Human trophoblastic stem cell line derived from complete hydatidiform mole and its application

Abstract

This invention belongs to the medical field and provides a human trophoblastic stem cell line derived from complete hydatidiform mole and its applications. The human trophoblastic stem cell line, with accession number CGMCC No. C2024411, can be induced to differentiate into syncytiotrophoblast and extravillous trophoblast, mimicking the differentiation and developmental defects of the trophoblastic lineage in hydatidiform mole. It can be used as an in vitro research model for complete hydatidiform mole and has broad application prospects in drug target research and genetic studies.

Description

Technical Field

[0001] This invention belongs to the medical field, specifically relating to the construction and application of a human trophoblastic stem cell line derived from complete hydatidiform mole. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Hydatidiform mole is a gestational trophoblastic disease characterized by abnormal proliferation of the trophoblast and chorionic villus edema. Globally, it occurs in approximately 1-10 cases per 1000 pregnancies, leading to recurrent implantation failure, miscarriage, stillbirth, or progression to invasive gestational trophoblastic neoplasms, severely impacting female fertility and health. Early treatment of hydatidiform mole can achieve a cure and preserve fertility. However, delayed diagnosis increases the risk of serious complications, including hyperthyroidism, hyperemesis gravidarum, early-onset preeclampsia, and life-threatening bleeding. Patients with a history of complete hydatidiform mole (CHM) have a significantly increased risk of developing gestational trophoblastic neoplasms (GTN, including invasive hydatidiform mole and choriocarcinoma), estimated to be approximately 1000 times higher than in normal pregnancies. Recent studies have found significant genomic abnormalities (such as mutations in maternal effector genes like NLRP7 and KHDC3L) and epigenetic reprogramming disorders (such as abnormal DNA methylation) in some patients with hydatidiform mole, suggesting that its pathogenesis is closely related to the regulation of early embryonic development. However, due to the lack of suitable animal and in vitro cell models, the pathogenesis of hydatidiform mole is not yet fully understood, which slows down the progress of strategies for the prevention, treatment, and recurrence prevention of hydatidiform mole.

[0004] Currently, several traditional human trophoblastic disease cell models exist, such as the JEG-3, BeWo, and JAR cell lines, primarily derived from choriocarcinoma, and are widely used to study the development and function of the human placenta. These cells possess strong proliferative capacity and a certain degree of differentiation ability, and are often used as models for studying the physiological functions of the trophoblast. For example, JEG-3 cells have the ability to spontaneously differentiate into extravillous trophoblast cells (EVTs), exhibiting strong invasiveness, but their immunomodulatory function and ability to differentiate into syncytial cells (STBs) are limited. BeWo cells can be induced to differentiate by drugs such as forskolin, exhibiting cell fusion and syncytial formation, accompanied by increased expression of endogenous retrovirus ERV-3 and the production of human chorionic gonadotropin (β-hCG), but they are difficult to differentiate into invasive EVT cells. JAR cells can form epithelial-like monolayers and express tight junction protein ZO-1 and cell adhesion molecule E-cadherin, but they lack significant cell polarity and differentiated apical surface cells. Furthermore, their monolayer structure is unstable, easily forming multilayered cell clumps, limiting their application as a model for transepithelial placental transport. In addition, these choriocarcinoma cell lines exhibit complex chromosomal abnormalities, typically showing near-triploid or tetraploid karyotypes, and extensive chromosomal rearrangements involving chromosomes 1, 3, 7, 9, 10, 12, and 21, lacking a stable karyotype. Complete hydatidiform moles are mostly male-derived uniparental diploids with stable karyotypes that differ significantly from the aforementioned choriocarcinoma cell lines.

[0005] In 2018, the Okea team induced trophoblast stem cells (hTSCs) from the placenta or blastocyst. These cells exhibited strong proliferative capacity and epithelial morphology, and possessed the ability to differentiate into STBs and EVTs, respectively. Furthermore, their gene expression was highly similar to that of primary trophoblast cells. Currently, the hTSCs established by this team are a classic cell line for studying trophoblast function. In 2019, the Okea team used the same method to establish a stem cell line derived from hydatidiform mole (TSC). mole ), discovered TS mole Cell contact inhibition sensitivity decreased. However, the most important characteristic of trophoblast stem cells is their differentiation capacity, as differentiated STBs and EVTs are the final functional cells in the placenta. This study did not utilize TS. mole Exploring the differentiation potential of the CHM trophoblast did not report any TS. moleThe abnormal differentiation of the trophoblastic layer significantly limits its practical application. Apart from this study, no other teams have reported the establishment of trophoblastic stem cells derived from hydatidiform mole. This is primarily due to ethical concerns regarding human subjects, which create obstacles to obtaining human placental tissue as research material. Secondly, the incidence of hydatidiform mole is low, making its pathological tissue relatively precious, which prevents the establishment of trophoblastic stem cell lines derived from hydatidiform mole for trophoblastic differentiation research. Summary of the Invention

