Category Archives: Science News

En Passage, an Approach to the Use and Provenance of Immortalized Cell Lines

This guest post is by Lisa Krugner-Higby, DVM, PhD.  Dr. Krugner-Higby is a scientist and also a research veterinarian within the Research Animal Resource Center at the University of Wisconsin-Madison. Dr. Krugner-Higby’s research is in development of extended-release formulations of analgesic and antimicrobial drugs. She previously worked in anti-HIV drug development.

I am always fascinated by the idea promoted by some animal rights activists – repeated in many versions and for many decades – that all preclinical biomedical research can be conducted using in vitro cell culture. I have never found one of them who has spent much time working with cell culture. On the other hand, I have spent approximately seven years of my life working with cell cultures, looking at the stainless steel back wall of a laminar flow work station day after day. One thing I can say about immortalized cell lines, or cells that reproduce indefinitely, is that they are not alive in the same way that a mouse is alive.

 

Cell culture

Cell culture

The first thing that a graduate student learns when they begin to work with cell culture is how to take cells that have overgrown the sterile plastic flask they inhabit and put them into a fresh flask with fresh growth medium. It’s called ‘splitting’ the number of cells and ‘passaging’ them into a new home. Split and passage, split and passage… I knew that with every passage, the cell line became a little more different than normal cells and even a little more different than the original cell line. The remedy for this type of genetic drift was to freeze low passage cells in liquid nitrogen and re-order the line from the repository when the low passage stocks were depleted. I was careful with my sterile technique, cleaned the laminar flow hood, and used a new sterile pipet every time in order to avoid contamination of my cells. Unfortunately, the day came when I opened the incubator door and the flasks were black and fuzzy with fungus, and all of my carefully tended cells were dead. An anguished conversation with the tissue culture core technician revealed that this happened every Spring in North Carolina when the physical plant turned on the air conditioning for the year, blowing a Winter’s worth of fungal spores out of the ductwork and into the air. She recommended doing other things for about 6 weeks until the spore load had blown out of the ducts. I have had other cell line disasters in my scientific career: the malfunctioning incubator thermostat that turned a colleague’s two months’ worth of cell culture growth into flasks of overheated goo or that generally reputable vendor that sold us a case of tissue culture flasks that were not properly sterilized resulting in clouds of bacteria in the warm, moist, nutrient-rich environment of the incubator.

I never thought to ask, in those early days, if the cells that I fussed, worried, and wept over, were actually the cells that they were supposed to be. Raji Cells, A549s, U937s, I knew them all, worked with them every day, and never doubted that they were the cells that I thought that they were. I knew that some cell lines had been contaminated with the HeLa cell line. HeLa cells are very hardy and could spread quite easily from one flask to another. But I thought that was in the past. It turns out that there was more to the story than I realized. Cell lines have a provenance, like paintings or other works of art. They have an origin, a laboratory where the line was first isolated and propagated. From there, it may have been distributed to other laboratories and to repositories like the American Type Culture Collection or ATCC. Some cell lines are used by only a few laboratories, and some become used very widely and in a large number of biomedical disciplines. Whereas some paintings are intentionally forged, many cell lines have now been shown to be unintentionally forged. A recent article in the journal Science estimated that 20% of all immortalized cell lines are not what they were thought to be1.

Download original file2400 × 1999 px jpg View in browser You can attribute the author Show me how Multiphoton fluorescence image of cultured HeLa cells with a fluorescent protein targeted to the Golgi apparatus (orange), microtubules (green) and counterstained for DNA (cyan). Nikon RTS2000MP custom laser scanning microscope. National Institutes of Health (NIH).


Multiphoton fluorescence image of cultured HeLa cells with a fluorescent protein targeted to the Golgi apparatus (orange), microtubules (green) and counterstained for DNA (cyan). Nikon RTS2000MP custom laser scanning microscope. National Institutes of Health (NIH).

We now have better methods of identifying cell lines by their DNA, called short tandem repeat (STR) profiling, and scientific journals are beginning to require this testing for cell lines prior to publication. Currently, 28 scientific journals require STR profiling to establish cell line provenance prior to publishing a manuscript from a particular laboratory. Some scientists are also beginning to create catalogs of contaminated cell lines in an attempt to quantitate the damage done by some misidentified, but widely studied, cell lines. The same Science article, notes that the International Cell Line Authentication Committee (ICLAC) maintains a database of misidentified cell lines that now numbers 475 different lines. A cell line geneticist, Dr. Christopher Korch, recently estimated that just two of the immortalized cell lines that were found to be misidentified, HEp-2 and INT 407, have generated 5,789 and 1,336 articles in scientific journals, respectively. These studies cost an estimated $713 million dollars and generated an estimated $3.5 billion in subsequent work based on those papers1. This is because the usual trajectory for testing a new therapeutic modality, especially in cancer research, is to test a compound or technique in cell culture. It will then be tested in mice that express a tumor derived from the cultured cancer cells. If those studies are successful, the compound and/or technique undergoes further toxicity testing in other animal models before entering its first Phase I trial in human volunteers.

A lot of compounds that show early promise in cell culture and in cell line-injected mice turn out not to have efficacy in animal models or in human patients. Sometimes this is simply a matter of the compound being too toxic to organs or cell types that are not represented in the initial cell culture. Often, the reason why particular compounds or strategies fail is not known, and most granting agencies are not keen to fund laboratories to find out why things don’t work. I have wondered if the failure of some compounds or techniques is in part due to misidentified cell lines. I have also wondered if it is a reason why testing in animal models, particularly in animal models with spontaneously-occurring tumors, is necessary.

Testing anti-cancer compounds in models of spontaneously-occurring tumors in animals and/or testing in human tumor cells taken directly from patients and injected into mice (the ‘mouse hospital’ approach) is more time and resource intensive than screening in immortalized tumor cell lines. This approach, however, has the advantage of knowing that the investigator is not just treating misidentified HeLa cells in error. It is always necessary to go from in vitro cell culture models to in vivo animal models to confirm the viability of a therapy.

