Petri dish preparation
Gelatin Plates: Add a sterile 0.2% gelatin solution to Petri dishes to cover the whole surface, then incubate plates for at least 20 min at 37ºC before use.
Mouse Embryonic Fibroblast (MEF) plates: Add a sterile 0.2% gelatin solution to the Petri dishes to cover the whole surface, then incubate plates for 20 min at 37ºC. Afterwards, place frozen irradiated MEF stock cryotubes in a water bath at 37ºC until ice is melted (1-2 min), dilute on MEF medium (see below) in a 15 ml tube, and centrifuge at 1400 rpm for 5 minutes. Resuspend the pellet on MEF medium accordingly (3 million cells counted before freezing is optimal per 10 cm petri dish), remove the gelatin from the plates and add a total of 8 ml volume to each plate of MEFs resuspended in medium, keep in 5% CO2 20% O2 incubator until use. If plates are kept for several days, it is recommended to exchange MEF medium every 48h.
Matrigel Plates: Add 5 ml P/S solution to 500 ml of DMEM (makes 1% solution) and take the required amount according to the number of plates to prepare (8 ml per 10 cm plate) and supplement with 0.5% Matrigel (Matrigel should be quickly resuspended in the medium after removing from -20 freezer to avoid polymerization). Mix well and distribute on the plates accordingly (make sure that the whole surface of the plate is covered). Keep plates in a 37ºC incubator until use within 5 days from the initial preparation.
RSeT: Medium is prepared according to manufacturer. Thaw the 10x RSeT supplement at room temperature, one bottle of supplement (50 ml) is added to the 450 ml RSeT medium bottle. 200 µl of Matrigel are rapidly added to the medium according to the manufacturer's instructions. After preparation, medium is stable for two weeks at 4C.
HENSM: Base medium was prepared by mixing 50% DMEM/F12 and 50% Neurobasal, supplemented with 1x N2 supplement and 1x B27 supplement, 1% Nonessential Amino Acids, 1% GlutaMAX, 1% P/S, 1% Sodium Pyruvate. Extra supplementation included 50 µg/ml Ascorbic Acid and 2% Geltrex or Matrigel. Small molecule and recombinant proteins are supplemented as follows: human Lif 10ng/ml, XAV939 2μM, Go6983 2μM, PD0325901 1-1.2 μM, CGP77675 1.2μM, BIRB0796 0.8μM, ROCKi Y27632 1.2 μM, and ACTIVIN A 5ng/ml. To assemble the medium, all components were mixed, Geltrex was added in the end after fast removal from the freezer to avoid polymerization.
*In this SEM protocol, a fixed version of HENSM was used to fit the requirements of the procedure, including fast growth and clean cultures of WIBR1 and WIBR3 human ESC lines. The medium composition may vary according to the cell line employed. Please refer to Bayer et al 20211 and Hanna lab full HENSM protocol posted on Hanna lab website ( https://www.weizmann.ac.il/molgen/hanna/ ) for further standardization (e.g. slight increase in ACTIVIN dose used or MEKi/ERKi used).
MEF medium: DMEM supplemented with 1% Nonessential Amino Acids, 1% GlutaMAX, 1% P/S, 1% Sodium Pyruvate and 20% heat inactivated Fetal Bovine Serum (FBS).
RCL PrE/ExEM priming from naïve PSCs in HENSM: RPMI 1640 supplemented with 1% GlutaMAX, 1x B27 minus insulin, 10ng/ml hLIF and 3 µM CHIR99021.
TE priming medium for differentiating from HENSM naive PSCs -BAP(J) regimen: Medium was prepared by mixing 50% DMEM/F12 with 50% neurobasal, supplemented with 1x N2 and 1X B27, 1% Nonessential Amino Acids, 1% GlutaMAX, 1% P/S, 1% Sodium Pyruvate, MEK/ERKi PD0325901 2µM and TGFRi A-83-01 2µM. This medium was used as a base (termed P2A2) and complemented with 10ng/ml human recombinant BMP4 (on day 1 only) OR with 1µM JAK1 inhibitor on (days 2 and 3 only).