[0006] To address the lack of complete hydatidiform mole cell models, this invention provides a human trophoblast stem cell line HM16-hTSCs derived from complete hydatidiform mole. This line is capable of inducing differentiation into syncytiotrophoblast and extravillous trophoblast, and can be used to study the differentiation defects of the trophoblast in hydatidiform mole.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A type of human trophoblast stem cell ( Homo sapiens HM16-TSCs were deposited at the China Center for Type Culture Collection (CCTCC) on December 25, 2024, with accession number CGMCC No. C2024411.

[0009] The aforementioned human trophoblastic stem cells were isolated from complete hydatidiform mole and possess typical physiological, pathological, and molecular biological characteristics of complete hydatidiform mole. They can be used as an in vitro model for studying human trophoblastic cell function and screening anti-fertility drugs or drugs for treating pregnancy-related diseases. When screening anti-fertility drugs, the aforementioned human trophoblastic stem cells are used as a control. When screening drugs for treating pregnancy-related diseases, the aforementioned human trophoblastic stem cells are used as target cells.

[0010] The present invention has the following advantages:

[0011] The human trophoblast stem cells provided by this invention are derived from complete hydatidiform mole and can be induced to differentiate into syncytiotrophoblast and extravillous trophoblast. They can simulate the differentiation and developmental defects of the trophoblast lineage of hydatidiform mole and can be used as an in vitro research model for complete hydatidiform mole. They have broad application prospects in drug target research and genetic research.

[0012] Biological Preservation Information

[0013] Human complete hydatidiform mole villus induced trophoblast stem cells ( Homo sapiens HM16-TSCs were deposited on December 25, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No: C2024411. Attached Figure Description

[0014] Figure 1 Morphology of placental tissue from hydatidiform mole (A), P57 immunostaining (B), and Ki67 immunostaining (C).

[0015] Figure 2 STR locus identification (A) and CNV analysis (B) for normal and hydatidiform placental tissues;

[0016] Figure 3 Morphology of normal and trophoblastic stem cells in hydatidiform mole (A) and immunofluorescence images of trophoblastic stem cell markers in hydatidiform mole (B);

[0017] Figure 4 Allele frequency analysis (A) and allele likelihood ratio analysis (B) of trophoblast stem cells of hydatidiform mole and normal trophoblast stem cells;

[0018] Figure 5 The changes in mRNA expression levels of downregulated genes (A), protein expression levels of TP63 (B), and upregulated genes (C) in trophoblast stem cells of hydatidiform mole.

[0019] Figure 6 The mRNA expression levels of CSH1, CSH2 and PAPPA after STB induction of normal and hydatidiform mole trophoblast stem cells (A), the protein expression of PAPPA and hPL (B), and the immunofluorescence staining of PAPPA and hCG after STB induction by normal TSC and normal TSC induction (C).

[0020] Figure 7 Immunofluorescence staining of normal and hydatidiform mole trophoblast stem cells after inducing EVT (A), mRNA expression levels of HLA-G, MMP2, ADAMTS20, ADAM19 and HTRA4 (B); and changes in MMP2 protein expression levels in normal TSC-induced EVT and hydatidiform mole TSC-induced EVT (C). Detailed Implementation

[0021] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0022] Example 1: Isolation and culture of hTSCs

[0023] 1. Identification of complete hydatidiform mole

[0024] Placental tissue from hydatidiform moles (8-16 weeks of gestation) and normal placental tissue (8-12 weeks of gestation) were collected; all donors voluntarily signed informed consent forms regarding the use of placental tissue for research.

[0025] All placental tissues of hydatidiform mole were identified by P57 and Ki67 staining, and P57-negative and Ki67-positive placental tissues of complete hydatidiform mole were screened. Figure 1 ).

