Preferably, the EOM-A culture medium (500 mL) in step (2) has the following formulation:

[0017] Basic culture medium: Advanced DMEM / F-12 500 mL;

[0018] Added factors (final concentration fluctuation range): Y-27632 5-10 μmol / L; A83-01 0.7-1.3 μmol / L; CHIR99021 2-4 μmol / L; EGF 15-25 ng / mL; WNT3A 50-150 ng / mL; RSPO1 2-10 nmol / L; Noggin 0.8-3.0 nmol / L; 1 × N2 Supplement-A; 1 × Vitamin A-free B27 and antibiotic mixture.

[0019] Culture conditions: 37℃, 5% CO2, continuous culture for 5-8 days; replace with fresh culture medium every 2-3 days. Early cystic structure and uniform proliferation can be observed during culture.

[0020] Preferably, the EOM-B culture medium in step (3) is based on EOM-A with time-adjusted formulation: CHIR99021 is removed; A83-01 is reduced to 0.2-0.5 μmol / L; estradiol-E2 10 is added. -6 -10 -8 mol / L; DAPT 1-10 μmol / L.

[0021] Specifically, the EOM-B medium (500 mL) formula is as follows:

[0022] Basic culture medium: Advanced DMEM / F-12 500 mL;

[0023] Added factors (final concentration fluctuation range): Y-27632 5-10 μmol / L; A83-01 0.2-0.5 μmol / L; EGF 15-25 ng / mL; WNT3A 50-150 ng / mL; RSPO1 2-10 nmol / L; Noggin 0.8-3.0 nmol / L; 1× N2 Supplement-A; 1× Vitamin A-free B27; Antibiotic mixture 1×; Estradiol-E2 10 -6 -10 -8 mol / L and DAPT 1-10 μmol / L.

[0024] Preferably, the culture conditions described in steps (1)-(3) are 37°C, 5% CO2, and medium change every 2-3 days.

[0025] Preferably, the differential two-stage digestion and purification step in step (1) includes:

[0026] (a) First digestion: treat with a mixture of 0.05-0.5% (w / v) trypsin and 0.2-2 mmol / L EDTA at 30-40°C for 20-40 seconds to selectively remove the first subpopulation of adherent cells;

[0027] (b) Second digestion: Treat with a mixture of 0.05-0.5% (w / v) trypsin and 0.2-2 mmol / L EDTA at 30-40°C for 5-10 minutes, and collect the second cell subpopulation.

[0028] More preferably, the trypsin concentration is 0.25% (w / v), the EDTA concentration is 1.0 mmol / L, the first digestion time is 30 seconds, and the second digestion time is 8 minutes.

[0029] Preferably, the flow cytometry or immunofluorescence identification in step (1) specifically involves screening the second cell subpopulation by flow cytometry or immunofluorescence to identify cell populations with a positive rate of SSEA-1 ≥ 90% (preferably ≥ 92%) and a positive rate of CD13 ≤ 5%, which will be used as the sole source cells for subsequent construction.

[0030] Preferably, in step (3), when the organoid cysts are stably formed and the average diameter reaches 150-200 μm, they are transferred to the EOM-B medium for directional induction differentiation.

[0031] Preferably, the determination of advanced endometrial organoids in step (3) must simultaneously meet the following requirements:

[0032] (a) Structural aspects: Cavitation rate ≥ 80% (image analysis);

[0033] (b) Molecular markers: FOXJ1 positive ciliated cells ≥20%-30%, and PAEP or FOXA2 expression levels upregulated ≥3-fold compared to the expansion phase;

[0034] (c) Polarization function: MUC1 polarization index ≥ 0.6.

[0035] The cavityization rate was determined by image analysis software, the positive rate and expression level were determined by immunofluorescence or qPCR, and the polarization index was determined by immunofluorescence.

