Culturing Transcriptomically Distinct Pluripotent mESCs
In Brief
Kolodziejczyk et al. describe a protocol that uses two different cocktails of small molecules to maintain heterogeneous populations of mESCs with different metabolisms. STO feeder cells were used for mESC maintenance.
The use of 2i medium results in a heterogeneous population of blastocyst-like cells with a stable or ‘ground state’ of pluripotency. Alternative 2i (a2i) medium also results in a heterogeneous population of blastocyst-like cells with a stable or ‘ground state’ of pluripotency, which is transcriptomically distinct from that obtained with 2i medium. A subpopulation of 2C-like cells is present with 2i medium. No differentiating cells are observed with either 2i or a2i.
Cocktails
| Serum-containing Medium | 2i Medium | a2i Medium | |||
|---|---|---|---|---|---|
| DMEM | N2B27 | N2B27 | |||
| 1X Pen/Strep | CHIR 99021 (Cat.No. 4423) | 3 μM | CHIR 99021 (Cat.No. 4423) | 3 μM | |
| 15% FBS | PD 0325901 (Cat.No. 4192) | 1 μM | CGP 77675 | ||
| Beta-Mercaptoethanol | 0.1 mM | rhLIF | 100 U/ml | rhLIF | 100 U/ml |
| rhLIF | 100 U/ml | ||||
Reference
Kolodziejczyk et al. (2015) Single cell RNA-sequencing of pluripotent states unlocks modular transcriptional variation. Cell Stem Cell 17 471. PMID: 26431182