Human Cell Atlas: The next frontier

Reprinted from the October 2016 AltTox Digest; used by permission.

Now that the human genome has been mapped, the next frontier is to map the human cellular phenotypes. An understanding of all the types of human cells and how they interact in the various tissues and organs will provide the new level of understanding of human biology needed to accomplish medical breakthroughs, understand physiological processes such as human development and aging, and understand pathophysiological processes such as disease and toxicity.

Cells are the basic building blocks of all human tissues and organs. Beginning in the embryo, cells divide and begin to specialize into the different cell types that make up the human body.

General estimates identify several hundred major cell types, however, new methods of characterizing cells show that even within what appears to be a homogenous population there is great variability.

The technique used to identify cells at the level of the single cell is single-cell messenger RNA sequencing (RNA-seq), where every messenger RNA species in a sample is sequenced and identified.  Credit: Genome Research Limited

The technique used to identify cells at the level of the single cell is single-cell messenger RNA sequencing (RNA-seq), where every messenger RNA species in a sample is sequenced and identified. Credit: Genome Research Limited

Recent government funding initiatives have spurred innovation and progress in studying cells at the level of the single cell. In 2014, the US National Institutes of Health (NIH) awarded $7.9 million to 25 projects studying various aspects of single cell analysis as part of the Single Cell Analysis Program (SCAP).

On October 13-14, 2016 an international group of renowned researchers met in London to discuss building the Human Cell Atlas. The Human Cell Atlas will be more than just a catalogue of static cell types. Like SCAP, it involves addressing the many challenges in characterizing human cell heterogeneity.

For more on single cell analysis and the new international effort to develop the Human Cell Atlas, see the entire article, “The Human Cell Atlas: An international effort,” on

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Animal-free skin allergy testing

With the recent approval of the human Cell Line Activation Test (h-CLAT) for skin sensitization (allergy), toxicologists now have a battery of methods that allows them to test for sensitization without using animals.mice

Testing a chemical substance for skin irritation or corrosion is pretty straight-forward: the substance is applied to a skin sample (there are many non-animal in vitro options) and damage will be seen relatively quickly. But to learn if the chemical has the potential to cause skin allergies, testing is more complicated. Skin sensitization is a two-stage process. In the first stage, a chemical exposure “primes” the immune system. Additional exposures then provoke an allergic response (inflammation, redness, itching, etc). Because of the biological complexity of the process, skin sensitization testing is usually conducted on mice or guinea pigs.

But mice and guinea pigs don’t always react the same way as humans would to potential skin allergens. To replace these animal tests with more human-relevant methods, toxicologists have long recommended developing a battery of in vitro tests that could be combined in an Integrated Testing Strategy – where each test method captures a different part of the skin sensitization process. The biological processes that underlie the skin allergy reaction are pretty well understood, and have been described (the Organisation on Economic Co-operation and Development (OECD) has published a description of this process – the Adverse Outcome Pathway leading to skin allergies). Two of the pieces of this strategy are already in place: the OECD approved the use of the Direct Peptide Reactivity Assay (DPRA) and the KeratinoSens test – each measuring a different step in the sensitization process. The DRPA assays measures whether a chemical can react with proteins in a way that causes the protein to become an allergen. The KeratinoSens assay measures activation of genes involved in the allergic reaction in skin cells (keratinocytes). The h-CLAT method completes the battery of tests: it detects biomarkers that indicate activation of immune (dendritic) cells in the skin.

Many countries require that chemicals used in manufacturing, agriculture, medicine, and cosmetics be tested for their potential to cause skin allergies in humans. Without approved in vitro options, REACH regulations in the EU alone would force industry to use hundreds of thousands of animals for skin sensitization testing. The animal-free, 3-test strategy is a “textbook” example of the mechanistic, pathway-based approach to chemical testing promoted by the Human Toxicology Project Consortium. Use of this strategy has the potential to greatly reduce the numbers of animals used for skin sensitization testing, while also reducing the cost and time it takes to produce human-relevant results.

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“Blood-brain barrier on-a-chip”

Scientists at the Wyss Institute have created a 3-dimensional in vitro model of the human blood-brain-barrier (BBB)-“on-a-chip.”  The device will make it possible for researchers to test drugs, chemicals, and disease factors that interact with the BBB, without using animals – and in a 3-dimensional environment that mimics that of the human BBB in vivo.

