New way for derivation / maintenance of naïve human PSCs

Newly added to the Stemolecule portfolio are three small molecules which support a new approach for the derivation and maintenance of naïve human pluripotent stem cells. These three newly identified small molecules, WH-4-023, SB590885 and IM-12, are all kinase inhibitors.

small-molecules-03

ReproCELL is currently the only stem cell reagent company able to supply all components of the 5i/L/A (5 inhibitors/human LIF/Activin A) media supplement formulation needed for the derivation and maintenance of a “naïve” or ground-state of pluripotency of human cells as referenced in the October 2014 Cell Stem Cell paper by Theunissen et al. out of Dr. Rudolf Jaenisch’s Lab at MIT’s Whitehead Institute. The paper is titled “Systematic identification of culture conditions for induction and maintenance of naïve human pluripotency.”

Stemgent Stemolecule WH-4-023 (SRC Inhibitor)IM-12 Structure

Stemgent Stemolecule SB590885 (BRAF Inhibitor)

Stemgent Stemolecule IM-12 (GSK-3β Inhibitor)

 

The addition of these three new small molecules consolidates a 5i/L/A media supplement offering in one place. 5i/L/A media supplement consists of 5 kinase inhibitors; WH-4-023 SRC inhibitor, SB590885 BRAF inhibitor, IM-12 GSK-3β inhibitor, PD0325901 MEK inhibitor, Y27632 ROCK inhibitor, recombinant human leukemia inhibitory factor (LIF) and Activin A. The 5i/L/A media supplement allows for the derivation of and conversion of human PSCs to a naïve state of pluripotency hypothesized to be the human equivalent to the mouse ground state of pluripotency.

The Theunissen paper demonstrates the conversion of traditional human epiblastic pluripotent stem cells to a ground state of pluripotency thought to be equivalent to the traditional mouse embryonic stem cell state. The human equivalent to the mouse ground state of pluripotency is termed naïve. Naïve human PSCs differ from traditional human epiblastic ESCs in that human ESCs are FGF/BMP signaling dependent (equivalent to mouse epiblastic stem cells). While naïve PSCs are LIF/Stat3 signaling dependent (equivalent to traditional mouse ESCs). Both traditional mouse ESCs and naïve human PSCs demonstrate higher single cell viability when passaging. Likewise both cell types demonstrate enhanced proliferation when compared to epiblastic stem cells. These attributes make naïve human pluripotent stem cells a good choice for genetic manipulation and gene targeting applications.

Related products used in the paper that researchers may be interested in include: Human recombinant FGF-basic growth factor, CHIR99021 and doxycycline.

The easy way to find your bioactive small molecules for stem cell biology

In addition to a plethora of cytokines and growth factors which can be used in stem cell biology to stimulate differentiation, proliferation, and maintenance of different cell types, there are a number of small molecules which are known to act in similar ways on stem cells. The crucial point, is that these molecules have to be produced and formulated in a way which keeps them bio-active. [Read more…]

Chemically synthesized mRNAs: now a reality

In vitro transcription has been a common protocol in RNA biology laboratories wishing to work directly with mRNA molecules to study phenomena such as mRNA translation. Commercially available kits have greatly facilitated the capping and polyadenylation and in vitro transcription of RNAs, but now there is another option:  ordering chemically synthesized mRNAs.

In vitro transcription kits such as the T7-FlashScribe™ Transcription Kit allow maximum RNA yields in 30 minutes. Subsequent processing of primary transcripts involves addition of the 5′ cap and 3′ poly(A) tail.

N-7001

m7GTP mRNA cap

N-7003

ARCA mRNA cap

Some of the most popular kits for mRNA processing include the ScriptCap™ m7G Capping System for capping and the A-Plus™ Poly(A) Polymerase Tailing Kit for tailing. Researchers wishing to optimize protein expression use chemically-modified mRNAs, such as those carrying an anti-reverse cap analog ARCA at the 5′ end. Again, kits such as the MessageMAX™ T7 ARCA-Capped Message Transcription Kit have made in vitro transcription of ARCA capped mRNAs routine in laboratories. When a standard m7 GTP cap is added to mRNAs in vitro, only about 1/2 of the cap is added in the correct orientation. The ARCA cap is modified with a methyl group to prevent capping in the “incorrect” orientation, thus resulting in a higher percentage of efficiently translated mRNA.

