Notes

1. For more information regarding hESCs lines in the NIH Human Embryonic Stem Cell Registry, visit http://stemcells.nih.gov/registry.

2. Frozen vials of mitomycin C-treated or untreated MEFs can be available from several companies (e.g., Speciality Media).

3. The original recipe for H1 medium from the provider (WiCell Research Institute) does not contain any antibiotics. However, addition of penicillin/streptomycin to hESCs medium was not found to substantially affect any character of self-renewing H1 cells, and even beneficial to avoid risk of bacterial contamination. Because penicillin/streptomycin has no effect on fungi or mycoplasma, careful sterile culture procedure is always critical for each culture step.

4. Add collagenase to hESCs medium to make a final concentration of 1 mg/mL, and dissolve in a 37°C water bath. Filter the collagenase solution using 0.2-|jm syringe filter.

5. It is recommended to prepare fresh Dispase solution for each passaging procedure. Alternatively, the solution can be kept at 4°C for several days.

6. It is quite important that the operator wear gloves and glasses during cell culture procedures to prevent any possible cell culture-related troubles (e.g., virus infection, explosion of a frozen vial after thawing).

7. Although we used Human Genome U133A and U133B chips that were separated, a combined gene array, Human Genome U133 2.0 Plus, that covers entire human genome in one array is currently available.

8. It is of note that, because chromosomal abnormality is reported to be occasionally found in high passaged hESCs in either culture method, regular karyotyping is recommended to ensure the condition of hESCs especially at higher passages (23).

9. It is recommended that researchers working on hESCs for the first time start by growing hESCs on MEFs feeder cells rather than using the feeder-free system because of the latter's complexity. Experience in growing mESCs is beneficial but not absolutely required, because maintenance of hESCs is far quirkier than that of mESCs. Special attention must be paid at each process, and careful daily observation of hESCs through the inverted microscope is essential to get used to maintaining hESCs in the best condition.

10. It is particularly important to thaw the frozen vial as quickly as possible to minimize damage to the cells. Usually it is completed within 1-2 min.

11. Ten minutes can be considered the maximum incubation period for Dispase. Beyond this time, viability and quality (undifferentiated state) of hESCs after passaging is drastically decreased. For BGN cells, use cell dissociation buffer as the provider (BresaGen) recommends. Cell dissociation buffer's dissociation activity appears to be stronger than that of Dispase solution. Most of cases, cell dissociation is completed within 5 min. We also tested cell dissociation buffer or trypsin for passaging H1 cells. We found, however, that H1 cells dissociated by these reagents were not kept in as good condition as H1 cells treated with Dispase, although the exact reason remained unclear.

12. Although hESCs sometimes tightly adhere to the culture surface and are refractory to detach from the surface, the pipetting procedure should be completed in the shortest period to minimize the time in which hESCs are exposed to Dispase solution. If necessary, we even leave a part of hESCs colony unharvested to prioritize quality over quantity.

13. It is usually recommended that hESC colonies should be kept as large as possible when being passaged to maintain their viability and undifferentiated state.

14. Although the exact reason is unclear, it could be due to selected growth of certain cell populations that acquired growth advantage through the chromosomal rearrangement under relatively higher selective pressure driven by the feeder-free condition.

15. It usually takes two to three passages until hESCs become free from contaminated MEFs in the feeder-free condition.

16. The gelatin solution supports the attachment of MEFs on culture vessels. This treatment is especially important when MEFs are fed with hESCs (for H1) medium that induces a substantial morphological change in MEFs.

17. CM can be used immediately after collection or can be stored at -80°C for several weeks.

18. Cryotubes should be prefrozen at -20°C before making aliquots to avoid gelation of Matrigel. Pipetting and aliquoting procedures should be done as quickly as possible to keep Matrigel cold.

19. Property of Matrigel appears to be slightly different from batch to batch. It is therefore quite important to predetermine the optimal concentration of Matrigel for each batch.

20. Optimally coated Matrigel demonstrates a well-formed meshlike structure under the inverted light microscope. When the coated Matrigel is too diluted, the meshlike structure is less formed, resulting in flattening and differentiation of hESCs on the Matrigel even in the presence of CM. If the coated Matrigel is too dense, Matrigel tends to remain aggregated even after rinsing with DMEM/F-12, and prevents hESCs from attaching and spreading on the surface.

21. Importantly, when evaluating factors involved in the undifferentiated state in the screening process, it is beneficial to dissociate hESC colonies into small aggregates because smaller colonies are more sensitive to the differentiation environment than larger ones which are resistant to the differentiation signal.

22. There are several ways to computationally adjust the obtained data to fit representative profiling. There is, however, no way to make up for fundamental blemish at the raw data level.

23. A satisfying result can be obtained without using Phase Lock Gel (PLG) that Affymetrix recommends for the cleanup process. However, because PLG appears to help efficient recovery of the sample and avoid contamination, it would be beneficial for the cleanup step.

24. During the procedure, the reagents should be kept at room temperature to avoid precipitation of DTT.

25. The quality of the obtained data can be briefly evaluated by visual inspection of Chip intensity data or checking out the several parameters in the Chip Report generated by MAS5.0. Scaling factor is usually between 1.0 and 2.0. Internal positive and negative controls and 375' ratio also help to determine the condition of the array data.

26. It is also strongly recommended to examine expression of the selected genes by real-time reverse transcription polymerase chain reaction to confirm differential transcriptional regulation of the enriched genes between the two conditions.

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