Materials and Reagents

Mouse embryonic stem cells (ESCs)
M15 cells (ECACC: catalog number: 95102517 )
Mouse embryonic fibroblast (MEF)
0.1% gelatin
PBS
Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco^®, catalog number: 11995-075)
DMEM low glucose (Gibco^®, catalog number: 11885-084 )
Fetal Bovine Serum (FBS) (Hyclone, catalog number: SH30310.03 )
200 mM L-glutamine (L-Gln) (Nacalai Tesque, catalog number: 16948-04 )
5,000 units/ml mixture of penicillin and streptomycin (PS) (Nacalai Tesque, catalog number: 26252-94 )
10 mM MEM Non-Essential Amino Acids Solution (NEAA) (Gibco^®, catalog number: 11140-050 )
2-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M7522 )
D-(+)-Glucose (Sigma-Aldrich, catalog number: G5146 )
KnockOut Serum Replacement (KSR) (Gibco^®, catalog number: 10828 )
Recombinant human LIF (Wako Pure Chemical Industries, catalog number: 129-05601 )
Recombinant human Activin A (R&D Systems, catalog number: 338-AC )
Recombinant human bFGF (Pepro Tech, catalog number: 100-18B )
BIO (Calbiochem^®, catalog number: 361550 )
DAPT (Wako Pure Chemical Industries, catalog number: 041-30983 )
Mitomycin C (MMC) (Sigma-Aldrich, catalog number: M4287 )
0.05% Trypsin-EDTA (Life Technologies, catalog number: 2014-11)
0.25% Trypsin-EDTA (Life Technologies, catalog number: 25200-072 )
EF medium (see Recipes)
Maintenance Medium (see Recipes)
EB medium (see Recipes)
Endoderm Medium (see Recipes)
Intestinal Medium (see Recipes)

Equipment

6 well plate (Corning, catalog number: 3516)
100 mm dish (Corning, catalog number: 430167 )
60 mm dish (BD Biosciences, Falcon^®, catalog number: 353004 )
Centrifuge
37 °C 5% CO₂ Cell culture incubator

Procedure

Preparation of M15 cells
1. M15 cells (5x10⁶) are plated to a 100 mm dish with 10 ml EF medium.
2. M15 cells are incubated in the 5% CO₂ incubator at 37 °C.
3. When M15 cells are confluent (for 2-3 days), passage from a 100 mm dish to five 100 mm dishes.
  1. M15 cells are washed by PBS.
  2. M15 cells are trypsinized with 1 ml 0.05% Trypsin-EDTA at 37 °C for 5 min.
  3. M15 cells are pelleted at 190 x g for 5 min.
  4. M15 cells are plated to five 100 mm dishes.
4. When M15 cells are confluent, M15 cells are incubated with 10 μg/ml MMC containing EF medium in the 5% CO₂ incubator at 37 °C for 2 h. M15 cells are treated with MMC to stop M15 cell from proliferation.
5. MMC-treated M15 cells are washed by PBS.
6. MMC-treated M15 cells are trypsinized with 1 ml 0.05% Trypsin-EDTA at 37 °C for 5 min.
7. M15 cells are pelleted at 190 x g for 5 min.
8. Resuspend and freeze MMC-treated M15 cells into cryovials in 22% FBS and 20% DMSO containing DMEM at 1 x 10⁷ cells per tube at -150 °C.
Preparation of MEF
1. MEF (5x10⁶) are plated to a 100 mm dish with 10 ml EF medium.
2. MEF are incubated in the 5% CO₂ incubator at 37 °C.
3. When MEF are confluent (for 2-3 days), passage from a 100 mm dish to five 100 mm dishes.
  1. MEF are washed by PBS.
  2. MEF are trypsinized with 1 ml 0.05% Trypsin-EDTA at 37 °C for 5 min.
  3. MEF are pelleted at 190 x g for 5 min.
  4. MEF cells are plated to five 100 mm dishes.
4. When MEF cells are confluent, MEF are incubated with 10 μg/ml MMC containing EF medium in the 5% CO₂ incubator at 37 °C for 2 h. MEF are treated with MMC to stop M15 cell from proliferation.
5. MMC-treated MEF are washed by PBS.
6. MMC-treated MEF are trypsinized with 1 ml 0.05% Trypsin-EDTA at 37 °C for 5 min.
7. M15 cells are pelleted at 190 x g for 5 min.
8. Resuspend and freeze MMC-treated M15 cells into cryovials in 22% FBS and 20% DMSO containing DMEM at 2 x 10⁶ cells per tube at -150 °C.
Maintenance of mouse ESCs
1. 2 x 10⁶ MMC-treated MEF cells are plated to a gelatin-precoated 60 mm dish with EF medium.
2. MMC-treated MEF cells are incubated in the 5% CO₂ incubator at 37 °C overnight.
3. Mouse ESCs (5x10⁵) are plated on MEF-coated dish with Maintenance Medium, and are incubated in the 5% CO₂ incubator at 37 °C (day 0) until passage (3 days).
4. Medium is changed every day.
Intestinal differentiation of mouse ESCs
1. 1 x 10⁷ MMC-treated M15 cells are plated to two gelatin-coated 6 well plates and are incubated in the 5% CO₂ incubator at 37 °C overnight.
2. ESCs plated on MEF-coated dishes (step B3) are washed by PBS.
3. ESCs are trypsinized with 1 ml 0.25% Trypsin-EDTA at 37 °C for 5 min.
4. ESC cells are pelleted at 190 x g for 5 min.
5. The supernatant is removed and 1 x 10⁶ cells/ml resuspended in the EB Medium.
6. 2 x 10⁴ ESCs in 2 ml EB medium are plated to a M15-pre-coated well (day 0).
7. Medium is changed to Endoderm Medium at day 1 and 3.
8. Medium is changed to the Intestinal Medium at day 5.
9. Medium is changed with 2 ml Intestinal Medium at day 7, 9, 11, 13, 15, 17 and 19 (Figure 1).
  
  Figure 1. Mouse ESC-derived Cdx2 (red)-expressing intestinal endoderm at day12 (left) and PAS-positive mucus secreting functional goblet cell at day 20 (Right). Scale bar indicates 100 μm.

Recipes

EF medium
DMEM high glucose containing 10% FBS
2 mM L-Gln
50 units/ml PS
Maintenance Medium
DMEM high glucose containing 10% FBS
2 mM L-Gln
50 units/ml PS
100 μM NEAA
100 μM 2-ME
LIF
EB Medium
DMEM high glucose containing 10% FBS
2 mM L-Gln
50 units/ml PS
100 μM NEAA
100 μM 2-ME
Endoderm Medium
EB medium with 10 ng/ml Activin A
5 ng/ml bFGF
Intestinal Medium
2 ml DMEM low glucose with 1 mg/ml D-(+)-Glucose
10% KSR
2 mM L-Gln
50 units/ml PS
100 μM NEAA
100 μM 2-ME
5 μM BIO
10 μM DAPT

Acknowledgments

This protocol is adapted from Ogaki et al. (2013). This work was supported by a Grant-in-Aid (#21390280 to S.K. and #21790671 to N.S.), and in part by a Global COE grant (Cell Fate Regulation Research and Education Unit, to S.K.), and a grant from the Project for Realization of Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan.

References

Ogaki, S., Shiraki, N., Kume, K. and Kume, S. (2013). Wnt and Notch signals guide embryonic stem cell differentiation into the intestinal lineages. Stem Cells 31(6): 1086-1096.