Science makes claim to no enduring wisdom, except of its method. Scientists only strive to be more right about something than we were yesterday, and efforts are underway to weed out misidentified cell lines. But the fundamental issues behind cell line misidentification highlight one of the reasons why we should not rely on immortalized cell lines without animal models, and why granting agencies should fund more studies to try to identify the disconnect between the results of in vitro and in vivo studies when things do not go as planned. That is a part of good science and part of creating better cell culture models to refine, reduce, and sometimes replace the use of animals in biomedical research.

Lisa Krugner-Higby, DVM, PhD

1) Line of Attack. Science. 2015. Vol. 347, pp. 938-940.

Interview with a Primate Researcher

In the last few months, Italian animal rights activists have conducted a campaign against animal research, in particular against primate research. This is despite the important role that primates have played in breakthroughs in stem cell research and neuroprosthetics, among other things. Nonetheless, activists continue to try to claim such research is useless. In particular, they targeted Prof. Roberto Caminiti, a leading neurophysiologist at the University La Sapienza in Rome, and his research team, accusing them of animal mistreatment. Earlier this year students and scientists at the University rallied round Prof. Roberto Caminiti, his team, and his important research.
To answer some of the activists accusations, Pro-Test Italia has produced a video with Prof. Caminiti to illustrate why primate research is so important in the field of neurophysiology and brain-computer interface, and why animal models remain essential for this kind of research. Pro-Test Italia have also made an English version of the video:

It’s important to spread this video outside of Italy to both explain to the public what is going on, and to encourage other primate researchers not to remain hidden but to be clear about the important research that they do. Researchers should be proud of the important work they do in contributing to medical developments for everyone.

Marco

American Psychological Association supports NIH primate researcher Stephen J. Suomi

Research conducted within the National Institutes of Health (NIH) intramural program has been the focus of a PETA campaign over the past several months. Elements of the campaign mirror tactics PETA has used elsewhere to generate media coverage, fundraising, and emails or phone calls to the NIH. The campaign recently reached beyond newspaper, bus, and metro advertisements to include a congressional request to NIH to provide a review of the research.

The American Psychological Association (APA) responded on January 22 with strong statement of support for the scientist and research under attack by PETA.

APA 01.22.15

APA’s letter to the congress members, in its entirety, reads:

“In December 2014 you were one of four members of Congress who sent a letter to Dr. Francis Collins, Director of the National Institutes of Health (NIH), requesting that his office commission a bioethics review of a research program directed by the world renowned researcher, Dr. Stephen J. Suomi. On behalf of the American Psychological Association and its Committee on Animal Research and Ethics, I am writing to provide a broader scientific perspective on this research. As you are likely aware, the request you received was a part of a sustained and well publicized campaign against Dr. Suomi’s laboratory by the organization, People for the Ethical Treatment of Animals (PETA), in support of its mission to put an end to research with nonhuman animals.

Your letter stated that prominent experts have raised concerns about the scientific and ethical justification for these experiments. We believe that the facts do not support PETA’s public statements about this research. Over the past three decades, Dr. Suomi and his collaborators have made significant contributions to the understanding of human and nonhuman animal health and behavior. Dr. Suomi’s work has been critical in understanding how the interactions between genes and the physical and social environments affect individual development, which in turn has enhanced our understanding of and treatments for mental illnesses such as depression, addiction, and autism.

Dr. Suomi and colleagues found that like humans, monkeys share similar variants of genes that make an individual more vulnerable to mood and personality disorders; however, genetics interact with experience in determining such disorders, and mother-infant dynamics in particular have a large influence on later development. Dr. Suomi has successfully produced monkey models of depression and excessive alcohol consumption and his studies provide insight into modes of treatment. Through his work on neonatal imitation, Dr. Suomi discovered potential early signs of atypical social development in monkeys, which has informed the search for screening methods and treatments for autism in human children. Further, through his work on the development of attachment behavior to a caregiver, which is crucial for infant survival in both humans and other animals, Dr. Suomi’s research has had a tremendous impact on the standards for the welfare of nonhuman animals in captivity.

Cover PNAS monkey pic 2

The specific study targeted by PETA was designed to investigate the long-term effects of fluoxetine (Prozac) in children. Given that drugs are typically tested only on adults, the effects of this commonly prescribed anti-depressant on children were unknown. Thus, in response to overwhelming concern raised by parents, physicians, and others involved in child and adolescent health about the safety of this medication for children, Dr. Suomi and his colleagues began a study with baby monkeys to elucidate the effects of fluoxetine in children. Contrary to PETA’s repeated claims that animal research has not improved human health and that modern non-animal research methods are more effective, there are, in fact, no viable non-animal alternatives for identifying the causes of and treatments for disorders that affect the brain and behavior. Studies with a wide variety of nonhuman animal species have been and continue to be integral to basic and applied research on health.

Laboratory animal models generally provide the most scientifically rigorous means of studying normal and abnormal behaviors in order to better understand their underlying mechanisms and to remedy disorders. Monkeys are the ideal model for the work that Dr. Suomi does, because they share approximately 93% of human DNA, they live in social groups with similar mother-infant dynamics as humans, and they develop more quickly than humans. Moreover, the monkeys in Dr. Suomi’s studies are treated humanely, following strict guidelines set forth by the Animal Welfare Act and overseen by numerous entities including the NIH Office for Laboratory Animal Welfare (OLAW), the United States Department of Agriculture (USDA), the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC), and institutional animal care and use committees. And given that Dr. Suomi is an intramural researcher at NIH, you can be certain that his research animals receive premier quality of care.

I understand that it may sometimes be difficult to weigh the qualifications and varying conclusions of “dueling experts,” but let me assure you that Dr. Suomi is a highly regarded member of the APA and the psychological science community at large, as well as a highly sought-after expert in the field of pediatric medicine. In addition to providing information to the U.S. Congress, Dr. Suomi has testified at the World Health Organization and addressed the British House of Commons about the implications of his scientific findings.

