single cell analysis
Posted on by Dr. Francis Collins
These colorful lights might look like a video vignette from one of the spectacular evening light shows taking place this holiday season. But they actually aren’t. These lights are illuminating the way to a much fuller understanding of the mammalian brain.
The video features a new research method called BARseq (Barcoded Anatomy Resolved by Sequencing). Created by a team of NIH-funded researchers led by Anthony Zador, Cold Spring Harbor Laboratory, NY, BARseq enables scientists to map in a matter of weeks the location of thousands of neurons in the mouse brain with greater precision than has ever been possible before.
How does it work? With BARseq, researchers generate uniquely identifying RNA barcodes and then tag one to each individual neuron within brain tissue. As reported recently in the journal Cell, those barcodes allow them to keep track of the location of an individual cell amid millions of neurons . This also enables researchers to map the tangled paths of individual neurons from one region of the mouse brain to the next.
The video shows how the researchers read the barcodes. Each twinkling light is a barcoded neuron within a thin slice of mouse brain tissue. The changing colors from frame to frame correspond to one of the four letters, or chemical bases, in RNA (A=purple, G=blue, U=yellow, and C=white). A neuron that flashes blue, purple, yellow, white is tagged with a barcode that reads GAUC, while yellow, white, white, white is UCCC.
By sequencing and reading the barcodes to distinguish among seemingly identical cells, the researchers mapped the connections of more than 3,500 neurons in a mouse’s auditory cortex, a part of the brain involved in hearing. In fact, they report they’re now able to map tens of thousands of individual neurons in a mouse in a matter of weeks.
What makes BARseq even better than the team’s previous mapping approach, called MAPseq, is its ability to read the barcodes at their original location in the brain tissue . As a result, they can produce maps with much finer resolution. It’s also possible to maintain other important information about each mapped neuron’s identity and function, including the expression of its genes.
Zador reports that they’re continuing to use BARseq to produce maps of other essential areas of the mouse brain with more detail than had previously been possible. Ultimately, these maps will provide a firm foundation for better understanding of human thought, consciousness, and decision-making, along with how such mental processes get altered in conditions such as autism spectrum disorder, schizophrenia, and depression.
Here’s wishing everyone a safe and happy holiday season. It’s been a fantastic year in science, and I look forward to bringing you more cool NIH-supported research in 2020!
 High-Throughput Mapping of Long-Range Neuronal Projection Using In Situ Sequencing. Chen X, Sun YC, Zhan H, Kebschull JM, Fischer S, Matho K, Huang ZJ, Gillis J, Zador AM. Cell. 2019 Oct 17;179(3):772-786.e19.
 High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA. Kebschull JM, Garcia da Silva P, Reid AP, Peikon ID, Albeanu DF, Zador AM. Neuron. 2016 Sep 7;91(5):975-987.
Zador Lab (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)
NIH Support: National Institute of Neurological Disorders and Stroke; National Institute on Drug Abuse; National Cancer Institute
Posted on by Dr. Francis Collins
A major aim of the NIH-led Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative is to develop new technologies that allow us to look at the brain in many different ways on many different scales. So, I’m especially pleased to highlight this winner of the initiative’s recent “Show Us Your Brain!” contest.
Here you get a close-up look at pyramidal neurons located in the hippocampus, a region of the mammalian brain involved in memory. While this tiny sample of mouse brain is densely packed with many pyramidal neurons, researchers used new ExLLSM technology to zero in on just three. This super-resolution, 3D view reveals the intricacies of each cell’s structure and branching patterns.
The group that created this award-winning visual includes the labs of X. William Yang at the University of California, Los Angeles, and Kwanghun Chung at the Massachusetts Institute of Technology, Cambridge. Chung’s team also produced another quite different “Show Us Your Brain!” winner, a colorful video featuring hundreds of neural cells and connections in a part of the brain essential to movement.
Pyramidal neurons in the hippocampus come in many different varieties. Some important differences in their functional roles may be related to differences in their physical shapes, in ways that aren’t yet well understood. So, BRAIN-supported researchers are now applying a variety of new tools and approaches in a more detailed effort to identify and characterize these neurons and their subtypes.
