Human LIF

human LIF

The current trend of human pluripotent stem cell (hPSC) culture systems is to replace animal-derived materials such as FBS, BSA and supportive matrices such a Matrigel/Geltrex, a mouse-derived extracellular matrix, with defined factors in the media.  At NovoHelix, our products are manufactured in the USA using chemically-defined components and, therefore, do not contain manufacturing components such as serum or animal-derived raw materials. Animal-derived materials are complex undefined mixtures and are recognized as significant risks for stable cultivation and clinical application since they have lot-to-lot variations and risk of immunogenic contamination. In addition, the undefined nature of these raw animal products makes it difficult to deconvolute the essential factors that drive growth, maintenance and differentiation programs.

Basic experimentation in early human development is hampered by the paucity of fetal material available to study typical developmental trajectories, by ethical issues associated with using human fetal tissues, and by a dearth of knowledge of the genetic and molecular programs that direct human embryogenesis and gametogenesis.  Therefore, cellular models are a critical resource to understand dysgenesis and to advance our understanding of human infertility.   NovoHelix’ approach to personalized medicine is to develop autologous cell models via epigenetic reprogramming of a patient’s own biopsied cells to pluripotency to create induced pluripotent stem cells (iPSCs). A refined reprogramming procedure using Sendai-viral RNA and pioneer factors such as OCT4/SOX2/KLF4 generates iPSCs akin to human embryonic stem cells (collectively known as hPSCs). When these hPSCs are bathed in media containing the appropriate supportive cues to differentiate into somatic and germ cell lineages, these cell-specific models yield enormous potential to better understand the mechanisms of cell specification.  NovoHelix has manufactured a line of xeno-free cell reagents to support the establishment of clinical-grade human PSCs and to recapitulate some of the dynamic media environments necessary to elucidate key factors to boost our understanding of human development.

Product

Catalog Nr

Size

Concentration


Pricing

Human LIF
P4543S
50 µg
1 mg/ml
Human LIF
P4543M
100 µg
1 mg/ml
Human LIF
P4543L
1 mg
1 mg/ml
Human LIF
P4543X
10 mg
1 mg/ml
Human LIF
P4543B
bulk
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Product Information
Product Literature & Protocol
FAQs & Troubleshooting
References
Supporting Documents
Product Information
Source: 
Recombinant hLIF was improved through protein engineering efforts including directed evolution and rational design.  The final engineered protein was synthesized as a codon-optimized minigene and expressed heterologously in an E. coli strain. The recombinant protein was tested and normalized to standard activity assays for growth of naïve mouse and human pluripotent stem cells.

Molecular Weight: 
9110.6 ± 2 Daltons 

Purity: 
>98 % by SDS-PAGE and analysis by protein mass spectrometry

Sequence Analysis: 
Purified recombinant protein was subjected to peptide mass fingerprinting (PMF) by MALDI-TOF/TOF (PMF + MS/MS).

Cross Reactivity:  
Human, rhesus, cynomolgus/cercopithecine, mouse/murine, rat/murine, pig/porcine, cow/bovine, goat/caprine, dog/canine, cat/feline, horse/equine

Biological Activity: 
Proliferation of a naïve pluripotent mouse embryonic stem cell line cultured under 5% oxygen tension on a recombinant laminin 521 matrix with a serum-free DMEM/F12 standard basal media containing FGF2, TGFβ, Activin A, transferrin, vitamin C (L-ascorbic acid-2-phosphate), HRG1β (heregulin-1β), recombinant 0.1% HSA and supplemented with human LIF (ED50 < 20 ng/ml). The hPSC line contains OCT4-P2A-tdTomato knock-in at the human POU5F1 locus to indicate which colonies are still pluripotent.   10 units is equivalent to approximately 100 μg of recombinant hLIF protein.

Endotoxin: 
Lipopolysaccharide (LPS) was determined by the standard LAL (Limulus amebocyte lysate) test to be < 0.5 EU/µg.  Due to potential environmental sustainability concerns (collection of the hemolymph used in pharmaceutical testing may negatively affect horseshoe crab populations), a quantitative and more sensitive endotoxin assay was developed using a pyrogen‐testing cell model with knock-in (targeted, single-copy integration) of TLR4/CD14/MD2 at the safe harbor locus AAVS1/PPP1R12C on human chromosome 19. The TLR4/CD14/MD2 assay is used as an orthogonal screen for LPS in lieu of the LAL method and has a detection limit of 0.005 EU/ml. 

Appearance: 
A sterile, aqueous, clear and colorless solution. 

Storage/Stability: 
Recombinant hLIF is supplied at 1 mg/ml in stabilization buffer and remains bioactive for 30 days if maintained refrigerated [2º to 8ºC (35º to 46ºF)]. Stock solutions of human LIF in its concentrated form can be stored at least 6 months at -20°C and at least 12 months at -80°C from the date of manufacture with no loss of activity on ES cells. For long term storage, add 0.1% w/v carrier protein such as recombinant human serum albumin (HSA) and avoid repeated freeze-thaw cycles by aliquoting.  Multiple freeze-thaw cycles reduce potency and, therefore, aliquoting working stocks is strongly recommended.

Reconstitution: 
Recombinant hLIF is supplied reconstituted at 1 mg/ml in a stabilization buffer containing 9 mg/mL benzyl alcohol, 50 mM sodium phosphate, 250 mM sodium chloride, 2 mg/mL polysorbate 20, 10% v/v glycerol at a pH of approximately 7.4. The reconstituted solution has been filter sterilized by passing through a 0.22 micron PES membrane and tested to be negative for mycoplasma contamination.