Based on the conviction that research with nonhuman animals is a necessary component of basic and applied research on health, APA strongly supports humanely conducted, ethically sound, and scientifically valid research with nonhuman animals. For nearly 100 years, through its Committee on Animal Research and Ethics, APA has promoted informed, serious, and civil dialogue about the role of nonhuman animal research in science. If you should be asked to take further action against Dr. Suomi, I hope you will make it a point to seek out additional information before making a decision. My staff stand ready to provide you with additional information, including assembling experts for a staff briefing or assisting you in any other way on this issue.”

***

The complete statement can be found here:  APA Suomi-letter 01.22.15

 

70 year old professor retires and closes lab, PETA claims victory

The retirement of a highly respected senior neuroscientist at the center of a sustained recent publicity campaign by an animal rights group generated a victory claim on Friday when PETA realized that their target had retired. The retirement came after a productive and award-winning 40 year research and teaching career. University of Wisconsin-Madison neuroscience Professor Tom Yin’s research led to breakthroughs in understanding how the brain processes and localizes sounds. The highly cited research was continuously funded by the US National Institutes of Health because it contributed fundamentally important new knowledge that is the necessary building block for advances in medicine and science that involves hearing. We have written about Professor Yin’s research previously, for more information see here, here, here.

Yin’s sound localization research was the target of a sustained and multi-dimensional attack by PETA over the past three years. The campaign had provided rich opportunities for stunts, attracted celebrities, generated media attention, and undoubtedly brought in many donors for animal rights groups.

Metro bus displaying PETA ad. Image: Wisconsin State Journal.

Metro bus displaying PETA ad. Image: Wisconsin State Journal.

The scientist’s retirement is unlikely to provide obstacles to PETA’s continued success in using the research for fundraising appeals, as was indicated by the group’s immediate response. Despite the obvious fact that the retirement of a 70 year old scientist is expected, rather than unusual, PETA promptly claimed responsibility and announced that they had secured a victorious end to their campaign.

PETA’s tactic may well serve as a model to other groups because it offers a solid opportunity to claim effectiveness of their campaigns. If so, we might expect to see other scientists seemingly within the realm of retirement age appear as targets of major campaigns that involve bus ads, celebrities, and stunts that misrepresent the research. (Or perhaps they could simply claim all retired scientists did so not as a result of age, or the natural conclusion of long and productive careers, but rather in response to campaigns by those opposed to the research.)

Despite the scientist’s retirement and the lab closing, it seems unlikely that PETA will retire the photos of research animals and misleading claims about Yin’s work that were the center of PETA’s campaign. It is more likely that the campaign will continue to be used by PETA to attract attention and donors, with the promise of more victories in ending research.

PETA also took a page from other animal rights groups that claim credit for the retirement of research animals, despite the fact that it is the scientists and research institutions that find adoptive homes and retire the animals. Like many research institutions, the University of Wisconsin-Madison finds adoptive homes for animals that are no longer in research and whose care and safety can be assured in a home setting. In this case, four of the five cats that were part of Professor Yin’s research were retired into private homes. This is in stark contrast to the PETA policy at its Norfolk, VA shelter of killing on average 2000 dogs and cats per year (http://www.nytimes.com/2013/07/07/us/peta-finds-itself-on-receiving-end-of-others-anger.html)

The university, like other research facilities, does not use those adoptions as a vehicle for media attention. By contrast, retired research animals are often featured as centerpieces in fundraising campaigns by animal rights groups. We have written about this previously in the context of a controversial campaign by Beagle Freedom, in which the animal rights group appropriates credit for research facilities’ successful adoption programs. In general, the focus of the adoption programs is on successfully placing the animals. Even the NIH and federal government, while providing over $30 million for retirement of research chimpanzees and committed to tens of millions more for their lifetime support, do so without sustained high-profile media campaigns. Similarly there are rarely press releases from the UW-Madison announcing the animal adoptions or the lab closing due to the scientist’s retirement.

PETA seized the opportunity for their own press release and claim of victory after they realized what had happened. How did they find out? Simply by reading the records that the university regularly sends to PETA and other animal rights groups in response to their regular open records requests. PETA was no doubt pleased by their discovery. Not only could they claim victory for the retirement of the 70 year old scientist, they could also continue to claim PETA themselves were responsible for the research animals’ retirement.

The victory claim is PETA’s central rationale for continued used of the images and claims that were at the center of their campaign. There is little doubt that they will not be retired; rather they are likely to be used for a long time to convey the impression of a success. The question is whether those who hear the victory claim might wonder whether there is anything surprising about the retirement of a 70 year old scientist. Others might be curious enough to learn more about the remarkable accomplishments of that scientist over his 40 year career (see here for more information). In light of current campaigns against other scientists, the question will also become whether PETA has highlighted a new path that paves the way for higher likelihood of being able to claim an unearned victory.

 

The Uniqueness of Human Suffering

Jeremy Bentham, an 18th century utilitarian philosopher, famously asked: “The question is not ‘can they reason?’ or ‘can they talk?’ but ‘can they suffer?’” A utilitarian philosopher of our times, Peter Singer, latched into that question to write his book Animal Liberation, and so the modern animal rights movement was born. Basically, Peter Singer and many other animal rights activists claim that animals suffer like humans and therefore they should be treated like humans. Put in a more sophisticated way, Peter Singer argues that the moral imperative of equality dictates “equal consideration of interests”, that is, that the interests of all beings receive the same consideration. Animals have an interest in avoiding pain, therefore egalitarianism demands that we respect that interest. It is argued further that claiming human superiority based on our superior intelligence, our ability to talk or our culture is just stacking the cards in our favor because those are the special attributes of our species. By the same token, an elephant may claim moral superiority based on the fact of having a trunk.