The video featured here took advantage of Chung’s new method for preserving brain tissue samples . Another secret to its powerful imagery was a novel suite of mouse models developed in the Yang lab. With some sophisticated genetics, these models make it possible to label, at random, just 1 to 5 percent of a given neuronal cell type, illuminating their full morphology in the brain . The result was this unprecedented view of three pyramidal neurons in exquisite 3D detail.
Ultimately, the goal of these and other BRAIN Initiative researchers is to produce a dynamic picture of the brain that, for the first time, shows how individual cells and complex neural circuits interact in both time and space. I look forward to their continued progress, which promises to revolutionize our understanding of how the human brain functions in both health and disease.
 Protection of tissue physicochemical properties using polyfunctional crosslinkers. Park YG, Sohn CH, Chen R, McCue M, Yun DH, Drummond GT, Ku T, Evans NB, Oak HC, Trieu W, Choi H, Jin X, Lilascharoen V, Wang J, Truttmann MC, Qi HW, Ploegh HL, Golub TR, Chen SC, Frosch MP, Kulik HJ, Lim BK, Chung K. Nat Biotechnol. 2018 Dec 17.
 Genetically-directed Sparse Neuronal Labeling in BAC Transgenic Mice through Mononucleotide Repeat Frameshift. Lu XH, Yang XW. Sci Rep. 2017 Mar 8;7:43915.
Chung Lab (Massachusetts Institute of Technology, Cambridge)
Yang Lab (University of California, Los Angeles)
Show Us Your Brain! (BRAIN Initiative/NIH)
NIH Support: National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; National Institute of Biomedical Imaging and Bioengineering
Posted on by Dr. Francis Collins
Courtesy of the Chen and Macosko labs
A few years ago, I highlighted a really cool technology called Drop-seq for simultaneously analyzing the gene expression activity inside thousands of individual cells. Today, one of its creators, Evan Macosko, reports significant progress in developing even better tools for single-cell analysis—with support from an NIH Director’s New Innovator Award.
In a paper in the journal Science, Macosko, Fei Chen, and colleagues at the Broad Institute of Harvard and Massachusetts Institute of Technology (MIT), Cambridge, recently unveiled another exciting creation called Slide-seq . This technology acts as a GPS-like system for mapping the exact location of each of the thousands of individual cells undergoing genomic analysis in a tissue sample.
This 3D video shows the exquisite precision of this new cellular form of GPS, which was used to generate a high-resolution map of the different cell types found in a tiny cube of mouse brain tissue. Specifically, it provides locations of the cell types and gene expression in the hippocampal regions called CA1 (green), CA2/3 (blue), and dentate gyrus (red).
Because using Slide-seq in the lab requires no specialized imaging equipment or skills, it should prove valuable to researchers across many different biomedical disciplines who want to look at cellular relationships or study gene activity in tissues, organs, or even whole organisms.
How does Slide-seq work? Macosko says one of the main innovations is an inexpensive rubber-coated glass slide nicknamed a puck. About 3 millimeters in diameter, pucks are studded with tens of thousands of 10 micron-sized beads, each one decorated with a random snippet of genetic material—an RNA barcode—that serves as its unique identifier of the bead.
The barcodes are sequenced en masse, and the exact location of each barcoded bead is indexed using innovative software developed by a team led by Chen, who is an NIH Director’s Early Independence awardee.
Then, the researchers place a sample of fresh-frozen tissue (typically, 10 micrometers, or 0.00039 inches, thick) on the puck and dissolve the tissue, lysing the cells and releasing their messenger RNA (mRNA). That leaves only the barcoded beads binding the mRNA transcripts expressed by the cells in the tissue—a biological record of the genes that were turned on at the time the sample was frozen.
The barcoded mRNA is then sequenced. The spatial position of each mRNA molecule can be inferred, using the reference index on the puck. This gives researchers a great deal of biological information about the cells in the tissue, often including their cell type and their gene expression pattern. All the data can then be mapped out in ways similar to those seen in this video, which was created using data from 66 pucks.
