Archive for the ‘BioBanking’ Category

Colon cancer and organoids

Larry H. Bernstein, MD, FCAP, Curator





Guts and Glory

An open mind and collaborative spirit have taken Hans Clevers on a journey from medicine to developmental biology, gastroenterology, cancer, and stem cells.

By Anna Azvolinsky

Ihave had to talk a lot about my science recently and it’s made me think about how science works,” says Hans Clevers. “Scientists are trained to think science is driven by hypotheses, but for [my lab], hypothesis-driven research has never worked. Instead, it has been about trying to be as open-minded as possible—which is not natural for our brains,” adds the Utrecht University molecular genetics professor. “The human mind is such that it tries to prove it’s right, so pursuing a hypothesis can result in disaster. My advice to my own team and others is to not preformulate an answer to a scientific question, but just observe and never be afraid of the unknown. What has worked well for us is to keep an open mind and do the experiments. And find a collaborator if it is outside our niche.”

“One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory.”

Clevers entered medical school at Utrecht University in The Netherlands in 1978 while simultaneously pursuing a master’s degree in biology. Drawn to working with people in the clinic, Clevers had a training position in pediatrics lined up after medical school, but then mentors persuaded him to spend an additional year converting the master’s degree to a PhD in immunology. “At the end of that year, looking back, I got more satisfaction from the research than from seeing patients.” Clevers also had an aptitude for benchwork, publishing four papers from his PhD year. “They were all projects I had made up myself. The department didn’t do the kind of research I was doing,” he says. “Now that I look back, it’s surprising that an inexperienced PhD student could come up with a project and publish independently.”

Clevers studied T- and B-cell signaling; he set up assays to visualize calcium ion flux and demonstrated that the ions act as messengers to activate human B cells, signaling through antibodies on the cell surface. “As soon as the experiment worked, I got T cells from the lab next door and did the same experiment. That was my strategy: as soon as something worked, I would apply it elsewhere and didn’t stop just because I was a B-cell biologist and not a T-cell biologist. What I learned then, that I have continued to benefit from, is that a lot of scientists tend to adhere to a niche. They cling to these niches and are not that flexible. You think scientists are, but really most are not.”

Here, Clevers talks about promoting a collaborative spirit in research, the art of doing a pilot experiment, and growing miniature organs in a dish.

Clevers Creates

Re-search? Clevers was born in Eindhoven, in the south of The Netherlands. The town was headquarters to Philips Electronics, where his father worked as a businessman, and his mother took care of Clevers and his three brothers. Clevers did well in school but his passion was sports, especially tennis and field hockey, “a big thing in Holland.” Then in 1975, at age 18, he moved to Utrecht University, where he entered an intensive, biology-focused program. “I knew I wanted to be a biology researcher since I was young. In Dutch, the word for research is ‘onderzoek’ and I knew the English word ‘research’ and had wondered why there was the ‘re’ in the word, because I wanted to search but I didn’t want to do re-search—to find what someone else had already found.”

Opportunity to travel. “I was very disappointed in my biology studies, which were old-fashioned and descriptive,” says Clevers. He thought medicine might be more interesting and enrolled in medical school while still pursuing a master’s degree in biology at Utrecht. For the master’s, Clevers had to do three rotations. He spent a year at the International Laboratory for Research on Animal Diseases (ILRAD) in Nairobi, Kenya, and six months in Bethesda, Maryland, at the National Institutes of Health. “Holland is really small, so everyone travels.” Clevers saw those two rotations more as travel explorations. In Nairobi, he went on safaris and explored the country in Land Rovers borrowed from the institute. While in Maryland in 1980, Clevers—with the consent of his advisor, who thought it was a good idea for him to get a feel for the U.S.—flew to Portland, Oregon, and drove back to Boston with a musician friend along the Canadian border. He met the fiancé of political activist and academic Angela Davis in New York City and even stayed in their empty apartment there.

Life and lab lessons. Back in Holland, Clevers joined Rudolf Eugène Ballieux’s lab at Utrecht University to pursue his PhD, for which he studied immune cell signaling. “I didn’t learn much science from him, but I learned that you always have to create trust and to trust people around you. This became a major theme in my own lab. We don’t distrust journals or reviewers or collaborators. We trust everyone and we share. There will be people who take advantage, but there have only been a few of those. So I learned from Ballieux to give everyone maximum trust and then change this strategy only if they fail that trust. We collaborate easily because we give out everything and we also easily get reagents and tools that we may need. It’s been valuable to me in my career. And it is fun!”

Clevers Concentrates

On a mission. “Once I decided to become a scientist, I knew I needed to train seriously. Up to that point, I was totally self-trained.” From an extensive reading of the immunology literature, Clevers became interested in how T cells recognize antigens, and headed off to spend a postdoc studying the problem in Cox Terhorst’s lab at Dana-Farber Cancer Institute in Boston. “Immunology was young, but it was very exciting and there was a lot to discover. I became a professional scientist there and experienced how tough science is.” In 1988, Clevers cloned and characterized the gene for a component of the T-cell receptor (TCR) called CD3-epsilon, which binds antigen and activates intracellular signaling pathways.

On the fast track in Holland. Clevers returned to Utrecht University in 1989 as a professor of immunology. Within one month of setting up his lab, he had two graduate students and a technician, and the lab had cloned the first T cell–specific transcription factor, which they called TCF-1, in human T cells. When his former thesis advisor retired, Clevers was asked, at age 33, to become head of the immunology department. While the appointment was high-risk for him and for the department, Clevers says, he was chosen because he was good at multitasking and because he got along well with everyone.

Problem-solving strategy. “My strategy in research has always been opportunistic. One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory. I think there is an art to doing pilot experiments. So we have always just set up systems in which something happens and then you try and try things until a pattern appears and maybe you formulate a small hypothesis. But as soon as it turns out not to be exactly right, you abandon it. It’s a very open-minded type of research where you question whether what you are seeing is a real phenomenon without spending a year on doing all of the proper controls.”

Trial and error. Clevers’s lab found that while TCF-1 bound to DNA, it did not alter gene expression, despite the researchers’ tinkering with promoter and enhancer assays. “For about five years this was a problem. My first PhD students were leaving and they thought the whole TCF project was a failure,” says Clevers. His lab meanwhile cloned TCF homologs from several model organisms and made many reagents including antibodies against these homologs. To try to figure out the function of TCF-1, the lab performed a two-hybrid screen and identified components of the Wnt signaling pathway as binding partners of TCF-1. “We started to read about Wnt and realized that you study Wnt not in T cells but in frogs and flies, so we rapidly transformed into a developmental biology lab. We showed that we held the key for a major issue in developmental biology, the final protein in the Wnt cascade: TCF-1 binds b-catenin when b-catenin becomes available and activates transcription.” In 1996, Clevers published the mechanism of how the TCF-1 homolog in Xenopus embryos, called XTcf-3, is integrated into the Wnt signaling pathway.

Clevers Catapults


3DCrypt building and colon cancer.

Clevers next collaborated with Bert Vogelstein’s lab at Johns Hopkins, linking TCF to Wnt signaling in colon cancer. In colon cancer cell lines with mutated forms of the tumor suppressor gene APC, the APC protein can’t rein in b-catenin, which accumulates in the cytoplasm, forms a complex with TCF-4 (later renamed TCF7L2) in the nucleus, and caninitiate colon cancer by changing gene expression. Then, the lab showed that Wnt signaling is necessary for self-renewal of adult stem cells, as mice missing TCF-4 do not have intestinal crypts, the site in the gut where stem cells reside. “This was the first time Wnt was shown to play a role in adults, not just during development, and to be crucial for adult stem cell maintenance,” says Clevers. “Then, when I started thinking about studying the gut, I realized it was by far the best way to study stem cells. And I also realized that almost no one in the world was studying the healthy gut. Almost everyone who researched the gut was studying a disease.” The main advantages of the murine model are rapid cell turnover and the presence of millions of stereotypic crypts throughout the entire intestine.

Against the grain. In 2007, Nick Barker, a senior scientist in the Clevers lab, identified the Wnt target gene Lgr5 as a unique marker of adult stem cells in several epithelial organs, including the intestine, hair follicle, and stomach. In the intestine, the gene codes for a plasma membrane protein on crypt stem cells that enable the intestinal epithelium to self-renew, but can also give rise to adenomas of the gut. Upon making mice with adult stem cell populations tagged with a fluorescent Lgr5-binding marker, the lab helped to overturn assumptions that “stem cells are rare, impossible to find, quiescent, and divide asymmetrically.”

On to organoids. Once the lab could identify adult stem cells within the crypts of the gut, postdoc Toshiro Sato discovered that a single stem cell, in the presence of Matrigel and just three growth factors, could generate a miniature crypt structure—what is now called an organoid. “Toshi is very Japanese and doesn’t always talk much,” says Clevers. “One day I had asked him, while he was at the microscope, if the gut stem cells were growing, and he said, ‘Yes.’ Then I looked under the microscope and saw the beautiful structures and said, ‘Why didn’t you tell me?’ and he said, ‘You didn’t ask.’ For three months he had been growing them!” The lab has since also grown mini-pancreases, -livers, -stomachs, and many other mini-organs.