However, the whole argument is based on the claim that animals suffer and, moreover, that they suffer like us. Singer and the other animal rightists just assume that they do. I think this is a faulty assumption that needs to be addressed head-on, but I understand why they take umbrage in it: the whole problem of defining suffering seems intractable at first sight. ‘Suffering’, like ‘happiness’ and ‘consciousness’, belong to a class of concepts that are at the same time abstract and fundamental, so that defining them in terms that are non-circular seems nearly impossible. If you look at dictionary definitions of ‘suffering’ you will find that they refer to pain, unpleasantness or perceptions of threat, which are just synonyms or examples of suffering. This does not represent a problem when the idea of suffering is applied to human beings, because we can get accurate descriptions of their suffering from other people. However, when we want to apply this concept to animals we need a clear idea of what we are talking about, otherwise we risk falling into one of two opposite pitfalls: self-serving callousness -choosing to think that animals do not suffer because this is convenient for us; and anthropomorphizing – thinking an animal suffers just because we would suffer if they did that same thing to us. The latter feels intuitively true because is based on empathy, a very powerful human emotion. However, it is not a rational conclusion.

Do all animals suffer? Do all animals suffer equally?

Do all animals suffer? Do all animals suffer equally?

Just like in the case of happiness and consciousness, the problem of suffering can be studied scientifically. In fact, there are a lot of scientific studies related to suffering because one important thing the public demand from scientists is to find solutions to pain and other forms of distress. Just like in the case of happiness and consciousness, science may not have come up (yet) with a complete description of suffering, but it certainly can tell us a lot of things about it. I think that this information can help us form an educated opinion about whether some particular animal suffers or not.

Most people would agree with the idea that not all living beings suffer. One of the most peculiar things about life is that it seems goal-directed: living beings strive towards keeping themselves alive and making more beings like them. However, this does not imply any form of consciousness or intentionality; it is just something that living beings do automatically because otherwise they wouldn’t be living anymore. It is important to underline this fact because this striving to stay alive can be easily confused with the “interest” that Peter Singer talks about. Yes, life perpetuates itself, but that doesn’t mean that living beings are conscious or that they have interests and plans like we do. To think otherwise would be to accept some magical vitalist concept of life that science rejected long ago. Therefore, we can conclude that plants do not suffer, although they grow, reproduce and even fight their enemies with chemical responses. Likewise, we should accept that animals that lack a nervous system (like sponges) or that have only a rudimentary nervous system (like worms) do not suffer.

What about animals that do have a complex nervous system? Do they suffer? Here we must consider that suffering and pain are often confused, but in fact are not identical. Pain produces suffering, but suffering can be produced by things other than pain, generally speaking by negative emotional states. That pain and suffering are not identical is also shown by the fact that people may experience pain and not suffer from it. For example, the pain experienced when practicing some sports, when eating spicy food and by sexual masochists induces positive feelings instead of suffering. Some drugs called dissociative anesthetics (like ketamine) can selectively turn off the emotional part of pain leaving intact its sensory component: we are still able to feel the pain, but just don’t care about it. Given the complexity of this subject, I chose to divide this discussion into two parts: suffering that comes from physical pain and emotional suffering. I will start with the first.

Pain scientists distinguish between three concepts: nociception, pain and suffering. This distinction is even recognized by the Humane Society of the United States, an animal rights organization. To understand nociception consider the case of a patient who is undergoing surgery under general anesthesia. As the skin and organs of this person are being cut, pain sensory nerves faithfully record the damage and send this information to the spinal cord, which continues to the brain. The normal traffic of noxious signals only stop at the cerebral cortex because the large parts of the brain cortex is turned off by the general anesthetic [1, 2]. This basic processing of noxious information is what we call nociception. Of course, in an awake person nociception leads to pain. The important idea, however, is that the processing of information concerning physiological damage involving millions of neurons and sophisticated neural pathways does not imply the existence of pain. In fact, nowadays pain is considered part sensation part emotion; because fundamental aspects of pain are its negative valence (we dislike it) and its salience (we cannot avoid paying attention to it). Pain requires a fairly complex nervous system capable of turning sensations into emotions. Based on these ideas, I think it is reasonable to infer that animals that lack a nervous system of enough complexity do not feel pain, they just have nociception. Behavior consisting in avoiding a noxious stimulus should not necessarily be taken as an indication of pain. After all, even plants react to noxious stimuli. It is difficult to draw the line between animals that have just nociception and those that experience pain. However, it is clear that many animals do not come even close to having a nervous system complex enough to produce the sensation of pain with its associated negative emotions. Animals like the pond snail (11,000 neurons) or the sea slug (28,000 neurons) just don’t have this capacity. By comparison, we have 100 millions neurons just in our gut (the enteric nervous system) and 86 billion neurons in our brain. Of the invertebrates, the only animal that comes close is the octopus, with 300 million neurons, comparable with the rat’s 200 million neurons. This is why countries like the UK and Canada now give cephalopods (octopi, squids and cuttlefish) the same protections given to vertebrates. Of course, the number of neurons is not the only metric to measure the complexity of a nervous system, but using other metrics like number of synapses or overall capacity to process information will give similar results. A table of the number of neurons in different animal species can be found here.

Cephalopods are protected in Canadian and EU regulations

“Countries like the UK and Canada now give cephalopods the same protections given to vertebrates”

But what most people are concerned about are the most complex animals, the mammals and the birds, which we eat, have as pets and use in scientific research. What about them? Do they feel pain? Do they suffer?

A lot can be learned about the relationship between pain and suffering in mammals by studying brain areas involved in the processing of pain in the brain. As I indicated above, pain has a sensory aspect and an emotional aspect. The sensory aspect of pain is processed by the somatosensory cortex, an area shaped like a hairband going from the top to the sides of the brain. It contains a detailed map of the body and processes pain and touch, telling us where these sensations originate (nowadays it is recognized that the dorsal posterior insula also contains a map of the body and is responsible for judgements on the localizations and intensity of pain). The somatosensory cortex is connected to the orbitofrontal cortex, located at the front end of the brain and whose function is to plan actions according with the information it receives. But neither the somatosensory nor the orbitofrontal cortex are responsible for the emotional component of pain. This function is assigned to two other areas of the cortex: the insula and the anterior cingulate cortex (ACC). Generally speaking, the function of the insula is to tell us how bad pain feels and to associate that emotion with a host of other emotions like sadness, fear, anger, joy, disgust and pleasure. Emotions can be understood as motivational states of the brain: they predispose us to act in a certain way, organizing everything we feel in a hierarchical way according to what we need to do. Pain is an emotion that motivates us to stop or escape from whatever is hurting us. This urgent motivational aspect of pain is processed by the ACC. So we could say that the insula and the ACC work together to turn pain into suffering by giving it its “I don’t like it” and “I want to stop it” qualities.