Slide-seq has been tested on a range of tissues from both mouse and human, replicating results from similar maps created using existing approaches, but also uncovering new biology. For example, in the mouse cerebellum, Slide-seq allowed the researchers to detect bands of variable gene activity across the tissues. This intriguing finding suggests that there may be subpopulations of cells in this part of the brain that have gene activity influenced by their physical locations.
Such results demonstrate the value of combining cell location with genomic information. In fact, Macosko now hopes to use Slide-seq to study the response of brain cells that are located near the buildup of damaged amyloid protein associated with the early-stage Alzheimer’s disease. Meanwhile, Chen is interested in pursuing cell lineage studies in a variety of tissues to see how and where changes in the molecular dynamics of tissues can lead to disease.
These are just a few examples of how Slide-seq will add to the investigative power of single-cell analysis in the years ahead. In meantime, the Macosko and Chen labs are working hard to develop even more innovative approaches to this rapidly emerging areas of biomedical research, so who knows what “seq” we will be talking about next?
 Slide-seq: A scalable technology for measuring genome-wide expression at high spatial resolution. Rodriques SG, Stickels RR, Goeva A, Martin CA, Murray E, Vanderburg CR, Welch J, Chen LM, Chen F, Macosko EZ. Science. 2019 Mar 29;363(6434):1463-1467.
Single Cell Analysis (NIH)
Macosko Lab (Broad Institute of Harvard and MIT, Cambridge)
Chen Lab (Broad Institute)
NIH Support: National Institute on Aging; Common Fund
Posted on by Dr. Francis Collins
A Happy New Year to one and all! While many of us were busy wrapping presents, the journal Science announced its much-anticipated scientific breakthroughs of 2018. In case you missed the announcement , it was another banner year for the biomedical sciences.
The 2018 Breakthrough of the Year went to biomedical science and its ability to track the development of life—one cell at a time—in a variety of model organisms. This newfound ability opens opportunities to understand the biological basis of life more systematically than ever before. Among Science’s “runner-up” breakthroughs, more than half had strong ties to the biomedical sciences and NIH-supported research.
Sound intriguing? Let’s take a closer look at some of the amazing science conducted in 2018, starting with Science’s Breakthrough of the Year.
Development Cell by Cell: For millennia, biologists have wondered how a single cell develops into a complete multicellular organism, such as a frog or a mouse. But solving that mystery was almost impossible without the needed tools to study development systematically, one cell at a time. That’s finally started to change within the last decade. I’ve highlighted the emergence of some of these powerful tools on my blog and the interesting ways that they were being applied to study development.
Over the past few years, all of this technological progress has come to a head. Researchers, many of them NIH-supported, used sophisticated cell labeling techniques, nucleic acid sequencing, and computational strategies to isolate thousands of cells from developing organisms, sequence their genetic material, and determine their location within that developing organism.
In 2018 alone, groundbreaking single-cell analysis papers were published that sequentially tracked the 20-plus cell types that arise from a fertilized zebrafish egg, the early formation of organs in a frog, and even the creation of a new limb in the Axolotl salamander. This is just the start of amazing discoveries that will help to inform us of the steps, or sometimes missteps, within human development—and suggest the best ways to prevent the missteps. In fact, efforts are now underway to gain this detailed information in people, cell by cell, including the international Human Cell Atlas and the NIH-supported Human BioMolecular Atlas Program.
An RNA Drug Enters the Clinic: Twenty years ago, researchers Andrew Fire and Craig Mello showed that certain small, noncoding RNA molecules can selectively block genes in our cells from turning “on” through a process called RNA interference (RNAi). This work, for the which these NIH grantees received the 2006 Nobel Prize in Physiology or Medicine, soon sparked a wave of commercial interest in various noncoding RNA molecules for their potential to silence the expression of a disease-causing gene.
After much hard work, the first gene-silencing RNA drug finally came to market in 2018. It’s called Onpattro™ (patisiran), and the drug uses RNAi to treat the peripheral nerve disease that can afflict adults with a rare disease called hereditary transthyretin-mediated amyloidosis. This hard-won success may spark further development of this novel class of biopharmaceuticals to treat a variety of conditions, from cancer to cardiovascular disorders, with potentially greater precision.
