Mouse iPSC Chemical Reprogramming Cocktails Kit plus
“VC6TF + EPZ004777+AM580”, “VC6TFZ+AM580+SGC0946+5-aza-dC” and “N2B27-2i+LIF” are three novel small molecules cocktails that enhanced the chemical reprogramming efficiency of an newly identified extraembryonic endoderm (XEN)-like state (i.e. an intermediate during the early stage of chemical reprogramming). [1]
Pluripotent stem cells are self-replicating cell that can be induced from somatic cells by nuclear transfer into oocytes, transgene delivery, or treatments with chemical compounds and then differentiate into three primary germ layers. [1]
There are 3 essential stages in the chemical reprogramming process. In stage 1, RA agonist-AM580 (A) and DOT1L inhibitor- EPZ004777 (E) enhances the formation of XEN-like colonies by 2- to 3-fold. In a cocktail of seven small molecules (VC6TFAE: VPA, CHIR99021, 616452, tranylcypromine, forskolin, AM580 and EPZ004777), the number of XEN-like colonies is increased by >5-fold. During stage 2, using another DOT1L inhibitor- SGC0946 (S) that replacing EPZ004777, the reprogramming efficiency is enhanced further by up to 5-fold , especially when an optimized 2i-medium (N2B27-2iL medium) is applied. In additions, CiPSC (chemically induced pluripotent cell) colonies generates in stage 3 only when supplemented with 5-aza-dC (D) in stage 2. Using the small-molecule cocktail VC6TFAZDS during stage 2 for 12 days induce ~100–600 CiPSC colonies from 50,000 re-plated cells at the final stage of chemical reprogramming. [1]
All six tested CiPSC lines can form teratoma after injection into SCID mice and produce chimeric mice after blastocyst injection. Four lines displays germline integration potential in chimeric mice, and germline transmission offspring are gained from chimeric mice. [1]
Reference:
1. Zhao Y, Zhao T, Guan J et al. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming. Cell. 2015 Dec 17;163(7):1678-91.
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Stages |
Time |
Procedures |
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Plate |
Day -1 |
MEFs were plated at 300,000 cells per 100 mm dish, or 50,000 cells per well of a 6-well plate. |
|
Stage 1 |
Day 0 |
The culture was changed into stage 1 medium (containing 100 ng/mL bFGF, 0.5 mM VPA, 20 μM CHIR99021, 10 μM 616452, 5 μM tranylcypromine, 50 μM forskolin, 0.05 μM AM580 and 5 μM EPZ004777). |
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Re-plate |
Day 12-16 |
On day 12, the cells were trypsinized, harvested and then re-plated at 50,000–200,000 cells per well of a 6-well plate (1:10–15). During days 12–16, concentrations of bFGF, CHIR, and forskolin were reduced to 25 ng/ml, 10 μM, and 10 μM, respectively. |
|
Stage 2 |
Day 16 |
XEN-like epithelial colonies were formed and the culture was changed into stage 2 medium (containing 25 ng/mL bFGF, 0.5 mM VPA, 10 μM CHIR99021, 10 μM 616452, 5 μM tranylcypromine, 10 μM forskolin, 0.05 μM AM580, 0.05 μM DZNep, 0.5 μM 5-aza-dC, and 5 μM SGC0946). |
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Stage 3 |
Day 28 |
The culture was transferred into stage 3 medium (N2B27-2iL medium with 3 μM CHIR99021, 1 μM PD0325901, and 1,000 U/mL LIF). |
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Pick up |
Day 36-40 |
After another 8–12 days, 2i-competent, ESC-like, and GFP-positive (if using pOct4-GFP reporter) CiPSC colonies emerged and were then picked up for expansion and characterization. |
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Reference: 1. Zhao Y, Zhao T, Guan J et al. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming. Cell. 2015 Dec 17;163(7):1678-91. |
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Related Biological Data
Related Biological Data
Related Biological Data
Related Biological Data
| Chemical Reprogramming Cocktail 1/2 | ||||||
| Cat No | Compound Name | Target | Cocktail 1 | Cocktail 2 | Size (for 100 mL medium) | Size (for 500 mL medium) |
| A4099 | Valproic acid sodium salt (Sodium valproate) | HDAC inhibitor | 0.5 mM | 0.5 mM | 10 mg | 50 mg |
| A3011 | CHIR-99021 (CT99021) | GSK-3 inhibitor | 20 μM | 10 μM | 1 mg | 5 mg |
| A3754 | RepSox (616452) | ALK5 inhibitor | 10 μM | 10 μM | 1 mg | 2 mg |
| B7514 | Tranylcypromine hydrochloride | LSD1/MAO inhibitor | 5 μM | 5 μM | 1 mg | 1 mg |
| B1421 | Forskolin | Adenylate cyclase activator | 50 μM | 10 μM | 2.5 mg | 12.5 mg |
| B4654 | AM580 | RARα agonist | 0.05 μM | 0.05 μM | 1 mg | 1 mg |
| A4170 | EPZ004777 | DOT1L inhibitor | 5 μM | N/A | 1 mg | 2 mg |
| A8182 | 3-Deazaneplanocin A | SAH and ENZ2 inhibitor | N/A | 0.05 μM | 1 mg | 1 mg |
| A1906 | Decitabine (NSC127716, 5AZA-CdR) | Cellular differentiation inducer | N/A | 0.5 μM | 1 mg | 1 mg |
| A4167 | SGC 0946 | DOT1L inhibitor | N/A | 5 μM | 1 mg | 2 mg |
| Dual Inhibition (2i) Medium Additive | ||||
| Cat No | Compound Name | Target | Final Concentration | Size (for 500 mL medium) |
| A3011 | CHIR-99021 (CT99021) | GSK-3 inhibitor | 3 μM | 1 mg |
| A3013 | PD0325901 | MEK inhibitor | 1 μM | 1 mg |
Kit contains Chemical Reprogramming Cocktail+ Dual Inhibition (2i) Medium Additive
Evaluation sample solution: ship with blue ice
All other available size: ship with RT, or blue ice upon request
For obtaining a higher solubility, please warm the tube at 37°C and shake it in the ultrasonic bath for a while. Stock solution can be stored below -20°C for several months.