pseudouridine

Depending on the application, researchers are seeing advantages of using other chemical modifications as well. Pseudouridine-5′-Triphosphate, a naturally occurring base, is used to decrease nuclease activity and TLR activation, and modified cytidine base (5-Methylcytidine-5′-Triphosphate) is used for similar reasons. Warren et al. 2010 (doi: 10.1016/j.stem.2010.08.012), for example, found that mRNAs carrying an ARCA cap and Pseudo-uridine and methyl-cytidine substitutions are very efficient for reprogramming of many human cell types and fail to activate the toll-like receptor innate immune pathways.
Similarly, the Immune Stimulation Transcription Nucleotide Set is available for those who want to purchase ARCA cap, Pseudouridine-5′-Triphosphate, and 5-Methylcytidine-5′-Triphosphate. Another modification gaining popularity is the modified uridine (2-Thiouridine-5′-Triphosphate), however this popularity may be a result only of intellectual property limiting the commercial use of Pseudo-uridine.

EGFPmRNA_expression

Commercially-available capped and polyadenylated mRNAs modified with pseudouridine and 5-methylcytidine include those encoding:

Gene
Pseudouridine
5-methylcytidine
Length (nucleotides)
EGFP mRNA (5meC, Ψ) + + 996
Oct4 mRNA (5meC, Ψ) + + 1,359
Klf4 mRNA (5meC, Ψ) + + 1,688
SOX2 mRNA (5meC, Ψ) + +  1,230
c-Myc mRNA (5meC, Ψ) + + 1,596
Lin28 mRNA (5meC, Ψ) + + 906
FLuc mRNA (5meC, Ψ) + + 1‚929
NLS-Cre mRNA (5meC, Ψ) + +  1‚350
β-gal mRNA (5meC, Ψ) + +  3,336
Factor IX mRNA (5meC, Ψ) + + 1,662
hAAT mRNA (5meC, Ψ) + + 1,530
mCherry mRNA (5meC, Ψ) + + 996
Eira CFP mRNA (5meC, Ψ) + +  978
Blaze YFP mRNA (5meC, Ψ) + + 987
Cas9 Nickase mRNA (5meC, Ψ) + + 4,341
EPO mRNA (5meC, Ψ) + + 858
CD8 mRNA (5meC, Ψ) + + coming soon
NGFR mRNA (5meC, Ψ) + + coming soon
Guassia Luciferase mRNA (5meC, Ψ) + + 834
Renilla Luciferase mRNA (5meC, Ψ) + + 1,212
Cas9 mRNA (5meC, Ψ) + + 4,509
EGFP mRNA (5meC) + 996
FLuc mRNA (5meC) + 1‚929
OVA mRNA (5meC, Ψ) + + 1,437
Cas9 mRNA (Ψ) + 4,509
EGFP mRNA 996
FLuc mRNA 1‚929
β-gal mRNA 3,336
OVA mRNA 1,437
Cyanine 5 FLuc mRNA (5meC, Ψ) + + 1‚929
Cyanine 5 EGFP mRNA (5meC, Ψ) + + 996

 

Clearly, many of these purified mRNAs encoding fluorescent proteins, luciferase, or other reporter proteins are intended as controls for users setting up assays on which disease-relevant mRNAs will be tested. Once the assays are established, users now have the choice to produce their mRNAs of interest themselves using the in vitro transcription, capping, and polyadenylation kits described above or to order high quality mRNAs produced by expert chemists. Chemically synthesized mRNAs can be synthesized to contain nearly any sequence and chemical modification desired and gram quantity yields are possible. For particularly long mRNAs (up to multiple kilobases), in vitro transcription steps may still be required, but experts at TriLink Biotechnologies are able to design custom strategies to optimize yield even for the most complicated custom mRNA production requirements.

European scientists interested in learning more about out-sourcing mRNA production are encouraged to contact the local TriLink Biotechnologies distributor, tebu-bio.

HSCI’s researchers switch skin cells into pain-sensing neurons

Human noxious stimulus–detecting (nociceptor) sensory neurons - (Credit: Elizabeth Buttermore, PhD).

