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 AltTox.org.

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Picture of video reel, by Coyau / Wikimedia Commons / CC-BY-SA-3.0

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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