Tumor Organoids. Clevers showed that organoids can be grown from diseased patients’ samples, a technique that could be used in the future to screen drugs. The lab is also building biobanks of organoidsderived from tumor samples and adjacent normal tissue, which could be especially useful for monitoring responses to chemotherapies. “It’s a similar approach to getting a bacterium cultured to identify which antibiotic to take. The most basic goal is not to give a toxic chemotherapy to a patient who will not respond anyway,” says Clevers. “Tumor organoids grow slower than healthy organoids, which seems counterintuitive, but with cancer cells, often they try to divide and often things go wrong because they don’t have normal numbers of chromosomes and [have] lots of mutations. So, I am not yet convinced that this approach will work for every patient. Sometimes, the tumor organoids may just grow too slowly.”

Selective memory. “When I received the Breakthrough Prize in 2013, I invited everyone who has ever worked with me to Amsterdam, about 100 people, and the lab organized a symposium where many of the researchers gave an account of what they had done in the lab,” says Clevers. “In my experience, my lab has been a straight line from cloning TCF-1 to where we are now. But when you hear them talk it was ‘Hans told me to try this and stop this’ and ‘Half of our knockout mice were never published,’ and I realized that the lab is an endless list of failures,” Clevers recalls. “The one thing we did well is that we would start something and, as soon as it didn’t look very good, we would stop it and try something else. And the few times when we seemed to hit gold, I would regroup my entire lab. We just tried a lot of things, and the 10 percent of what worked, those are the things I remember.”

Greatest Hits

  • Cloned the first T cell–specific transcription factor, TCF-1, and identified homologous genes in model organisms including the fruit fly, frog, and worm
  • Found that transcriptional activation by the abundant β-catenin/TCF-4 [TCF7L2] complex drives cancer initiation in colon cells missing the tumor suppressor protein APC
  • First to extend the role of Wnt signaling from developmental biology to adult stem cells by showing that the two Wnt pathway transcription factors, TCF-1 and TCF-4, are necessary for maintaining the stem cell compartments in the thymus and in the crypt structures of the small intestine, respectively
  • Identified Lgr5 as an adult stem cell marker of many epithelial stem cells including those of the colon, small intestine, hair follicle, and stomach, and found that Lgr5-expressing crypt cells in the small intestine divide constantly and symmetrically, disproving the common belief that stem cell division is asymmetrical and uncommon
  • Established a three-dimensional, stable model, the “organoid,” grown from adult stem cells, to study diseased patients’ tissues from the gut, stomach, liver, and prostate
 Regenerative Medicine Comes of Age   
“Anti-Aging Medicine” Sounds Vaguely Disreputable, So Serious Scientists Prefer to Speak of “Regenerative Medicine”
  • Induced pluripotent stem cells (iPSCs) and genome-editing techniques have facilitated manipulation of living organisms in innumerable ways at the cellular and genetic levels, respectively, and will underpin many aspects of regenerative medicine as it continues to evolve.

    An attitudinal change is also occurring. Experts in regenerative medicine have increasingly begun to embrace the view that comprehensively repairing the damage of aging is a practical and feasible goal.

    A notable proponent of this view is Aubrey de Grey, Ph.D., a biomedical gerontologist who has pioneered an regenerative medicine approach called Strategies for Engineered Negligible Senescence (SENS). He works to “develop, promote, and ensure widespread access to regenerative medicine solutions to the disabilities and diseases of aging” as CSO and co-founder of the SENS Research Foundation. He is also the editor-in-chief of Rejuvenation Research, published by Mary Ann Liebert.

    Dr. de Grey points out that stem cell treatments for age-related conditions such as Parkinson’s are already in clinical trials, and immune therapies to remove molecular waste products in the extracellular space, such as amyloid in Alzheimer’s, have succeeded in such trials. Recently, there has been progress in animal models in removing toxic cells that the body is failing to kill. The most encouraging work is in cancer immunotherapy, which is rapidly advancing after decades in the doldrums.

    Many damage-repair strategies are at an  early stage of research. Although these strategies look promising, they are handicapped by a lack of funding. If that does not change soon, the scientific community is at risk of failing to capitalize on the relevant technological advances.

    Regenerative medicine has moved beyond boutique applications. In degenerative disease, cells lose their function or suffer elimination because they harbor genetic defects. iPSC therapies have the potential to be curative, replacing the defective cells and eliminating symptoms in their entirety. One of the biggest hurdles to commercialization of iPSC therapies is manufacturing.

  • Building Stem Cell Factories

    Cellular Dynamics International (CDI) has been developing clinically compatible induced pluripotent stem cells (iPSCs) and iPSC-derived human retinal pigment epithelial (RPE) cells. CDI’s MyCell Retinal Pigment Epithelial Cells are part of a possible therapy for macular degeneration. They can be grown on bioengineered, nanofibrous scaffolds, and then the RPE cell–enriched scaffolds can be transplanted into patients’ eyes. In this pseudo-colored image, RPE cells are shown growing over the nanofibers. Each cell has thousands of “tongue” and “rod” protrusions that could naturally support rod and cone cells in the eye.

    “Now that an infrastructure is being developed to make unlimited cells for the tools business, new opportunities are being created. These cells can be employed in a therapeutic context, and they can be used to understand the efficacy and safety of drugs,” asserts Chris Parker, executive vice president and CBO, Cellular Dynamics International (CDI). “CDI has the capability to make a lot of cells from a single iPSC line that represents one person (a capability termed scale-up) as well as the capability to do it in parallel for multiple individuals (a capability termed scale-out).”

    Minimally manipulated adult stem cells have progressed relatively quickly to the clinic. In this scenario, cells are taken out of the body, expanded unchanged, then reintroduced. More preclinical rigor applies to potential iPSC therapy. In this case, hematopoietic blood cells are used to make stem cells, which are manufactured into the cell type of interest before reintroduction. Preclinical tests must demonstrate that iPSC-derived cells perform as intended, are safe, and possess little or no off-target activity.

    For example, CDI developed a Parkinsonian model in which iPSC-derived dopaminergic neurons were introduced to primates. The model showed engraftment and enervation, and it appeared to be free of proliferative stem cells.

    • “You will see iPSCs first used in clinical trials as a surrogate to understand efficacy and safety,” notes Mr. Parker. “In an ongoing drug-repurposing trial with GlaxoSmithKline and Harvard University, iPSC-derived motor neurons will be produced from patients with amyotrophic lateral sclerosis and tested in parallel with the drug.” CDI has three cell-therapy programs in their commercialization pipeline focusing on macular degeneration, Parkinson’s disease, and postmyocardial infarction.

    • Keeping an Eye on Aging Eyes

      The California Project to Cure Blindness is evaluating a stem cell–based treatment strategy for age-related macular degeneration. The strategy involves growing retinal pigment epithelium (RPE) cells on a biostable, synthetic scaffold, then implanting the RPE cell–enriched scaffold to replace RPE cells that are dying or dysfunctional. One of the project’s directors, Dennis Clegg, Ph.D., a researcher at the University of California, Santa Barbara, provided this image, which shows stem cell–derived RPE cells. Cell borders are green, and nuclei are red.

      The eye has multiple advantages over other organ systems for regenerative medicine. Advanced surgical methods can access the back of the eye, noninvasive imaging methods can follow the transplanted cells, good outcome parameters exist, and relatively few cells are needed.

      These advantages have attracted many groups to tackle ocular disease, in particular age-related macular degeneration, the leading cause of blindness in the elderly in the United States. Most cases of age-related macular degeneration are thought to be due to the death or dysfunction of cells in the retinal pigment epithelium (RPE). RPE cells are crucial support cells for the rods, cones, and photoreceptors. When RPE cells stop working or die, the photoreceptors die and a vision deficit results.

      A regenerated and restored RPE might prevent the irreversible loss of photoreceptors, possibly via the the transplantation of functionally polarized RPE monolayers derived from human embryonic stem cells. This approach is being explored by the California Project to Cure Blindness, a collaborative effort involving the University of Southern California (USC), the University of California, Santa Barbara (UCSB), the California Institute of Technology, City of Hope, and Regenerative Patch Technologies.

      The project, which is funded by the California Institute of Regenerative Medicine (CIRM), started in 2010, and an IND was filed early 2015. Clinical trial recruitment has begun.

      One of the project’s leaders is Dennis Clegg, Ph.D., Wilcox Family Chair in BioMedicine, UCSB. His laboratory developed the protocol to turn undifferentiated H9 embryonic stem cells into a homogenous population of RPE cells.

      “These are not easy experiments,” remarks Dr. Clegg. “Figuring out the biology and how to make the cell of interest is a challenge that everyone in regenerative medicine faces. About 100,000 RPE cells will be grown as a sheet on a 3 × 5 mm biostable, synthetic scaffold, and then implanted in the patients to replace the cells that are dying or dysfunctional. The idea is to preserve the photoreceptors and to halt disease progression.”

