EpiGenie | Epigenetics, Stem Cell, and Synthetic Biology News http://epigenie.com Scientific News, Technology, and Product Information Mon, 06 Jul 2015 21:11:01 +0000 en-US hourly 1 http://wordpress.org/?v=4.2.2 CRISPR-Display: For the lncRNA Enthusiast that has Everything http://epigenie.com/crispr-display-for-the-lncrna-enthusiast-that-has-everything/ http://epigenie.com/crispr-display-for-the-lncrna-enthusiast-that-has-everything/#respond Mon, 06 Jul 2015 21:10:13 +0000 http://epigenie.com/?p=23686 From wall hung TVs to the latest wearable; displays have been a key technology differentiator for years. Now we’d like to introduce you to CRISPR-Display, one of the latest applications of CRISPR technology for the lncRNA lover that wants to elevate their functional game.

Interest in lncRNAs continues to surge, but like other uncharted areas of research, loads of functional studies are still needed. Now, by utilizing dCas9 and some designer sgRNA, researchers have access to a lncRNA delivery system with more localization power than the last 4 digits on your zip code.

CRISPR-Display comes to you from the talented team of lncRNA researchers in the Rinn lab at Harvard.

Here are the specs on CRISPR-Display:

  • The sgRNA is upgraded with custom RNA domains (outside targeting sequence) to create the substrate needed for 3 possible distinct functional domains that protrude from the CRISPR/sgRNA complex
    • These domains are located 5’, center, and 3’.
  • dCas9 and sgRNAs containing the modifications are delivered via lentiviral vector to mammalian cells (HEK293FT cells).
  • The system was tested using a VP64 activation domain to drive luciferase expression.
    • The results were also confirmed using RNA Immunoprecipiation (RIP)
  • The custom RNA cargo put into the domains can form functional secondary structure to be ‘displayed’ and can be up to 4.8 kb.
  • The functionality of the loop domains was demonstrated in displays that included natural lncRNAs, protein-binding cassettes that allow for bridged activation, artificial aptamers, and pools of random sequences.
  • Multiple display functions can be carried out at different loci in a single cell.

CRISPR-Display enables new experimentation by allowing for the precision targeting of functional RNA domains and ribonucleoproteins to loci of interest. Ultimately, CRISPR-Display enables not only the study of natural RNA domains but also synthetic domains for custom RNA devices, which effectively brings ncRNA into the CRISPR renaissance.

Go see the whole display at Nature Methods, June 2015

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DNA Methylation Steps Up to Protect Against the Impact of Environmental Stress http://epigenie.com/dna-methylation-steps-up-to-protect-against-the-impact-of-environmental-stressors/ http://epigenie.com/dna-methylation-steps-up-to-protect-against-the-impact-of-environmental-stressors/#respond Fri, 03 Jul 2015 17:40:24 +0000 http://epigenie.com/?p=23681 It rears its head around tax time and lunges at you when your experiments fail. It can be good for you, but usually stinks….STRESS. It goes without saying that stress in any form is best avoided. This is especially true if you happen to be an expecting mother who’s currently eating for two, as the impact of stress on the development of offspring in animals is well documented. Mounting evidence suggests that prenatal maternal stress (PNMS) is associated with childhood obesity and other metabolic related complications in humans. Sources of prenatal stress are vast, and may include rare extreme environmental conditions, such as the Canadian 1998 Ice Storm.

Remember “Project Ice Storm”? Well, in case you have forgotten here’s a refresher. A team of Scientists from Canada’s McGill University studied a cohort of women who were pregnant during the “greatest natural disaster in Canadian history”— the 1998 Ice Storm.

Last year the group reported that the ice storm, which left many people without power and other amenities for a long time, created extremely stressful conditions for the pregnant women. Additionally, the environmentally induced stress was found to be associated with DNA methylation at numerous CpG sites, affecting genes involved in immunity and other important biological functions.

This time around Cao-Lei and colleagues decided to extend their study to children born to women affected by the 1998 ice storm.

To accomplish this, the team examined the impact of prenatal “objective exposure and subjective distress” on the BMI and central adiposity (waist-to-height ratio) of children at age 13.5. The team also wanted to determine the role of DNA methylation on mediating prenatal maternal stress and its concomitant impact on growth.

