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Trophoblast stem cell-based organoid models of the human placental barrier – Nature Communications

Ethical approval

TS cells and human placentas were used based on the approval the Ethics Committee of Tohoku University School of Medicine (Research license 2014-1-879).

Fabrication of agarose microwell plates

Agarose microwell plates (61 wells) were fabricated using a 3D-printed mold and a polydimethylsiloxane (PDMS) mold, as illustrated in Supplementary Fig. S2. First, a microwell plate was designed using SolidWorks 2019 (Dassault Systèmes SolidWorks Corporation). A 3D-printed mold of a microwell plate was made with a printer (QIDI TECH Shadow 5.5S printer), poly(ethylene glycol) diacrylate (PEGDA, Mn: 250) as a resin, 1%(w/w) photoinitiator (Omnirad 819), and 1% (w/w) photosensitizer (2-Isopropylthioxanthone). The mold was immersed in ca.100 % ethanol for more than 1 min and was exposed to UV light for 4 min, then heated at 80 °C overnight. The mold was coated with Parylene (DPXC, CAS No. 28804-46-8; Parylane Japan) using a PDS-2010 Labcoter 2 (Specialty Coating Systems Inc.). Parylene was used for making PDMS (Silpot184; Toray-Dow Corning) easy to peel off. A PDMS mold was fabricated by conventional soft lithography. PDMS (elastomer: curing agent = 10:1) was cast into the parylene-coated molds and heated to solidify them. The solidified object was taken off from the molds. Agarose (2%) (SeaKem GTG Agarose, Lonza) was poured into the PDMS mold. After solidifying the agarose, the agarose microwell plate was removed from the mold and then immersed in TS basal medium until use.

Fabrication of a column-type device for barrier models

Column inserts for culturing TS cells were fabricated by modifying columns that contained a vitrified collagen membrane (ad-MED Vitrigel 2 for 24-well plates, Kanto chemical co., Inc.) as reported by Takezawa et al.69. The collagen vitrigel membrane functions as a permeable substrate. As shown in Fig. 3A, a ring of polyimide film (t, 5 μm; outer diameter, 8 mm; inner diameter, 3 mm) was bonded to the collagen membrane. To place cell suspension on the collagen membrane, a silicone ring (t, 0.5 mm; outer diameter, 8 mm; inner diameter, 5 mm) was bound onto the polyimide ring using PDMS solution. The column was heated at 60 °C overnight to solidify the PDSM solution and exposed to UV for about 20 min.

Cells and culture conditions

Human TS cells were established in our previous studies21, which were derived from CT cells isolated and purified from first-trimester placentas (6–9 weeks gestation). Experiments using human placentas were conducted according to protocols approved by the Ethics Committee of Tohoku University School of Medicine (Research license 2014-1-879). TS cells were cultured based on a previous method21. Briefly, TS cells were maintained in TS medium (TSM; DMEM/F12 supplemented with 0.15% BSA, 50 units/mL penicillin, 50 μg/mL streptomycin, 1% ITS-X, 1% KSR, 0.2 mM L-ascorbic acid, 2.5 μM Y-27632, 25 ng/mL EGF, 0.8 mM VPA, 5 μM A83-01, and 2 μM CHIR99021) containing 0.25 μg/mL iMatrix-511. Experiments were conducted using a cell line CT27, except for experiments with CT29 and CT30 to confirm reproducibility. Green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) were purchased from Angio-Proteomie (cAP-0001GFP) and cultured in endothelial cell growth medium 2 (EGM-2, Lonza). All cells were cultured at 37 °C with 5% CO2.

