Highly Efficient Generation of CiPSCs from MEFs

This is intended as a guide only; for full experimental details please read the reference provided.

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.

https://resources.tocris.com/images/protocols/mefs-to-cipscs.jpg

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    
https://resources.tocris.com/images/protocols/mefs-to-cipscs-timeline.jpg

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

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