Culture of mouse embryonic stem cells and mouse embryonic stem cells transiently expressing Gata4
1. We cultured mouse ESCs in N2B27/2iLIF on gelatinised, tissue culture-grade plates in an incubator kept at 37°C with 5% CO2 and 21% O2.
2. When the culture was 70/80% confluent, or the colonies had begun to touch and merge, or they had started flattening, we passaged the cells.
3. 1 mL of gelatine was added to a clean well of a tissue culture dish in preparation for replating. We incubated the plate at room temperature in the biosafety cabinet until needed (at least 5 min).
4. To passage, we washed the culture once with 1 mL of 1x PBS and then added 500 uL-1ml of Trypsin; we incubated the plate in the incubator for 4 minutes.
5. We stopped the trypsinisation reaction by adding 1-2 volumes of FC medium and then we resuspended the cell suspension 2-3 times by gentle pipetting with a P1000 to break the cells colonies.
6. We transferred the cell suspension to a 15 mL Falcon tube and centrifuged it at 0.2 x g (RCF) for 4 minutes.
7. Following centrifugation, we carefully aspirated the medium as not to disturb the cell pellet and then washed the cells with 1 mL of PBS.
8. To wash, we gently resuspended the culture twice with the P1000 and then centrifuged them one more time at 0.2 x g.
9. After centrifugation, we carefully aspirated the PBS and gently resuspended the cell culture in 1 mL of N2B27/2iLIF by pipetting twice with the P1000.
10. We aspirated the gelatine from the plate and added 2 mL of N2B27/2iLIF to the well. We plated the cells at a dilution of either 1:5, 1:10, 1:20. In our experience, we found that a confluent culture plated at 1:5 will need to be re-passaged on the following day; a culture plated at 1:10 will be ready after 2 days and a culture plated at 1:20 will be confluent after 3 days.
11. We mixed the cells evenly by gently moving the plate side to side and front-back three times (or in a figure of 8 motion) and placing the plate in the incubator.
Culture and passaging of TSCs
We passaged TSCs similarly to ESCs but with the following differences:
12. Six hours before passaging, we replaced the FC medium of a well coated with inactive MEFs with 2 mL of TSF4H medium. We find that medium conditioning prevents differentiation of TSCs.
13. After 6 hours, we aspirated the medium from TSCs and washed them twice with PBS.
14. We added 500 uL-1ml of Trypsin and incubated the plate at 37°C for 4 minutes.
15. We stopped the trypsinization reaction with 2 mL of TS medium.
16. We dissociated TS cells by gentle pipetting with the P1000 for 3-4 times.
17. We transferred the cell suspension to a 15 mL Falcon tube and centrifuged it for 4 minutes at 0.2 x g (RCF).
18. Following centrifugation, the supernatant was carefully aspirated without disturbing the pellet.
19. We added 1 mL of TSF4H and resuspended the cells by gentle pipetting with the P1000.
20. We plated TS cells either at 1:10 or 1:20. Denser or sparser dilutions may cause differentiation of the cells. Cells plated at 1:20 will general be confluent again after 4 days; cells plated at 1:10 will generally be confluent after 3 days.
21. After the passage, we changed media on the cells on the following day; after that, the medium was topped up by adding 2 mL of TSF4H.
Preparation and seeding of cells for ETiX-embryoid formation
22. On the day before the experiment, tetO-Gata4 ESCs were passaged as described earlier and plated at a dilution of 1:5. This was to ensure and homogenous response to doxycycline.
23. On the day of the experiment, we provided fresh media to the ESCs and tetO-Gata4 ESCs. Six hours before the start of the experiment, we added Dox (1:1000 dilution) to the tetO-Gata4 ESCs and we topped up the medium on TSCs. We also changed the medium on one plate of MEFs to TSF4H to allow them to condition it. This was then used for propagation of the TSC line.
24. After six hours of induction with Dox, we began the experiment.
25. 500 mL of rinsing solution was added to the desired number of AggreWells, the plate was then centrifuged at 2000 x g (RCF) for 5min and incubated at room temperature in the biosafety cabinet until the cell suspensions were prepared and quantified. The plate was checked for bubbles and if present, the plate was centrifuged again for an additional 5 min.
26. We placed the appropriate amount of FC medium to equilibrate in a Falcon tube with a loose cap in the incubator. Each AggreWell requires 1.5 mL of medium in total. 0.5ml of this is placed in the AggreWell first and the remaining 1ml is supplemented with Rock inhibitor and used for the resuspension of cells (see later steps 33 and 34).
27. We also put 1 mL of gelatine in a well of a tissue culture treated 6 well plate, which we incubated at room temperature. This is required for MEF depletion from the TSC suspension.