Rapid Chemical Structure Determination: Last October, two research teams released papers almost simultaneously that described an incredibly fast new imaging technique to determine the structure of smaller organic chemical compounds, or “small molecules“ at atomic resolution. Small molecules are essential components of molecular biology, pharmacology, and drug development. In fact, most of our current medicines are small molecules.
News of these papers had many researchers buzzing, and I highlighted one of them on my blog. It described a technique called microcrystal electron diffraction, or MicroED. It enabled these NIH-supported researchers to take a powder form of small molecules (progesterone was one example) and generate high-resolution data on their chemical structures in less than a half-hour! The ease and speed of MicroED could revolutionize not only how researchers study various disease processes, but aid in pinpointing which of the vast number of small molecules can become successful therapeutics.
How Cells Marshal Their Contents: About a decade ago, researchers discovered that many proteins in our cells, especially when stressed, condense into circumscribed aqueous droplets. This so-called phase separation allows proteins to gather in higher concentrations and promote reactions with other proteins. The NIH soon began supporting several research teams in their groundbreaking efforts to explore the effects of phase separation on cell biology.
Over the past few years, work on phase separation has taken off. The research suggests that this phenomenon is critical in compartmentalizing chemical reactions within the cell without the need of partitioning membranes. In 2018 alone, several major papers were published, and the progress already has some suggesting that phase separation is not only a basic organizing principle of the cell, it’s one of the major recent breakthroughs in biology.
Forensic Genealogy Comes of Age: Last April, police in Sacramento, CA announced that they had arrested a suspect in the decades-long hunt for the notorious Golden State Killer. As exciting as the news was, doubly interesting was how they caught the accused killer. The police had the Golden Gate Killer’s DNA, but they couldn’t determine his identity, that is, until they got a hit on a DNA profile uploaded by one of his relatives to a public genealogy database.
Though forensic genealogy falls a little outside of our mission, NIH has helped to advance the gathering of family histories and using DNA to study genealogy. In fact, my blog featured NIH-supported work that succeeded in crowdsourcing 600 years of human history.
The researchers, using the online profiles of 86 million genealogy hobbyists with their permission, assembled more than 5 million family trees. The largest totaled more than 13 million people! By merging each tree from the crowd-sourced and public data, they were able to go back about 11 generations—to the 15th century and the days of Christopher Columbus. Though they may not have caught an accused killer, these large datasets provided some novel insights into our family structures, genes, and longevity.
An Ancient Human Hybrid: Every year, researchers excavate thousands of bone fragments from the remote Denisova Cave in Siberia. One such find would later be called Denisova 11, or “Denny” for short.
Oh, what a fascinating genomic tale Denny’s sliver of bone had to tell. Denny was at least 13 years old and lived in Siberia roughly 90,000 years ago. A few years ago, an international research team found that DNA from the mitochondria in Denny’s cells came from a Neanderthal, an extinct human relative.
In 2018, Denny’s family tree got even more interesting. The team published new data showing that Denny was female and, more importantly, she was a first generation mix of a Neanderthal mother and a father who belonged to another extinct human relative called the Denisovans. The Denisovans, by the way, are the first human relatives characterized almost completely on the basis of genomics. They diverged from Neanderthals about 390,000 years ago. Until about 40,000 years ago, the two occupied the Eurasian continent—Neanderthals to the west, and Denisovans to the east.
Denny’s unique genealogy makes her the first direct descendant ever discovered of two different groups of early humans. While NIH didn’t directly support this research, the sequencing of the Neanderthal genome provided an essential resource.
As exciting as these breakthroughs are, they only scratch the surface of ongoing progress in biomedical research. Every field of science is generating compelling breakthroughs filled with hope and the promise to improve the lives of millions of Americans. So let’s get started with 2019 and finish out this decade with more truly amazing science!
 “2018 Breakthrough of the Year,” Science, 21 December 2018.
NIH Support: These breakthroughs represent the culmination of years of research involving many investigators and the support of multiple NIH institutes.