ES cell derivation, culture conditions, and characteristics
Cell line . | Derivation . | Serum . | Feeder . | Cell harvest/passaging* . | Characteristics/comments . |
|---|---|---|---|---|---|
| PES | Frozen blastocyst, prior to August 9, 2001 | Yes | No (CM) | RNA prepared from pooled samples of GE01, GE07, and GE09 cell lines. Every fourth day with trypsin | Expression of undifferentiated markers, Xu et al19 |
| BG01 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every third day with collagenase | Expression of undifferentiated markers, Zeng et al20 |
| BG02 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every third day with collagenase | Expression of undifferentiated markers, Zeng et al20 |
| GE01 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every fifth day with collagenase | Expression of undifferentiated markers, Thomson et al4 |
| GE09 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every third day with collagenase | Expression of undifferentiated markers, Amit and Iskovitz-Eldor21 |
| TE06 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every third day with collagenase | Expression of undifferentiated markers, Amit and Iskovitz-Eldor21 |
| EB | Prepared from PES | Yes | No (CM) | Embryoid bodies prepared from 3 independent experiments from 3 different cell lines | Carpenter et al22,23 |
Cell line . | Derivation . | Serum . | Feeder . | Cell harvest/passaging* . | Characteristics/comments . |
|---|---|---|---|---|---|
| PES | Frozen blastocyst, prior to August 9, 2001 | Yes | No (CM) | RNA prepared from pooled samples of GE01, GE07, and GE09 cell lines. Every fourth day with trypsin | Expression of undifferentiated markers, Xu et al19 |
| BG01 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every third day with collagenase | Expression of undifferentiated markers, Zeng et al20 |
| BG02 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every third day with collagenase | Expression of undifferentiated markers, Zeng et al20 |
| GE01 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every fifth day with collagenase | Expression of undifferentiated markers, Thomson et al4 |
| GE09 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every third day with collagenase | Expression of undifferentiated markers, Amit and Iskovitz-Eldor21 |
| TE06 | Frozen blastocyst, prior to August 9, 2001 | Yes | MEF | Every third day with collagenase | Expression of undifferentiated markers, Amit and Iskovitz-Eldor21 |
| EB | Prepared from PES | Yes | No (CM) | Embryoid bodies prepared from 3 independent experiments from 3 different cell lines | Carpenter et al22,23 |
To produce conditioned media, MEFs were grown as described20 for less than 5 passages and then growth arrested with mitomycin C or irradiation. Medium was replaced with ES growth medium and the supernatant was collected after 48 hours. The medium was pooled and used fresh or frozen in pooled aliquots. SR indicates serum; EB, embryoid body; MEF, mouse embryonic fibroblast; and CM, conditioned medium.
Cells were harvested after digesting with enzymes as indicated for each cell line and maintained as described. Prior to harvesting for microarray analysis, expression of ES cell markers and the absence of markers of differentiation were assessed by RT-PCR and immunocytochemistry (data not shown). RNA of EB for EST enumeration analysis was prepared by pooling samples from 3 different (GE01, GE07, and GE09) cell lines.