Recent discoveries have revealed that during the evolution of primates (monkeys, apes and humans) there was a reconfiguration of the brain pathways that process pain, culminating with the appearance of completely new pain processing areas in the human brain [2, 3]. To convey the importance of these changes, I must quickly summarize the neural pathways that carry pain signals from the body to the cerebral cortex. Noxious signals are carried by specialized fibers in the nerves from any part of the body to the dorsal horn of the spinal cord (see figure below). From there, the signals travel to the parabrachial nucleus in the brain stem, where they branch out to different nuclei of the thalamus and the forebrain [3]. Located in the middle of the brain, the thalamus function as the central relay of all sensory information, with its different parts or nuclei handling visual, auditory, gustatory, tactile and pain information. Different thalamic nuclei send pain signals to the four areas of the cortex described above: the somatosensory cortex, the orbitofrontal cortex, the insula and the ACC. These pain pathways are present in all mammals, but in primates a new additional pathway emerged that directly links the spinal cord with the nucleus of the thalamus connected to the insula, bypassing the parabrachial nucleus. This means that pain sensations are able to directly reach the part of the cortex where feelings are created. In humans, the size of this direct pathway between the thalamus and the insula is much larger anatomically, and much larger and more complex than in monkeys.

Spinal Cord Diagram Pain

“Noxious signals are carried by specialized fibers in the nerves from any part of the body to the dorsal horn of the spinal cord”

But is there another change in the brain that is unique to humans and a small number of other species including elephants and cetaeceans, but not being found in monkeys: a new part of the insula called the anterior insula [4, 5]. A.D. Craig, a scientist who has studied these changes by mapping the brains of monkeys, apes and humans, thinks that the posterior insula serves to create an emotional map of the state of the body in each moment. The anterior insula, on the other hand, serves to model the state of the body as it was in the past or in hypothetical situations in the future: “if this were to happen, that’s what I would feel”. Craig thinks that this gives us self-awareness by modeling feelings that represent the interior state of the body through time. The representation of hypothetical states of the body performed by the anterior insula is probably also responsible for empathy, the ability to feel what another person is feeling by simulating his body state in our own brain. In relation to suffering, we can see how growing relevance of the insula in generating the negative emotions associated with pain progressively increase the depth of suffering. This process culminates with humans; we are not only able to experience the pain of the present but are also aware of ourselves as beings that have suffered in the past and that may suffer in the future. Animals that lack an anterior insula would not be able to experience this type of suffering. The gradual appearance of the anterior insula in apes like bonobos and chimpanzees seems to correlate with the development of empathy and positive social emotions [4, 6].

In summary, we need to take a gradualist approach when considering the presence of pain and suffering in animals. Invertebrates, with the possible exception of cephalopods, do not appear to have a nervous system complex enough to feel pain, let alone suffering. Their behavior can be explained by simple responses to nociceptive signals. Vertebrates, particularly the ones with highly complex nervous systems like mammals and birds, do experience pain and quite probably suffer from it. However, the deep suffering that we experience as humans beings, rooted in our memory and our capacity to imagine the future, does not seem to exist other than in a rudimentary form in other mammals. Although animals have memories, without an anterior insula they cannot use them to construct a vivid representation of their past suffering, like we do. A measure of self-awareness and deep suffering exists in elephants and cetaceans, which also have a highly developed anterior insula and ACC with von Economo neurons.

Jeremy Bentham and Peter Singer failed to understand the true nature of suffering when they came up with the idea of speciesism. Just like we do not give the same moral status to animals and plants, we cannot give the same moral status to all animal species. When deciding how we should treat them we need to take into consideration whether they can feel pain and, if they do, how they suffer from that pain. The suffering of a mouse, a dog, a monkey and a chimpanzee are not equivalent. By the same token, human suffering has to be given a higher ethical consideration than the suffering of other animals. There is a moral imperative to diminish suffering in all sentient beings, but when difficult choices have to be made, human suffering has to come first. If saying this makes me a speciecist, I will wear that label with pride. But I’d rather call myself a humanist, because for me the priority is to decrease human suffering.

Juan Carlos Marvizon, Ph.D.

The author wishes to thank Dr. Bud Craig for his helpful comments

References:

  1. Craig, A.D., Topographically organized projection to posterior insular cortex from the posterior portion of the ventral medial nucleus in the long-tailed macaque monkey. J Comp Neurol, 2014. 522(1): p. 36-63.
  2. Craig, A.D., The sentient self. Brain Struct Funct, 2010. 214(5-6): p. 563-77.
  3. Craig, A.D., Interoception: the sense of the physiological condition of the body. Current Opinion in Neurobiology, 2003. 13(4): p. 500-505.
  4. Bauernfeind, A.L., et al., A volumetric comparison of the insular cortex and its subregions in primates. J Hum Evol, 2013. 64(4): p. 263-79.
  5. Craig, A.D., Significance of the insula for the evolution of human awareness of feelings from the body. Ann N Y Acad Sci, 2011. 1225: p. 72-82.
  6. Rilling, J.K., et al., Differences between chimpanzees and bonobos in neural systems supporting social cognition. Soc Cogn Affect Neurosci, 2012. 7(4): p. 369-79.