28. We dissociated ESCs and TSCs as described earlier. At the end of the dissociation, TSCs were resuspended in 2 mL of TSF4H, and plated in the well that was coated with gelatine (in step 27).
29. We incubated the TSC suspension for 20-30 minutes to allow for the MEFs to stick to the plate and leave an enriched suspension of TSCs in the supernatant. After 20 minutes, the cell suspension was collected for the experiment. If needed, a fraction of this suspension was also replated onto inactive MEFs for propagation (see step 23).
30. At the end of the dissociation and after a wash in PBS, ESCs and tetO-Gata4 ESCs were resuspended in 1-2 mL of FC medium. 10ul of this suspension were collected for quantification and the remainder of cells were placed in the incubator until needed.
31. We quantified the cell suspensions using a haemocytometer.
32. We transferred the volumes yielding 6,000 ESCs, 6,000 tetO-Gata4 ESCs and 19,200 TSCs to a 15 mL Falcon tube and centrifuged them for 4 minutes at 0.2 x g (RCF).
33. The rinsing solution was aspirated from the AggreWell and we rinsed the wells with 1 mL of PBS twice and then twice with 1 mL of FC. After these washes, we added 500 uL/well FC that was equilibrated in step 26. We placed the AggreWell plate back in the incubator until the cells were ready to be plated.
34. We added Rock inhibitor at a final concentration of 0.5 nM to the remaining medium in the incubator and used it to resuspend the cells at the end of the centrifugation (for a 5uM stock we used Rock inhibitor at 1:10000). 1 mL of medium with Rock inhibitor is added for each well to be plated.
35. We added cells to the AggreWell dropwise on the centre of each well.
36. We centrifuged the plate for 3 minutes at 100 x g (RCF), and placed the plate in the incubator (Day 0).
37. On the next day (Day 1), media change was performed twice by removing 1ml of medium from each well and adding 1ml of fresh FC medium without ROCK inhibitor. The media changes need to be performed very gently exercising great care as not to disturb the structures forming within the wells. Media is removed and added slowly by placing the pipette tip against the side of the wells, avoiding formation of bubbles.
38. On D2, media change was performed once to replace 1ml of medium with 1ml of fresh FC medium.
39. On D3, 1 ml of medium was removed from each well and 1.5ml of IVC-20% FBS was added, following its equilibration for 20 minutes in the incubator.
40. On D4, ETiX-embryoids in the AggreWell were transferred to CELLSTAR 6 well multiwell plate for suspension culture (Greiner Bio-One 657185) with 5ml of IVC-30% FBS per well.
41. All ETiX-embryoids were collected from AggreWell for analysis at 4 days of development and analysed under a stereomicroscope. We selected ETiX-embryoids with cylindrical morphology and two clearly defined cellular compartments (an ESC compartment and TSC compartment) surrounded by an outer cell layer, the VE-like layer. We expect the ESC compartment to be epithelialized with a lumen. The TSC compartment is more variable in appearance and therefore, even though one would also want an epithelial-looking TSC compartment similar to the extra-embryonic ectoderm of natural embryos, we select a wider range of appearances for the TSC compartment. Since the majority of ETiX-embryoids were generated by using wild-type, unlabelled stem cell lines, the selection was based on morphology alone. ETiX-embryoids with the correct body plan of ESC and TSC compartments surrounded by a VE-like layer were then transferred to equilibrated media to continue their culture. At day 4, we were able to collect 10%-15% of the structures formed in the pyramidal microwells.
42. When selecting at D5, however, we included additional criteria: i) we expect the lumen of the ESC and TSC compartment to be merged; ii) ideally we can observe the beginning of gastrulation on one side of the ETiX-embryoids; iii) we expect the AVE to have migrated to the ESC-TSC boundary and be opposite to the forming streak; iv) ETiX-embryoids with the AVE stuck at the tip of the structure or not at the boundary were excluded. From day 4 to day 5 we cultured 20% of the structures collected at day 4.
43. On Day 5 each ETiX-embryoid was transferred to a single well of 48-well, non-adherent dish with 250 uL of DRH or EUCM medium.
44. On day 6 each ETiX-embryoid was fed with an additional 250 uL of DRH/EUCM.
45. A supplement of 3.0 mg/mL of D-(+)-Glucose (Sigma G8644) was added to DRH/EUCM medium on Day7 when the ETiX-embryoids were moved to the rotating bottle culture chamber apparatus. Each rotating bottle contained 2 mL of medium and 3 ETiX-embryoids.
46. On day 8 DRH/EUCM medium was further supplemented with 3.5 mg/mL of D-(+)-Glucose. In each rotating bottle 2 ETiX-embryoids were cultured with 3 mL of medium.
47. At the end of day 8, we dissected and fixed the structures in 4% PFA for 20 minutes at room temperature in preparation for downstream analyses.