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Salk Cores supporting science and technology

A software program calculates the distance between brain cell terminals in a sample to determine the number of synaptic connections (yellow).

Courtesy of the Allen lab.

It's feeding time at the microscopic zoo. In a small, brightly lit room, scientists sit or stand at counters, carefully pouring liquid food into round petri dishes. The dishes, which contain tiny specks of stem cells, turn yellow (a result of the cells' waste) when it's time to feed them. The dishes turn a happier crimson when the cells are given their daily cocktail of vitamins and sugars. Stem cells are incredibly fickle, requiring precise, often daily, care to survive. But the effort has a potentially big payoff, as the cells are a powerful way to study diseases ranging from autism to cancer and hold the key to lifesaving regenerative therapies.

The most sophisticated research technologies—from stem cell protocols to genome sequencers—demand dedicated expertise and thorough understanding of their complexities, limitations and possibilities. This specialized knowledge is often beyond the purview of the average research lab, as are the expenses associated with purchasing, operating and maintaining the very latest in equipment and supplies. That's where shared scientific facilities called cores, like the Salk Stem Cell Core, come in. As part of Salk's first major fundraising campaign, the Institute has secured support to expand its core facilities in recent years. The Institute currently has 13 core facilities providing leading- edge scientific technologies and expertise. Far from being just service stations, the cores are idea factories spurring collaboration and pushing the limits of technology to enable breakthroughs.

Travis Berggren and Inder Verma

"To do big science, you need big technology," says Travis Berggren, senior director of Salk's scientific core facilities. "The continued advancement and cost of technology, as well as the need for trained experts, are the drivers for creating these structured, shared resources between the labs."

Inder Verma, a Salk professor and faculty advisor for the Stem Cell and Next Generation Sequencing cores, argues that research in modern biological sciences requires expertise in a range of pioneering technologies, including imaging, genomic sequencing, proteomics and much more.

"No single scientist can have these specialized technologies in their laboratory," says Verma. "The most efficient and productive way to do cutting-edge science is to have the resources to not only run, but continuously upgrade, shared facilities. We are very fortunate here at Salk to have state-of-the-art cores."

While many academic institutions have shared facilities, Salk's cores have been particularly effective at catalyzing discovery because of both the size and structure of the Institute. "At other institutes, this kind of equipment is only accessible to a select group of influential researchers. We're at the optimal size to have great resources and share them equally," says Berggren. Indeed, all 48 active labs are able to use the cores for needs that include biophotonic expertise, stem cell culturing and genomic sequencing, to name a few.

But perhaps even more important than equal access, Salk's cores serve as connecting ligaments between the diverse and varied scientific labs. By working with many researchers, the core staff has the perspective to see what works and what doesn't. The cores also advise labs on best practices; offer scientific consultations; engage in teaching, training and outreach to scientific and lay audiences; and standardize procedures across the Institute. This support bolsters the Institute's foundational and translational science and helps make groundbreaking discoveries that point to ways to tackle some of today's most devastating diseases.


Leah Boyer, director of Salk??s Stem Cell Core, uses a pipette to transfer medium into stem cell dishes.

"Stem cells play hard to get," says Leah Boyer, director of Salk's Stem Cell Core, which opened in 2008 and serves approximately half of the labs at Salk. Many of these users are in neuroscience, a field that has benefited greatly from the ability to grow neurons from stem cells derived from skin cells—a technological advance developed less than a decade ago. Boyer's own interest in neuroscience began, in part, after witnessing her grandmother's battle with Parkinson's disease.

"For 20 years I watched my grandmother's disease unfold, transforming her from an independent pharmacist, keen on crosswords and puzzles, into a woman shamed by the helmet and kneepads she had to wear in public," says Boyer. "She is the reason why I am in San Diego, a neuroscience stronghold, and why I am a stem cell biologist."

Developments in stem cell science have the ability to model and potentially treat a wide range of diseases: not only Parkinson's and other neurological disorders, but also threats ranging from cancer to organ failure. Labs that study cancer mechanics, cellular aging and many other topics rely on stem cell research to make discoveries.

