Materials and Reagents

Tissue culture plate, 24 well (Corning, Falcon^®, catalog number: 353047 )
15 ml conical tube (Corning, Falcon^®, catalog number: 352196 )
MEF feeder cells (University of Michigan, Transgenic Animal Model Core, https://www.med.umich.edu/tamc/list.html#reagents)
0.2% gelatin solution (Sigma-Aldrich, catalog number: G1890 )
PBS
Collagenase type IV (Thermo Fisher Scientific, Gibco^TM, catalog number: 17104-019 )
0.05% trypsin (Thermo Fisher Scientific, Gibco^TM, catalog number: 25300-054 )
The Vector^TM alkaline phosphatase (AP) staining kit (Vector Laboratories, catalog number: SK-5100 )
Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11995-065 )
Fetal bovine serum (FBS) (Atlas Biologicals, catalog number: F-0500-D )
L-glutamine (200 mM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030-081 )
100x non-essential amino acids (Thermo Fisher Scientific, Gibco^TM, catalog number: 11140-050 )
100x sodium pyruvate (Thermo Fisher Scientific, Gibco^TM, catalog number: 11360-070 )
2-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
Note: This product has been discontinued.
KnockOut^TM D-MEM (Thermo Fisher Scientific, Gibco^TM, catalog number: 10829018 )
KnockOut^TM serum replacement (KSR) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10828028 )
Glutamax (Thermo Fisher Scientific, Gibco^TM, catalog number: 35050061 )
Fibroblast growth factor basic (FGF2), human recombinant (R&D Systems, catalog number: 233-FB )
ESGRO^® leukemia inhibitory factor (LIF) (EMD Millipore, catalog number: ESG1107 )
MM-401
DMSO
Mouse embryonic fibroblasts (MEFs) culture medium (see Recipes)
EpiSCs culture medium (see Recipes)
Embryonic stem cells (ESCs) culture medium (see Recipes)
100 mM MM-401 (see Recipes)
MM-401 reversion medium (see Recipes)
MM-401 maintenance medium (see Recipes)

Equipment

1 ml pipette
Tissue culture incubator (Isotemp^TM Microbiological Incubator) (Fisher Scientific, model: Fisher Scientific^TM Isotempcatalog^TM Microbiological Incubator, catalog number: S28670 ), humidified, 37 °C and 5% CO₂ in air
Stereomicroscope (Olympus, model: DP73 )
Centrifuge (Eppendorf, model: 5810 R ; Swing-bucket rotor: Eppendorf, model: A-4-81 )

Procedure

A schematic summary of the reprogramming procedure described in this protocol can be found in Figure 1.

Figure 1. The schematic view of general procedures for the reversion of EpiSCs by MM-401 treatment

Preparation of MEF feeder cells: Irradiated MEF feeder cells are seeded at 1 x 10⁵ cell/well in MEF medium in a 24-well plate (pre-coated with 0.2% gelatin solution).
EpiSC culture: EpiSCs are routinely cultured following previously described protocol (Chenoweth and Tesar, 2010). For chemical treatment experiments, EpiSCs are cultured in the 24-well plate and enzymatically passaged every third day (see Figure 2).

Figure 2. Representative phase image of EpiSCs at the third day of culture before splitting. Scale bar = 50 μm.

To passage,
1. Remove EpiSC culture medium from each well and wash briefly with PBS. Discard PBS and add 150 μl of collagenase type IV or 0.05% trypsin to each well, and incubate at room temperature for up to 4 min.
2. Add 1 ml of EpiSC culture medium (without FGF2) to each well and gently pipet with a 1-ml pipette. Combine suspensions from 1 to 2 wells into a 15-ml conical tube. Add EpiSC culture medium (without FGF2) to a final volume of 5 ml to each tube for centrifugation.
3. Separate EpiSCs from MEFs (Optional):
Plating of EpiSCs for reversion: EpiSCs were passaged in single cell or small clumps on MEF feeder cells (as described in step 1) in MM-401 reversion medium.
1. Centrifuge the EpiSCs (from step 2b or 2c), aspirate and discard the supernatant. Resuspend EpiSC pellet in MM-401 reversion medium. For the 24-well plate, add 400 μl MM-401 reversion medium to each well.
2. For EpiSCs passaging in small cluster, replate cells to a new well with 1:4-1:5 dilutions. For EpiSCs passaging in single cells, plate the cells at 20,000/cm² (approximately 4 x 10⁴ cell/well for the 24-well plate, see Figure 4).
  