Aggregation medium: Medium was prepared by mixing 50% DMEM/F12 with 50% Neurobasal, supplemented with 1x N2 and 1X B27, 1% Nonessential Amino Acids, 1% GlutaMAX, 1% P/S, 1% Sodium Pyruvate, accompanied by the addition of 2.25ml of BSA solution (which is critical to reduce aggregate stickiness), and 1 ml (50mM) beta-mercaptoethanol.
hEUCM2 medium: Advanced DMEM/F12, 1 mM GlutaMAX, 1% P/S, 1x ITS-X supplement, 8 nM Beta-Estradiol, 200 ng/ml Progesterone, 25 µM N-acetyl-L-cysteine, and optionally 1 mg/mL D(+)-Glucose Monohydrate (J.T. Baker - 0113). Fetal Bovine Serum was supplemented on a final concentration range from 20-50% according to the protocol (see below). Filtrate before use.
Conversion and maintenance of PSC in RSeT medium
To maintain PSC in a state that allows fast, stable, and homogeneous feeder-free culture that enables fast conversion when transferred to HENSM conditions and avoiding the high DNA hypomethylation that occurs by extended passaging in naïve ground state conditions, we keep cells maintained in Naïve-like RSeT medium on Matrigel-coated plates up to 20 passages, and from the latter, we take a fraction as a source for HENSM conversion.
For this, primed PSC stocks were directly thawed on Matrigel coated plates in RSeT medium.
1.- Prepare RSeT medium and Matrigel coated plates as mentioned above.
2.- Remove a frozen cryovial of selected cells from the liquid nitrogen storage and place it in a 37ºC water bath for 1-2 min or until most of the ice is melted.
3.- Transfer to a 15 ml tube filled with MEF medium and centrifuge at 1300 rpm for 3 minutes.
4.- Remove supernatant and resuspend the pellet in 1 ml of RSeT medium.
5.- Take from the incubator a previously prepared Matrigel plate, remove medium, and place 7-9 ml of RSeT medium.
6.- Place previously resuspended cells in the plate and supplement with 10 µM ROCKi.
7.- Place in a hypoxia 5% O2 and 5% CO2 37C tissue culture incubator.
8.- Replace medium every 24h.
*Cells should be passaged every 3-4 days depending on confluency (80% approx.). PSCs in RSeT naive-like conditions generally show a dome shape colony morphology at a lower confluency and tend to flatten as higher confluences are reached (Figure 1).
For passaging RSeT cells, TrypLE is used as follows:
1.- Remove cells form the incubator and subtract the medium.
2.- Add 3 ml of TrypLE and incubate for 3 min at 37ºC on a CO2 incubator.
3.- Gently remove TrypLE from the plate ensuring that cells still adhere to the plate.
4.- Incubate for two minutes at room temperature in the tissue culture hood.
5.- Add 3ml of PBS to the plate and use this volume to collect the cells through gentle pipetting with a P1000 pipette, once all cells are in suspension transfer them to a 15ml tube (fill the tube up to 15 ml with PBS to ensure adequate pellet formation during the centrifugation step).
6.- Centrifuge at 1300 rpm for 3 minutes.
7.- Resuspend the pellet in 1 ml of RSeT medium and prepare new plates as mentioned above. (At this point cells could be seeded directly on HENSM medium for conversion)
*Usually cells are passaged 1:8 – 1:10 for maintenance and 1:6 – 1:8 for conversion from NHSM/RSeT naive-like to a naive state in HENSM.
Conversion and maintenance of naïve human PSCs in HENSM
*To convert cells to naïve state in HENSM conditions, RSeT cells were used and kept at least two passages (around 8-10 days) before proceeding.
* Conversion can be performed under MEF coated plates or on Matrigel feeder-free conditions depending on the experimental needs. For formation of SEMs, we have noticed higher efficiencies when cells were expanded in Matrigel coated plates in HENSM (which was selected to be used in the published manuscript), but MEF derived HENSM PSCs also contributed to adequate structures after including a MEF depletion step to pertinent cells.
*TE induction requires feeder-free conditions during the induction, since MEF supplementation significantly decreased the conversion efficiency. However, HENSM cells could be expanded on MEFs and switched to feeder free conditions only 24h before starting the induction if required.