Primate research and twenty years of stem cell firsts

This guest post is by Jordana Lenon, B.S., B.A., Senior Editor, Wisconsin National Primate Research Center and University of Wisconsin-Madison Stem Cell and Regenerative Medicine Center. The research will also be featured this evening in a public talk at UW-Madison’s Wednesday Nite at the Lab. WN@tL: “Twenty Years of Stem Cell Milestones at the UW.”  Details and link are below. Update 1/8/15:  Dr. William Murphy’s talk  can now be viewed at:  http://www.biotech.wisc.edu/webcams?lecture=20150107_1900

As we enter 2015, the 20th anniversary of the first successful isolation and culture of primate pluripotent stem cells in the world, it’s time to look back and see how far we’ve come. Thanks to a young reproductive biologist who came from the University of Pennsylvania’s VMD/PhD program to the Wisconsin National Primate Research Center at the University of Wisconsin-Madison in 1991, and to those whose research his groundbreaking discoveries informed, the fields of cell biology and regenerative medicine will never be the same.

stem cell colonies

Pluripotent stem cells are right now being used around the world to grow different types of cells—heart muscle cells, brain cells, pancreatic cells, liver cells, retinal cells, blood cells, bone cells, immune cells and much more.

Cultures of these cells are right now being used to test new drugs for toxicity and effectiveness.

More and more of these powerful cells are right now moving out of the lab and into preclinical (animal) trials and early human clinical trials to treat disease. The results are being published in peer-reviewed scientific journal articles on stem cell transplant, injection and infusion, reprogramming, immunology, virology and tissue engineering.

Pluripotent stem cells and their derivatives are right now being studied to learn more about reproduction and development, birth defects, and the genetic origins of disease.

Embryonic, induced pluripotent, tissue specific (adult), and other types of stem cells and genetically reprogrammed cells are all being used by researchers due to the open and collaborative environment of scientific and medical enterprises in the U.S. and around the world.

All of this is happening right now because of discoveries made 20 years ago by researchers at the Wisconsin National Primate Research Center.

Here is a brief timeline of stem cell breakthroughs by WNPRC scientists:

  • 1995-James Thomson becomes the first to successfully isolate and culture rhesus monkey embyronic stem cells (ES cells) at the Wisconsin Regional Primate Research Center (PNAS)
  • 1996-Thomson repeats this feat with common marmoset ES cells (Biol Reprod).
  • 1998-Thomson publishes the neural differentiation of rhesus ES cells (APMIS).
  • 1998-Thomson’s famous breakthrough growing human ES (hES) cells is published in Science. (This research occurred off campus, with private funding.)

Many subsequent stem cell “firsts” were accomplished by scientists who conducted lengthy training with James Thomson or Ted Golos, reproduction and development scientists at the Wisconsin National Primate Research Center. These highlights include the following accomplishments by Primate Center researchers:

  • 2003-WNPRC Post-doctoral trainee Thomas Zwaka achieves homologous recombination with hES cells. A method for recombining segments of DNA within stem cells, the technique makes it possible to manipulate any part of the human genome to study gene function and mimic human disease in the laboratory dish (Nature Biotechnology).
  • 2004-WNPRC Post-doctoral trainee Behzad Gerami-Naini develops an hES model that mimics the formation of the placenta, giving researchers a new window on early development (Endocrinology).
  • 2005- WNPRC scientist Igor Slukvin and post-doc Maxim Vodyanik become the first to culture lymphocytes and dendritic cells from human ES cells (Blood, J Immunol).
  • 2005-WiCell’s Ren-He Xu, who completed his post-doctoral research at the WNPRC, grows hES cells in the absence of mouse-derived feeder cells (Nature Methods).
  • 2006-WiCell’s Tenneille Ludwig, a graduate student/post-doc/assistant scientist through the Primate Center with Barry Bavister, then James Thomson, formulates a media that supports hES cells without the need for contaminating animal products (Nature Biotechnology). Co-authoring the work is another former Primate Center post-doc, Mark Levenstein.
  • 2007-Junying Yu, WNPRC and Genome Center, in Jamie Thomson’s lab, grows induced pluripotent stem cells, or iPS cells. (Science). These are genetically reprogrammed mature cells that act like embryonic stem cells, but without the need to destroy the embryo.

Researchers at all of the National Primate Research Centers continue to make advances in this remarkable field of research and medicine. A few more milestones include the following:

  • 2007- Shoukhrat Mitalipov at the Oregon National Primate Research Center successfully converted adult rhesus monkey skin cells to embryonic stem cells using somatic cell nuclear transfer (Nature)
  • 2012- Shoukhrat Mitalipov at the Oregon National Primate Research Center generation chimeric rhesus monkeys using embryonic cells (Cell)
  • 2012-Alice Tarantal at the California NPRC successfully transplants human embryonic stem cells differentiated toward kidney lineages into fetal rhesus macaques.
  • 2013-Qiang Shi at the Texas Biomedical Research Institute and Gerald Shatten at the University of Pittsburgh – and previously with the Oregon National Primate Research Center and Wisconsin National Primate Research Center – genetically programs baboon embryonic stem cells to restore a severely damaged artery.
  • 2013-Shoukhrat Mitalipov at the Oregon National Primate Research Center produces human embryonic stem cells through therapeutic cloning, or somatic cell nuclear transfer (Cell)

NPRC Stem Cell Timeline 01.06.15

Before all of this happened, we must note that non-primate mammalian embryonic stem cells were first successfully isolated and cultured in 1981, by Martin Evans and Matthew Kaufman at the University of Cambridge, England. That breakthrough occurred almost 35 years ago. Jamie Thomson studied mouse embryonic stem cells in Pennsylvania before working on primate cells.

Even before that, in 1961, Ernest McCulloch and James Till at the Ontario Cancer Institute in Canada discovered the first adult stem cells, also called somatic stem cells or tissue-specific stem cells, in human bone marrow. That was 55 years ago.

So first it was human stem cells, then mouse, then monkey, then back to humans again. Science speaks back and forth. It reaches into the past, makes promises in the present, and comes to fruition in the future.

In every early talk I saw Jamie Thomson give about his seminal stem cell discoveries in the late 1990s and early 2000s – to staff, scientists, to the public, to Congress, to the news media – he would explain why he came to UW-Madison in the early 1990s to try to advance embryonic stem cell research. In large part, he said, it was because we had a National Primate Research Center here at UW-Madison, and also that we had leading experts in transplant and surgery at our medical school. After he joined the WNPRC as a staff pathologist and set up his lab, first he used rhesus and then marmoset embryos before expanding to cultures using human IVF patient-donated embryos off campus with private funding from Geron Corporation in Menlo Park, California.