      Moving therapies such as this RPE treatment from concept to clinic is a huge team effort and requires various kinds of expertise. Besides benefitting from Dr. Clegg’s contribution, the RPE project incorporates the work of Mark Humayun, M.D., Ph.D., co-director of the USC Eye Institute and director of the USC Institute for Biomedical Therapeutics and recipient of the National Medal of Technology and Innovation, and David Hinton, Ph.D., a researcher at USC who has studied how actvated RPE cells can alter the local retinal microenvironment.


Read Full Post »

What a brain is this?

Larry H. Bernstein, MD, FCAP, Curator



New cryopreservation procedure wins Brain Preservation Prize

First preservation of the connectome demonstrated in a whole brain
February 9, 2016


(Left): Control rabbit brain, showing neuropil near the CA1 band in the hippocampus. (Right): Vitrified rabbit brain, same location. Synapses, vesicles, and microfilaments are clear. The myelinated axon shows excellent preservation. (credit: Robert L. McIntyre and Gregory M. Fahy/Cryobiology)


The Brain Preservation Foundation (BPF) has announced that a team at 21st Century Medicine led by Robert McIntyre, PhD., has won the Small Mammal Brain Preservation Prize, which carries an award of $26,735.

The Small Mammalian Brain Preservation Prize was awarded after the determination that the protocol developed by McIntyre, termed Aldehyde-Stabilized Cryopreservation, was able to preserve an entire rabbit brain with well-preserved ultrastructure, including cell membranes, synapses, and intracellular structures such as synaptic vesicles (full protocol here).

The judges for the prize were Kenneth Hayworth, PhD., Brain Preservation Foundation President and neuroscientist at the Howard Hughes Medical Institute; and Prof. Sebastian Seung, PhD., Princeton Neuroscience Institute and Computer Science Department.

First preservation of the connectome

“This is a milestone in the development of brain preservation techniques: it is the first time that the preservation of the connectome has been demonstrated in a whole brain (prior to this only small parts of brains have been preserved to this level of detail),” said the BPF announcement.

“Current models of the brain suggest that the connectome contains all the information necessary for personal identity (i.e., memory and personality). This technique is not the same as conventional cryonics (rapidly freezing the brain), which has never demonstrated preservation of the ultrastructure of the brain. Thus the winning of this prize represents a significant advance in the methods available for large scale studies of the connectome and could lead to procedures that preserve a complete human brain.

Kenneth Hayworth (KH) (President of the Brain Preservation Foundation (BPF)) and Michael Shermer (member of BPF advisory board) witnessed (on Sept. 25, 2015) the full Aldehyde Stabilized Cryopreservation surgical procedure performed on this rabbit at the laboratories of 21 Century Medicine under the direction of 21CM lead researcher Robert McIntyre. This included the live rabbit’s carotid arteries being perfused with glutaraldehyde and subsequent perfusion with cryoprotectant agent (CPA). KH witnessed this rabbit brain being put in -135 degrees C storage, removal from storage the following day (verifying that it had vitrified solid), and KH witnessed all subsequent tissue processing steps involved in the evaluation process. (credit: The Brain Preservation Foundation)

“The key breakthrough was the rapid perfusion of a deadly chemical fixative (glutaraldehyde) through the brain’s vascular system, instantly stopping metabolic decay and fixing all proteins in place by covalent crosslinks. This stabilized the tissue and vasculature so that cryoprotectant could be perfused at an optimal temperature and rate. The result was an intact rabbit brain filled with such a high concentration of cryoprotectants that it could be stored as a solid ‘vitrified’ block at a temperature of ­-135 degrees Celsius.”

Winning the award also required that the procedure be published in a peer reviewed scientific publication. McIntyre satisfied this requirement and published the protocol in an open-access paper in the Journal of Cryobiology.


3D microscope evaluation of the rabbit brain tissue preservation (credit: Brain Preservation Foundation)


The Brain Preservation Foundation plans to continue to promote the idea that brain preservation following legal death, by using scientifically validated techniques, is a reasonable choice for consenting individuals to make. Focus now shifts to the final Large Mammal phase of the contest, which requires an intact pig brain to be preserved with similar fidelity in a manner that could be directly adapted to terminal patients in a hospital setting.

The 21st Century Medicine team has recently submitted to the BPF such a preserved pig brain for official evaluation. Lead researcher Robert McIntyre has started Nectome to further develop this method.

“Of course, [the demonstrated brain preservation procedure] is only useful if you think all the relevant information is preserved in the fixation,” said Anders Sandberg, PhD., of the Future of Humanity Institute/Oxford Martin School. “Protein states and small molecule chemical information may be messed up.”

GPA | Will You Preserve Your Brain?


Background and significance (statement by BPF)

Proponents of cryonics have long sought a technique that could put terminal patients into long­term stasis, the goal being a form of medical time travel in which patients are stabilized against decay with the hope of being biologically revived and cured by future technologies. Despite decades of research, this goal of reversible cryopreservation remains far out of reach — too much damage occurs during the cryopreservation itself.

This has led a new generation of researchers to focus on a more achievable and demonstrable goal–preservation of brain structure only. Specifically preservation of the delicate pattern of synaptic connections (the “connectome”) which neuroscience contends encodes a person’s memory and identity. Instead of biological revival, these new researchers often envision a future “synthetic revival” comprising nanometer-­scale scanning of the preserved brain to serve as the basis for mind uploading.

This shift in focus toward “synthetic” revival has completely transformed the cryonics debate, opening up new avenues of research and bringing it squarely within the purview of today’s scientific investigation. Hundreds of neuroscience papers have detailed how memory and personality are encoded structurally in synaptic connections, and recent advances in connectome imaging and brain simulation can be seen as a preview of the synthetic revival technologies to come.

Until now, the crucial unanswered questions were “How well does cryonics preserve the brain’s connectome?” and “Are there alternatives/modifications to cryonics that might preserve the connectome better and in a manner that could be demonstrated today?” The Brain Preservation Prize was put forward in 2010 to spur research that could definitively answer these questions. Now, five years later, these questions have been answered: Traditional cryonics procedures were not able to demonstrate (to the BPF’s satisfaction) preservation of the connectome, but the newly invented “Aldehyde­-Stabilized Cryopreservation” technique was.

This result directly answers what has for decades been the main skeptical and scientific criticism against cryonics –that it does not provably preserve the delicate synaptic circuitry of the brain. As such, this research sets the stage for renewed interest within the scientific community, and offers a potential challenge to medical researchers to develop a human surgical procedure based on these successful animal experiments.


Abstract of Aldehyde-stabilized cryopreservation

We describe here a new cryobiological and neurobiological technique, aldehyde-stabilized cryopreservation (ASC), which demonstrates the relevance and utility of advanced cryopreservation science for the neurobiological research community. ASC is a new brain-banking technique designed to facilitate neuroanatomic research such as connectomics research, and has the unique ability to combine stable long term ice-free sample storage with excellent anatomical resolution. To demonstrate the feasibility of ASC, we perfuse-fixed rabbit and pig brains with a glutaraldehyde-based fixative, then slowly perfused increasing concentrations of ethylene glycol over several hours in a manner similar to techniques used for whole organ cryopreservation. Once 65% w/v ethylene glycol was reached, we vitrified brains at −135 °C for indefinite long-term storage. Vitrified brains were rewarmed and the cryoprotectant removed either by perfusion or gradual diffusion from brain slices. We evaluated ASC-processed brains by electron microscopy of multiple regions across the whole brain and by Focused Ion Beam Milling and Scanning Electron Microscopy (FIB-SEM) imaging of selected brain volumes. Preservation was uniformly excellent: processes were easily traceable and synapses were crisp in both species. Aldehyde-stabilized cryopreservation has many advantages over other brain-banking techniques: chemicals are delivered via perfusion, which enables easy scaling to brains of any size; vitrification ensures that the ultrastructure of the brain will not degrade even over very long storage times; and the cryoprotectant can be removed, yielding a perfusable aldehyde-preserved brain which is suitable for a wide variety of brain assays.


Ion Christopher –

Totally weird – IOW those “covalent bonds” act like a preservation matrix. So this brain indeed has been “fixed” – just at a smaller scale and level.

A couple of other factors:

* Quite a lot of the brain that counts (memory) may be on a larger scale than this – and may be preserved. While it is not, per the Connetome idea, at the macro axon scale – it is a general idea that at the molecular scale, something “plays” through the consciousness mechanism (Search = Hameroff Memory.)
I personally suspect a DNA like encoding in an as yet unproven language software. Perhaps even multiple “scale” functionality that would be a combination of organelle specialization (perhaps time perception) and THEN the inter-connectedness.

* As for personality, I know that that is entirely reproducible – in spite of such extreme complexity – but that is a proof for another day.

Just for kicks, note how the “search” code above results in prefabricated libraries being sent to your mind.


Gorden Russell –

You had me until I got to this part: “…a deadly chemical fixative (glutaraldehyde) through the brain’s vascular system…”

So this process perfectly preserves your brain after killer it dead.

So in the future it can be scanned and printed out into a perfect copy — but the copy won’t be you, it’ll be somebody else who is just like you. You will still be dead.

I’d rather be a live brain in a jar atop a robot wired into the spinal column so that I could still have all of my senses while awaiting the time a human body can be regrown.



