Differentiation medium (DM): GMEM supplemented with 10% knockout serum replacement (KSR), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 100 unit/mL penicillin potassium, and 100 μg/mL streptomycin sulfate and 55 μM monothioglycerol.
Epithelial differentiation medium (EDM): DM and CnT-PR w/o; EGF and FGF2 (1:1) containing 20 ng/mL KGF and 10 μM Y-27632, 100 unit/mL penicillin potassium and 100 μg/mL streptomycin sulfate.
Ocular surface epithelial differentiation medium (OSEM): DMEM/F12 containing 2% B-27 supplement, 20 ng/mL KGF, 10 μM Y-27632, 100 unit/mL penicillin potassium and 100 μg/mL streptomycin sulfate.
Lacrimal gland culture medium (LGM): DMEM/F12 containing 2% B-27 supplement, 10 ng/mL EGF, 10 μM Y-27632, 100 unit/mL penicillin potassium and 100 μg/mL streptomycin sulfate.
KCM medium: DMEM without glutamine and Nutrient Mixture F-12 Ham (3:1) supplemented with 5% FBS, 0.4 µg/ml hydrocortisone succinate, 2 nM 3,3',5-Triiodo-L-thyronine sodium salt, 1 nM cholera toxin, 2.25 µg /ml bovine transferrin HOLO form, 2 mM L-glutamine, 0.5% insulin transferrin selenium solution, and 100 unit/mL penicillin potassium and 100 μg/mL streptomycin sulfate.
The use of an appropriate hPSC clone is important to generate lacrimal gland-like tissue organoids. In our hands hiPSC lines 201B7 and YZWJs524, and hESC line KhES-1 all were able to generate lacrimal gland-like organoids. Initially, hPSCs are cultured in StemFitTM medium on LN511E8-coated 6-well culture plates.
1. Coat new 6-well culture plates with LN511E8 at 0.5 µg/cm2 in 1.5 ml PBS by incubation at 37°C for at least 60 min.
2. Add 0.7 mL StemFitTM medium to the culture plates.
3. Remove the mixed solution.
4. Immediately add 1.5 mL StemFitTM medium containing 10 µM Y-27632 and incubate at 37°C in 5% CO2.
1. Remove the medium.
2. Wash hPSCs once with 1 mL PBS.
3. Add 300 µL dissociation solution (50% TrypLE select and 50% 0.5 mM EDTA solution) and incubate for 4-5 min at 37°C.
4. Remove the dissociation solution carefully (hPSCs are still attached to the culture plates at this point).
5. Wash the hPSCs once with 2 mL PBS, and then add 1 mL StemFit™ medium containing 10 µM Y-27632.
6. Harvest the hPSCs with a Cell Scraper.
7. Pipette the hPSCs about 10 times with a 200 or 1000µL micropipette, then collect the dissociated hPSCs in a 1.5 mL tube.
8. Count the collected hPSCs using a Countess® Machine after staining with Trypan blue (settings for Countess®; Sensitivity: 5, Minimium size: 8, Maximum size: 30, Circularity: 75).
9. Seed hPSCs on LN511E8 coated 6-well culture plates at 13,000 cells/well.
10. Incubate the plates at 37°C in 5% CO2.
11. The next day, change the medium to fresh StemFitTM medium without Y-27632 and incubate at 37°C in 5% CO2 up to day seven. The medium should be changed every two days.
Critical point: If hPSCs are maintained under other conditions (e.g. co-cultivation with MEF feeder layers) after removal of the feeder cells, the dissociated hPSCs must be seeded in the StemFitTM/LN511E8 culture system at 13,000 cells/well and be passaged at least five times to adapt to the conditions.
Preparing hPSCs for differentiation (Day -10)
1. Coat a 6-well or 12-well culture plate with LN511E8 at 0.5 µg/cm2 as previously indicated.
2. Harvest hPSCs using the same method (above) as that employed for hPSC passaging.
3. Seed dissociated hPSCs on 6-well plates at 3000-6000 cells/well, or on 12-well plates at 1000-2000 cells/well in 1.5 mL (6-well plate) or 0.6 mL (12-well plate) of StemFitTM medium containing 10 µM Y-27632.
4. Incubate the plates at 37°C in 5% CO2.
5. The next day, change the medium to fresh StemFitTM medium without Y-27632.
6. Incubate the plates at 37°C in 5% CO2 for 10 days in total. The medium should be changed every two days before day seven, and every day thereafter.