[0026] Complete hydatidiform mole samples were screened using PowerPlex. ® The 21-system (Promega) identifies STR loci in DNA extracted from fetal chorionic villus tissue and maternal blood. PowerPlex ® The 21 system can perform multiplex amplification and detection of 21 loci (20 STR loci and 1 sex-determining locus, Amelogenin), including D1S1656, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, Amelogenin, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, and vWA. Following the provided instructions, add the following components sequentially to form the PCR mixture: PowerPlex ® 21 5× primer pair mixture, PowerPlex ® 21 5× master mixture, template DNA, and water (amplification grade). The DNA extract was then placed in a thermal cycler for amplification. The amplification products were then compared with a WEN Internal Lane Standard 500 and Hi-Di... TM After mixing with formamide, the amplified fragments were detected by electrophoresis on an AppliedBiosystems 3500 genetic analyzer. GeneMapper was used. ® ID-X software (version 3.0) analysis results were used to calculate the allele ratio based on peak height. STR genotyping confirmed the absence of maternal alleles in the villi of complete hydatidiform mole. Figure 2 (A)

[0027] Simultaneously, copy number variation (CNV) analysis was performed on complete hydatidiform mole samples: the tissue was ground and treated with RNase and proteinase K to digest the tissue until no particles remained. DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen) according to the manufacturer's instructions. After measuring DNA concentration using Qubit 4.0, a DNA library was constructed using the Fetal Chromosomal Aneuploidy Detection Kit (Annoroad), and the final library product was obtained by PCR amplification and purification. The quality-controlled library was then sequenced using NestSeq550AR (Annoroad). The results showed that the complete hydatidiform mole was a uniparental diploid pattern, while the control placenta was a diparental diploid pattern. Figure 2 (Middle B). These results confirm the uniparental origin of complete hydatidiform mole, with all complete hydatidiform moles having a 46,XX karyotype.

[0028] 2. Isolation and culture of trophoblast stem cells

[0029] Placental tissue was collected, washed three times in PBS, and blood clots and fine blood vessels were gently removed with curved forceps. The villi were separated from the basement membrane, and the villi tissue was minced and transferred to 50 mL centrifuge tubes. Digestion solution was prepared using a 1:1 mixture of TrypLE and Accumax. The tissue was digested at 37°C for 60 minutes, with continuous rotation and shaking of the centrifuge tube to ensure adequate contact between the villi tissue and the digestion solution. Digestion was terminated by adding culture medium containing 5% serum to the centrifuge tube. The solution was filtered through a 70 μm mesh filter, and the collected cell suspension was centrifuged at 300 g for 5 minutes. After removing the supernatant, the suspension was resuspended in EasySep Buffer (STEMCELL, 20144).

[0030] Count the cells, centrifuge to remove the supernatant, and adjust the cell suspension volume according to the total cell count: if the total cell count is less than 10 million, resuspend in 100 μL EasySep Buffer and estimate the total volume of the cell suspension; if the total cell count is greater than 10 million, add an appropriate amount of EasySep Buffer to make the cell concentration 1 × 10⁻⁶. 8 Cells / mL. Cells were screened using the PE Positive Selection Kit II (STEMCELL, 17684).

[0031] Add 100 μL of FcR blocker (STEMCELL, 19856) per mL of suspension to block non-specific antigens. Then, add 2.5 μL of ITGA6 antibody (Invitrogen, 12-0495-82) per mL of suspension to specifically select trophoblast stem cells. Mix well by pipetting and incubate at room temperature for 15 minutes in the dark. Add 10 volumes of EasySep Buffer and mix well by pipetting. Centrifuge at 300g for 10 minutes, remove the supernatant, and resuspend in 100 μL of EasySep Buffer. Add 10 μL of Selection Cocktail, mix well by pipetting, and incubate at room temperature for 15 minutes in the dark. Vortex RapidSpheres for 30 seconds, then add 5 μL of the mixture, mix well by pipetting, and incubate at room temperature for 10 minutes in the dark.

[0032] Transfer the cell suspension to a round-bottom polystyrene tube (STEMCELL, 38007), add EasySep Buffer to a total suspension volume of 2.5 mL, and mix by pipetting. Place the polystyrene tube in an EasySep magnet (STEMCELL, 18000) and incubate at room temperature for 5 minutes, then pour off the liquid. Repeat the above steps 3 times.

[0033] Human TSC culture medium was prepared by adding the following components to DMEM / F12 (Thermo Fisher Scientific, 21041025): 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific, 21985023); 0.50% penicillin-streptomycin (Thermo Fisher Scientific, 15140122); 100×ITS-X additive (Gibco, 51500056); 0.20% FBS (Thermo Fisher Scientific, 16141-079); 0.30% BSA (Sigma, A9418); 1.5 μg / mL L-ascorbic acid (Sigma-Aldrich, A4403-100MG); 50 ng / mL EGF (PeproTech, 315-09-100); 2 μM CHIR99021 (selleck, S2924); 0.5 μM A83-01 (MCE, HY-10432); 1μM SB431542 (MCE, HY-10431); 0.8mM VPA (MCE, HY-10585); 5μM Y27632 (Selleckchem, S1049).

