Description
General description
Two stages of mouse pluripotency have been stabilized to date through in vitro culture. ‘Naive’ represents the earliest stage, which can be enriched for a purer population, termed ‘ground state’. Naive most closely approximates embryonic epiblast in the preimplantation blastocyst. The primed state most closely approximates in vivo immediate postimplantation epiblast. There are clear differences between these stages in culture, expression, epigenetics (including appropriate X-inactivation) and developmental competence. The fact that naive mESCs can generate whole animals through tetraploid complementation while primed mESCs cannot, is a particularly compelling reason for heightened expectations from naive hESCs.
Mouse Leukemia Inhibitory Factor (LIF) is a critical component in mouse ES cell culture media where it promotes the self-renewal and maintains the pluripotency of mouse embryonic stem (ES) cells. Recent studies have determined that inhibitors that block MAPK/Erk pathway, in combination with the glycogen synthase kinase 3 (GSK3) inhibitor, protect mouse ES cells from differentiation-inducing signals, allowing for self-renewal even in the absence of LIF. Mouse ES cells cultured in these conditions maintain their LIF responsiveness and studies have shown that adding LIF to the 2i culture conditions led better colony propagation and enhanced the general health of ES colonies. Supports a naive/ground pluripotent state.
Application
Suggested Protocol for Medium Preparation:
1. Thaw MEK/GSK3 inhibitor supplement and mLIF at room temperature.
2. Each vial of supplement is sufficient to make 500 mL of complete medium for serum-free or serum dependent mouse ES culture.
3. Aseptically transfer the contents of the vials containing the supplements at 1000x dilution to the pre-warmed bottle of serum free basal medium such as ESGRO Complete Basal Medium (Cat. No. SF002-500) or serum containing medium and mix.
4. The complete medium can be filtered through 0.22 µm filter (Cat. No. S2GVU05RE).
5. After supplements are added to the medium, store in the dark at 4°C for up to ten days.
The ESGRO®-2i Supplement Kit (1000x) contains LIF a selective GSK3β & Mek 1/2 inhibitors to enhance viability of mouse embryonic stem & iPS cells in the absence of serum & feeder cells. Supports a naive/ground pluripotent state.
Packaging
Provided in ready to use aliquots sufficient for supplementing 10 x 500 mL mouse ES cell culture medium for a total of 5 liters complete medium.
Physical form
Both supplements were formulated at 1000x concentration in solution. Mouse LIF is prepared in phosphate buffered saline with 1% w/v bovine serum albumin BSA as a carrier for stability. MEK/GSK3 inhibitor supplement is prepared in DMSO.
Preparation Note
ESGRO supplement is supplied 0.22 micron filtered, and tested negative in mycoplasma tests
Store at -20°C. Upon thaw, ESGRO Supplement should be stored at 2-8°C. Do not refreeze ESGRO Supplement (part no. CS210511). Refer to label for expiration date.
Analysis Note
Specific Activity: ESGRO Supplement is assessed both on mouse ES cells and on murine M1 myeloid leukemic cells. A standard of 50 Units is defined as the concentration of ESGRO supplement in 1.0 mL of tissue culture medium that induces the differentiation of 50% of M1 colonies (Metcalf, 1988).
Embryonic Stem Cell Assay: Differentiation inhibition at 1000 units/mL
Murine myeloid leukemic, M1 Assay: Specific Activity is greater than or equal to 10⁸ units/mg
Other Notes
ESGRO® Supplement: (Part No. CS210511): Mouse recombinant Leukemia Inhibitory Factor (LIF), 0.5 x 106U/mL. Ten (10) 0.5 mL vials.
MEK/GS3 Inhibitor Supplement: (Cat. No. CS210510-500UL): Ten (10) 0.5 mL vials.
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
ESGRO is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
LABEL LICENSE - USE RESTRICTED TO RESEARCH USE ONLY
Leukemia Inhibitory Factor ("LIF") is protected by US Patents No. 5,443,825 and 5,750,654 and Canada Patent No. 1,341,581 and 1,341,469 owned by AMRAD Corporation Ltd. and licensed exclusively to Millipore for research uses. The use of this product is limited for research uses only and not for any commercial purposes, such as making or developing other research products from this product or use of this product as a component in a research kit. This product is not to be used in humans or for any diagnostic purposes.
The purchaser may refuse to accept the conditions of this label license by returning this product unopened and will receive a full refund of the purchase price paid.
For-profit entities or commercial research entities purchasing this product shall be required to execute a separate commercial license with Millipore within three months of purchase. Researchers may not transfer this product or its derivatives to researchers performing commercial research at other Nonprofit Organizations, or to For-Profit Organizations, without the express written consent of Millipore, and without those entities having an express license from Millipore for the use of the product. Other than as specifically provided herein, a transferee of this product shall have no right to transfer this product, or its derivatives to any other person or entity.