[0036] The technical solution of this invention has the following characteristics:

[0037] 1. Differential two-stage digestion without magnetic beads / flow: Stepwise digestion at 37°C with 0.25% Trypsin / 1 mmol / L EDTA (20-40 s + 2-4 min), using 1 mm 3 By adjusting the particle size and using a 100-mesh sieve, simple, rapid, and low-cost purification of heESCs can be achieved.

[0038] 2. Two-stage closed-region formulation and timing control: EOM-A focuses on amplification (EGF / HGF + A83-01 + CHIR99021 + low / optimized WNT axis), EOM-B involves withdrawing CHIR 99021 / lowering A83-01 + estradiol-E2 (10 -6 -10 -8 M) and Notch inhibition pulses (DAPT 1-10 μM, 24-72 h) promote ciliary and polarization maturation.

[0039] 3. Threshold-based quality control system: "Advanced" judgment criteria are constructed using quantifiable indicators such as cavity rate, FOXJ1, PAEP / FOXA2, and MUC1 polarization index, which can be compared between batches and across donors.

[0040] 4. User-friendly operation and resistance to batch fluctuations: The concentration of key factors provides an optimal range, with "window redundancy" for donor differences, and personnel differences have little impact on the results.

[0041] 5. Cryopreservation / thawing and multi-platform compatibility: Compatible with readout systems such as high-content imaging, ELISA, single-cell sequencing / spatial omics; facilitates integration with pharmacodynamics / toxicology workflows.

[0042] Based on the unique origin (SSEA-1) + With its heESCs, two-stage controllable induction (low / optimized WNT axis + timed withdrawal / pulse), and thresholding for "higher organoids," this invention demonstrates significant advantages over existing technologies in terms of definable origin, controllable differentiation, editability, and scalability. Combined with the aforementioned experimental data and standardized operating window, this invention provides a verifiable and scalable in vitro model and methodological foundation for the study of endometrial-related disease mechanisms, drug screening, and personalized applications.

[0043] The technical principle and effect of this invention are as follows:

[0044] 1. By using a sort-free differential two-stage digestion method, the separation of epithelial and stromal cells can be controlled in terms of time window and shear / digestion intensity. The phenotypic threshold of SSEA-1 / CD13 is used as a verifiable standard for unique origin, which significantly reduces the heterogeneity of origin.

[0045] 2. A two-stage strategy of combining low / optimized WNT axis with EGF / HGF + TGF-β inhibition (A83-01) (with timing control of withdrawal / pulse) was used to achieve on-demand induction, which improved ciliary differentiation and secretory function while maintaining cavitation / polarization.

[0046] 3. Ensure process reproducibility and batch-to-batch comparability by using closed-range parameters (concentration / time / temperature / volume).

[0047] In summary, this invention overcomes the shortcomings of existing technologies in three aspects: definable origin, controllable differentiation, and verifiable results, through threshold limitation of unique origin, two-stage dose / timing control, and quantitative determination of higher organoids.

[0048] The technical problem solved and the technical objective achieved by this invention are as follows:

[0049] 1. The source cell cannot be defined, and the model has poor reproducibility.

[0050] Problems to be solved: Existing technologies mostly use direct three-dimensional culture of primary tissue fragments, resulting in unclear origin cells for organoids, large batch-to-batch variations, and difficulties in standardization and traceability.

[0051] Objective of this invention: SSEA-1 defined by phenotypic thresholds + heESCs are the sole origin, and by limiting the testable thresholds of the initial cell population (such as the quantitative definition of epithelial / mesenchymal markers), the reproducibility and standardization of the model are improved.

[0052] 2. Heterogeneous traits and uncontrolled differentiation; lack of a unified standard for classifying "higher organoids".

[0053] Problems to be solved: The existing system has uneven cell traits, lacks fine-grained regulation of the timing and intensity of differentiation, and lacks quantitative criteria for the determination of "higher organoids".

