Enhancing Stem Cell Differentiation with a Transient DMSO Treatment

1. Stem Cell Maintenance

NOTE: The cell maintenance protocol described below applies to pluripotent stem cells (PSCs) maintained in an adherent monolayer. Media, other reagents, and cell culture plates used prior to dimethyl sulfoxide (DMSO) treatment can be adjusted as needed. For all the following protocols in this manuscript, cells should be handled under a biological safety cabinet.

1. Coat sterile, 6 well, tissue culture-treated plates with a pluripotent stem cell-qualified matrix or substrate prepared per the manufacturer's instructions and incubate for at least 1 h in a CO₂ incubator (5% CO₂, humid atmosphere). Coated plates can be film wrapped and stored at 4 °C for up to one week.
2. Thaw the cryopreserved PSCs in a 37 °C water bath. Sterilize vial with ethanol prior to introduction to the biological safety cabinet, then immediately transfer the cells by pipetting to a sterile conical tube containing 5-10 volumes of prewarmed stem cell media.
3. Centrifuge the cells at 300 x g for 5 min at room temperature (RT).
4. Aspirate the media and gently resuspend the cell pellet in 1 mL of stem cell media supplemented with a 10 µM rho-associated protein kinase (ROCK) inhibitor, such as Y-27632.
5. Aspirate the culture matrix from the plate and seed the cells at the desired density, typically 0.5-1 x 10⁶ cells per well in at least 2 mL of stem cell media per well.
   NOTE: The plating density can vary across different cell lines and clones and should be optimized accordingly.
6. Maintain the cells by replacing with prewarmed stem cell media daily. Split the cells at roughly 70%-80% confluency or when the cell colonies begin to make contact.
7. For splitting cells, aspirate the media and wash the cells once with sterile phosphate-buffered saline (PBS). Incubate the cells with 1 mL of a dissociation enzyme solution per well for 5-10 minutes at 37 °C.
8. Wash and resuspend the cells with prewarmed stem cell media and transfer to a sterile conical tube with 5-10 volumes of stem cell media. Follow steps 1.3-1.7 to plate the cells.

2. DMSO Pretreatment

NOTE: When plating the cells for DMSO pretreatment prior to differentiation, the starting plating cell density should be optimized with consideration of the typical growth rate of the stem cell line as well as the differentiation protocol being used. Validate pluripotency using conventional markers, as necessary. Cells should be passaged at least 1x-2x after initial thawing prior to differentiation.

1. 2D culture differentiation
   1. When the cells reach an appropriate confluency, prepare coated plates, dissociate the cells, and prepare a single-cell suspension as described above.
   2. Count the live cells using a hemocytometer or automatic cell counter including trypan blue or another viability marker.
   3. Plate the cells onto a coated 6 well plate at 0.5-1 x 10⁶ cells per well in stem cell media with the 10 µM ROCK inhibitor.         
      NOTE: For the cell lines tested in our laboratory, these densities typically resulted in 80%-90% confluent cells within the 24 h DMSO pretreatment.
   4. Allow cells to incubate for 24 h at 37 °C in a CO₂ incubator (5% CO₂, humid atmosphere).
   5. Prepare 1%-2% DMSO in prewarmed stem cell media (e.g., 100 µL of DMSO in 10 mL of media = 1% DMSO solution, or 200 µL DMSO in 10 mL of media = 2% DMSO solution).
   6. After 24 h incubation, aspirate the media from cells and replace it with DMSO solution.
   7. Allow the cells to incubate for 24 h to 48 h at 37 °C in a CO₂ incubator (5% CO₂, humid atmosphere) prior to differentiation.        
      NOTE: Typically, a 24 h DMSO treatment is sufficient across a majority of human embryonic and induced pluripotent stem cell (ESC and iPSC) lines. Cell lines with very slow growth rates (long doubling times) can benefit from the 48 h incubation with DMSO. For a 48 h incubation with DMSO, media can be replaced with fresh stem cell media with 1%-2% DMSO after the first 24 h of treatment.

3. Differentiation to Primary Germ Layers

NOTE: The following describes methods previously shown to be effective in our laboratory for PSCs grown in a monolayer on 6 well plates. Any differentiation protocol of choice should be used after the DMSO treatment to promote differentiation into desired lineages. Remove DMSO solution after a 24-48 h treatment and proceed with differentiation following standard protocols.

1. Ectoderm differentiation
   1. Pretreat the cells with DMSO as described above for 2D cultures.
   2. Prepare Noggin and SB431542 stock solutions.
   3. Prepare ectodermal differentiation base media by dissolving knockout serum replacement (KOSR) to a final concentration of 10% in knockout Dulbecco's Modified Eagle Medium (DMEM).         
      NOTE: Prepare enough base media for 3-4 days of media change.
   4. Prepare ectodermal differentiation media by adding Noggin to a final concentration of 500 ng/mL and SB431542 to a final concentration of 10 µM to the appropriate volume of prewarmed KOSR/knockout DMEM.
   5. After DMSO pretreatment, aspirate media from cells and replace with differentiation media (e.g., 2 mL per well of a 6 well plate).
   6. Allow cells to incubate for 3-4 days at 37 °C in a CO₂ incubator (5% CO₂, humid atmosphere), replacing media daily with freshly added differentiation factors.