  Figure 4. Representative image of single EpiSCs plated at the density of 20,000 cell/cm². Scale bar = 200 μm.
Establish MM-401-reverted ESC line

Morphologically distinct reverted naïve colonies become evident over the next 3-6 days. AKP is used to stain the colonies to evaluate reversion efficiency. Strong AKP staining distinguishes the reverted naïve colonies from EpiSCs that have weak AKP staining (see Figure 5). AKP activity is detected using Vector^TM alkaline phosphatase (AP) staining kit according to the manufacturer’s instructions.

Figure 5. AKP staining of EpiSCs and reverted naïve colonies after MM-401 treatment as indicated on top. Left: Flat EpiSC clones with weak AKP staining before MM401 induction; Right: Two representative images of EpiSCs taken at 72 h of 100 μM MM-401 treatment. The reverted cells (indicated by arrows) are morphologically distinct from EpiSCs. The colonies are dome-shaped with intense AKP staining. Scale bar = 100 μm
The reversion culture is passaged by a brief exposure (4-5 min) to 0.05% trypsin/EDTA with gentle pipetting into single cells. Cells can be re-seeded at 20,000-50,000/cm². MM-401 is replenished to reach 50-100 μM final concentrations in each passage.
Reverted ESC (rESC) lines can be established by clone picking or en masse passaging. The established rESC line can be maintained, passaged, cryopreserved and thawed by the standard protocol for conventional mESCs.
rESC line can be expanded in MM-401 maintenance medium or ESC culture medium without MM-401 after passage 6.

Notes

For reversion, MM-401 (50-100 μM final concentration) can be added to culture medium immediately or 2-3 days after EpiSCs forming clones.
Mediums containing MM-401 are recommended to be pre-warmed at room temperature (RT) for 15 min.
For MM-401 request: please contact yalid@med.umich.edu.

Recipes

Mouse embryonic fibroblasts (MEFs) medium
DMEM supplemented with:
10% FBS
2 mM 1x L-glutamine
1x nonessential amino acids
1x sodium pyruvate
0.1 mM 2-mercaptoethanol
EpiSC culture medium
KnockOut^TM D-MEM supplemented with:
20% KSR
2 mM Glutamax
1x nonessential amino acids
1x sodium pyruvate
0.1 mM 2-mercaptoethanol
10 ng/ml FGF2
Note: The medium can be stored at 4 °C for up to 2 weeks.
Embryonic stem cells (ESCs) culture medium
KnockOut^TM D-MEM supplemented with:
20% FBS
2 mM Glutamax
1x nonessential amino acids
1x sodium pyruvate
0.1 mM 2-mercaptoethanol
10³ U/ml LIF
Note: The medium can be stored at 4 °C for up to 2 weeks.
100 mM MM-401
MM-401 (Molecular Mass: 700.75) is dissolved in DMSO at the final concentration of 100 mM
Note: It is stored at -20 °C or -80 °C.
MM-401 reversion medium
ESC culture medium supplemented with 100 μM MM-401
Note: The medium can be stored at 4 °C for up to 2 weeks.
MM-401 maintenance medium
ESC culture medium supplemented with 20 μM MM-401
Note: The medium can be stored at 4 °C for up to 2 weeks.

Acknowledgments

This protocol was originally published as part of Zhang et al. (2016). The authors wish to thank all present and past members developing/functional testing MM-401 in the Dr. Yali Dou, Dr. Sundeep Kalantry and Dr. Shaomeng Wang laboratories. Special thanks are obligated to Dr. Saunders and Ms. Hughes at the University of Michigan for technical support, and Dr. Brady and Dr. Tesar for sharing materials.
This work is supported by National Institute of General Medical Sciences (NIGMS) (GM082856), Leukemia and Lymphoma Society and the National Cancer Institute (CA117307-04) to Dr. Yali Dou.

References

Chenoweth, J. G. and Tesar, P. J. (2010). Isolation and maintenance of mouse epiblast stem cells. Methods Mol Biol 636: 25-44.
Zhang, H., Gayen, S., Xiong, J., Zhou, B., Shanmugam, A. K., Sun, Y., Karatas, H., Liu, L., Rao, R. C., Wang, S., Nesvizhskii, A. I., Kalantry, S. and Dou, Y. (2016). MLL1 inhibition reprograms epiblast stem cells to naive pluripotency. Cell Stem Cell 18(4): 481-494.