Insertion of 7xGFP11 into the endogenous SDC1 locus

The puromycin-resistant gene (PuroR) and Cas9 were PCR amplified from pGIPZ vector (Open Biosystems) and Alt-R S.p. Cas9 Expression Plasmid (IDT), respectively. PuroR, a T2A element, and Cas9 were cloned into the CS-CA-MCS plasmid (kindly provided by H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) using the In-Fusion HD Cloning kit (Takara). The resulting vector was designated as pCS-CA-PuroR-T2A-Cas9. The kanamycin/neomycin resistance gene and the pUC origin were amplified from pCAG-HIVgp (kindly provided by H. Miyoshi), and the human U6 (hU6) promoter was amplified from the pCS-hU6 vector65. The kanamycin/neomycin resistance gene, the pUC origin, the hU6 promoter, 7xGFP1170, and a loxP sequence were assembled using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB). A sgRNA targeting the last exon of SDC1 (target sequence: 5′- GAG CCG AAA CAA GCC AAC GG −3′), a loxP sequence, and a gRNA target sequence (5′-CCC CCG TTG GCT TGT TTC GGC TC −3′; the PAM sequence is underlined), were inserted between the hU6 promoter and 7xGFP11 to generate pU6-sgSDC1-loxP-7xGFP11-loxP. The Cre recombinase gene was amplified from the Cre Vector (Santa Cruz) and cloned into the pCS-3G vector65 to generate pCS-3G-Cre. GFP1070 was chemically synthesized and cloned into the CS-CA-MCS plasmid to generate pCS-CA-GFP10. Sequences of the primers used for the vector construction are shown in Supplementary Data 1.

pCS-CA-PuroR-T2A-Cas9 was cotransfected with pCMV-VSV-G-RSV-Rev and pCAG-HIVgp (kindly provided by H. Miyoshi) into 293T cells and a lentivirus expressing PuroR-T2A-Cas9 was prepared. A lentivirus expressing GFP10 was also prepared from pCS-CA-GFP10 in the same way. These lentiviruses were transduced into CT27 cells, and Cas9-expressing cells were selected with 2 mg/mL puromycin for two days. The pU6-sgSDC1-loxP-7xGFP11-loxP vector was transfected into the Cas9-expressing CT27 cells using an electroporator (CUY21Vitro-EX; BEX) with the following setting: Poration pulse [125 V, 10 ms]; Driving pulse [20 V, 50 ms, 5 pulses]. After 200 mg/ml G418 selection for 5 days, lentivirus expressing Cre was transduced into the cells to delete the kanamycin/neomycin resistant gene, the hU6 promoter, and the sgRNA sequence. Then, single-cell cloning was performed, and clones with an in-frame insertion of 7xGFP11 were identified by Sanger sequencing. One of the obtained clones was used for the experiments. This knock-in strategy is based on homology-independent targeted integration71.

Isolation of TS cell clones expressing EGFP and Kusabira Orange (KSB)

EGFP was PCR amplified from pEGFP-N1 (Clontech) and cloned into the CS-CA-MCS plasmid to generate pCS-CA-EGFP. KSB was PCR amplified from Kusabira Orange-N172 (a gift from Michael Davidson & Atsushi Miyawaki; Addgene plasmid # 54793; http://n2t.net/addgene:54793; RRID: Addgene_54793) and cloned into the CS-CA-MCS plasmid to generate pCS-CA-KSB. Lentiviruses expressing EGFP or KSB were prepared as described above and transduced into CT27 cells. Then, single-cell cloning was performed to isolate EGFP- or KSB-expressing CT27 cell clones.

Generation of spherical trophoblast organoids

TS cells or SDC1-GFP TS cells were seeded at 1.1 ×105 cells/mL ×200 μL on the agarose microwell plate and centrifuged at 160 × g for 20 sec. Then 2 mL of a pre-culture medium (PreM; DMEM/F12 supplemented with 0.15% BSA, 50 units/mL penicillin, 50 μg/mL streptomycin, 1% ITS-X, 1% KSR, 0.2 mM L-ascorbic acid, 2.5 μM Y-27632, 25 ng/mL EGF, 20 ng/mL BMP4, 50 ng/mL bFGF, 0.1 μg/mL heparin, 2 μM CHIR99021, and 2 μM SB202190) was added to each well of 12-well plates. The culture medium was replaced with 2 mL/well of a weak differentiation medium (W-DM; DMEM/F12 supplemented with 0.15% BSA, 50 units/mL penicillin, 50 μg/mL streptomycin, 1% ITS-X, 1% KSR, 0.2 mM L-ascorbic acid, 2.5 μM Y-27632, 25 ng/mL EGF, 20 ng/mL BMP4, 50 ng/mL bFGF, 0.1 μg/mL heparin, 0.5 μM CHIR99021, and 2 μM SB202190) on days 2 and 4. Then culture media were replaced with 2 mL/well of a strong differentiation medium (S-DM; DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin or DMEM/F12 supplemented with 10% FBS, 50 units/mL penicillin, and 50 μg/mL streptomycin) on days 5 and 7. On day 8, spherical trophoblast organoids were obtained.