[0054] The purpose of this invention is to establish a two-stage culture strategy (amplification stage → differentiation / advanced differentiation stage) to achieve on-demand induction under low / optimized WNT axis dosage and controllable timing (including addition / withdrawal / pulsation); and to propose a threshold-based judgment system combining structure-molecule-function multi-indicators (such as cavityization ratio, ciliary / polarization markers and secretion profile, etc.) to unify the criteria for judging "advanced organoids".

[0055] 3. Unfavorable to gene editing and personalized modification: The primary fragmentation method has complex sources and weak controllability, which poses obstacles to targeted modification and mechanism verification by lentiviruses / CRISPR, etc.

[0056] The purpose of this invention is to provide an editable and traceable construction path based on heESCs with definable sources and a controllable two-stage culture process, which facilitates gene editing, lineage tracing and individualized modification at the organoid level, and expands its application space in disease models and precision medicine.

[0057] By achieving the above objectives, the present invention obtains the following beneficial effects:

[0058] 1. Definable source and homogeneous traits

[0059] heESCs were obtained by differential two-stage digestion without sorting, and the SSEA-1 positivity rate ≥90% and CD13 ≤5% were used as the sole origin phenotype thresholds, which significantly reduced origin heterogeneity and improved the model reproducibility and standardization.

[0060] 2. Differentiation is controllable and standards are detectable.

[0061] Through a two-stage culture process of EOM-A amplification → EOM-B advanced differentiation, combined with a low-intensity WNT axis and a timed Notch inhibition pulse, on-demand induction was achieved with a low WNT burden; the resulting advanced organoids simultaneously met the following requirements:

[0062] Structural characteristics: Cavitation rate ≥ 80% (image analysis);

[0063] Molecular markers: FOXJ1 positive ciliated cells ≥20%-30%, and PAEP or FOXA2 expression levels upregulated ≥3-fold compared to the expansion phase;

[0064] Polarization function: MUC1 polarization index ≥ 0.6;

[0065] A unified quantitative standard for determining "advanced organoids" has been established.

[0066] 3. Better long-term stability and genetic safety.

[0067] Optimized EOM-A / B formulations and closed-interval operating parameters (temperature, time, volume) support continuous passage of organoids for ≥10 generations while maintaining karyotype and whole-exome genetic stability.

[0068] 4. Editability and scalability

[0069] It is a single-source and controllable process, compatible with gene modification such as lentivirus / CRISPR, with a transduction efficiency of ≥90%, which facilitates lineage tracing and functional gene research, and meets the needs of high-throughput drug screening and personalized mechanism analysis. Attached Figure Description

[0070] Figure 1 heESCs were obtained by differential digestion. Among them, (A): Comparison of clonal morphology of heESCs at the P0 and P1 stages (immunofluorescence), with green arrows indicating heESC clonal clusters; (B): Flow cytometry analysis of P0 mixed cells; (CD): Immunofluorescence purity test of P1 heESCs after differential digestion; (EF): Flow cytometry results of mixed cells from P0 to P1; (G): CCK-8 proliferation curves of P1 heESCs from three donors.

[0071] Figure 2 EOM-A can stably culture heESC organoids. Among them, (AB): screening of heESC organoid culture conditions by small molecule regulation; (CD): Ki67 immunofluorescence detection of heESC organoid cell viability after freeze-thaw and passage; (E): heatmap of the top 20 CNV genes in three donor organoids and endometrial tissue; (FG): reconstruction of the somatic mutation spectrum and clonal structure of heESC organoids from the source endometrial tissue (correlation analysis).

[0072] Figure 3The organoids of heESCs can differentiate in response to steroid hormones. Among them, (A): the cavitation of heESCs organoids and the formation of advanced endometrial organoids in the EOM-B system; (B): the cavitation rate of endometrial organoids is <10% in EOM-A, while it is >80% in the EOM-B system; (C): immunohistochemical expression of PAEP, FOXJ1 and MUC1 in advanced endometrial organoids; (D): transmission electron microscopy comparison of advanced endometrial organoids and primary endometrial tissue (structures such as glandular epithelium, cilia, and intracellular vacuoles).