Human And Mouse EmbryoIn these early talks, Jamie included images (see above) showing how very differently the mouse blastocyst (a days-old embryo, before implantation stage) is structured from the nonhuman primate and human primate blastocysts concerning germ layer organization and early development (ectoderm, mesoderm and endoderm). He also was able to show for the first time how differently stem cells derived from these early embryos grow in culture. In contrast to the mouse ES cells, the monkey cells, especially those of the rhesus monkey, grow in culture almost identically to human cells.

At the time, Thomson predicted that more scientists would study human ES cells in their labs over monkey ES cells, if human ES cells could become more standardized and available. Yet he emphasized that the NPRCs and nonhuman primate models would continue to play a critical role in this research, especially when it would advance to the point when animal models would be needed for preclinical research before attempting to transplant cells and tissues grown from ES cells. Both predictions have come true.

Jamie closed his talks, and still does, with this quotation:

“In the long run, the greatest legacy for human ES cells may be not as a source of tissue for transplantation medicine, but as a basic research tool to understand the human body.”

This simply and elegantly reminds us how basic research works: Many medical advances another 20 years from now will have an important link to the discoveries of today, which have their underpinnings in that early research in Jamie Thomson’s lab 20 years ago. It will become easy to forget where it all started, when many diseases of today, if not completely cured, will become so preventable, treatable and manageable that those diagnosed with them will spend more time living their lives than thinking about how to survive another day.

Just as I did not have to worry about polio, and my children did not have to worry about chicken pox, my grandchildren will hopefully see a world where leukemia, blindness, diabetes and mental illness do not have the disabling effects or claim as many young lives as they do today.

***

_______________________________________________________

WN@tL “Twenty Years of Stem Cell Milestones at the UW”

http://www.uwalumni.com/event/wntl-twenty-years-of-stem-cell-milestones-at-the-uw/

January 7 – 7:00PM – 8:15PM CT
Location: UW Biotechnology Center 425 Henry Mall, Room 1111, Madison, WI 53706
Cost: Free

Speaker: William L. Murphy, Stem Cell and Regenerative Medicine Centerwnatl_williammurphy

Don’t miss this fascinating talk covering stem cell milestones at the UW. Professor Murphy will talk about the work of his team at the Stem Cell and Regenerative Medicine Center, where they are creating biological materials that could radically change how doctors treat a wide range of diseases.

Bio: Murphy is the Harvey D. Spangler Professor of Engineering and a co-director of the Stem Cell and Regenerative Medicine Center. His work includes developing biomaterials for stem cell research. Specifically, Murphy uses biomaterials to define stem cell microenvironments and develop new approaches for drug delivery and gene therapy. His lab also uses bio-inspired approaches to address a variety of regenerative medicine challenges, including stem-cell differentiation, tissue regeneration and controlled drug delivery. Murphy has published more than 100 scientific manuscripts and filed more than 20 patent applications.

Peritoneal Carcinosis and HIPEC: A second chance for patients, thanks to animal research

When we hear the phrase ‘animal research’ we tend to think about the development of new drugs for the clinical practice, or studying molecular pathways involved in the progression of disease; but we must also remember that the techniques used in the operation room are a consequence of biomedical research, including the use of animals. It is not just the creation of these techniques but also for the prior steps necessary for us to consider a surgical technique as an option when faced with a disease. An example of this is research into a type of cancer known as Peritoneal Carcinosis (PC) and the development of a technique, known as HIPEC, that may dramatically improve the prognosis for patients with this type of cancer.

What is the definition of Peritoneal Carcinosis? We describe this medical condition as the presence of neoplastic nodules caused by the spreading of a primary or secondary tumor in the peritoneal cavity. The peritoneal cavity, also called the abdominal cavity, is the largest body cavity and contains many of the major organs – such as the liver, kidneys, stomach and intestines – surrounded by a protective membrane known as the peritoneum.

Although PC is sometimes seen in primary tumours, such as peritoneal mesothelioma or Pseudomyxoma peritoneii, it is more frequently observed as a metastatic diffusion of gastroenteric (stomach and colon, primary) or gynaecologic (ovarian) tumors. In the second situation, we could see it as an advanced manifestation present at the same time as the primary neoplastic disease or appearing in the years following treatment of the tumour. This condition is often associated with a poor prognosis (about 6 months), depending on the site to which it spreads, the involvement of abdominal organs (like colon or liver) and how aggressive is the tumor at the moment of diagnose.

Peritoneal Carcinosis viewed by laparoscopy. Image: www.cancersurgery.us

Peritoneal Carcinosis viewed by laparoscopy. Image: http://www.cancersurgery.us

In the past, physicians have had only two options when combating the disease: systemic chemotherapy or palliative surgical therapy to debulk the tumor masses- removing as much as possible of tumors which cannot be entirely removed –  and prevent severe conditions such as bowel obstruction. Recently, surgical research developed another therapeutic approach, known as Cytoreduction (CR) associated with Hyperthermic intraperitoneal Chemotherapy (HIPEC). This technique consists of a two-part operation: during the first part, the surgeon debulks as much of the neoplastic nodules in the peritoneal cavity as possible, and in the second stage the peritoneal cavity is washed with a hyperthermic chemotherapy solution, where a solution containing a high concentration of chemotherapy drugs is heated to above body temperature (usually 41.5°-42.5°C) which increases absorption of the drugs by the target tumor and therefor their effectiveness.