To demonstrate the fusion of ST cells, KSR-expression TS cells and GFP-expressing TS cells were mixed at the ratio of 1:2 (KSR:GFP), seeded at 1.1 ×105 cells/mL ×200 μL on the agarose microwell plate, and cultured in the same manner described above.

Generation of ST barrier models

The column-type device was used to develop placenta barrier models (Fig. 3A, B). Each column was placed with a collagen membrane on top. To promote cell attachment and growth, the collagen membrane was coated with 40 μL of 0.2 mg/mL Matrigel/PBS(-) at 37 °C for 30 min, followed by washing the membrane with PBS(-) twice. TS cells were seeded at 1.0 ×106 cells/mL ×40 μL/column on the collagen membrane as shown in Fig. 3A. The cells were incubated for 5 h at 37 °C and 5% CO2 to allow cells to attach to the membrane. Then, columns were inserted into each well of a 24-well plate, and PreM was added to each column (200 μL) and well (800 μL). Cells were cultured using the three kinds of medium according to the culture schedule illustrated in Fig. 3D. For a generation of ST/HUVEC barrier models, TS cells were seeded on Day 0 on the collagen membrane from the outside of the column insert. On day 1, HUVEC were seeded at 0.2 ×106 cells/mL ×200 μL/column within the column insert. TS cells were cultured along with the 3-step culture schedule with PreM, W-DM, and S-DM and HUVECs were cultured within the insert with EGM-2.

To increase the ST coverage in the column device, we modified the culture condition for ST barrier models. Human fibronectin (Promo Cell) (0.05 mg/mL) was mixed in the coating solution of 0.2 mg/mL Matrigel/PBS(-), applied to the column, and incubated at 37 °C for over 1 h. Following removal of the coating solution, TS cells were seeded at 0.8 ×106 cells/mL ×40 μL/column to the collagen membrane. After preculturing for 5 h, columns were inserted into each well of a 24-well plate, and PreM was added to each column (200 μL) and well (800 μL). After 2 days, the medium was replaced with W-DM for each column (200 μL) and well (1300 μL). Following 2 days, cells were cultured with the same amount of S-DM2 containing 2 μM forskolin for 2 days. For co-cultures, GFP-HUVEC was seeded at 0.2–0.3 ×106 cells/mL ×200 μL/column to the column insert 2 days after seeding of TS cells. TS cells were cultured along with the 3-step culture schedule with PreM, W-DM, and S-DM2, and HUVECs were cultured within the insert with EGM-2.

Preparation of sections

For frozen sections, TS organoids were fixed with 4% paraformaldehyde (PFA) for 1 h and then permeabilized with 0.3% Triton X-100 for 1 h. Cells were washed with PBS(-) twice and treated with 15% sucrose/PBS(-) at 4 °C for over 5 h. Then, the cells were treated with 30% sucrose/PBS(-) at 4 °C overnight. Cell samples were embedded in Tissue-Tek OCT compound (Sakura Finetek Japan Co., Ltd.), frozen on dry ice, stored at −20 °C, and then sectioned using a cryostat (Leica CM1950 or Tissue-Tek Polar-D) at a thickness of about 12 μm. The frozen sections were mounted on glass slides, air-dried for 1 h, and stored at 4 °C until use.

For hematoxylin-eosin (H&E) sections, TS organoids were fixed with 4% paraformaldehyde (PFA) for 60 min and stored in 1% formalin neutral buffer solution (Fujifilm Wako), followed by conventional paraffin embedding and H&E staining (Advantec).