We have to differentiate on how we define “me” or “you”. Do we mean our memories (data) or consciousness (process). Our memories, personality, knowledge… alone (e.g. while we sleep and are unconscious)… are like fixed data until the brain (or a computer) begins to run and consciousness comes into existence .
We could copy the data to a computer (through scanning), which in the next step (after the simulation is beginning to operate) would create consciousness as well (defining itself as “me” or “you”). It wouldn’t be the same consciousness (process) due to other environmental inputs (and over time other memory/data- background). But the same is true for a biological based consciousness. My consciousness right now is not the consciousness anymore I had last year. It’s always a unique set-up.
From my point of view, the sentiment that there is some kind of metaphysical soul over an entire lifetime is an illusion based on the fact that we have memories, knowledge and personality (which we would have after the scanning process of our brain as well), that were formed in the past, and we are able to (subjectively altered) recreate it (and remember it) in our current state of consciousness. As a result we conclude, that we are/ have the same state of consciousness as the past me, which is (as I see it) an illusion.
So if we would be able to make a perfect copy of our brain that is able to create consciousness (in any kind of computer substrate, digital, analog or quantum) it wouldn’t be more or less the me (the consciousness) at the present than my future me in 5 minutes or years would be (in its biological form). From my point of view, the status quo wouldn’t change.


It is a copy because maybe one day they can do it without killing the original. The only way out of this conundrum was explained to me on this web site a while back in comments: if they substituted every neuron in my brain one at a time over a certain timescale so that eventually my brain would be synthetic, ‘”I” probably wouldn’t even notice.


But you are dreaming during your sleep.

Glutaraldehyde will put an end to all of your dreams.

A printed copy of you may have similar dreams, but not your dreams.




Read Full Post »

World’s Largest Public Stem Cell Bank is been launched by Coriell Institute

Reporter: Aviva Lev-Ari, PhD, RN

NEW YORK(GenomeWeb) – The Coriell Institute today announced the launch of the world’s largest publicly available human pluripotent stem cell (hPSC) bank, the California Institute for Regenerative Medicine (CIRM) hPSC Repository.

The Coriell Institute also said that 300 induced pluripotent stem cell (iPSC) lines will be available in September and up to 750 iPSC lines will be available by February 2016 at the repository.

Coriell originally announced the establishment of the new stem cell bank in March 2014 after receiving a $10 million grant from CIRM, which also awarded $16 million to Madison, Wisconsin-based Cellular Dynamics to support the creation of iPSCs for the bank.

The iPSC lines in the CIRM hPSC Repository are created using donor samples submitted by approximately 3,000 volunteers and are then screened by a consortium of researchers at elite California academic centers, including the University of California, Los Angeles, Stanford University, UC San Diego, and UC San Francisco. Samples from donors are reprogrammed by Cellular Dynamics before being banked in Coriell’s California facility within the Buck Institute in Novato. All specimens are accompanied by comprehensive demographic and clinical data, along with patient consents, and are securely stored and distributed to researchers by Coriell.

The new resource contains iPSC lines representing eleven diseases of interest including autism, epilepsy, blinding eye diseases, heart disease, lung disease, liver diseases, and Alzheimer’s disease. “This new stem cell collection will allow researchers to study molecular mechanisms of disease within specific tissues of interest, leading to better understandings of disease and possibilities for prevention,” Michael Christman, president and CEO of Coriell Institute, said in a statement.

In March, the Coriell Institute also received a $14 million grant from the National Institutes of Health to continue biobanking efforts and support the National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository.


Other related articles published in this Open Access Online Scientific Journal include the following:

Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3

Part 3 UPDATED on 5/16/2013 The Bank Where Doctors Can Stash Your Genome A new company offers a “gene vault” for […]

LIVE — April 23, 1:55PM – CLINICAL UTILITY OF GENOME VARIATION @ Cambridge HealthTech Institute’s 14th Annual Meeting BioIT World – Conference & Expo ’15, April 21 – 23, 2015 @Seaport World Trade Center, Boston, MA

Whole-Genome Sequencing Data will be Stored in Coriell’s Spin off For-Profit Entity

Nation’s Biobanks: Academic institutions, Research institutes and Hospitals – vary by Collections Size, Types of Specimens and Applications: Regulations are Needed


Read Full Post »

Ralph Brinster, ‘Father of Transgenesis’


Larry H. Bernstein, MD, FCAP, Curator
Leaders in Pharmaceutical Innovation
Series E. 2; 7.7

A candid conversation with veterinarian Ralph Brinster, ‘Father of Transgenesis’

For the past five decades, Ralph Brinster, VMD, PhD, Richard King Mellon Professor of Reproductive Physiology at the University of Pennsylvania’s School of Veterinary Medicine, has been working on furthering our understanding of the mammalian germ line. During that time, he helped forge the path that today’s biologists—including those working on transgenics and stem cell research—are following.

Brinster is often referred to as the “Father of Transgenesis,” the study of the experimental transfer of individual genes or DNA into the germ line of an animal, which then transmits the genetic alteration to offspring and successive generations. Many may remember the famous Nature cover story of 1982 that showed a normal-sized white mouse being dwarfed by his giant mouse sibling. That was Brinster’s work, along with his colleague Richard Palmiter of the University of Washington. Through painstaking experimentation, during which they inserted new genes into the germ line, Brinster and Palmiter demonstrated, in a dramatic and unequivocal manner, the true promise of transgenics. The “giant mouse” research was reported on the front page of most major newspapers around the globe.

For his groundbreaking work, Brinster was named one of seven scientists to win the National Medal of Science in 2010, a highly prestigious award that is given annually by the president of the United States. Brinster is the first veterinarian to be so honored and only the eighth from the University of Pennsylvania in the 50-year history of the medal.


Penn researcher shares insights of 50 years studying mammalian germ line.

Nov 01, 2012

For the past five decades, Ralph Brinster, VMD, PhD, Richard King Mellon Professor of Reproductive Physiology at the University of Pennsylvania’s School of Veterinary Medicine, has been working on furthering our understanding of the mammalian germ line. During that time, he helped forge the path that today’s biologists—including those working on transgenics and stem cell research—are following.

Ralph Brinster, VMD, PhD

Brinster was recently recognized for his 50 years of dedicated service as a faculty member in the Department of Animal Biology. The university and school held a two-day symposium in his honor, and research scientists from around the world participated. Michael Brown, a Nobel Laureate in Physiology or Medicine, presented the keynote lecture at the symposium.Brinster is often referred to as the “Father of Transgenesis,” the study of the experimental transfer of individual genes or DNA into the germ line of an animal, which then transmits the genetic alteration to offspring and successive generations. Many may remember the famous Nature cover story of 1982 that showed a normal-sized white mouse being dwarfed by his giant mouse sibling. That was Brinster’s work, along with his colleague Richard Palmiter of the University of Washington. Through painstaking experimentation, during which they inserted new genes into the germ line, Brinster and Palmiter demonstrated, in a dramatic and unequivocal manner, the true promise of transgenics. The “giant mouse” research was reported on the front page of most major newspapers around the globe.

An exceptional honor: Brinster is the only veterinarian to receive the National Medal of Science, presented last year by President Obama.

For his groundbreaking work, Brinster was named one of seven scientists to win the National Medal of Science in 2010, a highly prestigious award that is given annually by the president of the United States. Brinster is the first veterinarian to be so honored and only the eighth from the University of Pennsylvania in the 50-year history of the medal. He received his medal in October 2011 from President Obama himself during a ceremony held at the White House. In August of this year, the Theriogenology Foundation presented the 2012 Career Excellence in Theriogenology Award for his contributions to the field of reproductive veterinary medicine.DVM Newsmagazine recently caught up with Dr. Brinster in his office at the University of Pennsylvania School of Veterinary Medicine in Philadelphia.

DVM: How did you decide to work on the mammalian germ line?

Brinster: I grew up on a farm where we raised purebred animals. Genetics was fundamental on the farm in terms of breeding success and the potential profit gained from that endeavor. I learned at an early age that you must improve animal germ lines to succeed.

Three separate stages are important to consider in studying germ lines: Pedigree tells you what you think you should get. Performance is what you actually get. And progeny is what can be passed on to offspring. For example, a horse can have a great pedigree but never win a race, or be a great racer but never sire winners. So in the end you depend on progeny to ultimately prove the power, quality and characteristics of the germ line of an animal.

DVM: What excited you about work with germ cells?

Brinster: They are the only cells that biology really cares about. What is important to the biology of the species is actually the DNA in the gametes or germ cells. Therefore, on the basis of my background on the farm, my animal science training at Rutgers and my education in veterinary medicine, I felt they were the most important cells in any animal.

DVM: You started using stem cells in the 1970s, demonstrating that non-embryo-injected cells become part of the developing mouse. Why did you start that research?

Brinster: I believed that the introduction of stem cells early in development would allow them to take part in maturation of tissues. These are the studies that resulted in the teratocarcinoma cells being injected into blastocysts.

While we were trying to explore techniques to get better teratocarcinoma cells or similar cells to enter the germ line, I began developing techniques that might allow the introduction of genes directly into fertilized eggs. At first we worked with chromosomes, but they were difficult to handle. Fortunately, other scientists were developing recombinant DNA techniques, which made pure populations of specific genes available.