The BBB is a semi-permeable cellular structure that allows some nutrients and substances to enter the blood flow in the brain, and keeps other elements (such as bacteria and potential toxins) out. Because it is so effective, it can also prevent useful treatments from reaching targets in the brain. Researchers need to understand how and why certain substances can pass through the barrier, in part so they can design therapeutic drugs accordingly, and so they can design other substances to prevent neurotoxicity.

From the Wyss Institute press release: "These fluorescence confocal microscopy images show both a high magnification view (left) of a region of the human brain capillary endothelium within the endothelium lined tube (shown at lower magnification at right) that, in combination with surrounding human pericytes and astrocytes, comprise the blood-brain barrier (cell junctions linking adjacent endothelial cells are shown in magenta). " Credit: Wyss Institute at Harvard University

From the Wyss Institute press release: “These fluorescence confocal microscopy images show both a high magnification view (left) of a region of the human brain capillary endothelium within the endothelium lined tube (shown at lower magnification at right) that, in combination with surrounding human pericytes and astrocytes, comprise the blood-brain barrier (cell junctions linking adjacent endothelial cells are shown in magenta). ” Credit: Wyss Institute at Harvard University

To create the device, the Wyss Institute team carved a tiny channel in a polymer chip and filled it with a gel matrix containing human astrocytes, the cells that comprise the extra-tight “barrier” around blood vessels in the brain. Another channel was tunneled through this matrix and seeded with human pericyte cells (contractile cells which control the “gaps” through which substances can enter the neurological bloodstream) and then with human endothelial cells (the cells that line the interior of a blood vessel). The cells “self-assembled” into the same layers and connections they exhibit in blood vessels in vivo.

To test the model, the team introduced a protein known to cause inflammation – one that has been associated with a number of central nervous system diseases and disorders, including Alzheimer’s, multiple sclerosis, and stroke (among others). The in vitro BBB responded by producing protective proteins. The device can thus be used to study neuroinflammation, and to test new treatments.

Read more in the Wyss Institute’s press release.

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Human Toxicology Project Consortium at the Society of Toxicology meeting in New Orleans

You’ll find the Human Toxicology Project Consortium at the Society of Toxicology’s annual meeting in New Orleans next week – in the ToxExpo center, poster sessions, workshops, and seminars.

  • Visit HTPC’s informational booth at ToxExpo, booth #1704.
  • HTPC is co-sponsoring a “hands-on” seminar, “Creating an Adverse Outcome Pathway in the AOP Wiki,” on Tuesday, March 15, from 5-7PM in the Hilton Riverside.  More details about the seminar can be found here.
  • HTPC is also once again co-sponsoring and presenting at the annual SOT Satellite Meeting, Updates on Activities Related to 21st Century Toxicology and Related Efforts: Invited Presentations and Open Microphone, on Thursday, March 17, 12:30 PM to 4:00 PM, Hilton New Orleans Riverside, Jefferson Ballroom. As always, this informative meeting features a number of invited presentations, and also allows time for an “open microphone” segment in which participants are welcome to give brief presentations on germane topics.

The draft program is as follows:

12:30 PM—Box Lunch (for pre-registered participants) and Welcome by Thomas Hartung, Johns Hopkins University

1:00 PM—Invited Speakers (10 minute presentations each followed by 5 minute of discussion)

ToxCast Update: Russell Thomas, US Environmental Protection Agency

EDSP21 Update: David Dix, US Environmental Protection Agency

Tox21 Update: Richard Paules, US National Toxicology Program

Hamner TT21C Update: Melvin Andersen, Hamner Institutes

NICEATM Update: Warren Casey, NICEATM

SEURAT/EU Tox-Risk Update: Michael Schwarz, University of Tuebingen

CAAT’s Read-Across Initiative and Human Toxome-Related Activity Update: Thomas Hartung, Johns Hopkins

Human Toxicology Project Consortium Update: Catherine Willett, HTPC

Evidence-Based Toxicology Update: Martin Stephens, Johns Hopkins

3:15 PM—Open Microphone for Additional Presentations and Discussion

4:00 PM—Adjourn

  • Kate Willett will also present a poster in the Regulation and Policy session, Wednesday, March 16, 1:15 PM to 4:45 PM: “Regulatory Acceptance of Non-standard Toxicological Methods through Increased use of Integrated Approaches to Testing and Assessment (IATA)” (Abstract #3003/Poster #P143).