1.-Harvest RSeT cells as mentioned above and passage them if maintaining the RSeT condition is required.
2.-Take a previously prepared MEF or Matrigel-coated plate (according to experimental needs), remove the medium and add 8 ml of HENSM supplemented with 10 µM ROCKi.
3.-Seed the cells on a 1:5-1:6 concentration (approx. 8x105 cells per 10 cm plate).
4.-Change medium daily without supplementing with the extra ROCKi, expand and maintain under hypoxic conditions.
5.-Passage the cells every 3-4 days using TrypLE as mentioned above with a 1:6 – 1:8 ratio.
*Cells are kept 2-3 passages in HENSM before using them for TE/ExEM/PrE priming inductions to reach the naïve state and clean them from residual differentiated cells (Figure 2). In case of not reaching adequate morphology, go to Troubleshooting
*Cells were kept in HENSM for no longer than 10 passages to avoid excessive DNA damage and loss of imprinting by global DNA hypomethylation, as previously described.1
Induction of PrE/ExEM-like lineages for aggregation and further analysis
To induce primitive endoderm and extraembryonic mesoderm cells in parallel and in suitable conditions for the formation of SEMs, a protocol modified from Linneberg et. al2 is employed, using RCL medium for 3 days. Also, for further analysis and quality control, cells are kept up to day 6 in the same conditions or switched to N2B27 (with similar results) and evaluated by the surface expression of PDGFRa by flow cytometry.
1.- Two days before the initiation of the aggregation, inspect the HENSM cells and corroborate a homogeneous culture of dome-shape-like colonies at a 40-50% confluency. Prepare fresh MEF coated plates and prepare fresh RCL medium.
2.- The day before the initiation of the induction, check the cells and verify quality, harvest the cells, and count them using a hemacytometer (count all cells, no counterstaining for viability was used). Seed the adequate number of HENSM PSCs (see below) on a MEF plate in HENSM supplemented with 10 µM ROCKi.
* Cell confluency during the RCL induction was found to be essential for high quality and reproducible results during SEM formation and needs to be calculated for each cell line and condition tested. In our case, for WIBR3 cell line grown in HENSM on Matrigel, plating 8 x105 cells per one 10 cm dish showed the best results in terms of the induction efficiency, while in the case of WIBR1 cell line, plating 2x106 HENSM PSCs per single 10cm dish showed optimal results. Cell confluency curves on day 6 using PDGFRa expression on FACS can give a good idea on optimal HENSM PSC cell numbers for each cell line to be initially plated for obtaining optimal induction efficiencies.
3.- At the starting day of the induction, remove HENSM medium from the plate, rinse twice with PBS (without harvesting the cells), replace with RCL medium, and keep the cells under 5% hypoxia conditions.
4.-On days 1 and 2 repeat the same procedure.
5.- On day 3, cells can be collected for aggregation (adequate morphology shown in Figure 3) or, alternatively, exchange RCL medium daily until day 6 for FACS analysis.
Induction of TE-like cells for aggregation and further analysis from HENSM conditions
For rapid and efficient priming of naïve PSCs towards TE lineage cells that are capable to dynamically contribute to the formation of SEMs, the protocol from Io et. al3 was adapted:
1.- Two days before the initiation of the aggregation, evaluate HENSM PSCs quality at 40-50% confluency. Usually, the same batch of HENSM PSC cells is pooled for PrE, TE parallel inductions, and PSC HENSM maintained fraction. Prepare new Matrigel coated plates and fresh P2A2 base medium.
2.- The day before starting the induction, check whether HENSM cells reached adequate confluency (80-90%) and morphology. Harvest the cells and count them using a hemacytometer (same as for PrE induction, cells can be pooled and counted together). Seed the adequate number of cells (see below) on a Matrigel-coated 10 cm plate in 8-10 ml of HENSM supplemented with 10 µM ROCKi.