It was difficult to obtain funding for these studies because the probability of success appeared very low. To obtain funding for this type of study, injecting eggs with nucleic acids, I began studies putting messages for proteins into eggs to study the mechanisms by which the egg produced the specific proteins. One of the proteins I planned to study was ova albumin, and Richard Palmiter had done excellent studies with this protein. I contacted him to obtain the messenger RNA. However, he had stopped working on ova albumin and was working with metallothionein genes. Although I was already working with several molecular biologists and trying to introduce new genes into eggs, I was interested in other possible genes, but the metallothionein gene was present in mice, and there was no good assay to distinguish it from the endogenous protein.

However, I used the ova albumin messenger RNA for my protein studies, and later described these studies to Richard. At that time, he told me he was fusing the metallothionein promoter to the herpes simplex virus thymidine kinase gene, for which I had an assay. So I asked Richard to send me the fusion gene, and we began collaborating on microinjection of the gene into eggs. In the spring of 1981, we obtained transgenic mice expressing the metallothionein thymidine kinase fusion gene, and Richard and I then published the results of these studies in Cell several months later.

DVM: Tell us about the giant-mouse experiment of the early 1980s.

Brinster: I was very interested in making changes in the biology of the mouse using the transgenic technology, and one of the changes I envisioned was a correction of genetic defects. Richard and I discussed this project, which eventually led to the use of the metallothionein-growth hormone fusion gene in an attempt to correct the genetic defect in the “little” mice. The experiment was a success and was published in Nature 30 years ago this December. The effect was dramatic, and the experiment catalyzed interest in the transgenic technique among scientists as well as the general public. A picture of a large mouse next to its normal-sized sibling was published on the cover of Natureand appeared on the front page of most newspapers throughout the world.

DVM: I remember that photo. I was stunned by it and the research it represented. That must have been exciting for you, to get that much recognition for your work.

Brinster: Yes, I was surprised. The phone did not stop ringing for the entire day the picture appeared on the Nature cover. It brought a great deal of recognition to the scientific area and to our work, as well as the experiments of others working and contributing in this area.

DVM: What are your thoughts on the state of veterinary stem cell research today?

Brinster: It is critically important to understand how tissues develop, both normally and abnormally. I am not surprised that researchers are now studying the stem cell basis of cancer. I felt in the 1970s, when I was studying the teratocarcinoma cell, that all cancers must have a stem cell basis. It seemed logical that the cancer, like any self-perpetuating tissue, must have a stem cell basis, as well as a differentiation process, which is unregulated in cancer. The study of stem cells, including those of cancer, is an important area of investigation within veterinary medicine. Many veterinary schools are currently involved in innovative stem cell research.

DVM: What are you working on now?

Brinster: I am still studying the mammalian germ line and germ line cells. One area of investigation is related to human spermatogonial stem cells. About 80 percent of children with cancer are cured, but almost one-third of the prepubertal boys that recover become infertile or severely subfertile, which is a serious quality-of-life issue. About one in 5,000 reproductive-age men currently are cancer survivors with seriously impaired fertility. One method to alleviate this problem is to obtain a testicular biopsy before cancer treatment begins, and then use the stem cells from this cryopreserved biopsy at a later time to correct infertility. To be successful one must be able to expand the number of stem cells in the biopsy. Therefore, one of the main areas on which we have been working, in collaboration with researchers from the Children’s Hospital of Philadelphia, is the cultivation of human spermatogonial stem cells. This has proven to be very difficult, but I am sure we will be successful.

DVM: You are the only veterinarian to ever receive the National Medal of Science. What was that day like for you, meeting President Obama in the White House?

Brinster: It was extremely rewarding to be recognized by such a distinguished jury as the one that selects National Medal of Science winners. The ceremony itself was exceptional, and winning the Medal of Science brought well-deserved recognition to the School of Veterinary Medicine, my department and Penn.

DVM: What do you think your legacy in the veterinary and the broader scientific community will be?

Brinster: I think I will be recognized for my work on transgenics, in part because it has been tremendously important as a scientific breakthrough. However, many believe that it represents more, because it provides a method by which man can experimentally modify the germ line of species and thus change the “program of life.” This ability is a major change in man’s relationship to other species.

DVM: What changes have you seen in your 50 years teaching in veterinary school?

Brinster: The diversity of opportunities for students in their veterinary education and the many areas in which they can use their training following graduation is now enormous. They can contribute to many aspects of society now, and veterinary medicine has become critical to a wide range of problems. One particular area in which veterinary medicine will be especially important is related to zoonotic diseases. Approximately 70 percent of new infectious diseases affecting humans currently arise from animals, which represent the reservoir. This is just one example of an area in which veterinarians are critically important to societal health.

Donna Loyle, MS, is a freelance writer in Philadelphia who specializes in medicine and veterinary science.


If you ask Ralph L. Brinster, VMD, PhD the secret to his success, he will say it is luck. But if you ask anyone else — including colleagues with whom he has worked for more than five decades — they will tell you it is much more than that — it is brilliance and unyielding curiosity. Dr. Brinster, the Richard King Mellon Professor of Reproductive Physiology at Penn Vet, was one of seven scientists to be honored by President Barack Obama in October 2011 with a 2010 National Medal of Science, the highest accolade bestowed by the United States government on scientists and engineers. Since the award was first established 50 years ago, Dr. Brinster is the first veterinarian and the eighth scientist from Penn to win the National Medal of Science. The reason for this highest of honors? Dr. Brinster is often regarded as the father of transgenesis, and it was his research on the manipulation of the mammalian germ line, the cells that give rise to sperm and eggs, for which he was honored. By inserting new genes into the germ line of a developing organism — the process known as transgenesis — researchers can produce animals with selected traits that are indispensible models in understanding life processes and disease. Penn President Amy Gutmann said, “Ralph Brinster is a trailblazer in the field of reproductive biology and genetics whose work has had inestimable influence in science and medicine. His early findings helped usher in the era of transgenic research and represent foundational aspects of techniques used in genetic engineering, in vitro fertilization and cloning. We are extraordinarily proud that he has received the National Medal of Science in recognition of more than five decades of scientific achievement.” Clearly, it took something more than simple luck. The Path to Discovery “I grew up on a small farm in northern New Jersey, and from my experiences there, I became interested in animal development and breeding, including fertility and transmission of genetic characteristics to progeny,” said Dr. Brinster. “Growing up on a farm was a good environment. You work hard and there are no vacations.” That environment and value system paved the way for Dr. Brinster’s long academic career and continued quest for understanding animal development. After earning a bachelor’s degree in animal science from Rutgers University in 1953, Dr. Brinster planned to continue his education, but the Korean War was underway.

Dr. Brinster became a second lieutenant in the United States Air Force and served a year in Korea, after which he finished his military commitment in Texas. Still, he did not lose sight of his intention to attend veterinary school. He started Penn Vet in 1956, putting the GI Bill benefits to good use financing his education. “I was not a great student as an undergraduate; therefore, I was fortunate to be accepted at Penn Vet,” recalled Dr. Brinster. “My intent was to work with large animals, but I became more interested in fertility of animals and germ cell biology; thus, following graduation I began PhD training in physiology at Penn Medicine.” Dr. Brinster earned his PhD in 1964, and made nearimmediate and long-lasting impacts in science.

Dr. Brinster’s first major breakthrough came from research leading to his PhD. It was this research in the early 1960s that led to the development of an effective and reliable system in which to observe and experiment on eggs and embryos outside of the body. By using a culture method that consisted of placing mouse embryos in culture medium under an oil layer, Dr. Brinster created a system that would be adopted by the scientific community almost immediately. The system is still used today – virtually unchanged – as the go-to technique for experiments involving mammalian eggs and embryos, including all transgenic work, embryonic stem cell research, in vitro fertilization in humans, cloning and knockout technology. But creating this system was just a first step for Dr. Brinster. Next he planned to manipulate the germ line and germ cells to further understand their development and regulation. Thinking back to his childhood on the farm and appreciating the need for producing quality livestock, Dr. Brinster said, “I never lost interest in animal breeding and eventually became more and more interested in fertility, specifically the germ line. I wanted to modify the germ line and germ cells to understand how they function.” Using a mouse model, a standard species in the field of genetics because of their short reproductive time of three weeks and their well-defined genetic background, Dr. Brinster in the early 1970s began his work towards transgenesis. By taking stem cells from mouse teratocarcinomas and injecting them into mouse blastocysts, Dr. Brinster was able to demonstrate, through a series of experiments, that the non-embryo injected cells amazingly became part of the developing mouse tissues and were present in the adult.