The core offers a "choose-your-own-adventure" experience for the labs, says Boyer, with access to reagents, project consultation, handson- training and additional space. It also generates pluripotent stem cell lines for Salk researchers and has developed over 300 stem cell lines with a 92 percent success rate—numbers that are jaw-dropping to other stem cell cores, many of which have developed 20-some stem cell lines, says Boyer. Additionally, the core tests novel protocols and buys reagents in bulk for the entire Institute, a process that helps keep all of the labs up to date on the latest advances in stem cell research at a fraction of the cost. And it does all of this with only a staff of three.

"We're like researchers' labs outside a lab," says Boyer, who joined the core after working in a Salk neuroscience lab and Harvard's Stem Cell Institute. "Small labs literally could not do this research if we weren't here."

Aside from assisting smaller labs that don't have experience in stem cell work, the core also helps larger stem cell labs that need access to more advanced equipment, qualified reagents and additional space.

In one instance, the lab of Juan Carlos Izpisua Belmonte was able to use custom resources provided by the Stem Cell Core to help prepare experimental stem cell conditions that led to the first reliable method for integrating human stem cells into nonviable mouse embryos in a dish. The work, published in the journal Nature in May 2015, garnered international attention for being a critical precursor to regenerative therapies.

"The core's state-of-the-art technical support for projects involved with the routine maintenance, characterization and differentiation of human pluripotent stem cells was critical to this work," says Jun Wu, first author of the Nature paper and postdoctoral researcher in the Izpisua Belmonte lab. "The customized basal media provided by the Stem Cell Core was one of the key components for generating these human region-selective pluripotent stem cells."

The discovery prompted much interest from other labs at Salk, to the point where Boyer acquired the chemicals and protocol for the technique so that everyone could use it. She is currently working with Wu to optimize and outline an easy-to-use protocol.

"The cores are facilitators of research and the hubs of collaboration—we make science more cost-effective, time-efficient and robust," says Boyer.


Manching Ku

Manching Ku holds out what looks like a glass business card with four pencil-thin lines running across it. Each line is a channel that can hold up to about 150 million fragments of DNA from samples such as, for example, a diseased brain and a healthy brain.

After one side of the double-stranded DNA is stripped off, the sample-laden card—known as a flow cell—is fed into a box-shaped machine called a sequencer. The sequencer reloads matching molecules, sequences of four nucleotides (adenine, thymine, cytosine and guanine) onto the DNA. But these replacement A, T, C and G nucleotides are tagged with colors, allowing a camera inside the machine to capture a snapshot of the newly colorized DNA and decode what exactly those sequences are.

Despite only being made up of four letters, so to speak, our DNA—and all the accompanying bits that hang onto it—is still a puzzle to read. It has been described as a book in which there are no spaces or punctuation marks and with letters thrown in at random. But decoding this book is the key to understanding disease and health, development and aging.

Next generation sequencing took off about 10 years ago, thanks to new technology that allowed comparisons of smaller, multiple samples. This high-throughput screening enables scientists to better understand diseases like cancer by showing what groups of genes are overexpressing on a DNA strand. Many areas of research, like studies in metabolism, epigenetics and plant biology, also look to next generation sequencing to uncover how and why disease progresses.

"We are like the glue of the Institute," says Ku, director of the Next Generation Sequencing Core. Ku came to Salk in 2013 after honing her expertise at the Broad Institute in Cambridge, Massachusetts. "We have a common technology that everyone is able to use no matter what type of organism or system they're studying, from fruit fly to human tissue samples."

Nearly all of the Salk labs use the sequencing core. One of the benefits of having it onsite and accessible to researchers is that Ku is constantly doing project consultations and advising researchers when something looks amiss.

"At other places you might send a sample offsite and not hear back for months," she says. "Here, we can troubleshoot from the beginning and give immediate feedback."

The core is also a knowledge platform, says Ku, who recommends (with permission) one researcher's technique to another. This open exchange makes science more efficient, adds Ku, because researchers don't have to waste time or resources trying attempts that others have already worked through. "Salk is already really collaborative, but the cores make it more so," she says. "No one is forced to reinvent the wheel."