[0073] Figure 4 : Constructing dual-labeled fluorescent heESCs and organoids. (A): ZsGreen expression in heESCs and organoids under a fluorescence microscope. (B): Luciferase activity of organoids in culture dishes and in rats. Detailed Implementation

[0074] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0075] Specific experimental conditions and methods are not specified in the following examples, and the technical means used are generally conventional means well known to those skilled in the art.

[0076] Example 1: Acquisition and purification of heESCs

[0077] 1. Materials and Reagents

[0078] (1) Source of donors

[0079] Inclusion criteria: female, age ≤ 40 years, regular menstrual cycle (21-35 days), no estrogen / progesterone use in the past 3 months, and signed informed consent.

[0080] Exclusion criteria: hysteroscopy showing endometritis / polyps / adhesions / septa; pathology showing endometritis / proliferative disorders / polyps; malignant tumors (such as endometrial cancer / cervical cancer); other reproductive diseases (PCOS / EMs / AM, etc.).

[0081] (2) Enzyme solution: 1% Collagenase I + 0.25% trypsin / 1 mM EDTA, freshly prepared at a volume ratio of 1:1.

[0082] (3) EEM (500 mL): Based on Advanced DMEM / F12, containing Y-27632 10 μmol / L, EGF 25 ng / mL, HGF 25 ng / mL, A83-01 1 μmol / L, CHIR99021 3 μmol / L, Nicotinamide 10 mmol / L, N-acetylcysteine ​​1 mmol / L, ascorbic acid 10 μg / mL, sodium pyruvate 1 mmol / L, penicillin and streptomycin 5 mg / mL, WNT3A 100 ng / mL, RSPO1 5 nmol / L, and Noggin 1 nmol / L.

[0083] 2. Operating Procedures

[0084] (1) Sampling and pretreatment: Collect approximately 0.5 mL of endometrial sample on day 10-11 of menstruation; cut into 1 mm pieces. 3 Add the mixed enzyme solution and digest at 37°C for 60 minutes, gently blowing intermittently.

[0085] (2) Sieving and erythrocyte lysis: Undigested tissue fragments were removed by filtration through a 100-mesh cell sieve, and the filtrate was collected. The filtrate was centrifuged at 300 × g for 5 min at 4°C, and the supernatant was discarded. 1 × erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2) was added to the precipitate, and the mixture was allowed to stand at room temperature for 3-5 min to complete lysis. Then, 10 times the volume of PBS was added to neutralize the reaction. The supernatant was removed by centrifugation, the cells were washed once with PBS, and finally, the cells were resuspended in EEM medium.

[0086] (3) Primary culture: The treated cell suspension was inoculated into EEM medium and cultured at 37℃ and 5% CO2. The medium was changed every 2-3 days.

[0087] (4) Differential two-stage digestion and purification without sorting

[0088] When the cells expand to P1, the following steps are performed sequentially:

[0089] ① First stage of rapid digestion: 0.25% trypsin / 1 mM EDTA, 37℃, 30 s, to remove adherent endometrial stromal cells; rinse with PBS.

[0090] ② Second digestion: 0.25% trypsin / 1 mM EDTA, 37℃, 8 min, to obtain epithelial stem cell-rich clusters.

[0091] (5) Phenotypic threshold confirmation: SSEA-1 ≥ 90% (preferably ≥ 92%) and CD13 ≤ 5% were identified by flow cytometry / immunofluorescence (IF) and were used as the sole starting cells for subsequent construction.

[0092] (6) Proliferation assessment: CCK-8, draw growth curves.