Corporate members and partners of HTPC will be presenting at SOT next week, as well.  Scientists from each of the member corporations are coauthors on the following posters:

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Educational Infographic produced by the Human Toxicology Project Consortium


A new infographic produced by the Human Toxicology Project Consortium shows in three sections how the future of toxicity testing promises a steady reduction in testing costs, increases in human relevance and confidence in safety assessments, and the eventual elimination of animal tests.

The first section provides a snapshot comparison of the current and future costs, efficiency and efficacy of toxicity testing, while the mid portion uses pesticide testing as a specific example of now, vs near-future, vs the optimal approach that, given the focus and resources necessary, will be envisioned within the decade.

The near-future and optimal approaches rely increasingly on our understanding of biology and using it to build a predictive systems biology platform that is comprised of an interrelated network of biological pathways. This platform is used to design and interpret tests that provide much more efficient and effective characterization of chemical activity that can be used to predict safe use of chemicals.

Finally, the results of this progression are captured in the summary graphic at the end – decreasing costs, animal use and time while human relevance and our confidence in safety decisions continue to improve.

As explained on our Project page, the Human Toxicology Project Consortium works on three areas critical for the successful, international implementation of a pathways-based approach to chemical safety testing: advancing the science, communicating the purpose and goals of pathway-based toxicology, and lobbying for funding and policy changes that will support pathway-based approaches in the US and around the world.

To advance our communication and education efforts, HTPC member organizations worked together to create this infographic, to quickly and effectively illustrate the differences between traditional animal-based toxicity testing and pathway-based testing in terms of predictive power, cost, and testing capacity.

Details on the numbers used in this comparison are available here (PDF).

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Researchers at HTPC partner organization CAAT create stem-cell derived mini-brains

CAAT's stem cell-derived "mini-brain"/image by Thomas Hartung (used with permission)

CAAT’s stem cell-derived “mini-brain”/image by Thomas Hartung (used with permission)

Researchers at Johns Hopkins’ Center for Alternatives to Animal Testing (CAAT) have developed a process to create “mini-brains” derived from stem cells reprogrammed from human skin cells. The resulting structures exhibit a number of cell types and cell functions of the human brain, and can be produced economically and in sufficient numbers to be especially useful for screening chemicals and drug candidates. The mini-brains will also be used to study Alzheimer’s disease, Parkinson’s disease, multiple sclerosis and autism.

From CAAT’s press release:

“[Principal investigator] Hartung and his colleagues created the brains using what are known as induced pluripotent stem cells (iPSCs). These are adult cells that have been genetically reprogrammed to an embryonic stem cell-like state and then are stimulated to grow into brain cells. Cells from the skin of several healthy adults were used to create the mini-brains, but Hartung says that cells from people with certain genetic traits or certain diseases can be used to create brains to study various types of pharmaceuticals. He says the brains can be used to study Alzheimer’s disease, Parkinson’s disease, multiple sclerosis and even autism. Projects to study viral infections, trauma and stroke have been started.

Hartung’s mini-brains are very small—at 350 micrometers in diameter, or about the size of the eye of a housefly, they are just visible to the human eye—and hundreds to thousands of exact copies can be produced in each batch. One hundred of them can grow easily in the same petri dish in the lab. After cultivating the mini-brains for about two months, the brains developed four types of neurons and two types of support cells: astrocytes and oligodendrocytes, the latter of which go on to create myelin, which insulates the neuron’s axons and allows them to communicate faster.

The researchers could watch the myelin developing and could see it begin to sheath the axons. The brains even showed spontaneous electrophysiological activity, which could be recorded with electrodes, similar to an electroencephalogram, also known as EEG. To test them, the researchers placed a mini-brain on an array of electrodes and listened to the spontaneous electrical communication of the neurons as test drugs were added.

“We don’t have the first brain model nor are we claiming to have the best one,” says Hartung, who also directs the School’s Center for Alternatives to Animal Testing.

“But this is the most standardized one. And when testing drugs, it is imperative that the cells being studied are as similar as possible to ensure the most comparable and accurate results.”

Hartung elaborated on this point to Gizmodo: “There are a handful of such models described over the last two years,” he said. “They show more fancy brain structures, but each and every one looks different, often with cells in the middle dying because of lack of oxygen as they have no blood vessels. We produce hundreds of identical mini-brains, every week. This is critical for testing and comparing substances. They have exactly the same size below a critical diameter.”

Learn more in the video embedded here.

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