* Cell confluency during the TE induction was previously shown to be essential for high quality, reproducibility (Io et. al3), and efficiency of SEM formation, and the optimal seeding cell number needs to be calculated for each cell line tested. In the case of WIBR3 hESC line grown on Matrigel, 1 x106 HENSM PSCs per one 10 cm plate showed the most optimal results of SEM formation in our conditions as measured by high frequency of aggregates being surrounded by TE-like cells at day 6 (wanted outcome >60%+). For WIBR1 hESC line, seeding 2x106 HENSM PSCs per one 10 cm dish yielded optimal results. Cell confluency curves and FACS staining on day 3 of BAP(J) induction using ENPEP/TACSD2 expression to obtain >80% double positive cells, is considered a good predictor for successful induction and subsequent correct localization of TE-like cells around the SEMs after aggregation (measured as indicated above).
3.- At the day of starting the induction, remove HENSM medium from the plate, rinse twice with PBS (without harvesting the cells), and replace it with BAP(J) base medium supplemented with 10 ng/ml hBMP4. Maintain cells in hypoxic 5% O2 5% CO2 37C tissue culture incubator.
4.- Next day (day 1 of induction), remove the medium and replace it with BAP(J) base medium supplemented with 1µM JAK inhibitor I.
5.- Repeat same procedure on day 2.
6.- On Day 3, cells are ready for aggregation and/or further analysis by FACS (e.g., with anti-ENPEP and anti-TACSD2 antibodies). Cells should show a flat colony morphology with big cytoplasm and some big dome shape structures as shown in Figure 3.
Preparation of materials for human SEM aggregation
1.-For initiating the aggregation, HENSM PSCs on day 3-4 after seeding, RCL day 3 induced cells, and BAP(J) day 3 induced cells are required (induced as detailed above). Notice that BAP(J) cells are required in bigger amounts since aggregation ratios are 1:1:3 (ES: RCL: BAP(J)). Typically, 2-3 10 cm plates of BAP(J) are prepared per single 10 cm plate of HENSM PSCs and one plate of RCL-induced cells.
2.-Prepare one gelatin-coated 10 cm plate per each RCL plate for MEF depletion.
3.-To prepare Aggrewell 400 24-well plate, first add 500 ul of rinsing solution per well.
4.-Centrifuge the plate at 1300g for 5 min.
5.-Examine the Aggrewell under the microscope. Repeat the centrifugation step if air bubbles are still trapped inside the microwells.
6.-Incubate for 30 min at room temperature in a sterile environment.
7.-Aspirate the rinsing solution and wash once with PBS.
8.-Add 500 µl of Aggregation medium per 24-well and place in hypoxia 37C incubator.
Aggregation procedure to obtain human SEMs
*Before starting make sure to have all pertinent materials, e.g., Gelatin plates, Aggrewell and mediums prepared (MEF medium, aggregation medium), and perform the procedure in the shortest time possible to improve cell viability.
Start the procedure by performing MEF depletion of the RCL cells:
1.-Remove the RCL medium from the 10 cm plate and add 3 ml of TrypLE.
2.-Incubate 3 min at 37ºC.
3.-Examine the plate under the microscope. If the cells are still attached, remove TrypLE, add 3 ml of PBS and gently resuspend. If the cells are found in suspension, collect the cells along with the enzyme (RCL cells tend to detach faster than other cells).
4.- Collect cells in a 15 ml tube, and centrifuge at 1300 rpm for 3 min.
5.- Take out a gelatin-coated 10 cm plate from the incubator, remove the gelatin, and add 7 ml of MEF medium.
6.- Remove the supernatant and resuspend the pellet in 1 ml of MEF medium.
7.- Add the cells to the gelatin-coated plate with MEF medium and incubate for 30 min at 37ºC inside the CO2 incubator.
8.- Collect supernatant without disturbing attached cells and pass through a 100 µM cell strainer.
9.- Centrifuge at 1300 rpm for 3 min and resuspend the pellet in 1 ml of the Aggregation medium.
Collect HENSM and BAP(J) cells:
1.- Remove the medium from the plates and add 3 ml of TrypLE.
2.- Incubate plates at 37ºC (3 min for HENSM cells and 5 min for BAP(J) cells as they are relatively more adherent).
3.- Remove TrypLE and incubate for 2 additional minutes at room temperature.
4.- Collect the cells with 3ml of PBS and centrifuge at 1300 rpm.