The role of the hyperthermic solution and the possibility of using a high-dose of chemotherapic agent was developed through research in rodents and dogs: these studies demostrated that the peritoneal barrier itself is not a barrier that prevents substances from pass through it. This is in agreement with observations made during surgery in human patients, when we remove the peritoneum (for example, when we debulk a neoplastic nodule on a peritoneal surface with a technique known as peritonectomy) the rate at which drugs are cleared from peritoneal cavity is not significantly affected. [1]

Studies in dogs and subsequently in human volunteers demonstrated that the high concentration of chemotherapeutic drugs in the peritoneal cavity is not related to a high concentration of these in the blood stream [2]. In particular a key study undertaken in dogs by Rubin et al. [3], consisted of studying the effects of removing portions of the perotineum such as the the omentum, the mesentery or the small bowel on the clearance of substances like glucose, urea and insulin from the peritoneal cavity. Surprisingly, this experiment indicated that these operations do not influence the clearance of these substances. On the base of these observation, clinical studies were started on clearance of drugs from the peritoneal compartment:. These clinical studies demonstrated that the process observed in dog with other substances occured also with drugs and that, in some cases, the concentration of a drug within the peritoneal cavity could be extremely high without having effects on the concentration in the bloodstream.

A natural consequence of this evidence is that we can use a high-dose chemotherapy drug against these nodules without having systemic adverse effects on the patient, a problem frequently observed in conventional systemic chemotherapy. These studies also led researchers to reconsider the spreading of a tumour in the peritoneal cavity not as a systemic dissemination but as a local disease, and that treatment might be able to cure it rather than just have a palliative impact. If the peritoneal barrier can selectively allow only some molecules to pass through, it could have also an active role on slowing the diffusion of metastatic cancer cells.

This evidence, together with the property of hyperthermia in helping drugs to penetrate cancer cells [4], and avoid the normal defences that a tumor cell has, led to development of this ambitious surgical technique.

The results of this combined technique is clear. Against primary tumors this technique shows a high survival-rate after 5 years (reaching 96% in some studies [5]). Against secondary spreading of gastroenteric or gynaecological tumours it shows a lower efficacy that may be related to the more diverse biological characteristics of the tumor cells, to the physiopathological features (diffusion, tumor already treated with chemotherapy etc.) and also to the characteristics of the patient (such as clinical status, age, concomitant diseases) [6],[7],[8],[9]. The 5-years survival rate for PC from colorectal cancer, for example, according to studies conducted by Dr. Paul Sugarbaker of the Washington Cancer Institute, one of the most important researcher on this field, is around 40%, when the cytoreduction is complete and the disease is not so diffuse in the peritoneal cavity. [7] Also, this surgical approach can be uses a second time, in case of a recurrence of PC, and, ultimately, as a palliative treatment to delay complications and reduce suffering of the cancer patients.

These numbers could seem low but we have to consider that we’re facing a disease that is often fatal within six months if left untreated. This technique gives patients another chance until very recently, they did not have. Why? Because of research that was built up, in part, thanks to animal research

These results are a direct effect of research in the fields of surgery and oncology, from the including the development of more effective chemotherapic agents, research that, as we have said many times, requires the study of animals for everything from the basic understanding of the processes involved to the preclinical testing a new therapy’s effectiveness and safety profile.

Marco Delli Zotti

[1] Michael F. Flessner “The transport barrier in intraperitoneal therapy” Am J Physiol Renal Physiol 288:F433-F442, 2005. http://www.ncbi.nlm.nih.gov/pubmed/15692055

[2] Pierre Jacquet, Andrew Averbach, Arvil D. Stephens, O. Anthony Stuart, David Chang, Paul H. Sugarbaker “Heated Intraoperative Intraperitoneal Mitomycin C and Early Postoperative Intraperitoneal 5-Fluorouracil: Pharmacokinetic Studies” Oncology 1998;55:130–138 http://www.ncbi.nlm.nih.gov/pubmed/9499187

[3] Rubin J, Jones Q, Planch A, Rushton F, Bower J. “The importance of the abdominal viscera to pertioneal transport during peritoneal dialysis in the dog.” Am J Med Sciences 1986;292:203– 208. http://www.ncbi.nlm.nih.gov/pubmed/3752166

[4] Elwood P. Armour, Donna McEachern, Zhenhua Wang, et al. “Sensitivity of Human Cells to Mild Hyperthermia” Cancer Res 1993;53:2740-2744. http://www.ncbi.nlm.nih.gov/pubmed/8504414

[5] Yan TD, Black D, Savady R et al. “Systematic review on the efficacy of cytoreductive surgery and perioperative intraperitoneal chemotherapy for pseudomyxoma peritonei.” Ann Surg Oncol 2007;14:484-92 http://www.ncbi.nlm.nih.gov/pubmed/17054002

[6] Franco Roviello, Daniele Marrelli, Alessandro Neri, Daniela Cerretani, Giovanni de Manzoni, Corrado Pedrazzani, MD, Tommaso Cioppa, MD, Giacomo Nastri, MD, Giorgio Giorgi, Enrico Pinto
“Treatment of Peritoneal Carcinomatosis by Cytoreductive Surgery and Intraperitoneal Hyperthermic Chemoperfusion (IHCP): Postoperative Outcome and Risk Factors for Morbidity” World J Surg (2006) 30: 2033–2040 http://www.ncbi.nlm.nih.gov/pubmed/17006608

[7] Paul H. Sugarbaker “Review of a personal experience in the Management of Carcinomatosis and Sarcomatosis” Jpn J Clin Oncol 2001; 31(12)573-583 http://www.ncbi.nlm.nih.gov/pubmed/11902487

[8] Zanon C, Bortolini M, Chiappino I et al. “Cytoreductive surgery combined with intraperitoneal chemohyperthermia for the treatment of advanced colon cancer.” World J Surg. 2006 Nov;30(11):2025-32. http://www.ncbi.nlm.nih.gov/pubmed/17058031

[9] Bijelic L, Jonson A, Sugarbaker PH “Systematic review of cytoreductive surgery and heated intraoperative intraperitoneal chemotherapy for treatment of peritoneal carcinomatosis in primary and recurrent ovarian cancer.” Ann Oncol 2007;18:1943-50 http://www.ncbi.nlm.nih.gov/pubmed/17496308

To learn more about the role of animal research in advancing human and veterinary medicine, and the threat posed to this progress by the animal rights lobby, follow us on Facebook or Twitter.