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Mouse iPSC Chemical Reprogramming Cocktails Kit

Catalog No.
K1022
Chemical reprogramming from somatic cells to pluripotent stem cells.
Grouped product items
SizePriceStock Qty
100ml
$276.00
In stock
500ml
$397.00
In stock

Tel: +1-832-696-8203

Email: [email protected]

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Description

A cocktail of seven small-molecule compounds (VC6TFZ+TTNPB) can be used to generate pluripotent stem cells from mouse somatic cells (such as MEFs, MNFs, MAFs or ADSCs) at a frequency up to 0.2%. This chemical reprogramming technology has potential use in generating functional desirable cell types for clinical applications. [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] To achieve complete chemical reprogramming without the Oct4-inducible system, these small molecules were further tested in the chemical reprogramming of OG-MEFs without transgenes. When DZNep was added 16 days after treatment with VC6TF (VC6TFZ), GFP-positive cells were obtained more frequently. The expression levels of most pluripotency marker genes were increased, but were still lower than in ESCs. After switching to 2i-medium with dual inhibition (2i) of glycogen synthase kinase-3 and mitogen-activated protein kinase signaling with CHIR-99021 (CT99021) and PD0325901, certain GFP-positive colonies developed an ESC-like morphology, which are chemically induced pluripotent stem cells (CiPSCs). An additional screen found a synthetic retinoic acid receptor ligand, TTNPB enhanced chemical reprogramming efficiency up to a frequency comparable to transcription factor–induced reprogramming (up to 0.2%). [1] In immunodeficient (SCID) mice injected with CiPSCs, the cells were able to differentiate into tissues of all three germ layers. When injected into eight-cell embryos or blastocysts, CiPSCs were capable of integration into organs of all three germ layers, including gonads and transmission to subsequent generations. The chimeric mice generated from CiPSCs were 100% viable and apparently healthy for up to 6 months, which suggested that the CiPSCs were fully reprogrammed into pluripotency. [1]

Reference:

1. Hou P, Li Y, Zhang X, Liu C, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013 Aug 9;341(6146):651-4.

Protocol

Stages

Time

Procedures

Plate

Day -1

The initial cells (MEFs, MNFs, MAFs or ADSCs) were seeded at a density of 50,000 cells per well of a 6-well plate or 300,000 cells per 100 mm dish.

Stage 1

Day 0

The original medium was replaced with chemical reprogramming medium containing the small-molecule cocktails. The small-molecule cocktails-containing medium was changed every 4 days.

Re-plate

Day 12-16

On day 12, these cells were washed in PBS and digested with 0.25% Trypsin-EDTA at 37°C for 3-5 min. After neutralization, the cell clumps were dissociated into single cells by thorough pipetting. The cells were harvested (300,000-1,000,000 cells per well of a 6-well plate) and replated at a density of 300,000-500,000 cells per well of a 6-well plate in the chemical reprogramming medium containing the small-molecule cocktails.

Stage 2

Day 16 or 20

DZNep was added to the cell cultures on day 16 or day 20.

Stage 3

Day 28

On day 28, the small-molecule cocktails including DZNep were removed. Meanwhile, the chemical reprogramming medium was replaced with 2i-medium (ESC culture medium supplemented with 2i (3 μM CHIR99021 and 1 μM PD0325901)).

Pick up

Day 36-40

2i-competent, ESC-like and GFP-positive colonies were counted as primary CiPSC colonies. For CiPSC induction from wild-type cells without OG reporter, 2i-competent and ESC-like colonies were counted as primary CiPSC colonies. These CiPSC colonies were picked up for expansion and characterization. Alternatively, CiPSCs could be induced without replating on day 12.

Reference:

1. Hou P, Li Y, Zhang X, Liu C, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013 Aug 9;341(6146):651-4.

Quality Control

Related Biological Data

K1022

Related Biological Data

K1022

Related Biological Data

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Related Biological Data

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Components and Storage

Chemical Reprogramming Cocktail
Cat No Compound Name Target Cocktail Size (for 100 mL medium) Size (for 500 mL medium)
A4099 Valproic acid sodium salt (Sodium valproate) HDAC inhibitor 0.5 mM 10 mg 50 mg
A3011 CHIR-99021 (CT99021) GSK-3 inhibitor 10-20 μM 1 mg 5 mg
A3754 RepSox (616452) ALK5 inhibitor 5-10 μM 1 mg 2 mg
B7514 Tranylcypromine hydrochloride LSD1/MAO inhibitor 5-10 μM 1 mg 1 mg
B1421 Forskolin Adenylate cyclase activator 10-50 μM 2.5 mg 12.5 mg
B2058 TTNPB (Arotinoid Acid) RAR agonist 1 μM 1 mg 1 mg
A8182 3-Deazaneplanocin A SAH and ENZ2 inhibitor 0.05-0.1 μM 1 mg 1 mg
Note: * For CiPSCs induction from MEFs, CHIR-99021 should be used at a concentration of 10 μM. For CiPSCs induction from MNFs, MAFs or ADSCs, and CiPSCs induction without replating, CHIR-99021 should be used at a concentration of 20 μM during day 0-12. ** For CiPSCs induction from MEFs, Forskolin should be used at a concentration of 10 μM. For CiPSCs induction from MNFs, MAFs or ADSCs, Forskolin should be used at a concentration of 50 μM during day 0-12.
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.