Human noxious stimulus–detecting (nociceptor) sensory neurons produced by converting skin cells with a set of five genes (Credit: Elizabeth Buttermore, PhD).

In a recent publication, Dr Clifford J. Woolf’s team (Harvard Stem Cell Institute (HSCI), USA) described a new experimental technique to produce in vitro pain-sensing neurons directly from mouse and human skin cells. For this, the authors have defined optimal cell culture conditions with five transcription factors to reprogram mouse or human fibroblasts into noxious stimulus–detecting (nociceptor) neurons.

Interestingly, these neural models displayed TRPV1 sensitization to PGE2 (an inflammatory mediator) and Oxaliplatin (a chemotherapeutic drug) modeling the mechanisms related to 1/ in inflammatory pain hypersensitivity and 2/ painful chemotherapy-induced neuropathy.

This in vitro neural cellular model will help the research community to better understand neuropathies and hypersensitivity to pain in fundamental and personalized medicine programs.

Sources:

  • Harvard Stem Cell Institute: Pain in a dish – Turning skin cells into pain-sensing neurons, November 24, 2014.
  • Woolf B.J. et al. “Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts” (2014) Nature Neuroscience. DOI: 10.1038/nn.3886

Pluripotency tests with validated antibodies

I’d like to share with you some input given to me by my colleagues working daily with researchers involved in Stem cell research. They regularly report positive feedback about antibodies used for pluripotency tests.

Which are the most popular antibodies made for Stem cell and iPSC research, validated by Life Scientists? Well, here you’ll find a compilation of the most cited markers and corresponding antibodies.

[Read more…]

FAQs on feeder cells for optimal human iPSC culture

Human Newborn Foreskin Fibroblasts (NuFF cells) are genuine feeder cells for Stem cell research. Mitotically inactive NuFF cells (by irradiation or Mitomycin C chemical treatment) can efficiently maintain healthy undifferentiated human pluripotent cell status and support cell reprogramming. In this post, you’ll find the answers to Frequently Asked Questions collected by tebu-bio’s cell biology experts regarding these feeder layers.

I hope these FAQs will help you in your Stem cell research! Have a good read.

[Read more…]

Cell reprogramming strategies for Stem cell research & cell sourcing

Cellular reprogramming is a process by which a fully differentiated cell type with specialized functions, is brought to be transformed into another cell type. This reprogrammed cell type exhibits different characteristics and functions it would not display initially under normal physiological conditions.

[Read more…]

mRNA reprogramming system, fastest & safest!

Human induced pluripotent stem (iPS) cells hold great promise for advancing our understanding of human biology and medicinal research. It has been shown that ectopic expression of just a few key transcription factors in somatic cells can induce an embryonic stem cell phenotype hallmarked by the ability to differentiate into any cell of the three basic germ layers (1,2). [Read more…]

Return of SIRT1 deacetylase – Why is SIRT1 so attractive in Drug discovery and Stem cell research?

SIRT1 is a NAD dependent class III Histone Deacetylase (HDAC). The tumor suppressor p53 is not the unique SIRT1 substrate. SIRT1 also deacetylates various key protein targets (PTEN, FOX, HIF-1, XPA, SMAD7 …) involved in the regulation of cellular survival, proliferation and angiogenesis.

SIRT1 – an attractive druggable target in oncologyPrimary cells, active enzymes and small molecules for stem cell and Drug discovery research

SIRT1 has rapidly been considered a valuable druggable target because of its implication in p53-dependent apoptosis in response to cellular stress and DNA damage. This enthusiasm was even more enhanced in 2011 when certain studies suggested that a natural dietary polyphenolic compound (Resveratrol) could promote healthy ageing and increase mouse and yeast life span through activation of sirtuins. (1)
Today, SIRT1 returns to the front of the stage with its possible involvement in cellular reprogramming and pluripotency.

[Read more…]

iPS cells: Bioactive small molecules make exogenous reprogamming genes dispensable!

Cell reprogramming of Mouse and Human somatic cells to induced Pluripotent Stem Cells (iPSC) have opened outstanding new opportunities for biomedical research. iPSC are now becoming unique cellular models for personalized therapies and regenerative medicine.

[Read more…]