[0093] 3. Results

[0094] To establish a high-purity method for isolating human endometrial epithelial stem cells (heESCs), we employed a two-step differential digestion method to digest and enrich primary tissues. Results showed that this method efficiently yielded heESCs: immunofluorescence analysis revealed typical heESC clonal clusters agglomerating in both the primary (P0) mixed cell population and the P1 generation cells after secondary enzymatic digestion. Figure 1 (A in the text). Flow cytometry analysis further showed that the P0 population mainly consisted of two distinct subpopulations: endometrial stromal cells and heESCs. Figure 1 (B in the text). Immunofluorescence images of P1 generation cells after differential digestion showed that endometrial stromal cells were effectively cleared, and cell purity was significantly improved. Figure 1 CD in the middle). Flow cytometry results further showed that from P0 to P1, the expression of cell surface markers underwent a fundamental change: the positive rate of the mesenchymal cell marker CD13 decreased from about 50% to close to 0%, while the positive rate of the stem cell marker SSEA-1 increased significantly from 50% to 98%, confirming the successful enrichment of high-purity heESCs (CD). Figure 1 In addition, CCK-8 cell proliferation experiments showed that P1 generation heESCs from three different donor sources all exhibited good in vitro proliferation activity, demonstrating that the isolation and culture procedure has good stability and reproducibility. Figure 1 (G in the middle).

[0095] Example 2: Three-dimensional construction of heESCs-organoids

[0096] 1. Materials and Reagents

[0097] (1) P3 heESCs (obtained in Example 1).

[0098] (2) Matrigel: Pre-cooled, 1 ×.

[0099] (3) EOM-A (amplification culture medium, 500 mL): Based on Advanced DMEM / F12, containing Y-2763210 μmol / L, A83-01 1 μmol / L, CHIR99021 3 μmol / L, EGF 25 ng / mL, WNT3A 100 ng / mL, RSPO1 5 nmol / L, Noggin 1 nmol / L, 1 × N2 Supplement-A, 1 × Vitamin A-free B27, and 1 × antibiotic mixture.

[0100] 2. Operating Procedures

[0101] (1) Cell preparation: Collect heESCs, resuspend the cells in 500 μL Matrigel, and adjust the cell concentration to 5 × 10⁻⁶. 5 Inoculate 12-well plate with 1 / mL of the sample.

[0102] (2) Three-dimensional culture

[0103] After solidification at 37℃, the culture medium was added for culturing. EOM-A was replaced every 3 days, and human endometrial organoids were obtained after 7-10 days of culturing. The control group consisted of EOM-A with or without various small molecules.

[0104] (3) Organoid cryo-thawing

[0105] ① Cold depolymerization of Matrigel: Place the culture plate on ice and aspirate the supernatant; add 1 mL of pre-cooled Cell Recovery Solution (product number 354253), and gently shake at 4℃ / ice bath for 30-45 min until the colloid is completely depolymerized; centrifuge at 400 g for 3-5 min and discard the supernatant; wash gently twice with PBS.

[0106] ② Cryopreservation: Resuspend organoid fragments in CryoStor® CS10 (Cat: 100-1061) at 0.5-1.0 mL / tube, with a total cell count per tube ≈ (1-3) × 10⁻⁶. 6 Set the temperature in a programmed cooling box (-1℃ / min), and transfer it to a liquid nitrogen tank (≤ -150℃) for long-term storage the next day.

[0107] ③Resuscitation: Take the frozen tube and thaw it quickly in a 37℃ water bath for 60-90 s. Add pre-warmed EOM-A, centrifuge at 200 g for 3 min, discard the supernatant, and continue culturing with EOM-A according to steps (1) and (2).

[0108] ④ WES (whole exon sequencing) was used to detect whether organoids could reproduce the mutation spectrum of endometrial tissue; Ki67 was used to detect the activity of organoids after repeated freeze-thaw cycles.