Immunofluorescent staining

Cells on column-type devices were fixed with 4% PFA for about 20–40 min at room temperature (RT). After washing cells with PBS(-), the cells were treated with 0.3% Triton X-100/PBS(-) for 40 min at RT. Cells were washed with PBS(-) and incubated with a primary antibody at 4 °C overnight. The following primary antibodies were diluted with PBS(-) containing 0.1% Tween 20 and 2% FBS. PE-conjugated anti-SDC1 (Clone 44F9) (Miltenyi Biotec) (1:500 dilution), anti-CDH1 (#24E10, CST; Cell Signaling Technology) (1:250-1:500), anti-CDH1 (Clone SHE78-7) (#M126, Takara) (1:800), anti-CGB (#IR508, Dako) (1:10), anti-GATA3 (#5852, CST) (1:200), anti-TP63 (#ab124762, Abcam)(1:400), anti-Ki-67 (#M7240, Dako)(1:200), rabbit IgG Isotype control (#3900, CST) (1 μg/mL), and mouse IgG Isotype control (#5415, CST) (1 μg/mL). After being washed with PBS(-), cells were treated with the following secondary antibodies at RT for 1 h. Alexa Fluor 488 conjugated anti-rabbit IgG (#4412, CST) (1:400), Alexa Fluor 555 conjugated anti-mouse IgG (#4409, CST) (1:400), and Alexa Fluor 647 conjugated anti-rabbit IgG (#4414, CST) (1:400) were used. Nuclei were stained with Hoechst 33258 (Dojindo) (1:1250) or Hoechst 33342 (Dojindo) (1:1000). Cells were analyzed using a BZ-X800 or X810 All-in-one microscope (×4, ×10, or ×20 objectives) (Keyence), a Zeiss LSM 700 confocal microscope (Carl Zeiss), or TCS SP8 (Leica). For staining F-actin, frozen sections were treated with 1 μg/mL phalloidin-FITC for 60 min at RT, followed by washing with PBS(-). For staining cells of frozen sections, cells were washed with PBS(-) twice and followed by the same staining procedure as cells on the column-type devices were subjected to. Samples of ST barrier models and section samples of spherical trophoblast organoids were stained with antibodies for SDC1, CGB, or E-cadherin in more than triplicates in over three independent experiments.

Scanning electron microscopy

Cell samples were fixed in 4% PFA for 40 min. The samples were washed overnight at 4 °C in 0.1 M phosphate buffer and post-fixed with 1% OsO4 buffered with 0.1 M phosphate buffer for 2 h. The samples were dehydrated in a graded series of ethanol and dried in a critical point drying apparatus (JCPD-5;JEOL) with liquid CO2. The cell samples were spatter-coated with platinum and examined by scanning electron microscope (JSM-7900F; JEOL, Tokyo, Japan).

Transmission electron microscopy

Cell samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 h, and washed with 0.1 M phosphate buffer, post-fixed in 1% OsO4 buffered with 0.1 M phosphate buffer for 2 h, dehydrated in a graded series of ethanol and embedded in Epon 812. Ultrathin sections, 70 nm, were collected on copper grids, double-stained with uranyl acetate and lead citrate. and then examined by a transmission electron microscope (JEM-1400Flash, JEOL, Japan).

TEER measurement

TEER was measured with an epithelial voltohmmeter (EVOM)2 (World Precision Instruments) and STX2 Chopstick electrodes to evaluate barrier integrity. The TEER values of cell samples were normalized to the values of controls without cells.

Measurement of hCG

TS cells were seeded at 1.2 ×106 cells/mL ×40 μL/column onto a membrane coated with Matrigel in column-type devices. Cells were cultured with three kinds of medium [PreM (2 days), W-DM (2 days), and S-DM (3 days)] as illustrated in Fig. 3D(a). Media were collected from columns and wells on days 2, 4, 6, and 7. For controls, the same number of TS cells were seeded onto a membrane coated with 0.3 mg/mL collagen (I-AC, Koken, Japan) and cultured in TSM for 2 or 4 days. The amount of secreted hCG was determined using an enzyme-linked immunosorbent assay (ELISA) kit (#KA4005, Abnova).

IgG and IgA incorporation analysis

ST barrier models were prepared according to the culture schedule illustrated in Fig. 3D(a). As controls, TS cells were seeded at 1.0 ×106 cells/mL ×40 μL/column on the collagen membrane coated with 0.3 mg/mL collagen solution and cultured for 1 day in TSM. IgG-FITC from human serum (#F9636, Merck) and FITC-conjugated ChromPure Human IgA (#099-090-011, Jackson Immuno Research) were diluted to 80 μg/mL with an assay buffer (HBSS containing 0.15% BSA, 1.6 g/L glucose, 2 mM L-glutamine) that was prepared with a modified ex vivo perfusion buffer73. Cell samples were washed once with the assay buffer, and then columns were inserted into wells of 24-well plates. The assay buffer was added to each column (200 μL), and buffer containing FITC-IgG or IgA was added to each well (1300 μL). Cells were incubated at 37 °C and 5% CO2 for 3 h. Cells were fixed with 4% PFA and washed with PBS(-).