This series of experiments illustrated that donor cells, which could be cultured in vitro and modified genetically, would become part of the adult mouse. Therefore, such cells could carry genetic change into the mouse and into its germ cells, thus permanently altering the germ line of the animal. “The germ cells are critical cells,” said Dr. Brinster. “They are the only cells in the body that will pass DNA to the next generation.” While he and other scientists continued to develop and perfect this approach with stem cells to alter the germ cells and germ line, Dr. Brinster began to explore and perfect another approach to germ line modification. He initiated these experiments by demonstrating in 1980 that fertilized one-cell mouse eggs could be injected with nucleic acids and survive. He and others then used this approach to introduce new genes into the adult mouse by injecting them into the fertilized egg. He and Richard Palmiter of the University of Washington published a foundation paper in 1981 demonstrating the integration and expression of a transgene in mice. The following year, they published the famous giant mouse experiment, which appeared as the cover story in the journal Nature in 1982 and was reported on the front page of newspapers throughout the world. In this transformational experiment, they demonstrated that the growth hormone transgene produced rapid growth and large size in the mouse, and the results catalyzed interest in transgenesis. A picture of the mice appears in most textbooks as representing the beginning of the transgenic revolution. “When we saw the giant mouse,” said Dr. Brinster, “we were surprised and delighted. The giant mouse experiment was a fantastic experiment. That is the experiment that made everybody, including us, stop and say, ‘This is incredibly powerful.’ That you could enter the germ line and make a change like that. It’s the first time man was able to experimentally modify the genetic code that will make the next individual.” The implications of this success are far-reaching and include the possibility of understanding the origin of animal and human diseases, as well as studying the mechanisms by which a single cell, the fertilized egg, develops into a complex animal.

Dr. Brinster has recently turned his attention to spermatogonial stem cells (SSCs), the foundation stem cells of the male germ line and spermatogenesis. SSCs self-renew and generate daughter cells to differentiate into spermatozoa throughout the entire lifespan of the male. “I started thinking about the male germ line, and I reasoned that if you took cells from a fertile testis and injected them into the seminiferous tubules of an infertile testis, they should be able to restore fertility to the animal,” said Dr. Brinster. Of all the testis cells transplanted, only the spermatogonial stem cells would colonize the testis and be able to regenerate complete spermatogenesis. “It was a simple concept; I am surprised no one did it before.” This transplantation system is now used worldwide to study and experiment on male germ line stem cells and spermatogenesis in all species. Dr. Brinster has used the transplantation system to develop cryopreservation and culture methods for spermatogonial stem cells of rodents and higher species, including primates. These techniques make individual male germ lines and their genetic content biologically immortal for all mammalian species. Clearly, the approaches Dr. Brinster has developed in the male mirror those that he introduced for the female back in the 1960s. They will be useful to preserve and genetically modify the germ lines of farm animals to increase productivity and health.


Stephen Williams, PhD, LPBI

Nice story and interview by Penn Vet. It’s good to read about the history, timeline of discovery, and important works that led to breakthroughs in biology.

Don’t forget about the “Mother of Transgenics” Dr. Bea Mintz, a brilliant developmental biologist in the same city of Philadelphia and discoverer of seminal findings in developmental biology. I have a posting which gives more of her bio at

“Heroes in Medical Research: Developing Models for Cancer Research”


Richard Palmiter
Professor of Biochemistry, Investigator, HHMI
PhD 1968 Stanford University
AB 1964 Duke University


  • 2004 Vern Chapman Lecture, 18th International Mouse Genome Meeting
  • 2004 Recipient of Julius Axelrod Medal
  • 1999 Tyner Eminent Scholar, Florida State University, Tallahassee, FL
  • 1999 Wallace Rowe Lecture, American Association of Laboratory Animal Sciences 50th Annual Meeting
  • 1998 Fourteenth von Euler Lecture, Karolinska Institute, Stockholm, Sweden
  • 1998 Second International Fellow of the Garvan Institute, Sydney, Australia
  • 1994 Charles-Leopold Mayer Award, French Academy of Sciences (shared with Dr. R. Brinster)
  • 1989 Distinguished Service Award of US Department of Agriculture.
  • 1988 Elected to National Academy of Sciences
  • 1988 Elected to American Academy of Arts and Sciences
  • 1987 Elected Fellow of American Association for the Advancement of Science
  • 1983 New York Academy of Sciences Award in Biological and Medical Sciences
  • 1982 George Thorn Award, Howard Hughes Medical Institute
  • 1988-1991 Co-Chairman of Four Mouse Molecular Genetics Meetings, Cold Spring Harbor & Heidelberg

Our group uses genetic techniques to study the role of neuromodulators in the development and function of the mammalian nervous system. Most neuromodulators are polypeptides or amino acid derivatives. They are packaged in synaptic vesicles and released into the synaptic cleft upon neuronal stimulation where they modulate the activity of neurons by binding to membrane receptors coupled to G-protein-linked signaling pathways. Our group has been studying the role of the catecholamines, norepinephrine and dopamine, by making mice in which enzymes required for their biosynthesis have been inactivated.Mice that cannot synthesize dopamine develop normally but they become hypoactive and die of starvation a few weeks after birth. Treatment with L-dopa restores dopamine and restores locomotion and feeding and most other behaviors for about 8 hours. Thus, it is possible to study the same mice in either a dopamine replete and dopamine depleted state. Using this model, we have been examining the roles of dopamine in motivation, reward and learning. We also use viral gene therapy strategies to restore dopamine signaling to particular brain regions to ask where dopamine is needed for particular behaviors. We have begun using genetic techniques to manipulate the activity of dopamine neurons. For example, we have removed NMDA receptors from dopamine neurons to reduce excitatory glutamatergic input and discovered that those mice cannot remember where pleasurable events occur. Next, we will be expressing genes into dopamine neurons that will allow pharmacological activation or inactivation of dopamine neuron activity to allow us more directly assess the role of dopamine neurons in various behaviors.

Another area of interest involves the role of hypothalamic neurons that express a neuropeptide called agouti-related protein (AgRP). This small population of neurons is involved in the regulation of appetite and metabolism. We devised a method to selectively kill these neurons and discovered that mice die of starvation. A few days after killing AgRP neurons, the mice neither initiate feeding voluntarily nor swallow much liquid diet even if it is introduced directly into their mouth. Thus, we believe sudden loss of these AgRP neurons disrupts the normal motivational and consummatory systems that control feeding behavior. In addition to AgRP, these neurons make neuropeptide Y and gamma-amino butyric acid (GABA). We have eliminated AgRP and NPY as being critical players in the starvation phenotype and are currently concentrating on the role of GABA.


Showing 5 most recent results. [Show All]

  1. Kim HW, Choi WS, Sorscher N, Park HJ, Tronche F, Palmiter RD, Xia Z. Genetic reduction of mitochondrial complex I function does not lead to loss of dopamine neurons in vivo. Neurobiol. Aging 2015 Sep; 36(9):2617-27. [PMID:26070241] [PMCID:PMC4523431]
  2. Denis RG, Joly-Amado A, Webber E, Langlet F, Schaeffer M, Padilla SL, Cansell C, Dehouck B, Castel J, Delbès AS, Martinez S, Lacombe A, Rouch C, Kassis N, Fehrentz JA, Martinez J, Verdié P, Hnasko TS, Palmiter RD, Krashes MJ, Güler AD, Magnan C, Luquet S. Palatability Can Drive Feeding Independent of AgRP Neurons. Cell Metab. 2015 Aug; [PMID:26278050]
  3. Han S, Soleiman MT, Soden ME, Zweifel LS, Palmiter RD. Elucidating an Affective Pain Circuit that Creates a Threat Memory. Cell 2015 Jul; 162(2):363-74. [PMID:26186190] [PMCID:PMC4512641]
  4. Garrett Morgan R, Gibbs JT, Melief EJ, Postupna NO, Sherfield EE, Wilson A, Dirk Keene C, Montine TJ, Palmiter RD, Darvas M. Relative contributions of severe dopaminergic neuron ablation and dopamine depletion to cognitive impairment. Exp. Neurol. 2015 Jun; 271:205-214. [PMID:26079646]
  5. Tong L, Strong MK, Kaur T, Juiz JM, Oesterle EC, Hume C, Warchol ME, Palmiter RD, Rubel EW. Selective deletion of cochlear hair cells causes rapid age-dependent changes in spiral ganglion and cochlear nucleus neurons. J. Neurosci. 2015 May; 35(20):7878-91. [PMID:25995473][PMCID:PMC4438131]

Richard Palmiter uses genetic and viral transduction techniques to discern neural circuits that control mouse behavior. He is particularly interested in neural circuits that control appetite.

 The central nervous system (CNS) integrates environmental sensory information (sight, sound, smell, taste, and touch) with signals from the body (sensory information from internal organs and hormones) to generate appropriate movements. The CNS can learn to associate particular sensory cues with subsequent events to facilitate appropriate responses—either approach or avoidance behaviors. Our laboratory uses genetic manipulations in the mouse to examine the neural circuits (the wiring diagrams) involved in these responses and the signaling molecules (neurotransmitters/neuromodulators) that are used by the neurons in the circuit. The neural circuits that mediate essential behaviors that do not require thought are likely to be hardwired, although still subject to modulation. Complete neural circuits have not been defined for most basic behaviors in mammals. However, genetic and viral tools are being developed that are promoting rapid progress. We are using these techniques to decipher neural circuits that promote or inhibit feeding behavior.

Neurons express agouti-related protein (AgRP) in the arcuate hypothalamus...