[0109] 3. Results

[0110] To establish a stable heESC organoid culture system, we systematically screened key small molecule combinations that regulate cell differentiation fate. We found that EOM-A medium conditions best supported the sustained growth of heESC-derived organoids. In addition to EOM-A medium, the absence of any one of the following components—EGF, Noggin, B27, N2 Supplement-A, RSPO1, WNT3A, CHIR99021, A83-01, or Y-27632—significantly reduced the average diameter of the organoids. Figure 2 (A and B in the text). Organoids cultured in this system maintained high proliferative activity after cryopreservation, thawing, and multiple passages; Ki67 immunofluorescence staining confirmed good cell viability. Figure 2 The CD in the middle). Further genomic comparison of three organoids from different donors with their corresponding endometrial tissues was performed. Copy number variation (CNV) heatmaps showed that the organoids and the original tissues had high similarity in the top 20 CNV genes (CD). Figure 2 More importantly, somatic mutation profile and clonal structure analysis showed that the heESCs organoids highly reproduced the genetic characteristics of their tissue origin, with a genome-wide correlation (Pearson) score of r = 0.94. Figure 2 The FG in the data suggests that the heESCs organoid model has good fidelity at the genome level.

[0111] Example 3: Advanced Differentiation of Endometrial Organoids

[0112] 1. Materials and Reagents

[0113] (1) EOM-B (differentiation phase medium, 500 mL): Based on Advanced DMEM / F12, containing Y-27632 10 μmol / L, A83-01 0.5 μmol / L, EGF 25 ng / mL, WNT3A 100 ng / mL, RSPO1 5 nmol / L, Noggin 1 nmol / L, 1 × N2 Supplement-A, 1 × Vitamin A-free B27, 1 × antibiotic mixture, and estradiol-E2 10 -6 mol / L, DAPT 10 μmol / L.

[0114] (2) Matrigel: 1 ×.

[0115] 2. Operating Procedures

[0116] (1) Cell preparation

[0117] heESCs were collected, suspended in EOM-A medium, and the cell concentration was adjusted to 1 × 10⁻⁶. 5 per mL.

[0118] (2) Three-dimensional culture

[0119] Resuspend the cells in 1 mL Matrigel, solidify them at 37°C, and then add them to EOM-A medium for culture. Replace the EOM-A medium every 3 days. Differentiated human endometrial organoids can be obtained after 7-10 days of culture.

[0120] (3) Differentiation induction

[0121] Once the organoid cysts have stabilized and reached an average diameter of approximately 150-200 μm, EOM-B is switched for directed differentiation. Cultured at 37°C and 5% CO2, with the medium changed every 3 days, after 14 days, advanced human endometrial organoids with cilia and glandular structures are obtained. A control group was used, culturing with EOM-A without switching to EOM-B.

[0122] (2) Phenotypic assessment: The morphology of the cavityd organoids was observed under light microscopy; the morphology of cilia, secretory gland epithelium, etc. was observed under transmission electron microscopy; and the expression of PAEP / FOXJ1 and the polarization of MUC1 were determined by immunohistochemistry.

[0123] 3. Results

[0124] To assess the differentiation potential of heESCs organoids, we cultured them in the EOM-B differentiation system containing steroid hormones. The results showed that heESCs organoids, cultured in EOM-B, could further develop into advanced endometrial organoids with distinct lumen-like structures. Figure 3 The cavity formation rate of organoids obtained using EOM-A culture is greater than 80%, while the cavity formation rate of organoids obtained using EOM-A culture is <10%, making it impossible to form advanced endometrial organoids. Figure 3 (B in the text). Immunohistochemistry detected clear expression of progesterone-related protein PAEP, ciliary marker FOXJ1, and epithelial membrane protein MUC1 in higher organoids. Figure 3 In the C-cell region, PAEP or FOXA2 expression was significantly upregulated compared to human endometrial organoids in the expansion phase, and the MUC1 polarization index was ≥0.6, confirming at the molecular phenotypic level that they successfully differentiated into endometrial glandular epithelial-like cells. Further transmission electron microscopy analysis showed that these higher-level organoids exhibited ultrastructural features highly similar to in vivo endometrial tissue, including typical glandular epithelial structures, cilia, and intracellular vacuoles. Figure 3 (D in the middle).

[0125] Example 4: Construction of dual-labeled endometrial organoids by transfecting heESCs with lentivirus

[0126] 1. Materials and Reagents

[0127] (1) heESCs: P2 heESCs obtained by in vitro expansion of primary endometrial tissue.