5.- Resuspend in 1 ml of aggregation medium then pass them through a 100um cell strainer.
Counting the cells and preparing for human SEM co-aggregation:
1. Count the cells of the three previously collected fractions using a hemacytometer (usually with a 1:10 dilution in PBS, no counterstaining is used).
2. Prepare 2x cell mix as follows:
a. Total cell number per microwell: 120 cells.
b. ~1 HENSM: 1 RCL: 3 BAP(J). [19.25% HENSM, 19.25% RCL and 61.5% BAP(J)]
c. As a single well in 24-well plate contains 1,200 microwells, a total of 144,00 cells are added per each single well of a 24-well plate. Prepare a 2X mix of cells: 288,000 cells per 1 ml. (12 ml are needed for a full 24-well plate, but preferably make at least 1 ml extra as a backup).
d. Supplement the 2X cell mix with 20 µM ROCKi.
*See supplementary cell calculator file for further information.
3. 500 µl of cell-mix suspension is gently added dropwise to each well of the previously prepared AggreWell plate (final yield per each well = 1 ml final volume with 10 µM final ROCKi concentration and 144,000 cells).
4. Centrifuge the plate at 100 g for 3 minutes.
5. Gently examine under the microscope to ensure that cells are homogeneously distributed through the microwells.
6. Gently place the plate back in the hypoxia incubator and incubate at 37ºC 5% O2 5% CO2.
Culture of SEMs from day 0 to day 3
After 24h of aggregation (at Day 1):
1.- Prewarm 1 ml of aggregation medium per well for 30-60 min at 37ºC water bath.
2.- Remove the plate gently from the incubator and examine it under the microscope to ensure the formation of an aggregate on each microwell.
3.- Carefully remove 800-900 µl of medium from each well without disturbing the aggregates.
4.- Gently add 1 ml of prewarmed Aggregation medium to each well and place it back into the hypoxia incubator.
*5.-On day 2 repeat the same steps.
Culture of SEMs between Day 3 and Day 8.
On Day 3 (72h after aggregation) SEMs are transferred to non-adherent six-well plates:
1.-Prepare 3 ml of hEUCM2 20% FBS per well of a non-adherent 6-well plate.
2.-Prewarm in a Normoxia 37ºC 20% O2 5% CO2 incubator for 30-60 min.
3.-Take out the 24-well Aggrewell plate containing the aggregates, and carefully remove most (nearly all (~1.2 ml)) of the aggregation medium (without disturbing the aggregates) and replace it with 1 ml of hEUCM2 20% from the 6-well plate (the goal is to transfer the aggregates from two wells of a 24-well plate to a single well of a 6-well plate), and distribute 2 ml out of total 3 ml hEUCM2 of a single well of a 6-well plate, into two wells of a 24-well plate (1 ml each per well).
4.- Using a 3 ml sterile Pasteur pipet, harvest the aggregates with slow up and down movements.
5.- Collect back to the 6-well (total volume of media per each well of the non-adherent 6-well plate should be 3 ml after finishing these transfers) and incubate on top of an orbital shaker at 60 rpm placed inside a Normoxia incubator.
*Note: it is important to not significantly deviate from a total of 3 ml volume per each well of the 6-well plate to avoid clumping of the aggregates.
On day 4, change medium to hEUCM2 30% FBS:
1.- Prepare 2 ml per well of hEUCM2 30% FBS and prewarm in a 37ºC water bath for 30-60 min.
2.- Gently remove 2 ml of each well without disturbing the aggregates.
3.- Gently add 2 ml of the previously prewarmed medium to each well of a 6-well plate and place it back on top of the shaker.
*Repeat the same procedure on day 5.
*Note: for initial calibration of the protocol, we recommend ending experiments on day 6 and examine the generated human SEM for the presence of the amniotic cavity, yolk sac, and trophectoderm on the outer layer. Once confident of success over multiple biological replicates, proceed to later stages up to day 8 of the protocol.
On day 6, replace 2 ml of hEUCM2 30% with 2 ml hEUCM2 50%.
1.- Repeat the same procedure on day 7.
2.- On day 8, harvest the aggregates that include human SEMs for further analysis.