Hu Cang uses a 3D printer to create his own flow cells for use in a hacked gene sequencer. The new technology aims to capture the spatial information of biological samples.

Aside from providing expertise and training, Ku also works with faculty to push the boundaries of the technology. In one collaboration, Ku and Hu Cang, assistant professor, are trying to find a way to capture the three-dimensional information in a cell by reverse-engineering a common genetic sequencer and combining it with a microscope. Their goal is to capture the spatial information of packages of DNA (chromatin) within a cell, the shape of which may have many implications for disease.

"In traditional sequencing, you break apart the cell and harvest the genetic material, but you lose all of the spatial information about that cell," says Cang, who was awarded a Waitt Advanced Biophotonics Imaging Tools and Technologies Seed Grant to pursue this work. "We want to be able to put the entire tissue onto a flow cell, amplify the genes of interest and do the sequencing without destroying its structure."

The duo used a 3D printer to make custom flow cells that would work with this hacked sequencer and hopefully retain—and image— the shape of the chromatin. Ideally, the hacked sequencer would be able to look at a tumor and record the individual transcription of each cell, showing how the cancer develops drug resistance.

"Right now the spatial information of those cells is very hard to quantify and measure," Cang says. He is also in talks with other potential collaborators to use this technique to quantify latent HIV genetic material inside cells, something that is currently hard to pinpoint.

A collaboration like this, adds Cang, wouldn't have happened anywhere else. "I don't know anyone who is pushing sequencing this far," he says. "You need someone with the biological and technical experience like Manching and the engineering background like me, and an environment that supports doing something risky but is also collaborative, like Salk."


Michael Adams and Nicola Allen

Salk Assistant Professor Nicola Allen wanted to find the answer to the question: how many neuronal connections are there in a developing brain? While it's easy enough to take a snapshot of dyed brain cells, figuring out the number of synapses—specialized connections that neurons use to communicate—was a much bigger computational problem, but one that could help to understand how the brain develops and what happens when things go awry.

She found a solution in a collaboration with Michael Adams, a Biophotonics Core staff scientist and physicist. Adams developed a software program to count the number of synaptic connections in a volume of brain tissue. This work will let the Allen lab ultimately investigate if altering the function of other brain cells, known as astrocytes, affects the formation of these synapses in developing and mature brains.

Brain cells called astrocytes are pictured in red, neuronal nuclei in blue.

Courtesy of the Allen lab.

"The Biophotonics Core helped us beyond what we could do on our own," says Allen, who has used most of the Salk cores to advance her neuroscience research. "It's not just about training you on how to use a confocal microscope; the core staff talks to you about your experiments and their design to do science most effectively."

Salk's Biophotonics Core was founded in 2011 as part of the Waitt Advanced Biophotonics Center, which was established with a $20 million gift from the Waitt Foundation. With a staff of only a few, this core remains one of the most heavily used shared resources at the Institute.

About 40 labs rely on the two dozen state-ofthe- art imaging platforms it houses, including light fluorescent and electron microscopy—a broad range of availability not common in a single core. This range allows what's called multi-scaled imaging: a researcher can not only image a whole organism (e.g., the brain of a worm) but can go down to single cells and molecules to understand what happens in genes and proteins and how they impact the whole organism in disease.

The core focuses not just on the development of new microscopes, but rather new computational methods to analyze images. The field of microscopy and imaging has become, like other technologies, data-driven science, as a single image can produce a terabyte or more of data to analyze.

"We very much see this as not a classic core where a staff of technicians assists scientists, but rather a center actively involved in developing new technology to enable people to do experiments that they couldn't otherwise do," says Martin Hetzer, Salk professor and faculty director of the biophotonics center. "Biophotonics is one of the hottest and most rapidly developing areas in biomedical research. Our ability to visualize things in animals and cells is improving through techniques in physics and optics, allowing us to dig deeper into cells."