[0128] (2) Matrigel: 1 ×.

[0129] (3) Culture media EEM, EOM-A.

[0130] (4) Lentiviral virus: pLenti-CBh-3xFLAG-Luc2-tCMV-mNeonGreen-F2A-Puro-WPRE. The specific construction method is described in: He W, Zhu X, Xin A, et al. Long-term maintenance of human endometrial epithelial stem cells and their therapeutic effects on intrauterine adhesion[J]. Cell & bioscience, 2022, 12(1):175.DOI:10.1186 / s13578-022-00905-4.

[0131] 2. Operating Procedures

[0132] (1) Viral transfection

[0133] P2 heESCs were cultured in 6-well plates. When the confluence reached 60%, 1.5 mL of EEM + 2 μL of pLenti-CBh-3xFLAG-Luc2-tCMV-mNeonGreen-F2A-Puro-WPRE was added to each well. After 24 hours, the medium was completely replaced and cultured again.

[0134] (2) Organoid culture

[0135] mNeonGreen expression was observed using fluorescence microscopy to determine viral transfection efficiency. Successfully transfected heESCs were further amplified and passaged, and organoids were constructed according to the method in Example 2.

[0136] (3) Evaluation: In vivo / in vitro luciferase imaging to verify expression

[0137] In vitro: Cells carrying the Luciferase reporter gene were seeded into 12-well plates and cultured until the cells were confluent / nearly confluent. The old culture medium was removed, and PBS containing 150 μg / mL D-luciferin was added according to the system. The plates were incubated at 37°C in the dark for about 5-10 min until the substrate was fully absorbed into the cells and a stable luminescence signal was generated. The 12-well plates were then placed in a luminescence imaging system / luminescence detection platform to acquire the signal.

[0138] In vivo: Rats weighing approximately 220 g were selected and anesthetized. A 1.5-2.0 cm incision was made in the midline of the lower abdomen, and organoids containing 500 μL of Matrigel-labeled (Luciferase / mNeonGreen) theESCs were infused into the right peritoneal cavity. The incision was then closed. After the model was established, luciferase substrate was administered via intraperitoneal injection (approximately 5-10 min). Once the luminescence signal stabilized, the animals were placed in a small animal in vivo bioluminescence imaging system to collect bioluminescence signals from the abdominal region for verification and quantitative analysis of Luciferase expression in vivo.

[0139] 3. Results

[0140] To construct tool-like organoids for real-time tracking and in vitro / in vivo detection, we used lentiviral transfection technology to label heESCs. The results showed that heESCs stably expressing the fluorescent protein mNeonGreen were successfully obtained, and the three-dimensional organoids further differentiated from them also exhibited a strong green fluorescent signal. Figure 4 The presence of A in the figure indicates that the fluorescent labeling is stably maintained during long-term culture. Furthermore, luciferase activity assays showed that significant luciferase activity was detected in these organoids both in vitro and after transplantation into rats. Figure 4 (B in the figure) verifies the application potential of this double-standard tool in live imaging.

[0141] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. A method using SSEA-1 + A method for the controlled construction and advanced differentiation of human endometrial organoids derived from human endometrial epithelial stem cells, characterized by... Includes the following steps: (1) Obtaining and purifying human endometrial epithelial stem cells: Human endometrial tissue was minced, enzyme-digested, sieved, and cleaved, and then seeded into EEM medium for culture; P1 generation cells were purified by differential two-stage digestion and identified by flow cytometry or immunofluorescence to obtain SSEA-1 cells. + Human endometrial epithelial stem cells; (2) Three-dimensional construction of human endometrial organoids: The SSEA-1 obtained in step (1) was used to construct the organoids. + Human endometrial epithelial stem cells were resuspended and solidified in Matrigel, added to EOM-A medium and cultured for a period of time, until human endometrial organoids were formed. (3) Advanced differentiation of human endometrial organoids: The organoids obtained from three-dimensional culture were transferred to EOM-B medium for directional induction, and continuously cultured in a conventional culture environment with regular medium changes until advanced human endometrial organoids with ciliated and glandular structures were obtained.