Immunostaining of human aggregates/SEMs
1.- Collect human SEMs in a 15 ml tube with a Pasteur pipette and let them sink at the bottom of the tube.
Note: through each one of the steps using 15 ml tubes, wait around 2 minutes for aggregates to settle down at the bottom before removing the supernatant.
2.- Carefully remove the medium without disturbing the SEMs and wash once with PBS.
3.- Remove the PBS and add 4% paraformaldehyde diluted in PBS.
4.- Incubate overnight at 4ºC or 1 hour at room temperature, by tilting the tube 45° on orbital shaker (50 rpm).
5.- Rinse 3 times with PBS (5 minutes each).
* At this stage, the procedure can be interrupted. Fixed embryos can be stored in PBS at 4ºC for a few days.
6.- Distribute SEMs on the glass spot plates according to desired experiments.
7.- Permeabilize SEMs in 200 µl of permeabilization solution (0.5% Triton X-100, 0.1 M glycine in PBS) for 30 min at room temperature on an orbital shaker (50 rpm).
8.- Rinse 3 times with PBS + 0.1% tween 20.
9.- Block for 1 hour at room temperature on a shaker (50 rpm) with blocking solution (10% Normal donkey serum, 0.1% BSA, 0.1% tween 20 in PBS).
10.- Dilute antibodies in the blocking solution to the desired working concentrations (between 1:100 or 1:400 depending on antibody used).
11.- Add 200 µl of antibody mix.
12.- Incubate overnight at 4ºC on shaker (50 rpm) inside a wet chamber to avoid evaporation. This can be a sealed box with a wetted paper in the bottom.
13.- Rinse 3 times with PBS + 0.1% tween 20.
14.- Prepare secondary antibody mix in a blocking solution (1:200 dilutions).
15.- Centrifuge antibody solution at 10,000 g for 1 min to reduce antibody precipitates.
16.- Add 200 µl of antibody mix and incubate 1h at room temperature on the shaker (50 rpm).
17.- Stain nuclei with DAPI 1:1000 for 10 min at room temperature.
18.- Rinse 3 times with PBS.
19.- SEMs are mounted on PBS or PBS with 0.1% Tween 20 by gently moving them with a plastic Pasteur pipette on a 35 mm petri dish with a 1.5 cm coverslip glass bottom.
20.- Disperse the SEMs homogenously at the coverslip and examine under the confocal or later light-sheet microscopes as described below.
Confocal Imaging of human SEMs/aggregates
To assess the quality of the experiment and select putative SEMs for further imaging, an overview of the majority of the SEMs is gathered with a tiled scanning implemented in Zeiss LSM 700 and LSM 800 inverted confocal microscopes. The SEMs representative of a single aggregation and immunofluorescence experiment, were mounted in a 35 mm glass bottom dish (Mattek, P35G-1.5-14-C) covered with PBS. To generate the overview images, a 10× EC Plan Neofluar air objective (0.3 NA) or 20x Plan-Apochromat air objective (1.0 NA) were employed using the Zen software black edition (ZEISS).
Confocal imaging of multiple SEMs/aggregates
1. After mounting the sample, the image acquisition parameters were established for each of the assessed wavelengths (405, 488, 568, 647), according to the used secondary antibodies.
2. Tiled images were obtained to sample a significant portion or most of the aggregates.
3. Since aggregates are dispersed in different directions and angles, Z-stacks were used to better understand their structure.
4. The efficiency of the experiment can be calculated after gathering the overview image (see below). In addition, the images of individual SEMs/aggregates can be taken by movement of the objective to the recorded positions within a tiled scanning.
5. For light-sheet microscopy, the SEMs were examined and picked with a mouth pipette (aspirator tube; Sigma, A5177), connected to a thinned glass capillary pulled from a glass microliter capillary (Blaubrand intraMark 708744) with an inner diameter above the diameter of the SEM. The SEM of interest is taken from the imaging plate under the binocular view, while trying to avoid disturbing other aggregates and without generating air bubbles. The picked SEM is placed in a new drop of PBS on a petri dish for further analysis.
Confocal imaging of individual SEMs/aggregates
1. The previously collected overview images of the experiment are used to select the SEMs based on the microscope stage position. Importantly, to maintain the same stage position, the SEMs should not be moved or disturbed during the entire imaging process. Hence, for every single imaging session, the new overview image is required.