The team discovered that:

  • Objective and subjective PNMS correlated with central adiposity, but only objective PNMS predicted BMI.
  • DNA methylation minimizes the impact of objective PNMS on both central adiposity and BMI by regulating key immune genes (LTA, NFKBIA, and PIK3CD), and type-1 and -2 diabetes mellitus pathways.

The authors conclude that “DNA methylation is a potential mechanism involved in the long-term adaptation and programming of the genome in response to early adverse environmental factors”.

Get protected at Epigenetics, June 2015.

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Book Review: Epigenetics – Current Research and Emerging Trends http://epigenie.com/book-review-epigenetics-current-research-and-emerging-trends/ http://epigenie.com/book-review-epigenetics-current-research-and-emerging-trends/#respond Thu, 02 Jul 2015 18:03:52 +0000 http://epigenie.com/?p=23676 Edited by Brian P. Chadwick and composed of 17 chapters from thought leaders in academia and industry, this is one text you don’t want to miss. It covers a wide breadth of topics ranging from DNA methylation to Chromatin to ncRNA, with insight from across the tree of life and related human disorders. The perspective is modern from both the aspects of fundamental biology and technical breakthroughs, offering up the latest insights into the molecular players of the epigenome from the pioneering experts themselves.

The Identification of Mammalian Proteins Involved in Epigenetics

Luke Isbel, Harry Oey, and Emma Whitelaw

Non-mammalian models have yielded great insight into the fundamentals of the epigenome, as highlighted by mutagenesis screens in Drosophila and Yeast. In fact those little flies and yeast have really made their mark in our understanding of heterochromatin boundaries as exemplified by the position-effect variegation. However, these model organisms have limitations, including the fact that some gene silencing mechanisms are essentially absent, with a prime example being DNA methylation in Drosophilla. Thus, mammalian models have always been a needed driving force of our understanding of DNA methylation and related proteins.

Two types of massive screening approaches have been utilized in mammalian models: Random mutagenesis screens in mice and also RNAi in cell lines. One of the first approaches was the Agouti mouse model. It enabled study of the position-effect variegation in a mouse model and gave birth to the concept of the metastable epiallele by allowing for an examination of heritable alterations that are independent of underlying sequence. The chapter then goes into the details of a screening technique known as Modifiers of Murine Metastable Epialleles and all the systematic insight it provided into epigenetic proteins (both known and unknown). After going into the history of screening for epigenetic proteins the authors then provide state of the art coverage of screening techniques and highlight the next challenge of putting the puzzle together to figure out how all these proteins interact.


Epigenetic Mechanisms in Rett Syndrome

Janine M. LaSalle

Rett syndrome (RTT) is a neurodevelopmental disorder that affects 1 in 10 000 people. It has many similarities to Autism Spectrum Disorders (ASDs) but it also happens to have one distinct difference, the molecular cause is known. RTT is caused by mutations to Methyl CpG Binding Protein 2 (MECP2). Interestingly, the nature of MeCP2 means RTT is an epigenetic disease at two levels. First, RTT has a strong gender bias, which is because it is an X-linked gene. Thus, due to the relatively stochastic nature of X-inactivation, the affected females are mosaics of wild-type and mutated MeCP2. However, a pattern of inheritance and expression related to X-inactivation isn’t always the case. Second, MECP2 encodes a methyl binding domain (MBD), which is a classic reader of the methylome. But the complexities of MeCP2 don’t end there as it wears many masks. MECP2 has multiple isoforms, undergoes extensive post-translational modification, can bind RNA and unmethylated DNA, and likes to party with quite a few co-factors that give this disordered protein some much needed structure. All of this diversity allows it to take on different functions depending on tissue and developmental stage. Thus, while originally thought to be a transcriptional repressor, MECP2 has emerged as a context dependent regulator of the epigenome that acts more like a histone mod than generic transcriptional repressor, as it can have long-range interaction with active genes at their CpG shores. By digging deeper into these functional molecular mechanisms and putting together the complex interactions we can begin to understand how the epigenome is read, how RTT progresses, and finally begin to develop effective therapies.