Neurons that express agouti-related protein in the arcuate hypothalamus…

Neurons in an area of the brain called the arcuate region of the hypothalamus (ARC) integrate hormonal (insulin, leptin, and ghrelin) and neuronal inputs to modulate food intake and metabolism as a means of maintaining adequate energy supplies for bodily needs. One population of neurons in the ARC that has received considerable attention expresses γ-aminobutyric acid (GABA), neuropeptide Y, and agouti-related protein (AgRP) as neurotransmitters/neuromodulators. Because AgRP is expressed exclusively in these neurons, we refer to them as AgRP neurons. These neurons become active when an animal is hungry and promote feeding behaviors by releasing their transmitters in various regions of the brain to activate the next neurons in the circuit. Genetic manipulation of AGRP neurons is relatively easy because one can target the expression of new genes to the Agrp gene locus by homologous recombination in embryonic stem cells and then derive mice carrying that genetic modification. If Cre recombinase is targeted to these AgRP neurons, then a virus carrying a Cre-dependent effector gene can be injected into the ARC and the effector protein will be expressed only in AgRP neurons. One useful effector gene is channelrhodopsin (ChR2),a light-activated ion channel that causes neurons to release transmitters when activated by a laser connected to an optic fiber inserted just above the ARC. Photo activation of AgRP neurons during the day when mice are usually sleeping stimulates robust feeding behaviors. Scott Sternson, a group leader HHMI’s Janelia Farm Research Campus, has exploited this clever technique. We performed a converse experiment by making mice that expressed the human diphtheria toxin receptor from the Agrp locus and then administered diphtheria toxin, which killed the AgRP neurons. As might be expected, if these neurons are important for feeding, the demise of AgRP neurons in adult mice led to starvation.

To understand why the mice starve after ablation of AgRP neurons, we assumed that loss of inhibitory signaling by these AgRP neurons resulted in hyperactivity of postsynaptic neurons elsewhere in the brain, which then promoted anorexia. Through a series of experiments, we discovered that hyperactivity of neurons in a brain region called the parabrachial nucleus (PBN) was responsible for the anorexia. We exploited the location of the hyperactive neurons in the PBN and coincidence with the expression pattern of the Calca gene encoding calcitonin gene–related protein (CGRP) to make a mouse that expresses Cre recombinase from the Calca locus. Viral delivery of Cre-dependent ChR2 to the PBN of the Calca-Cre mice followed by photoactivation revealed that excitation of these CGRP-expressing neurons inhibited feeding by hungry mice. Furthermore, we used another strategy to chronically inhibit these neurons and prevented starvation after ablation of AgRP neurons, providing strong evidence that loss of AgRP neurons promotes starvation by activating the CGRP neurons.

These CGRP neurons are known to relay sensory information to the forebrain. They are normally activated by visceral malaise (e.g., food poisoning), nausea (e.g., motion sickness), satiety, and probably many other conditions that lead to anorexia. Thus, activation of these neurons provides a brake on normal feeding activity and presumably protects mice from dangerous environmental events. The phenomenon of conditioned taste aversion, in which ingestion of a novel food is followed by visceral illness and consequent aversion to consuming that food in the future, depends on this circuit. This is a long-lasting, one-trial learning experience that is of obvious value to a foraging animal. Photoactivation of CGRP neurons coincident with presentation of a novel food is sufficient to establish an aversion to eating that food. The CGRP neurons also mediate the aversive effects of a foot shock: blockade of CGRP neuron function attenuates the ability of a mouse to associate the foot shock with the location in the environment where it occurred.

By selective expression of ChR2-mCherry in CGRP neurons, we visualized axon projections to the bed nucleus of the stria terminalis (BNST) and to the lateral capsule region of the central nucleus of the amygdala (lcCeA). Photoactivation of the terminals in the lcCeA inhibited feeding by hungry mice, whereas activation of terminals in the BNST had no effect. Current efforts are directed toward identifying the target neurons in the lcCeA and determining where they project their axons. We also intend to identify neurons in the hindbrain that directly activate CGRP neurons. These efforts should help to define the neural circuit that leads from the viscera to the amygdala and beyond.

Ablation of AgRP neurons in adult mice results in starvation by activating the CGRP neurons. We have discovered numerous ways to prevent the hyperactivation of CGRP neurons and thus prevent starvation, including genetic downregulation of glutamatergic signaling onto CGRP neurons, pharmacological activation of GABA signaling onto CGRP neurons, and prior treatment with lithium chloride. Remarkably, after a week or so of these interventions, the mice survive without their AgRP neurons, suggesting that some form of adaptation has taken place such that the appetite-enhancing role of the AgRP neurons is no longer necessary for adequate feeding. We suspect that adaptation involves synaptic plasticity within the CGRP neurons and plan to use electrophysiological techniques to discern the mechanisms.

Read Full Post »

Comparison of Biotech in the US and in Europe

Reporter: Aviva Lev-Ari, PhD, RN



Read Full Post »

Icelandic Population Genomic Study Results by deCODE Genetics come to Fruition: Curation of Current genomic studies

Reporter/Curator: Stephen J. Williams, Ph.D.


UPDATED on 9/6/2017

On 9/6/2017, Aviva Lev-Ari, PhD, RN had attend a talk by Paul Nioi, PhD, Amgen, at HMS, Harvard BioTechnology Club (GSAS).

Nioi discussed his 2016 paper in NEJM, 2016, 374:2131-2141

Variant ASGR1 Associated with a Reduced Risk of Coronary Artery Disease

Paul Nioi, Ph.D., Asgeir Sigurdsson, B.Sc., Gudmar Thorleifsson, Ph.D., Hannes Helgason, Ph.D., Arna B. Agustsdottir, B.Sc., Gudmundur L. Norddahl, Ph.D., Anna Helgadottir, M.D., Audur Magnusdottir, Ph.D., Aslaug Jonasdottir, M.Sc., Solveig Gretarsdottir, Ph.D., Ingileif Jonsdottir, Ph.D., Valgerdur Steinthorsdottir, Ph.D., Thorunn Rafnar, Ph.D., Dorine W. Swinkels, M.D., Ph.D., Tessel E. Galesloot, Ph.D., Niels Grarup, Ph.D., Torben Jørgensen, D.M.Sc., Henrik Vestergaard, D.M.Sc., Torben Hansen, Ph.D., Torsten Lauritzen, D.M.Sc., Allan Linneberg, Ph.D., Nele Friedrich, Ph.D., Nikolaj T. Krarup, Ph.D., Mogens Fenger, Ph.D., Ulrik Abildgaard, D.M.Sc., Peter R. Hansen, D.M.Sc., Anders M. Galløe, Ph.D., Peter S. Braund, Ph.D., Christopher P. Nelson, Ph.D., Alistair S. Hall, F.R.C.P., Michael J.A. Williams, M.D., Andre M. van Rij, M.D., Gregory T. Jones, Ph.D., Riyaz S. Patel, M.D., Allan I. Levey, M.D., Ph.D., Salim Hayek, M.D., Svati H. Shah, M.D., Muredach Reilly, M.B., B.Ch., Gudmundur I. Eyjolfsson, M.D., Olof Sigurdardottir, M.D., Ph.D., Isleifur Olafsson, M.D., Ph.D., Lambertus A. Kiemeney, Ph.D., Arshed A. Quyyumi, F.R.C.P., Daniel J. Rader, M.D., William E. Kraus, M.D., Nilesh J. Samani, F.R.C.P., Oluf Pedersen, D.M.Sc., Gudmundur Thorgeirsson, M.D., Ph.D., Gisli Masson, Ph.D., Hilma Holm, M.D., Daniel Gudbjartsson, Ph.D., Patrick Sulem, M.D., Unnur Thorsteinsdottir, Ph.D., and Kari Stefansson, M.D., Ph.D.

N Engl J Med 2016; 374:2131-2141June 2, 2016DOI: 10.1056/NEJMoa1508419

Citing Articles (22)


Several sequence variants are known to have effects on serum levels of non–high-density lipoprotein (HDL) cholesterol that alter the risk of coronary artery disease.


We sequenced the genomes of 2636 Icelanders and found variants that we then imputed into the genomes of approximately 398,000 Icelanders. We tested for association between these imputed variants and non-HDL cholesterol levels in 119,146 samples. We then performed replication testing in two populations of European descent. We assessed the effects of an implicated loss-of-function variant on the risk of coronary artery disease in 42,524 case patients and 249,414 controls from five European ancestry populations. An augmented set of genomes was screened for additional loss-of-function variants in a target gene. We evaluated the effect of an implicated variant on protein stability.


We found a rare noncoding 12-base-pair (bp) deletion (del12) in intron 4 of ASGR1, which encodes a subunit of the asialoglycoprotein receptor, a lectin that plays a role in the homeostasis of circulating glycoproteins. The del12 mutation activates a cryptic splice site, leading to a frameshift mutation and a premature stop codon that renders a truncated protein prone to degradation. Heterozygous carriers of the mutation (1 in 120 persons in our study population) had a lower level of non-HDL cholesterol than noncarriers, a difference of 15.3 mg per deciliter (0.40 mmol per liter) (P=1.0×10−16), and a lower risk of coronary artery disease (by 34%; 95% confidence interval, 21 to 45; P=4.0×10−6). In a larger set of sequenced samples from Icelanders, we found another loss-of-function ASGR1 variant (p.W158X, carried by 1 in 1850 persons) that was also associated with lower levels of non-HDL cholesterol (P=1.8×10−3).