[0034] Primary TSC culture medium was prepared by mixing human TSC culture medium with Penicillin-Streptomycin and Anti-Anti at a volume ratio of 100:1:1. The polystyrene tubes were removed from the EasySep magnetic poles, and 1 mL of primary culture medium was added to wash the tube walls. The mixture was then pipetted and thoroughly mixed. The selected cells were evenly seeded onto mouse feeder cells, and primary TSC culture medium was added until the volume of culture medium in each well was 2 mL.

[0035] HM16-TSCs are routinely cultured on mouse feeder cells under the following conditions: 37°C, 5% CO2, 20% O2, using human TSC culture medium. The culture medium is changed every two days, and the cells are cultured until TSC cell colonies appear. These cell colonies represent the P0 generation of the HM16-TSC cell line.

[0036] After stable growth, the cells were passaged once when the cell density reached 70-90% at a passage ratio of 1:3. The specific passage procedure was as follows: The old culture medium in the wells was removed, the cells were washed with DPBS, and then digested with TrypLE for 5 minutes. Digestion was stopped by adding culture medium containing 5% serum. After mixing thoroughly, the cells were transferred to 15 mL centrifuge tubes and centrifuged at 300 g for 5 minutes. The supernatant was removed, and the cells were resuspended in 1 mL of human TSC culture medium. The cells were then evenly seeded onto 3-well mouse feeder cells, and human TSC culture medium was added to bring the culture medium volume to 2 mL per well. Normal passages were performed, and after reaching P5-10, the cells were identified as exhibiting hTSC characteristics, thus obtaining the HM16-TSCs cell line. This cell line maintained its proliferative capacity and stemness after 30 passages, without showing signs of senescence. Using this method, three HM-hTSCs derived from complete hydatidiform mole were obtained. These cell lines have similar characteristics. One HM16-hTSC with better proliferation ability was selected for cell preservation, and its preservation number is CCTCC No: C2024411.

[0037] Example 2 Identification of trophoblast stem cells

[0038] HM-TSCs are characterized by clonal, polygonal epithelial-like cells, conforming to the morphological criteria of trophoblast stem cells. Figure 3 (A)

[0039] 5×10 4 Cells were inoculated into 35mm culture dishes pre-coated with feeder cells. Three cells were randomly selected daily from each well for counting, and growth curves were plotted. Results showed that the HM-TSCs doubled in approximately 36 hours, reached confluence on day 6, and exhibited good proliferation capacity.

[0040] 1×10 4Cells were inoculated into eight-well chambers (Ibidi) and cultured normally for three days. Cells were then fixed with 4% PFA, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA. Immunofluorescence staining showed that HM-TSCs exhibited GATA3 (+) in the nucleus, Cytokeratin-7 (+), TP63 (+), and E-Cadherin (+) in the cell membrane, demonstrating expression of trophoblast stem cell markers consistent with normal TSCs, confirming that HM-TSCs possess the characteristics of trophoblast stem cells. Figure 3 (B)

[0041] Example 3: Identification of uniparental diploidity

[0042] Allelic analysis of HM-TSCs: Adherent cells were digested using TrypLE, washed three times with PBS, centrifuged, and the cell pellet was retained. Cells were then treated with RNase and proteinase K to digest the cells until no particles remained. DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen) according to the manufacturer's instructions. After measuring DNA concentration using Qubit 4.0, a DNA library was constructed using the Fetal Chromosomal Aneuploidy Detection Kit (Annoroad), and the final library product was obtained through PCR amplification and purification. The quality-controlled library was then sequenced using a NestSeq550AR (Annoroad). Results showed that HM-TSCs were uniparental diploid, while control TSCs were diparental diploid. Figure 4 These results confirm that the genotype of HM-TSCs is uniparental diploid, consistent with the genotype of complete hydatidiform mole.

[0043] Example 4: Gene expression characteristics of HM-TSCs

[0044] Total RNA was extracted from hTSCs using Trizol reagent (Takara), and RNA concentration and purity were determined using a Qubit® 2.0 fluorometer (ThermoFisher Scientific Life Sciences, MA, USA). 1000 ng of RNA was reverse transcribed into cDNA using PrimeScript™ RT Master Mix (Takara) according to the manufacturer's instructions. q-PCR analysis of the target gene was performed using TB Green Premix® Ex Taq. TM The assay was performed on a quantitative PCR system. GAPDH was used as an internal control for gene expression, and each primer was used at a concentration of 1 µM.