Environment and the Epigenetic Transgenerational Inheritance of Disease

Ingrid Sadler-Riggleman and Michael K. Skinner

Transgenerational inheritance is one of epigenetics’ hottest topics in epigenetics and explains a large chunk of modern disease. It all started with Skinner’s work on endocrine disruptors used as fungicides and pesticides on common crops. Since then the field has exploded and found the effects from other common chemicals like more pesticides, plastic, and jet fuel.

This chapter dives deep into non-Mendelian inheritance and starts with Skinner’s work as well as the Agouti mouse model. It then offers comprehensive coverage of the environmental toxicants capable of inducing transgenerational effects including Vinclozolin, Methoxychlor, DEET, Dioxin, BPA, Phthalates, and Tributlyin. The chapter doesn’t just end there as they also examine other exposures including caloric restriction, high fat diets, stress, folate, drought, heat/salt stress, prediabetes, smoking, and alcohol consumption. Next, the chapter examines the molecular and evolutionary mechanisms behind alterations to the germ-line, which result in somatic-lines that exhibit different impacts from the same exposure. This chapter then ties into the next by Jaclyn M. Goodrich and Dana C. Dolinoy, which covers exposures from a public health perspective and offers up insight into other common exposures including metals (Mercury, Cadmium, and Lead) and persistent organic pollutants, like polychlorinated biphenyls (PCBs).


Read On Friends

Check out the list of topics/authors, and maybe even grab a copy of Epigenetics: Current Research and Emerging Trends at the Horizon Press website. Also be sure to check out our coverage of the earlier related texts: Epigenetics: A Reference Manual and Epigenetics.

**EpiGenie would like to thank Ben Laufer from the Singh Lab at the University of Western Ontario for the contributing this book summary**


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5fC is Stable in Mammalian Brains http://epigenie.com/5fc-is-stable-in-mammalian-brains/ http://epigenie.com/5fc-is-stable-in-mammalian-brains/#respond Thu, 25 Jun 2015 00:26:57 +0000 http://epigenie.com/?p=23661 DNA methylation has been undergoing dramatic change lately. 6mA shook things up by showing Cytosine can’t claim all the glory and now a talented team from the UK have shown 5-formylcytosine (5fC) is starting to follow in 5hmC’s independent footsteps.

The team profiled 5hmC, 5fC, and 5caC using:

  • in vivo isotope labeling of methionine to penetrate the one carbon pool and be deposited as radioactive methylation. Using these isotopes, they determined the turnover rate by examining the uptake of isotopes.
  • nano high-performance liquid chromatography–tandem high-resolution mass spectrometry (nanoHPLC-MS/HRMS) to discriminate the rare bases at their low level.

All this fancy technology was used to analyze C57BL/6J mice at newborn, adolescent, and adult life stages. Interestingly, 5hmC levels depend on tissue proliferation rate and thus age. The team also analyzed different tissues like the brain, heart, liver, kidney, and colon.

Finally, they also examined embryos lacking thymine-DNA glycosylase (TDG), which is involved in removing 5fC and 5caC.

Here’s what the interdisciplinary set-up revealed:

  • 5fC is present in all mouse tissues at all stages but is most abundant in the brain, just like 5hmC.
  • In cultured embryonic cells 5fC differs from 5hmC and 5mC by taking longer to reduce its turnover rate.
  • Interestingly, the turnover rate of 5fC is lower in the developing brain when compared to other developing tissues.
  • 5fC also has a lower turnover rate in adult tissues, when compared to 5mC and 5hmC, which attests to its stability.
  • 5fC appears to have no turnover in adult brains, unlike 5mC and 5hmC, and as assessed by 5mC RNA labeling control.

The findings leave the authors hypothesizing that 5fC is present in two distinct cell populations; one that is not dividing (the brain) and one (the other tissues) with lower levels that is actively dividing.

Since 5fC was stable, this experimentation suggests that it could be creating a unique epigenetic landscape. This leaves us wondering if 5fC is being repurposed in the brain and how long it will be before 5caC is caught in the act of being more stable.

Learn more about this stable modification in Nature Chemical Biology, June 2015

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Gene Editing Future Brightens with paCas9 http://epigenie.com/gene-editing-future-brightens-with-pacas9/ http://epigenie.com/gene-editing-future-brightens-with-pacas9/#respond Tue, 23 Jun 2015 02:58:41 +0000 http://epigenie.com/?p=23650 We knew it was coming.  No, not another Jurassic Park sequel.  No, not another installment of Star Wars.  More inevitable than the revival of the Terminator franchise: we now have photoactivatable CRISPR/Cas9.