ASGR1 haploinsufficiency was associated with reduced levels of non-HDL cholesterol and a reduced risk of coronary artery disease. (Funded by the National Institutes of Health and others.)


Amgen’s deCODE Genetics Publishes Largest Human Genome Population Study to Date

Mark Terry, Breaking News Staff reported on results of one of the largest genome sequencing efforts to date, sequencing of the genomes of 2,636 people from Iceland by deCODE genetics, Inc., a division of Thousand Oaks, Calif.-based Amgen (AMGN).

Amgen had bought deCODE genetics Inc. in 2012, saving the company from bankruptcy.

There were a total of four studies, published on March 25, 2015 on the online version of Nature Genetics; titled “Large-scale whole-genome sequencing of the Icelandic population[1],” “Identification of a large set of rare complete human knockouts[2],” “The Y-chromosome point mutation rate in humans[3]” and “Loss-of-function variants in ABCA7 confer risk of Alzheimer’s disease[4].”

The project identified some new genetic variants which increase risk of Alzheimer’s disease and confirmed some variants known to increase risk of diabetes and atrial fibrillation. A more in-depth post will curate these findings but there was an interesting discrete geographic distribution of certain rare variants located around Iceland. The dataset offers a treasure trove of meaningful genetic information not only about the Icelandic population but offers numerous new targets for breast, ovarian cancer as well as Alzheimer’s disease.

View Mark Terry’s article here on

“This work is a demonstration of the unique power sequencing gives us for learning more about the history of our species,” said Kari Stefansson, founder and chief executive officer of deCode and one of the lead authors in a statement, “and for contributing to new means of diagnosing, treating and preventing disease.”

The scale and ambition of the study is impressive, but perhaps more important, the research identified a new genetic variant that increases the risk of Alzheimer’s disease and already had identified an APP variant that is associated with decreased risk of Alzheimer’s Disease. It also confirmed variants that increase the risk of diabetes and a variant that results in atrial fibrillation.
The database of human genetic variation (dbSNP) contained over 50 million unique sequence variants yet this database only represents a small proportion of single nucleotide variants which is thought to exist. These “private” or rare variants undoubtedly contribute to important phenotypes, such as disease susceptibility. Non-SNV variants, like indels and structural variants, are also under-represented in public databases. The only way to fully elucidate the genetic basis of a trait is to consider all of these types of variants, and the only way to find them is by large-scale sequencing.

Curation of Population Genomic Sequencing Programs/Corporate Partnerships

Click on “Curation of genomic studies” below for full Table

Curation of genomic studies
Study Partners Population Enrolled Disease areas Analysis
Icelandic Genome


deCODE/Amgen Icelandic 2,636 Variants related to: Alzheimer’s, cardiovascular, diabetes WES + EMR; blood samples
Genome Sequencing Study Geisinger Health System/Regeneron Northeast PA, USA 100,000 Variants related to hypercholestemia, autism, obesity, other diseases WES +EMR +MyCode;

– Blood samples

The 100,000 Genomes Project National Health Service/NHS Genome Centers/ 10 companies forming Gene Consortium including Abbvie, Alexion, AstraZeneca, Biogen, Dimension, GSK, Helomics, Roche,   Takeda, UCB Rare disorders population UK Starting to recruit 100,000 Initially rare diseases, cancer, infectious diseases WES of blood, saliva and tissue samples

Ref paper

Saudi Human Genome Program 7 centers across Saudi Arabia in conjunction with King Abdulaziz City Science & Tech., King Faisal Hospital & Research Centre/Life Technologies General population Saudi Arabia 20,000 genomes over three years First focus on rare severe early onset diseases: diabetes, deafness, cardiovascular, skeletal deformation Whole genome sequence blood samples + EMR
Genome of the Netherlands (GoNL) Consortium consortium of the UMCG,LUMCErasmus MCVU university and UMCU. Samples where contributed by LifeLinesThe Leiden Longevity StudyThe Netherlands Twin Registry (NTR), The Rotterdam studies, and The Genetic Research in Isolated Populations program. All the sequencing work is done by BGI Hong Kong. Families in Netherlands 769 Variants, SNV, indels, deletions from apparently healthy individuals, family trios Whole genome NGS of whole blood no EMR

Ref paper in Nat. Genetics

Ref paper describing project

Faroese FarGen project Privately funded Faroe Islands Faroese population 50,000 Small population allows for family analysis Combine NGS with EMR and genealogy reports
Personal Genome Project Canada $4000.00 fee from participants; collaboration with University of Toronto and SickKids Organization; technical assistance with Harvard Canadian Health System Goal: 100,000 ? just started no defined analysis goals yet Whole exome and medical records
Singapore Sequencing Malay Project (SSMP) Singapore Genome Variation Project

Singapore Pharmacogenomics Project

Malaysian 100 healthy Malays from Singapore Pop. Health Study Variant analysis Deep whole genome sequencing
GenomeDenmark four Danish universities (KU, AU, DTU and AAU), two hospitals (Herlev and Vendsyssel) and two private firms (Bavarian Nordic and BGI-Europe). 150 complete genomes; first 30 published in Nature Comm. ? See link
Neuromics Consortium University of Tübingen and 18 academic and industrial partners (see link for description) European and Australian 1,100 patients with neuro-

degenerative and neuro-

muscular disease

Moved from SNP to whole exome analysis Whole Exome, RNASeq


  1. Gudbjartsson DF, Helgason H, Gudjonsson SA, Zink F, Oddson A, Gylfason A, Besenbacher S, Magnusson G, Halldorsson BV, Hjartarson E et al: Large-scale whole-genome sequencing of the Icelandic population. Nature genetics 2015, advance online publication.
  2. Sulem P, Helgason H, Oddson A, Stefansson H, Gudjonsson SA, Zink F, Hjartarson E, Sigurdsson GT, Jonasdottir A, Jonasdottir A et al: Identification of a large set of rare complete human knockouts. Nature genetics 2015, advance online publication.
  3. Helgason A, Einarsson AW, Gumundsdottir VB, Sigursson A, Gunnarsdottir ED, Jagadeesan A, Ebenesersdottir SS, Kong A, Stefansson K: The Y-chromosome point mutation rate in humans. Nature genetics 2015, advance online publication.
  4. Steinberg S, Stefansson H, Jonsson T, Johannsdottir H, Ingason A, Helgason H, Sulem P, Magnusson OT, Gudjonsson SA, Unnsteinsdottir U et al: Loss-of-function variants in ABCA7 confer risk of Alzheimer’s disease. Nature genetics 2015, advance online publication.

Other post related to DECODE, population genomics, and NGS on this site include:

Illumina Says 228,000 Human Genomes Will Be Sequenced in 2014

CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics

CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics and Computational Genomics – Part IIB

Human genome: UK to become world number 1 in DNA testing

Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious Depression

Sequencing the exomes of 1,100 patients with neurodegenerative and neuromuscular diseases: A consortium of 18 European and Australian institutions

University of California Santa Cruz’s Genomics Institute will create a Map of Human Genetic Variations

Three Ancestral Populations Contributed to Modern-day Europeans: Ancient Genome Analysis

Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations

Read Full Post »

Presentations Content for All Business Track Panels and the Scientific Panel on Immunotherapy @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

 REAL TIME Curator: Aviva Lev-Ari, PhD, RN

9:00 am – 9:30 am 3/26/2015  Welcome Remarks & MassBio Board Elections @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

9:30 am – 10:15 am 3/26/2015, Keynote: Kathy Giusti, Founder & Executive Chairman of the Multiple Myeloma Research Foundation, @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

10:30 am – 11:30 am, 3/26/2015 – LIVE Better Business Track: It’s Not Your Grandfather’s Manufacturing @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

11:45 am – 1:30 pm, 3/26/2015,  LIVE – The MassBio Annual Awards Luncheon @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

1:30 pm – 2:20 pm 3/26/2015, Precision Medicine: Who’s Paying? @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

2:25 pm – 3:15 pm 3/26/2015 Better Business Track: Externalizing Pharma R&D @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

3:30 pm – 4:20 pm LIVE Trends in Science Track: Immunotherapy – Oncology and Beyond @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

8:40 am – 9:30 am   3/27/2015  Better Business Track: Innovative Ways to Fund Your Early-Stage Company @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

9:40 am – 10:30 am 3/27/2015 Better Business Track: The Evolving Reimbursement Landscape @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

11:00 am – 12:00 pm, 3/27/2015 Defining Value @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

12:45 pm – 1:30 pm  3/27/2015  LIVE Keynote: Andrew Lo, Director of the MIT Laboratory for Financial Engineering  @ MassBio Annual Meeting 2015, Cambridge, MA, Sonesta Hotel, 3/26 – 3/27, 2015

Read Full Post »

« Newer Posts - Older Posts »