2. The method according to claim 1, characterized in that, The formulation of the EEM medium mentioned in step (1) is as follows: based on Advanced DMEM / F12, and containing Y-27632 5-10 μmol / L, EGF 15-25 ng / mL, HGF 15-25 ng / mL, A83-01 0.7-1.3 μmol / L, CHIR99021 2-4 μmol / L, Nicotinamide 5-10 mmol / L, N-acetylcysteine ​​1-1.5 mmol / L, ascorbic acid 10-15 μg / mL, sodium pyruvate 1 mmol / L, penicillin and streptomycin 5 mg / mL, WNT3A 50-150 ng / mL, RSPO1 2-10 nmol / L and Noggin 0.8-3.0 nmol / L.

3. The method according to claim 1, characterized in that, The formulation of EOM-A medium in step (2) is as follows: based on Advanced DMEM / F12, and containing Y-27632 5-10 μmol / L, A83-01 0.7-1.3 μmol / L, CHIR99021 2-4 μmol / L, EGF 15-25 ng / mL, WNT3A 50-150 ng / mL, RSPO1 2-10 nmol / L, Noggin 0.8-3.0 nmol / L, 1 × N2 Supplement-A, 1 × Vitamin A-free B27 and 1 × antibiotic mixture.

4. The method according to claim 1, characterized in that, The formulation of EOM-B medium in step (3) is as follows: Based on Advanced DMEM / F12, and containing Y-27632 5-10 μmol / L, A83-01 0.2-0.5 μmol / L, EGF 15-25 ng / mL, WNT3A 50-150 ng / mL, RSPO1 2-10 nmol / L, Noggin 0.8-3.0 nmol / L, 1 × N2 Supplement-A, 1 × Vitamin A-free B27, 1 × antibiotic mixture, and estradiol-E2 10 -6 -10 -8 mol / L and DAPT 1-10 μmol / L.

5. The method according to claim 1, characterized in that, The culture conditions described in steps (1)-(3) are 37℃, 5% CO2, and medium change every 2-3 days.

6. The method according to claim 1, characterized in that, The differential two-stage digestion and purification steps described in step (1) include: (a) First digestion: treat with a mixture of 0.05-0.5% w / v trypsin and 0.2-2 mmol / L EDTA at 30-40°C for 20-40 seconds to selectively remove the first subpopulation of adherent cells; (b) Second digestion: Treat with a mixture of 0.05-0.5% w / v trypsin and 0.2-2 mmol / L EDTA at 30-40°C for 5-10 minutes, and collect the second cell subpopulation.

7. The method according to claim 6, characterized in that, The trypsin concentration was 0.25% w / v, the EDTA concentration was 1.0 mmol / L, the first digestion time was 30 seconds, and the second digestion time was 8 minutes.

8. The method according to claim 1, characterized in that, The flow cytometry or immunofluorescence identification in step (1) specifically involves screening the second cell subpopulation by flow cytometry or immunofluorescence to identify cell populations with a positive rate of SSEA-1 ≥ 90% and a positive rate of CD13 ≤ 5%, which will be used as the sole source cells for subsequent construction.

9. The method according to claim 1, characterized in that, In step (3), when the organoid cysts are stably formed and the average diameter reaches 150-200 μm, they are transferred to the EOM-B medium for directional induction differentiation.

10. The method according to claim 1, characterized in that, In step (3), the determination of advanced endometrial organoids requires the following conditions to be met simultaneously: (a) Structural aspects: Cavitation rate ≥ 80%; (b) Molecular markers: FOXJ1 positive ciliated cells ≥20%-30%, and PAEP or FOXA2 expression levels upregulated ≥3-fold compared to the expansion phase; (c) Polarization function: MUC1 polarization index ≥ 0.6.

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