Yes, as surely as the sun rises, scientists have developed a way to use that light (or at least the blue part) to activate Cas9 gene editing.  The new photoactivatable Cas9, developed by Yuta Nihongaki and colleagues in the lab of Moritoshi Sato at the University of Tokyo, builds on another recent photoactivated Cas9, enabling gene editing in specific cells at specific times.

The Dawn of paCas9

We may have known photoactivatable Cas9 was coming, but that doesn’t mean it was easy.  Previously, catalytically dead dCas9 was made into a photo-induced transcriptional activator by using a photo-dimerization system (CRY2/CIB1) to make it reversibly bind activator domains.  That let Cas9 turn genes on, but not edit them.  In other recent work, fully active Cas9 was made photoactivatable by replacing a key lysine with photocaged lysine.  However, that required a special genetic code-expanding, amber-suppressing tRNA, which could potentially cause side effects for genes no longer stopping at the TAG codon.

In the new paper, a split Cas9 was generated that reversibly dimerizes when hit with light, activating its full gene-editing ability.  This took a bit of optimizing:

  • First, the team screened potential split sites using a rapamycin-induced dimerization system, finding a good fragment pair
  • Next, they fused this pair to the photo-dimerizing Cry2/CIB1 domains, producing… nothing
  • Then the team tried another (smaller) photo-dimerization pair: the Magnet system, which worked!
  • But there was still some background gene cutting in the dark, so they tried another version of Magnets, which was just as active in the light, but this time stayed off in the dark

paCas9, Hey, What’s it Good For?

Ok, so we have photoactivatable CRISPR.  Does it do everything the regular version can?  Fortunately, Nihongaki and colleagues performed a tour-de-force of demonstrations, including:

  • Light-induced mutation by non-homologous end joining (NHEJ)
  • Light-induced gene replacement by homologous recombination
  • Light-induced nicking
  • Reversible Cas9 activity
  • Light-induced (and reversible) transcriptional repression
  • Gene editing in a narrow illuminated stripe
  • Gene editing in multiple cell lines (HEK293T and HELA)


The system isn’t quite perfect; it’s only about 60% as active as regular Cas9, but it extends the precision of CRISPR to space and time.  As a bonus, the Magnet-CRISPR fragments are small, making them easier to shove into a virus for more efficient delivery.
Read more in Nature Biotechnology, June 2015

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Stem Cell Reprogramming – Leaner, Quicker, Cheaper, Better http://epigenie.com/stem-cell-reprogramming-leaner-quicker-cheaper-better/ http://epigenie.com/stem-cell-reprogramming-leaner-quicker-cheaper-better/#respond Sat, 20 Jun 2015 16:05:06 +0000 http://epigenie.jeffpayne.net/?p=23644 “More for less” is a phrase that generally gets most people excited, and scientists trying to make induced pluripotent stem cells (iPSCs) are no exception. The reprogramming of somatic cells to generate iPSCs aims to bring patient-specific cell replacement therapies to the masses, but the process currently suffers from obstacles common to new technologies – slow pace, low efficiency, high costs, and back breaking effort.

However, a recent report from the laboratory Guokai Chen in Scientific Reports has described a new reprogramming protocol which promises cheap, quick, and easy iPSC generation for all.

The improvements described in this new study include:

  • Spin transduction-mediated reprogramming using the CytoTune™-iPS 2.0 Sendai RNA virus Reprogramming Kit (Life Technologies)
    • Utilizes three different non-integrative vectors (polycistronic Klf4–Oct4–Sox2, cMyc, and Klf4)
    • One kit can reprogram 24-48 samples generating 600 iPSC colonies per sample
    • Low reagent cytotoxicity
    • Small number of input cells (1 x 104) required
  • A streamlined reprogramming schedule, which allows the initiation and synchronization of 20 simultaneous reprogramming experiments
    • Morphological changes at day 2 allows the evaluation of transduction success
  • Expansion of colonies using a serial dilution method onto plates directly coated with Matrigel in chemically defined E8 xeno-free feeder-free medium (Life Technologies)
    • Allows passaging of single colonies by EDTA/PBS with ROCK inhibitors to reduce time
    • Generates high purity cells for expansion/preservation at passage two.
  • Highly efficiency media usage to reduces costs further
  • Provision for multiple backups of parental cells and iPSCs, allowing for quick experiment repetition
  • Cryopreservation of iPSCs directly on culture plates, to reduce costs and time, and expand preservation capacity.

