Table 2.
Details of mouse pluripotent stem cell culture conditions. This table highlights that there are many variations on culture conditions for maintaining cells in distinct pluripotent states, including subtle differences in basal medium and cytokine concentrations that are often not emphasized. All culture medium formulations contain amino acids and 2-mercaptoethanol as standard. Pluripotency markers include Oct4, Sox2. Naïve pluripotency markers include Nanog, Rex1, Stella, Klf4, Esrrb. Primed pluripotency markers include Fgf5. Lineage markers include Brachyury, FoxA2, Eomes, Gata6, Gata4, Sox17. ‘Embryo-derived’ refers to cell lines that are directly derived from embryos, whereas ‘culture-derived’ refers to cell lines that are interconverted from different states e.g. changing the culture conditions of ESCs or EpiSCs
| Cell type | Culture condition | Added factors | Basal medium | Embryo-derived | Culture- derived | Gene expression | X status | Functional tests | Ref. |
|---|---|---|---|---|---|---|---|---|---|
| Embryonic stem cells (ESCs) |
Feeders/ serum | - | MEFs/ DMEM/ 20% serum | ✓ | Little transcriptional data | ✓ | IVD, teratomas, pre-imp. Chimaeras, GLT | [4, 12] | |
| Serum/LIF‡ | 1000 U/ml LIF | GMEM or DMEM/ 10% serum | ✓ | Naïve markers. Heterogeneous lineage markers. | ✓ | IVD, teratomas, pre-imp. Chimaeras, GLT. | [27, 28] | ||
| BMP/LIF | 10 ng/ml BMP4 OR 200 ng/ml GDF6 + 1000 U/ml LIF |
N2B27 | ✓ | ✓ | Little transcriptional data. Homogeneous Oct4 | Pre-imp. Chimaeras, GLT. | [29] | ||
| 2i* | 1 μm PD032/ 3 μM CHIR/ (+/− LIF) | N2B27 | ✓ | ✓ | Homogeneous naïve markers. Reduced lineage markers (rel. to SL). | ✓ | IVD, pre-imp. Chimaeras, GLT. | [91, 97] | |
| KOSR/LIF | 1000 U/ml LIF | Knockout DMEM/ 20% KOSR | ✓ | ✓ | Naïve and PrE markers | ? | IVD, pre-imp. Chimaeras, GLT. | [46, 101] | |
| Epiblast stem cells (EpiSCs) | F/A‡ | 5-12 ng/ml FGF2 + 10-20 ng/ml Activin | MEFs, Knockout DMEM/ 20% KOSR or “CDM” [207] | ✓ | ✓ | Primed markers. Heterogeneous lineage markers. | ✗ | IVD, teratoma formation, rare pre-imp. Chimaeras, post-imp. Chimaeras. | [3, 17] |
| F4-EpiSC | 25 ng/ml FGF4 | DMEM/ 20% serum | ✓ | Homogeneous Oct4. Primed and lineage markers. | ✗ | Teratomas, pre-imp. Chimaeras. | [160] | ||
| F/A/XAV* (WiEpiSC) |
5 ng/ml FGF2/ 10 ng/ml Activin/ 10 μM XAV | DMEM/20% KOSR | ✓ | ✓ | Homogeneous pluri and primed markers. Reduced lineage markers (relative to FA). | ? | Post-imp. Chimaeras –bias towards extraembryonic mesoderm. | [156] | |
| F/A/IWP2* (WiEpiSC) |
12 ng/ml FGF2, 20 ng/ml Activin, 2 μM IWP2 | Derivation in DMEM/ 18% KOSR/ 2% serum ➔ N2B27 at P2 | ✓ | Pluri markers. Reduced lineage markers (rel. to FA). | ? | Enhanced ESC reversion, pre-imp. Chimaeras. | [127] | ||
| XAV/CHIR | 3 μM CHIR/ 2 μM XAV | GMEM/ 10% serum | ✓ | ✓ | Reduced naïve markers (rel. to ESCs). Primed markers. | ✗ | Teratomas formation. | [20] | |
| Potential intermediate states | EpiLCs | 12 ng/ml FGF2, 20 ng/ml Activin | N2B27/ 1% KOSR | ✓ | Reduced pluri/naïve markers. Increased primed markers (rel. to ESCs). Reduced lineage markers (rel. to EpiSCs) | ✗ | Efficient PGCLC generation. | [145] | |
| Transient Epi state | 10-12 ng/ml FGF2 | N2B27 (+/− 1% KOSR) | ✓ | Decreased Rex1, Increased Fgf5. | ? | In vitro NMPs. | [154, 155] | ||
| INTPSC | 12 ng/ml FGF2, 10 ng/ml Activin, 3 μM CHIR | N2B27/ 1% KOSR | ✓ | Intermediate expression of pluri, naïve, primed and lineage markers. | ✓ | Teratoma formation, pre-imp. Chimaeras. | [155] | ||
| FAB-SCs | 1 ng/ml FGF2, 50 ng/ml Activin, 0.5 μM BIO, 100 ng/ml LIF blocking antibody | MEFS/ DMEM/ 15% KOSR | ✓ | Pluri markers. Naïve miRNAs. Reduced naïve/germ cell markers (rel. to ESCs). Little/no primed expression. | ? | Not functionally pluripotent | [161] | ||
| IESCS | 12 ng/ml Activin | GMEM/ 10% serum | ✓ | Intermediate expression of pluri, naïve, primed and lineage markers. | ? | IVD, teratoma formation – incorporate into pre-imp. Embryos but negative impact on development. | [162] | ||
| EPL | +/− 1000 U/ml LIF | 50% MEDII conditioned medium/ 50% DMEM/ 10% serum | ✓ | Pluri markers. Reduced naïve and increased primed markers (rel. to ESCs). | ? | Not functionally pluripotent |
X status refers to the activation status of the X chromosome, ✗ = inactive X chromosome, ✓ = active X chromosome. DR downregulated, UR upregulated, Epi epiblast, PrE primitive endoderm, IVD in vitro differentiation, pre-imp. pre-implantation, post-imp. post-implantation, GLT germline transmission, pluri pluripotency genes. ‡ = standard culture condition, * = “ground state” of self-renewal, rel. to = relative to