[0045] q-PCR results showed that genes related to trophoblast stemness, such as TP63, BCAM, EGFR, and YAP1, were downregulated in HM-TSCs. Figure 5 In the middle A), the maternally expressed imprinted genes H19 and CDKN1C were downregulated ( Figure 5 In the middle A), downregulation of epigenetic-related DNMT3A and NLRP2 ( Figure 5 In the middle A), HLA-G, which is associated with trophoblast differentiation, TBX3 is upregulated ( Figure 5 (Middle B). It can be seen that HM-TSCs can serve as a disease model for complete hydatidiform mole, and can be used to study the regulation of gene expression related to abnormal development of the trophoblast in hydatidiform mole.

[0046] Example 5: Induction of differentiation of trophoblast stem cells

[0047] 1. Syntrophoblast (STB) induced differentiation

[0048] Preparation of TS culture medium:

[0049] Add 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific, 21985023), 0.5% penicillin-streptomycin (Thermo Fisher Scientific, 15140122), 0.3% BSA (Sigma, A9418), 1% ITS-X (Gibco, 51500056), 2.5 μM Y27632 (Selleck, S1049), 2 μM saliva extract (MCE, HY-15371) and 4% KSR (Gibco, 10828010) to DMEM / F12.

[0050] 80% confluent hTSCs were dissociated into single cells using TrypLE, totaling 1.5 × 10⁻⁶ cells. 6 Cells were seeded into 6-well plates pre-coated with 5 μg / mL type IV collagen. The cells were then cultured in 1 mL of standard STB medium, and the cell culture was analyzed on day 3.

[0051] After inducing TSCs to differentiate into syncytiotrophoblasts (STBs), quantitative PCR analysis revealed that, compared with the control group STBs (STBs...), Control Compared to STB HM The expression levels of CSH1, CSH2, and PAPPA were significantly reduced. Figure 6 (A). Western blot results further confirmed that STB HM The expression of PAPPA and human placental prolactin (hPL) was significantly lower than that of STB. Control ( Figure 6 (Middle B). Immunofluorescence staining also showed that STB...HM PAPPA expression is higher than STB. Control reduce( Figure 6 (C). In summary, these results suggest that hydatidiform mole may be associated with impaired differentiation of SCT-Mature1 subtype cells, accompanied by hormonal imbalances.

[0052] 2. Extratrophoblastic layer (EVT) induced differentiation

[0053] Preparation of EVT culture medium:

[0054] The following ingredients were added to DMEM / F12: 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific, 21985023), 0.5% penicillin-streptomycin (Thermo Fisher Scientific, 15140122), 0.3% BSA (Sigma, A9418), 1% ITS-X supplement (Gibco, 51500056), 100 ng / mL NRG1 (MCE, HY-P7365), 7.5 μM A83-01 (MCE, HY-10432), 2.5 μM Y27632 (Selleck, S1049), and 4% KSR (Gibco, 10828010).

[0055] 80% confluent hTSCs were dissociated into single cells using TrypLE. A total of 1.5 × 10⁻⁶ cells were collected. 6 Cells were seeded into new 6-well plates pre-coated with 1 μg / mL type IV collagen. The cells were then cultured in 2 mL of EVT medium. Additionally, Matrigel (Corning, 354234) was added to the cell-containing medium to a final concentration of 2%. On day 3, the medium was replaced with EVT, and Matrigel was added to a final concentration of 0.5%. The cultures were analyzed starting on day 6 of the experiment.

[0056] After inducing TSCs to differentiate into extravillous trophoblast cells (EVT), immunofluorescence analysis showed that the MMP2 fluorescence intensity of HLA-G(+) cells in EVT-HM was increased. Figure 7 (A). Quantitative PCR further confirmed that, compared with the EVT-Control group, the expression levels of genes related to EVT invasion (HLA-G, MMP2, ADAMTS20, ADAM19, and HTRA4) were significantly upregulated in the EVT-HM group. Figure 7 (B) Western blot analysis showed that the expression level of MMP2 protein in the EVT-HM group was significantly higher than that in the EVT-Control group (B). Figure 7 (C). In summary, these results suggest that the invasive potential of EVT cells is enhanced under HM conditions.

Claims

1. A type of human complete hydatidiform mole villus-induced trophoblastic stem cells HM16-TSCs ( Homo-sapiens ), characterized in that, The accession number is CGMCCNo.C2024411.

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