Overall, the group has found that their enhancements can:

  • Generate hundreds of iPSCs lines in a short time period
  • Decrease experimental scale by 50-fold
  • Increase number of reprogramed samples by 10-fold
  • Decrease medium usage by 70%
  • Decreased reagent cost by more than 80% per sample
  • Drastically reduce cell culture workload

The streamlining of this previously daunting process will hopefully bring this exciting technology to labs great and small across the globe. See how you can apply these techniques to your iPSC experiments today! – Sci Rep, June, 2015.

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Nukes Aid Stem Cell Research in the Heart http://epigenie.com/nukes-aid-stem-cell-research-in-the-heart/ http://epigenie.com/nukes-aid-stem-cell-research-in-the-heart/#respond Sat, 20 Jun 2015 15:56:55 +0000 http://epigenie.jeffpayne.net/?p=23641 Researchers from the laboratory of Jonas Frisén at the Karolinska Institutet in Sweden have recently put radioactive carbon from nuclear bomb tests to good use, testing whether the adult human heart has the ability to repair itself. So how does that work? Well, a sharp rise in atmospheric radioactive carbon in the 1950-60s due to nuclear testing and its subsequent incorporation into genomic DNA provides a method to measure cell age and turnover.

Previous studies have successfully applied this technique to the study of cell regeneration in the human heart, although this still remains a contentious point. This resolution of the quandary is important, as if cardiomyocytes, the cells which make up the contractile tissue of the heart, have some level of endogenous regenerative capacity, we may be able to exogenously promote repair of the damaged heart.

In this new study, the researchers provided an integrated model of heart cell generation and turnover in humans through the study of heart tissue from 29 deceased individuals of various ages.

This demonstrated that:

  • Cardiomyocyte number actually remains constant over the human lifespan
    • Cardiomyocyte turnover is highest in early childhood and decreases gradually throughout life to <1% per year in adulthood, with similar turnover rates throughout the myocardium.
    • Hearts grow in size with age due to cells increasing in size, rather than in number
    • Overall, heart cells regeneration occurs on a modest scale, with only 40% replaced during a lifetime.
  • Endothelial and mesenchymal cell number increase substantially from childhood into adulthood
    • Endothelial cells have a high turnover rate (>15% per year)
    • Mesenchymal cells have a more limited adult renewal rate (<4% per year).

The authors note that these findings should aid the development of new therapeutic strategies aiming to treat heart attacks and heart disease, and hopefully this will put an end to the uncertainty surrounding the regenerative capacity of the adult human heart. So, researchers in the field take heart! Read this explosive study here at Cell, June, 2015.

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Advances in ChIP-based Technologies for Profiling Epigenomic Landscapes and Gene Regulatory Networks http://epigenie.com/advances-in-chip-based-technologies-for-profiling-epigenomic-landscapes-and-gene-regulatory-networks/ http://epigenie.com/advances-in-chip-based-technologies-for-profiling-epigenomic-landscapes-and-gene-regulatory-networks/#respond Thu, 18 Jun 2015 05:05:37 +0000 http://epigenie.jeffpayne.net/?p=23635


Gene expression is regulated by a combination of transcription factor binding and the distribution of epigenetic modifications across regulatory regions. Much of what we know about the epigenome and gene regulation stems from our ability to determine the genome-wide distribution of histone modifications and transcription factors using chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). As sequencing technology has advanced, genome-wide epigenetic assays have evolved to take advantage of high read numbers at decreasing costs. However as we ask more complex questions the limitations of traditional ChIP have impeded our scientific advancements. Active Motif has developed a variety of tools and services to overcome many of these challenges, which will be presented in this webinar including:

• A Spike-In normalization method for preserving biological differences between ChIP samples.
• ChIP-exo: A method for determining the precise region of DNA bound by your factor
• A unique epitope tag for studying difficult-to-ChIP transcription factors
• Methods for reducing the number of cells required for ChIP

Lastly, we will discuss new approaches we have taken to understand gene regulation by elucidating proteins in complex with your factor of interest using Rapid Immunoprecipitation Mass Spec of Endogenous proteins (RIME), developed in collaboration with the Carroll lab at the Cambridge Research Institute. Taken together, all of the above advancements in ChIP-based assays help us to gain a deeper understanding of the complex mechanisms that regulate our genomes.