2. Redirect the stage to one of the chosen positions, and switch to a higher magnification objective and recalibrate acquisition parameters for each channel.
3. Use the digital zoom and rotation to center the SEM as desired. Importantly, ensure the entire SEM is fit into the imaging frame when examining morphology.
4. If 3D volumes are aimed, set the beginning and end frames to cover the SEM and select the slice spacing according to the desired sampling along the Z-axis.
5. Optionally, use the laser intensity adjustment (implemented in Zeiss LSM) to reduce decay of the signal along the Z-axis.
6. Acquire the image.
Quantification of the SEM structure experiment efficiency
Quantification of the efficiency of the experiment is performed based on the overview tile scanned images (see “Confocal imaging of multiple SEMs/aggregates”). The cell-type markers for immunostaining should be selected prior to the experiment depending on the defined efficiency criteria. For assessing the efficiency and quality of the experiments, we recommend using markers for the main three lineages: epiblast (OCT4, SOX2), (secondary) yolk-sac (SOX17), and trophoblast (GATA3, CK7 or SDC1).
1. Using FIJI software, open the previously acquired overview image.
2. Using the Cell Counter tool, manually count the total number of aggregates that entirely fit into the image.
3. Subsequently, select the aggregates that meet the desired criteria using another Cell Counter tool. Calculate the efficiency of the experiment by dividing the number of adequate SEMs by the total number of structures. Multiply by 100 to get percentage values.
Other efficienies can be conducted similarly by choosing the right immunostaining markers for the embryonic structures/lineages/cell types being co-quantified.
The immunofluorescence images are acquired using Zeiss Z7 light-sheet microscope, equipped with 405 nm, 488 nm, 561 nm, and 638 nm lasers, using a single water 20x Plan-Apochromat (numerical aperture 1.0) detection objective (Zeiss) and two air 10x Plan-Apochromat (numerical aperture 0.2) illumination objectives (Zeiss).
1. Prior to imaging, mount the sample in a glass capillary filled with 1% low-melting temperature agarose prepared in PBS. Depending on the size of the SEM, use a glass capillary with an inner diameter of 0.5 – 2 mm. Mount the capillary inside the sample holder of the Z7 light-sheet microscope.
2. Upon solidification, pull the agarose out of the capillary with a custom plunger to hang the mounted sample into the imaging chamber filled with PBS.
3. Acquire the image of the sample from several angles using multiview acquisition. Light-sheet volumes along the Z-axis are acquired in a dual scanning mode, using a pivot scan.
4. The light-sheets for left- and right-side illuminations can be adjusted independently inside the sample volume for each channel based on the signal intensity in the focal plane of the detection lens.
Light-sheet image processing is performed in ZEN 3.5 software.
1. Dual side images are fused based on the maximum intensity signal.
2. Multiview fusion is performed using interactive registration of the brightest channel (typically DAPI) or each channel independently in front and side views.
3. The image deconvolution is applied for single-view images prior to their fusion in the "Fast Iterative" or “Constrained Iterative” settings.
Immunostaining of cells for flow cytometry analysis
To corroborate the quality of the inductions before aggregation, flow cytometry analysis can be performed for PDGFRa (for RCL) and ENPEP/TACSD2 (BAP(J) it is expected to find around 50-60% PDGFRa cells (by day 6) and 85-90% double-positive cells in the TE (day 3), on optimal inductions.
To stain the cells for these markers:
1.- Collect the cells with TrypLE as mentioned above.
2.- Wash the cells once with PBS by resuspending the pellet.
3.- Prepare blocking solution (PBS + 0.5% BSA).
4.- Dilute antibodies in the blocking solution to have a total volume of 100 µl per sample (1x106 cells, with a 1:20 antibody concentration).
5.- Incubate for 30 minutes on ice and covered from the light.
6.- Wash two times with PBS.
7.-Resuspend with 500 µl of PBS.
8.- Pass through a 40 µm cell strainer.
9.- Read out with the Flow Cytometer. Ensure having adequate controls, including negative control samples and unstained induced cells.