FITC-dextran permeability assay

ST barrier models were prepared according to the culture schedule illustrated in Fig. 4B. To prepare TS barrier models, TS cells were seeded at 0.8 ×106 cells/mL ×40 μL/column on the collagen membrane coated with the mixture of human fibronectin (0.05 mg/mL) and Matrigel/PBS(-) (0.2 mg/mL) and cultured for 3 days in TSM with 0.25 μg/mL iMatrix-511. Culture mediums were changed every two days for each ST or TS model. Each barrier model was washed once with the assay buffer mentioned above. FITC-dextran (4 kDa and 70 kDa, Sigma Aldrich) was prepared at 0.4 mg/mL with the assay buffer. The fresh assay buffer was added to each column (200 μL), and buffer containing FITC-dextran was added to each well (1300 μL). Cells were incubated at 37 °C and 5% CO2 for 1.5 h. Then, FITC-dextran in each insert column was collected. FITC-dextran in the buffer samples was quantified by measuring FITC using a fluorescence microplate reader (SpectraMax Gemini XPS).

Translocation analysis

HUVEC, ST, and ST/HUVEC barrier models were prepared. After washing cells, the assay buffer was added to each insert (200 μL), and buffer containing 100 μg/mL antipyrine (Fujifilm Wako chemicals), 200 μM caffeine (Fujifilm Wako chemicals), or 200 μM glyphosate (Fujifilm Wako chemicals) was added to each well (1300 μL). After 30 min, the buffers were collected for HPLC analysis. For time course experiments, cells were exposed to 100 μg/mL antipyrine for 2, 30, 60, 120, 180 min.

The apparent permeability coefficient (Papp) was calculated as follows: Papp (cm/s) = (dQ/dt)/(A × C0), where dQ/dt is the rate of the substrate appearance in the column insert (nmol/s), A is the surface area of the basement collagen membrane (cm2), and C0 is the initial concentration of the substrate in the well (μM)19.

HPLC analysis

Analysis of antipyrine was performed using Agilent 1200. The Quaternary Pump G1311A was used in combination with a Variable Wavelength Detector (VWD) G1314B and TCC (Thermostatted Column Compartment) G1316A, and ALS G1329A autosampler (Agilent Technologies). The column was a Zorbax eclipse XDB C-18 (4.6 × 100 mm, 3.5 μm). A gradient analysis was performed using methanol and 0.1% formic acid, which started from 20% methanol for 7 min, followed by 28% methanol for 4 min and 75% methanol for 2 min. The flow rate was 1 mL/min and detection was 245 nm. Analyses of caffein were performed with the same instrument as the antipyrine analysis. The analysis was performed using a mixture of 85% water and 15% acetonitrile.

LC-MS/MS

Glyphosate was analyzed using an Agilent 1200 device (Agilent Technologies International Japan) coupled to an AB Sciex TripleTOF 5600+ (AB Sciex, Framingham). Chromatographic separation was performed with hydrophilic interaction liquid chromatography (HILIC) column (InertSustain Amide, 100 mm × 3.0 mm i. d., 3 μm; GL Sciences Inc.). The mobile phase was 0.1% formic acid–acetonitrile (80:20, v/v) at 0.2 mL/min and the injection volume was 2 μL. The column was kept at 40 °C in the column oven. An AB Sciex 5600+ triple quadrupole mass spectrometer was used for mass spectrometry determination. The software AB Sciex Analyst was used for data collection. Ionization was performed in negative mode. Mass spectrometry data were collected in the multiple reaction monitoring (MRM) mode. The MRM parameters for the target analytes are shown in Supplementary Data 2. Instrument settings for ESI analysis were as follows: Heater temperature, 350 °C; curtain gas, 25 psi; nebulizer gas, 50 psi; heater gas, 50 psi; ionspray voltage, −4500 V; dwell time, 250 ms.

Statistical analysis

Statistical analysis was performed using StatView version 5.0.1 (SAS Institute Inc.). A value of P < 0.05 was considered statistically significant. All tests were two-sided.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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