Disclaimer & Precautions: 
This product is solely for research and development use only and may be subject to conditional use and licensing restrictions. The product shall not be used as an advanced pharmaceutical intermediate (API) or investigational drug or a biologic. This product is not intended to be used as a therapeutic agent or facilitate clinical diagnosis or be used as an in vitro diagnostic (IVD) product. 

The Food and Drug Administration (FDA) and Center for Biologics Evaluation and Research (CBER) define an IVD as: 

“In vitro diagnostic products are those reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions, including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or its sequelae. Such products are intended for use in the collection, preparation, and examination of specimens taken from the human body. These products are devices as defined in section 201(h)of the Federal Food, Drug, and Cosmetic Act (the act), and may also be biological products subject to section 351 of the Public Health Service Act. Title 21, Code of Federal Regulations (CFR), section 809.3(a).” 

This product may not be used or formulated in any agricultural, pesticidal, veterinary or animal products, food additives or household chemicals or any other unspecified use.  Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. NovoHelix distributes products for basic and translational research use only. NovoHelix will report any unspecified use to respective regulatory authorities for enforcement to ensure safeguarding of our research products from potential abuse.

Notice to purchaser: 
The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patents or applications owned by NovoHelix to use this product. No other license under these patents or applications is conveyed expressly or by implication by purchase of this product.
Product Literature & Protocol


  • HiFi DNA Assembly Protocol

                                                                                                Recommended Amount of Fragments Used for Assembly

 2–3 Fragment Assembly*4–6 Fragment Assembly**
Positive Control✝
Recommended DNA Molar Ratio
 vector:insert = 1:2 vector:insert = 1:1
Total Amount of Fragments
0.03–0.2 pmols*
X μl
0.2–0.5 pmols**
X μl
10 μl
NovoHelix
HiFi DNA Assembly Master Mix
 10 μl10 μl
10 μl
 Deionized H2O10-X μl
 10-X μl0
 Total Volume20 μl✝✝
 20 μl✝✝ 20 μl
Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.
Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%.

**To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each).

Control reagents are provided for 5 experiments.

†† If greater numbers of fragments are assembled, increase the volume of the reaction linearly by using additional NovoHelix HiFi DNA Assembly Master Mix. Alternatively, pool the DNA fragments into an equimolar mix first and then re-purify these pooled equimolar fragments over a micro-column and elute with a minimum volume (~10-µl). The eluate may be reapplied to the same micro-column  membrane to improve elution of large DNA fragments without increasing the final volume..

Recommended Storage Condition:   
This assembly mixture can be stored at -20 °C for at least one year.  The enzymes remain active following at least 10 freeze-thaw cycles.
FAQs & Troubleshooting
References
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1: Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988 Dec 15;336(6200):688-90. PubMed PMID: 3143917.

1: Gearing DP, Nicola NA, Metcalf D, Foote S, Willson TA, Gough NM, Williams RL. Production of leukemia inhibitory factor in Escherichia coli by a novel procedure and its use in maintaining embryonic stem cells in culture. Nat Biotechnol. 1989 Nov 1;7(11):1157-1161. doi: 10.1038/nbt1189-1157 website link:  https://www.nature.com/articles/nbt1189-1157

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1: Guo G, von Meyenn F, Santos F, Chen Y, Reik W, Bertone P, Smith A, Nichols J.  Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass. Stem Cell Reports. 2016 Apr 12;6(4):437-446. doi: 10.1016/j.stemcr.2016.02.005. Epub 2016 Mar 3. PubMed PMID: 26947977; PubMed Central PMCID: PMC4834040.

1: Bredenkamp N, Yang J, Clarke J, Stirparo GG, von Meyenn F, Dietmann S, Baker D, Drummond R, Ren Y, Li D, Wu C, Rostovskaya M, Eminli-Meissner S, Smith A, Guo  G. Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency. Stem Cell Reports. 2019 Nov 1. pii: S2213-6711(19)30370-4. doi: 10.1016/j.stemcr.2019.10.009. [Epub ahead of print] PubMed PMID: 31708477.

1: Weinberger L, Ayyash M, Novershtern N, Hanna JH. Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat Rev Mol Cell Biol. 2016 Mar;17(3):155-69. doi: 10.1038/nrm.2015.28. Epub 2016 Feb 10. Review. PubMed PMID: 26860365.

1: Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben-Yosef D, Kalma Y,  Viukov S, Maza I, Zviran A, Rais Y, Shipony Z, Mukamel Z, Krupalnik V, Zerbib M,  Geula S, Caspi I, Schneir D, Shwartz T, Gilad S, Amann-Zalcenstein D, Benjamin S, Amit I, Tanay A, Massarwa R, Novershtern N, Hanna JH. Derivation of novel human ground state naive pluripotent stem cells. Nature. 2013 Dec 12;504(7479):282-6. doi: 10.1038/nature12745. Epub 2013 Oct 30. Erratum in: Nature. 2015 Apr 30;520(7549):710. PubMed PMID: 24172903.

1: Di Stefano B, Ueda M, Sabri S, Brumbaugh J, Huebner AJ, Sahakyan A, Clement K, Clowers KJ, Erickson AR, Shioda K, Gygi SP, Gu H, Shioda T, Meissner A, Takashima Y, Plath K, Hochedlinger K. Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells. Nat Methods. 2018 Sep;15(9):732-740. doi: 10.1038/s41592-018-0104-1. Epub 2018 Aug 20. PubMed PMID: 30127506; PubMed Central PMCID: PMC6127858.

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Supporting Documents

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