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Locus-Specific Biochemical Analysis of Genome Functions Using enChIP with CRISPR http://epigenie.com/upcoming-webinar-locus-specific-biochemical-analysis-of-genome-functions-using-enchip-an-application-of-crisprcas-on-purification-of-specific-genomic-regions/ http://epigenie.com/upcoming-webinar-locus-specific-biochemical-analysis-of-genome-functions-using-enchip-an-application-of-crisprcas-on-purification-of-specific-genomic-regions/#respond Wed, 17 Jun 2015 13:25:57 +0000 http://epigenie.com/?p=22852


Elucidation of molecular mechanisms of genome functions such as transcription and epigenetic regulation requires identification of components mediating the genome functions. To this end, we recently developed the locus-specific chromatin immunoprecipitation (ChIP) (locus-specific ChIP) technologies to identify molecules interacting with a given genomic region of interest in vivo.

Locus-specific ChIP consists of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) using transcription activator-like (TAL) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) system. Basically, locus-specific ChIP consists of locus tagging and affinity purification and can be combined with down-stream analyses such as mass spectrometry (MS) (iChIP-MS and enChIP-MS) and RNA sequencing (RNA-Seq) (iChIP-RNA-Seq and enChIP-RNA-Seq) to identify proteins and RNAs associated with the target genomic region.

Among others, enChIP using CRISPR is the most flexible and easy-to-use method in locus-specific ChIP because any genomic regions of interest can be easily targeted with guide RNAs. In this webinar, an overview of locus-specific ChIP will be given. In addition, applications of locus-specific ChIP will be introduced. Furthermore, step-by-step protocols of enChIP using CRISPR will be presented.


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KeyGenes Helps Predict Differentiated Humans iPSCs http://epigenie.com/is-it-a-nerve-cell-is-it-brain-keygenes-to-the-rescue/ http://epigenie.com/is-it-a-nerve-cell-is-it-brain-keygenes-to-the-rescue/#respond Mon, 15 Jun 2015 22:47:56 +0000 http://epigenie.jeffpayne.net/?p=23167 Cells differentiated from embryonic stem cells are generally phenotypically immature, so identifying what cell type they are can be a bit like trying to figure out what exactly the picture your child drew for you in kindergarten is supposed to be. “That’s lovely darling but…” Luckily, for biologists at least, a team of Dutch researchers led by Susana Chuva de Sousa Lopes have come up with a solution.

KeyGenes is an algorithm that can predict the identity of a test tissue from its transcriptional profile based on next generation sequencing (NGS) data of 21 human fetal and extra-embryonic tissues from the first and second trimester of development. First, Lopes’ team used a training set of 76 tissues to identify so called ‘classifier’ genes. These genes were highly expressed throughout development in the tissues they characterize and were either not expressed or only lowly expressed in several other tissues, which helped to define a sort of transcriptional “barcode” for each tissue. Then, the classifier genes were used to predict the identity of samples in a test set.

  • KeyGenes performed extremely well in the first test set, correctly identifying 38/39 fetal tissues.
  • More rigorous testing based on published microarray and NGS datasets showed that the developmental classifier genes could accurately predict the identity of their adult organ counterparts.
  • When applied to cells in culture, KeyGenes correctly predicted the differentiation of human pluripotent cells to a specific cell type. Moreover, the identity ‘score’ depended on the number of passages in culture, showing that KeyGenes can be used to optimize the quality of differentiated stem cells.

This is not the first algorithm to predict stem cell fate based on transcriptional profiling, but as Lopes points out, previous algorithms were based on the analysis of adult tissues with microarrays, which often excludes many classifier genes and are less relevant to early development.

Besides optimizing protocols for stem cell differentiation, KeyGenes may also help to researchers to understand how and why development goes wrong in some cases by identifying genes that are expressed in the wrong place or at the wrong time.

Check out the divining powers of KeyGenes at Stem Cell Reports, May 2015.

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