Ocular surface epithelial cell differentiation culture (Day 0-)
7. After 10 days culture in StemFitTM medium, change the medium to 2 mL (6-well plate) of DM (Day 0).
8. Incubate the plates at 37°C in 5% CO2 for four weeks, changing the medium every 2-3 days (i.e. three times per week). At around three weeks, hPSC colonies should appear as multi-cellular colonies formed of 3-4 concentric zones (this is called a “SEAM”; Self-formed Ectodermal Autonomous Multi-zone)9,10. Cells in the third zone from the centre of the colony are ocular surface ectodermal cells, which we have shown can differentiate into corneal and conjunctival epithelium.9-12
EDM culture (Day 28-)
9. After four weeks of incubation in DM, change the medium to 2 mL (6-well plate) of EDM.
10. Incubate the plates for an additional four weeks at 37°C in 5% CO2 (i.e. eight weeks in total). The medium should be changed every 2-3 days (three times per week). Optional: To promote retinal cell differentiation, the medium can be changed to OSEM at day 28. After incubation in OSEM for an additional 2-3 weeks (i.e. 6-7 weeks in total), cells with characteristics of pigmented retinal pigment epithelial (RPE) cells are frequently observed.
Manual pipetting to remove non-epithelial cells (Day 49-)
During cultivation in EDM, manual pipetting should be performed to remove non-epithelial cells (e.g. neuronal cells, retinal cells and/or lens cells) from the SEAMs. We have found that this significantly enhances the yield of corneal epithelial cells on subsequent cell sorting compared to the yield without manual pipetting.
11. At around three weeks of incubation in EDM (i.e. after seven weeks in total), perform manual pipetting using a PIPETMANTM (1000 or 200 µL) on a clean bench.
12. After pipetting, change the medium (which will contain the detached cells) to fresh EDM.
13. Incubate the plates for an additional one week in EDM (i.e. eight weeks in total).
OSEM culture (Day 56-)
14. After EDM culture for four weeks (i.e. eight weeks overall) change the medium to 2 mL (6-well plate) of OSEM.
15. Incubate the plates for either two weeks (i.e. 10 weeks in total) or four weeks as a maximum (i.e. 12 weeks in total) at 37°C in 5% CO2. The medium should be changed every 2-3 days (three times per week).
FACS for isolating ocular surface epithelial stem cells (Day 70-)
16. After OSEM culture, wash the hPSCs once with PBS.
17. Add AccutaseTM to the differentiated hPSCs and incubate for 45 min at 37°C.
18. Perform pipetting several times and again incubate for 15 min at 37°C.
19. Collect and re-suspend the dissociated cells in ice-cold KCM medium in a STEMFULLTM 15 mL tube.
20. Filter the cells using a Cell Strainer (40 µm) and count the cell number.
21. Centrifuge the cells at 1200 rpm for 8 min.
22. Aspirate the supernatant and re-suspend the cells in ice-cold KCM containing FITC-conjugated SSEA-4 (MC813-70), PE-conjugated CD104 (ITGB4; 58BX4) and PE-Cy7-conjugated anti-CD200 (OX-104) antibodies.
23. Incubate the cells for 1 hr at 4°C (agitate the cells every 20 min).
24. Wash the cells twice with PBS.
25. Resuspend the cells in PBS by filtration using a Cell strainer (40 µm). Cells should be stained with non-specific isotype IgG (for SSEA-4, CD104 and CD200) as controls. For each fluorescent probe, single-colour stained cells should be prepared for colour compensation during flow cytometry.
26. Set up the FACSAriaII or SH800 cell sorter.
27. Subject stained cells to sorting in the FACSAriaII or SH800.
28. Perform compensation between the detectors for FITC, PE, and PE-Cy7 using each of the single stained cells.
29. Analyze the triple colour-stained cells.
30. Sort the SSEA-4+, CD104+, and CD200- population (i.e. the ocular surface epithelial stem cell fraction) to 8 mL of KCM in a STEMFULLTM 15 ml tube. Typically, in CD200 negative cells, around 30-50% of the cells are detected as the ocular surface epithelial stem cell fraction.
31. Collect the ocular surface epithelial stem cells by centrifugation at 1200 rpm for 8 min.
32. Re-suspend the cells in LGM on ice.
Formation of lacrimal gland organoid (Day 70-)
33. Cultivate the sorted ocular surface epithelial stem cells (i.e. CD200−, SSEA-4+, ITGB4+) in OSEM in the wells of a non-adhesive, round-bottomed 96-well plate for one day at 1 × 105 cells per well. This results in the formation of a spheroid of lacrimal gland-like progenitors.
34. Embed spheroids in 50% (vol/vol) of Matrigel (growth factor reduced) diluted in LGM, and culture at 37°C in 5% in CO2 for approximately 20 days. The medium should be changed every 2-3 days (three times per week).