hPSCs were likely damaged while they were harvested. hPSCs may have been mechanically harvested by scraping or were pipetted too much or too vigorously. Lift Solution D1-treated hPSCs only by pipetting them up and down up to 15 times. The mixing of hPSCs with SeV may have also been done too strongly. Gently rock the plate several times to mix hPSCs with SeV.

It is not recommended to start mRNA-based differentiation induction because the differentiation efficiency is expected to be quite low. For successful differentiation using our mRNA-based methods including Mesendoderm Booster, hPSCs should be evenly distributed in the well. It is likely that the plate was shaken too much, and cells were accumulated in the center of the well while they were floating. Next time, please handle the plate more gently or it can be left on a flat bench top without vibration for 15 min before it is put inside a CO₂ incubator.

Unless cells are accumulated in the center of the well, the instructions can be followed since this is an issue of coating. We have observed that 4-well plates available from Nunc sometimes do not allow cells to settle down in the periphery of the well when our coating conditions are applied. Next time, please use a 24-well plate and try the following: 1) incubate the plate with Coating Material A at 4°C overnight instead of 37°C for 2 hours, 2) do not aspirate the supernatant completely or leave the supernatant just enough to cover the well bottom surface after coating with Coating Material A is completed and 3) do not touch the bottom of wells with fingers when handling the plate.

hPSCs were likely damaged while they were harvested. Ideally, hPSCs should be lifted only by pipetting several times using Solution D1 as instructed in the user guide. Even if pipetting 15 times does not lift all of the cells in the culture, do not attempt to do more pipetting but collect only cells that come off. Instead rinse the culture with PBS and start Solution D1 treatment again but with longer incubation (up to 20 minutes). Next time please reduce the concentration of the substrate used to coat the dish/well for the maintenance of the source hPSCs to 50-70%.

It could be that Coating Material A was diluted at room temperature or diluted using PBS warmed to room temperature. Keep both Coating Material A and PBS on ice before dilution.

The purpose of flicking the tube containing cells and SeV every 2 minutes during the 10 minute incubation is to prevent cells from settling down and forming aggregates. If the orbital shaker can do so, then it is possible to use it.

Our kits are not optimized with these substrates. However, if your hPSC differentiation protocols are already optimized with such a substrate, you may try using one of these with our kit at your own risk.

No. If you’re following the instructions given in the user guide, the hPSCs will be harvested in a differentiation medium so the leftover hPSCs cannot be maintained.

After harvesting hPSCs, cells should not be left in the collection tube for more than 5 minutes. Leaving cells for a longer period may cause the formation of cellular clumps which interferes with infection. Single cells are best for infection.

The concentration of the substrate used to coat the dish/well was likely too high, so Solution D1 treatment requires longer incubation than 10 minutes (up to 20 minutes). Next time, please reduce the concentration of the substrate to 50-70%.

hPSCs should be adapted to passaging as single cells prior to differentiation. Our protocols have only been tested with StemFit/StemFlex, so we can’t guarantee results using other culture conditions.

Before beginning differentiation, cultures should be healthy and displaying typical morphologies (i.e., round colonies with tightly packed cells exhibiting a small cytoplasmic area), few spontaneously differentiating cells, and normal growth rates (i.e., doubling once a day). We recommend growing the cells for at least 14 days post-thaw before differentiating and not allowing the cultures to reach more than 80% confluency.