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Mouse iPSC Chemical Reprogramming Cocktails Kit plus

Catalog No.
K1021
Chemical reprogramming from somatic cells to pluripotent stem cells
Grouped product items
SizePriceStock Qty
100ml
$362.00
In stock
500ml
$513.00
In stock

Tel: +1-832-696-8203

Email: [email protected]

Worldwide Distributors

Description

“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.

Protocol

Stages

Time

Procedures

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).

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).

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).

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.

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.

Quality Control

Related Biological Data

K1021
Phase and fluorescence images of primary CiPSC colonies in stage 3 (day 40) with treatment with VC6TFAZ plus 5-aza-dC and SGC0946 in stage 2. Scale bar, 100 mm.

Related Biological Data

K1021
Numbers and phase images of XEN-like colonies after treatment with control cocktail (VC6TF with CHIR, 20 μM) and that with additional small molecule EPZ004777 (E) and AM580 (A) for 16 days. Cells were re-plated at day 12 by 1:2. Scale bar, 100 mm.

Related Biological Data

K1021
Numbers of CiPSC colonies at day 44 induced with a control cocktail (VC6TFAZ) and with 5-azadC, 5-aza-dC plus EPZ004777 (EPZ), or SGC0946 (SGC) in stage 2 of 12 days.

Related Biological Data

K1021
Imaging tracing of the formation process of two CiPSC colonies (stage 3) in stage 1 and stage 2 during chemical reprogramming. Scale bars, 100 mm.
 

Components and Storage

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

Specialty

Kit contains Chemical Reprogramming Cocktail+ Dual Inhibition (2i) Medium Additive

Solubility

Soluble in DMSO >10 mM

Storage

Store at -20°C

Shipping Condition

Evaluation sample solution: ship with blue ice

All other available size: ship with RT, or blue ice upon request

General tips

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.