Highly Efficient Generation of CiPSCs from MEFs
In Brief
Zhao et al. describe a protocol for the highly efficient reprogramming of mouse embryonic fibroblasts (MEFs) to chemically-induced pluripotent stem cells (CiPSCs) in 16-20 days using a small molecule cocktail.
Briefly, MEFs were grown in MEF culture medium. On day 0 the medium was changed to optimized stage I medium. On day 4-6 small extraembryonic endoderm (XEN)-like colonies appeared and stage I medium was exchanged for optimized N2B27-SII medium. Cells were cultured for a further 4-6 days, then on day 8 or 12 N2B27-SII medium was exchanged for stage III medium containing 500 μM Valproic Acid (VPA). VPA was removed on day 12 or 16, and cells were cultured for a further 4-10 days in stage III medium, when CiPSC colonies emerged.
Cocktails
| Optimized Stage I Medium (FBS/KSR-based medium) | Optimized N2B27-SII Medium (N2B27-based medium ) | Stage III Medium (N2B27-based medium) | |||
|---|---|---|---|---|---|
| Valproic acid (Cat.No. 2815) | 100 μM | Valproic acid (Cat.No. 2815) | 1 mM | CHIR 99021 (Cat.No. 4423) | 3 μM |
| CHIR 99021 (Cat.No. 4423) | 40 μM | CHIR 99021 (Cat.No. 4423) | 10 μM | PD 0325901 (Cat.No. 4192) | 1 μM |
| RepSox (Cat.No. 3742) | 10 μM | RepSox (Cat.No. 3742) | 10 μM | LIF | 10 ng/ml |
| Tranylcypromine (Cat.No. 3852) | 5 μM | Tranylcypromine (Cat.No. 3852) | 5 μM | L-Ascorbic Acid (Cat.No. 4055) | 50 μg/ml |
| Forskolin (Cat.No. 1099) | 10 μM | Forskolin (Cat.No. 1099) | 10 μM | ||
| Ch 55 (Cat.No. 2020) | 1 μM | Ch 55 (Cat.No. 2020) | 1 μM | ||
| EPZ 004777 (Cat.No. 5567) | 5 μM | 3-Deazaneplanocin A (Cat.No. 4703) | 0.05 μM | ||
| Decitabine (Cat.No. 2624) | 0.5 μM | ||||
| SGC 0946 (Cat.No. 4541) | 5 μM | ||||
| L-Ascorbic Acid (Cat.No. 4055) | 50 μg/ml | ||||
| LIF | 10 ng/ml | ||||
| bFGF | 25 ng/ml | ||||
Reference
Zhao et al. (2018) Single-cell RNA-seq reveals dynamic early embryonic-like programs during chemical reprogramming. Cell Stem Cell. 23 31 PMID: 29937202