This core also has a unique model: it operates under the Waitt Advanced Biophotonics Center, which includes a group of award-winning faculty whose specialties span many fields: biomedicine, physics, chemistry and engineering.

"The center really enabled the core to take off," says Hetzer. "The idea was to have a core with the broadest impact on the Institute, while the biophotonics faculty—Hu Cang, Björn Lillemeier and Axel Nimmerjahn—push the new technology and develop imaging approaches that don't exist yet."


In this network representation of genes involved in lymphoma, each circle is a gene and the connections indicate a variety of biological interactions. Darker colors indicate a stronger association with the disease.

Image courtesy of Max Chang, Integrated Genomics Core

As technology becomes more advanced, so does the need to contextualize new data. The Integrative Genomics and Bioinformatics Core, started in 2012, processes raw data from sequencing and develops techniques to combine different types of genomics data, such as linking mutations to the molecular pathways and epigenetic changes involved in research. In addition to providing technological tools, it also offers innovative methods in bioinformatics, network analysis, molecular dynamics and computational data integration.

"Over the course of the last three years, I've learned how to analyze sequencing data through this core," says Julie Law, a Salk assistant professor who studies the genome of plants to better understand epigenetic regulation and DNA packaging. "I started with a cursory understanding of genomics analyses, and after working with the core my lab is now at a point where we can do more sophisticated interpretations of sequencing data to maximize what we can learn from the experiments we do."

Researchers who are doing anything with big data—whether it's next generation sequencing, mass spectrometry, proteomics or biophotonics— need new ways to interpret the reams and reams of information. For scientists that have huge data sets, a regular, off-the-shelf computer cannot process—let alone organize— the information within a reasonable timeframe. "Expanding in this area, and relatedly, high-performance computing in general, is one of our biggest needs for the future," says Berggren.

A data resource center at Salk has already been constructed, with high-end fiber optic data transmission cables and other infrastructure that supports moving and analyzing data for the cores and labs. The next steps, says Berggren, are to secure equipment and, most importantly, staff who not only understand biological research questions, but are also able to leverage the latest approaches in computer science and informatics.

"We have a truly dedicated group of some 40 scientists and core staff researchers that routinely give that extra effort," says Berggren. "It's that extra effort, combined with powerful new technologies, that makes core facilities at Salk such exceptional standouts."


Waitt Advanced Biophotonics Center Core
Provides technical and logistical access for advanced fixed and live cell fluorescence imaging and charged particle imaging methods

Behavior Testing Core
Offers comprehensive resource for behavioral phenotyping and standardized neurobehavioral testing

Flow Cytometry Core
Advances research projects requiring cell sorting and/or analysis of cell populations by flow cytometry

Functional Genomics Core
Provides gene expression profiling using standard Affymetrix arrays and custom cDNA or oligo arrays

Genome Manipulation Core
Generates homologous recombination-base gene targeted mouse embryonic stem cell lines

Gene Transfer, Targeting and Therapeutics Core
Contributes design, consultation and production services for retrovirus, lentivirus, adeno-associated virus, adenovirus, rabies virus and vesicular stomatitis virus-based viral vector systems

Razavi Newman Integrative Genomics and Bioinformatics Core
Focuses on the analysis of next-generation sequencing and other genomics data as well as develops novel analysis algorithms

Mass Spectrometry Core for Proteomics and Metabolomics
Offers an array of services from protein identification and proteomic profiling to more complex studies using state-of-the-art instrumentation

Media Preparation Core
Provides routine and specialized cell culture media

Peptide Synthesis Core
Carries out the synthesis of inexpensive peptides and provides unmodified, biotinylated, acetylated or phosphorylated peptides

The H.A. and Mary K. Chapman Charitable Foundations Genomic Sequencing Core
Provides low-cost, rapid-turnaround, high-throughput sequencing services, offers consultation and training in next-generation sequencing-based methods and assists in method development

Helmsley Stem Cell Core
Supports the needs for human ES and reprogrammed iPS cell culture and offers training and the physical space to carry out experiments using pluripotent cell types

Transgenic Core
Supplies services to create transgenic and knockout mouse models